Description:FIELD OF INVENTION:
The present invention relates to the field of green synthesis. Particularly, the present invention relates to a method using aqueous leaf extract of Murraya koenigii (Curry leaves) to synthesize silver-silver chloride (Ag-AgCl) nanocomposites (NCs). Further, the present invention provides a silver-silver chloride (Ag-AgCl) nanocomposites (NCs) effective against Candida albicans at very low concentrations.

BACKGROUND OF THE INVENTION:
The formulation of Ag-AgCl NC addresses the challenge of inhibiting C. albicans biofilm formation. C. albicans is a type of yeast that commonly resides in the human body, but it can cause infections, particularly in individuals with weakened immune systems or those undergoing certain medical treatments. One of the ways C. albicans can cause infections is by forming biofilms, which are complex communities of microorganisms encased in a protective matrix. Biofilms are notoriously difficult to eradicate and can be resistant to conventional antimicrobial treatments.
Currently, there is a lack of research specifically focused on the green synthesis of Ag-AgCl NC (Ag-AgCl NC) and their potential as antifungal agents to inhibit the virulence biofilm of C. albicans. However, there are few reports which suggested the antifungal potential of Ag-AgCl NC, Geomar F. Cruz and colleagues (2020) developed a method for producing photochemically-generated Ag-AgCl NC, which are stabilized by a peptide inhibitor of cell division, and assessed their antimicrobial properties. In this study, Geomar F. Cruz and colleagues synthesized plasmonic Ag-AgCl NC stabilized by peptides via a rapid and environmentally friendly method under blue light irradiation. Electron microscopy revealed a peptide layer enveloping Ag-AgCl NC, ensuring colloidal stability. X-ray diffraction analysis confirmed the crystalline nature of the Ag-AgCl NC, with a minor presence of metallic silver. Nevertheless, Ag-AgCl NC displayed notable antibacterial efficacy against the Gram-positive bacterium B. megaterium.

In another study, Yun Ok Kang et al. developed the chitosan oligomer stabilized Ag-AgCl NC display antimicrobial properties. The study demonstrated the straightforward and eco-friendly approach to produce chitosan oligomer-stabilized Ag-AgCl NC (CHI-Ag-AgCl NC), serving as a source of chloride ions and a stabilizing agent, with anticipated synergistic effects. As a result of combining CHI and Ag-AgCl NC, the synergistic impact of CHI-Ag-AgCl NC on antibacterial activity was significantly enhanced. This investigation offers further understanding of the role played by CHI in the formation of CHI-Ag-AgCl NC.

Ag-AgCl NC have shown antimicrobial properties, including activity against C. albicans. By formulating Ag-AgCl NC, there arise a need to enhance the efficacy of Ag-AgCl NC in inhibiting C. albicans biofilm formation. Ag-AgCl NC may offer several advantages over bulk silver chloride or other forms of Ag-AgCl NCs, such as improved stability, controlled release of silver-chloride ions, and enhanced interaction with microbial cells.

Partha et al., Inorganic Chemistry Communications, Volume 152, June 2023, 110676 discloses green synthesis of silver nanoparticles using Murraya koenigii leaf extract with efficient catalytic, antimicrobial, and sensing properties towards heavy metal ions. The document discloses to synthesize silver nanoparticles (AgNPs) using aqueous Murraya Koenigii leaf extract through a simple easy and green approach.
S. R. Bonde,D. P. Rathod,A. P. Ingle,R. B. Ade,A. K. Gade &M. K. Rai
Pages 25-36, Published online: 15 Mar 2012, discloses Murraya koenigii-mediated synthesis of silver nanoparticles and its activity against three human pathogenic bacteria. The document discloses synthesis of silver nanoparticles (Ag NPs) by the leaf extract of Murraya koenigii (Indian curry leaf tree) is reported in this study. The colour of the leaf extract prepared by grinding turned from green to brown after treatment with AgNO3 (1 mM).

V. Gopinath, S. Priyadarshini, N. Meera Priyadharsshini, K. Pandian, P. Velusamy Biogenic synthesis of antibacterial silver chloride nanoparticles using leaf extracts of Cissus quadrangularis Linn discloses single step method for the synthesis of silver chloride nanoparticles (AgCl-NPs) using leaf extract of Cissus quadrangularis. The AgCl-NPs were characterized by UV–Vis absorption spectroscopy and Fourier transform infra-red spectrum analysis.
Nisa et al, January 2024, Results in Chemistry 7:101287, January 20247:101287 discloses green synthesis of silver/silver chloride nanoparticles derived from Elaeocarpus Floribundus leaf extract and study of its anticancer potential against EAC and MCF-7 Cells with Antioxidant and Antibacterial Properties.
Shazia et al., Molecules 2023, 28(6), 2500 discloses a novel based synthesis of silver/silver chloride nanoparticles from stachys emodi efficiently controls Erwinia carotovora, the causal agent of blackleg and soft rot of potato.

The inhibitory action of Ag-AgCl NC on C. albicans biofilm formation could potentially lead to the development of novel strategies for preventing or treating C. albicans infections, particularly those associated with biofilm formation. This could be of significant clinical importance, especially in settings such as hospitals where biofilm-associated infections can pose serious challenges to patient care and treatment outcomes.

There arises a need to synthesize Ag-AgCl NC with curry leaves, which inhibits Candida albicans at a very low concentration (2.7 µg/mL).

Given the above, there arises a need to develop an improved arrangement and a process that eliminates the problems associated with the earlier processes. The present invention provides a better solution for the same.

OBJECTIVE OF THE INVENTION:
An objective of the present invention is to provide a silver-silver chloride (Ag-AgCl) nanocomposite (NCs).
Another object of the present invention is to provide a process for the preparation of silver-silver chloride (Ag-AgCl) nanocomposites (NCs).
Another object of the present invention is to provide an application of Ag/AgCl NC as a potential antifungal against Candida albicans.

SUMMARY OF THE INVENTION:
Accordingly, the present invention provides a process for the preparation of silver chloride (Ag-AgCl) nanocomposite comprising obtaining a curry leaf extract; obtaining a silver nitrate solution; mixing the curry leaf extract with silver nitrate solution; centrifuging and washing to obtain Ag-AgCl nanocomposites.

In an embodiment, the present invention provides that the curry leaf extract is obtained by boiling clean chopped curry leaves, cooling and filtering the curry leaves.

In an embodiment, the present invention provides that the curry leaves and water is 1:8-1:12.

In an embodiment, the present invention provides that the curry leaves are boiled in water for 20-60 min.

In an embodiment, the present invention provides that the boiled curry leaves are filtered with Whatman filter paper 1.

In an embodiment, the present invention provides that the silver nitrate solution has a concentration of 1 mM.

In an embodiment, the present invention provides that 10 mL of the prepared curry leaf extract is added to 120 mL of the 1 mM silver nitrate solution.

In an embodiment, the present invention provides that the mixture of the curry leaf extract and the at room temperature for 2-6 hours.

In an embodiment, the present invention provides that the curry leaf extract facilitate the reduction of silver ions to form Ag-AgCl NC.

In an embodiment, the present invention provides that the colloidal solution containing the synthesized Ag-AgCl NC is subjected to centrifugation at 12000 rpm for 20 to 30 minutes.

In an embodiment, the present invention provides that the antifungal activity of Ag-AgCl NC of bio film formation in C. albicans ranges from 15 µg/mL - 1.79 µg/mL respectively at which a 50% reduction in adhesion (MIC50).

In an embodiment, the present invention provides that the Ag-AgCl nanocomposite has a diameter ranging from 10 to 80 nm.

BRIEF DESCRIPTION OF DRAWINGS:
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read concerning the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

Fig. 1 Preparation of aqueous leaf extract of Curry leaves (Murraya koenigii) and synthesis of Ag-AgCl NC;

Fig. 2 Characterization of Ag-AgCl Nc (a) UV-vis spectrum of Ag-AgCl NC and aqueous extract of M. koenigii;

Fig 3: (a-c) TEM micrographs (a, b and c) revealed of monodispersed Ag-AgCl NC at different magnifications with diameters ranging from 10 to 80 nm, enveloped by a faint, thin layer of a biomolecule corona (a layer of biomolecules attached to the Ag-AgCl NC). (d) The selected area electron diffraction (SAED) pattern (d) of the Ag-AgCl NC displayed circular fringes, indicating their polycrystalline nature;

Fig 4: EDX Elemental analysis spectrum of the Ag-AgCl NC. The spectrum displayed notable peaks for Cl and Ag at 2.6 keV and 3 keV, respectively, confirming the presence of these elements;

Fig 5: Antifungal assays of Ag-AgCl NC on C. albicans
(a) The inhibitory potential of Ag-AgCl NC against the planktonic growth of C. albicans.
(b) The adhesion inhibition capacity of Ag-AgCl NCs against C. albicans was investigated by monitoring cell adhesion on polystyrene surfaces.
(c) Observations under a field emission electron microscope (10000X magnification) revealed that the presence of Ag-AgCl NC inhibited morphogenesis, as evidenced by the prevalence of oval-shaped cells. (i) Control (ii) fluconazole treated (iii) to viii) treated with 0.46 to 15 µg/mL of Ag-AgCl NC;

Fig 6: Biofilm inhibition assay and RT-PCR analysis (a) Ag-AgCl NCs exhibited inhibition of biofilm formation that depended on their concentration (b) Confocal microscopy images of biofilm inhibition treated with Ag-AgCl NC and fluconazole at 20X magnification (i) Control (ii) fluconazole treated (iii to viii) treated with 0.46 to 15 µg/mL of Ag-AgCl NC; and

Fig 7: Cytotoxicity testing of Ag-AgCl NCs was conducted using 3T3L1 fibroblast cells derived from mouse embryos.

Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. Furthermore, the drawings may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.

DETAILED DESCRIPTION:
To promote an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment”, and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting. Embodiments of the present invention will be described below in detail concerning the accompanying drawings.

The present invention relates to a method using aqueous leaf extract of Murraya koenigii (Curry leaves) to synthesize silver-silver chloride (Ag-AgCl) nanocomposites (NCs). The Ag-AgCl NCs are evaluated for their potential to inhibit planktonic growth, morphogenesis, adhesion, and biofilm formation in Candida albicans (C. albicans). The present invention provides an effectiveness of green-synthesized Ag-AgCl NCs as potent antifungal agents with a minimum inhibitory concentration (MIC50) of 2.7 µg/mL against biofilm-forming C. albicans. The present invention provides that these green-synthesized Ag-AgCl NCs could be developed into antifungal nano formulations for treating C. albicans infections and mitigating their virulence.

Green synthesis and characterization of Ag-AgCl NC to inhibit the virulence of biofilm formation in C. albicans, is to offer a sustainable and effective solution for combating biofilm formation by C. albicans. By using green synthesis methods to produce Ag-AgCl NC, the invention aims to leverage the antimicrobial properties of silver chloride while minimizing environmental impact. Characterization of these nanoparticles ensures their stability and efficacy. Ultimately, this invention seeks to provide a novel approach for inhibiting the virulence of C. albicans biofilm formation, with potential benefits for medical, pharmaceutical, and environmental applications.

M. koenigii leaves exhibit promising antifungal properties. Consequently, the present invention provides the utilization of aqueous leaf extract of M. koenigii as a reducing agent for the eco-friendly synthesis of Ag-AgCl NC. The reduction of silver nitrate in aqueous solutions using a green reducing agent represents the predominant method for producing Ag-AgCl NC in an environmentally sustainable manner. The process of synthesizing Ag-AgCl NC through green synthesis is characterized by cleanliness, safety, cost-effectiveness, and environmental friendliness.

Synthesis and characterization of Ag-AgCl NC using curry leaf extract involves a green and eco-friendly approach, utilizing the reducing and capping properties of phytochemicals present in curry leaves. The process typically consists of several steps including preparation of the curry leaf extract, synthesis of Ag-AgCl NC, and characterization of the synthesized NC.

The process for the synthesis comprises:
A) The preparation of curry leaf extract for the synthesis of Ag-AgCl NC:
1. Acquisition of Fresh Curry Leaves: Fresh curry leaves are obtained from a local market in Pune, India. It's important to ensure that the leaves are fresh and free from any visible signs of spoilage.
2. Cleaning and Washing of Curry Leaves: The acquired curry leaves are thoroughly cleaned to remove any dirt, dust, or other contaminants. They are washed three times with deionized water to ensure cleanliness.
3. Chopping of Curry Leaves: After cleaning, the curry leaves are chopped into small pieces or crushed. This process increases the surface area of the leaves, facilitating the extraction of phytochemicals during boiling.
4. Boiling of Curry Leaves: The 10 gm chopped curry leaves are then transferred to a container, and a measured volume of deionized water (100 mL) is added to them. The mixture is then boiled for 30 minutes. Boiling helps extract the bioactive compounds present in the curry leaves, which will serve as reducing and capping agents during the synthesis of Ag-AgCl NC.
5. Cooling and Filtration: After boiling, the leaf broth is allowed to cool down to room temperature. Once cooled, the mixture is filtered through Whatman No. 1 filter paper. This filtration process helps remove any solid residues or insoluble impurities, yielding a clear curry leaf extract rich in phytochemicals.

Following these steps, the prepared curry leaf extract is ready to be used as a green and eco-friendly reducing and capping agent in the synthesis of Ag-AgCl NC. This approach offers a sustainable alternative to traditional chemical methods, utilizing natural plant extracts for nanoparticle synthesis.

B) The synthesis of Ag-AgCl NC using the curry leaf extract involves the reduction of silver ions (Ag⁺) from silver nitrate (AgNO₃) solution by the phytochemicals present in the curry leaf extract.

1. Preparation of Silver Nitrate Solution: An aqueous solution of silver nitrate (AgNO₃) is prepared with a concentration of 1 mM. This solution serves as the precursor for the synthesis of Ag-AgCl NC.

2. Reaction with Curry Leaf Broth:
10 mL of the prepared curry leaf broth is added to 120 mL of the 1 mM silver nitrate solution.
The mixture is allowed to react at room temperature for 4 hours. During this time, the reducing agents present in the curry leaf extract facilitate the reduction of silver ions to form Ag-AgCl NC. The colour change of the reaction mixture from transparent yellow to dark brown indicates the formation of Ag-AgCl NC. This change in colour is often attributed to the surface plasmon resonance phenomenon exhibited by the nanoparticles.

3. Centrifugation and Washing:
After the completion of the reaction, the colloidal solution containing the synthesized Ag-AgCl NC is subjected to centrifugation at 12000 rpm for 30 minutes. Centrifugation helps separate the nanoparticles from the reaction mixture. Following centrifugation, the colloidal solution is washed thrice with distilled water. Washing helps remove any unreacted reactants, by-products, or excess curry leaf extract from the surface of the nanoparticles.

After centrifugation and washing steps, the purified Ag-AgCl NC can be characterized using various analytical techniques to determine their size, morphology, stability, and other physicochemical properties. These nanoparticles hold promise for various applications in fields such as catalysis, sensing, biomedical engineering, and environmental remediation.

The following examples define the invention by way of illustration which does not limit the scope of the invention.

Example 1:
Synthesis, characterization and anti-candidal application of Ag-AgCl NC
The characterization of Ag-AgCl NC involves a variety of analytical techniques to understand their size, morphology, crystalline structure, surface functional groups, and surface charge.

The UV-Vis spectrum of the reaction medium was measured after 4 hours to monitor the formation of Ag-AgCl NC. Spectral analysis is conducted using a UV-1800 Spectrophotometer, with distilled water as the baseline reference as illustrated in Fig. 2. The UV-Vis spectrum of the Ag-AgCl NC colloidal solution was analysed between 200 nm and 800 nm. Additionally, X-ray powder diffraction (XRD) measurements were performed using a Philips X'Pert PRO diffractometer to identify the phases of the Ag-AgCl NC. To analyse the functional groups and capping molecules present on the surfaces of Ag-AgCl NC, Fourier transform infrared (FTIR) spectroscopy was conducted. The FTIR spectroscopy studies covered a spectral range from 4000 to 400 cm-1. Zeta potential values, providing insights into surface charges on Ag-AgCl NC, were measured using a zeta potential analyser (Brookhaven Instruments, USA). High-resolution morphological studies and electron diffraction to determine morphology, size, and fringes spacing of Ag-AgCl NC were performed using a transmission electron microscope (TEM, FEI Technonai T20, Netherlands). The distribution of Ag and Cl elements was observed using energy dispersive X-ray spectroscopy (EDX, Bruker Flash 6I30) Fig. 4.

Example 2:
Application of Ag-AgCl NC to inhibit the formation of biofilm in C. albicans:
2.1. Medium and Culture Conditions
The C. albicans culture was maintained on YPD agar medium and stored at 4°C. A sterile 20 mL flask containing YPD broth was inoculated with a single colony of C. albicans isolated from a YPD agar plate. The flask was then incubated at 30°C on an orbital shaking incubator (120 rpm) overnight. Cells were harvested by centrifugation at 2000 rpm, washed three times with 0.1M phosphate-buffered saline (pH 7.4), and the cell density was measured using a hemacytometer.

2.2. Effect of Ag-AgCl NC on the planktonic Growth of C. albicans:
To assess the impact of different concentrations of Ag-AgCl NC on the growth of C. albicans in a planktonic state, we employed a standard broth microdilution method following Clinical Laboratory Standards Institute guidelines. Various concentrations of Ag-AgCl NC, ranging from 0.46 to 15 µg/mL, were prepared in 96-well plates using RPMI-1640 medium. Fluconazole (0.46 to 15 µg/mL) served as the standard antifungal drug for comparison (Fig. 5). Each well received RPMI-1640 medium containing a C. albicans cell suspension (1x104 cells/mL), with the total volume adjusted to 200 µL of RPMI-1640. Control wells lacked Ag-AgCl NC. The plates were then incubated at 37°C for 24 hours, and the absorbance at 620 nm was measured using a microplate reader (Hidex, Germany). The minimum inhibitory concentration (MIC50) was defined as the concentration of Ag-AgCl NC at which cell growth was reduced by 50% compared to the control condition. All experiments were conducted in triplicate.

2.3. Adhesion Assay:
The adhesion of cells to the polystyrene surface was assessed using 96-well plates. Each well was prepared with double dilutions of various concentrations (ranging from 0.46 to 15 µg/mL) of Ag-AgCl NC in phosphate-buffered saline (PBS). A 50 µL cell suspension was added to each well to achieve a cell density of 1x107 cells/mL, while control wells were not treated with Ag-AgCl NC. Fluconazole (0.46 to 15 µg/mL) served as the standard antifungal drug for comparison. The plates were then incubated at 37°C for 90 minutes. After incubation, the wells were washed with sterile PBS to remove non-adherent cells. The MTT metabolic assay was employed to determine the density of adhered cells, with color formation measured at 520 nm using a microplate reader (Hidex, Germany). The concentration of Ag-AgCl NC at which a 50% reduction in adhesion was observed compared to the control was considered the MIC50. All experiments were conducted in triplicate.

2.4. Inhibition of Morphogenesis Assay:
The yeast-to-hyphal form transition assay was conducted in 24-well plates. Morphogenesis inhibition was assessed in a 20% fetal bovine serum (FBS) medium. Various concentrations of Ag-AgCl NC (ranging from 0.46 to 15 µg/mL) were individually incubated with 1x106 cells/mL in 20 mL of 20% FBS at 37°C for 90 minutes. Fluconazole (0.46 to 15 µg/mL) served as the standard antifungal drug for comparison. After incubation, all samples were centrifuged separately at 2000 rpm, 28°C. Following centrifugation, the supernatant was discarded, and cells were washed twice with sterile distilled water. Field emission scanning electron microscopy (FEI Nova Nano SEM 450) was utilized to study morphogenesis, particularly the induction of germ tubes. Inhibition of germ tube formation in Ag-AgCl NC-treated samples was considered indicative of morphogenesis inhibition compared to controls (without Ag-AgCl NC).

2.5. Field emission scanning electron microscopy (FESEM) for Morphogenesis study:
Sterile silicon wafer pieces were utilized to drop-cast Ag-AgCl NC, fluconazole-treated, and control samples from the morphogenesis inhibition assay. Subsequently, the drop-casted silicon wafer pieces were allowed to air-dry. Following this, all cells drop-casted onto the silicon wafer pieces were fixed for 24 hours at 4°C in 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline. Post-fixation of the silicon wafer pieces was achieved by applying a 2% osmium tetraoxide aqueous solution for 4 hours, followed by dehydration in a series of graded alcohol solutions, and subsequent drying. All samples were then gold-coated for 15 minutes using an automated gold coater. Images were captured using a field emission scanning electron microscope (FESEM) at 10,000X magnification.

2.6. C. albicans Biofilm Inhibition Assay:
The biofilm inhibition assay was conducted in polystyrene-treated 96-well plates following standard protocols. Each well was initially seeded with 100 µL of cell suspension (1 x 107 cells/mL) and allowed to adhere to the polystyrene surface for 90 minutes at 37°C. After adhesion, the cells were washed with phosphate-buffered saline (PBS). In a separate 96-well plate, Ag-AgCl NC concentrations ranging from 0.46 to 15 µg/mL were prepared in RPMI-1640 medium. These concentrations (200 µL each) were added to the wells containing adhered cells, and the plates were then incubated at 37°C for 24 hours. Fluconazole (0.46 to 15 µg/mL) served as the standard antifungal drug for comparison. After 24 hours, non-adhered cells were removed, and the biofilms were washed with PBS. Subsequently, a 50μL solution of MTT dye (2 mg/mL) was added to all wells and kept at room temperature for 4 hours in the dark. Following incubation, 100 μL of DMSO was added to each well, and optical absorption was measured at 580 nm using a microplate reader (Hidex, Germany). The biofilm formation was quantified using the MTT metabolic assay, and the MIC50, representing the concentration of Ag-AgCl NC where a 50% reduction in adhesion occurred compared to the control, was determined. All experiments were performed in triplicate.

2.7. Confocal Microscopy of C. albicans Biofilm Inhibition Assay:
Fluorescent staining was employed to visualize fungal biofilm formation at various concentrations of Ag-AgCl NC, ranging from 0.46 to 15 µg/mL. After washing the biofilm with PBS, 25 μL of Con-A Alexa (1 μg/mL) was added to each well. Incubation proceeded for 45 minutes, followed by repeated washings to remove excess stain. Samples were then placed for observation under a confocal microscope (NIKON A1 R) at a magnification of 20X, with excitation and emission wavelengths of 480 nm and 520 nm, respectively. Control samples underwent the same staining procedure but without the presence of Ag-AgCl NC. The standard drug fluconazole (MIC50 concentration) was used to assess biofilm inhibition.

Example 3:
In-vitro toxicity investigation of Ag-AgCl NC
In-vitro cell cytotoxicity investigation of Ag-AgCl NC on 3T3-L1 fibroblast cell line:
A cytotoxicity study (Fig. 7) of Ag-AgCl NC was conducted using the 3T3-L1 fibroblast cell line isolated from mouse embryos, obtained from NCCS Pune. The 3T3-L1 cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and incubated in a 5% CO2 incubator at 37°C. For the assay, 100 μL of 3T3-L1 cells (1x105 cells/well) were seeded into 96-well plates and treated with various concentrations of Ag-AgCl NC (ranging from 0.46 to 7.5 µg/mL) for 24 hours. Subsequently, 50 µL of MTT solution (2 mg/mL) per well was added and incubated for an additional 4 hours at 37°C with a 5% CO2 supply. After incubation, the plate was centrifuged at 2000 rpm for 10 minutes, and 100 µL of DMSO was added to each well. The optical density of the samples was measured at 570 nm using a microplate reader (Hidex, Germany).

Advantages of the Invention:
The present invention provides that curry leaf can be used for the synthesis of Ag-AgCl NC and that it has antifungal activity against C. albicans at very low concentrations. In present study we have synthesized Ag-AgCl NC utilizing an aqueous leaf extract derived from Curry leaves (Murraya koenigii). This marks the inaugural documentation showcasing the utilization of Curry leaf extract for the synthesis of Ag-AgCl NC. While previous literature on Ag-AgCl NC as antifungal agents inhibiting fungal growth is limited, our research represents the pioneer endeavour exploring the efficacy of Ag-AgCl NC as an antifungal agent to impede the virulent biofilm formation in C. albicans. Moreover, we have identified, for the first time, the capacity of Ag-AgCl NC to hinder the planktonic growth of C. albicans at both MIC (Minimum Inhibitory Concentration) and MIC50 levels, measuring at 15 µg/mL and 1.79 µg/mL respectively. The M. koenigii-mediated Ag-AgCl NCs exhibited superior antiadhesion properties compared to the standard drug fluconazole. The MIC50 of Ag-AgCl NC and fluconazole was found to be 3.2 μg/mL and 13 μg/mL, respectively. Additionally, our synthesized Ag-AgCl NC effectively curtail the yeast-to-hyphae morphogenic transition at a low concentration of 0.46 µg/mL in C. albicans. Notably, our findings are first report which reveal the inhibitory effect of Ag-AgCl NC on biofilm formation in C. albicans at MIC and MIC50 concentrations of 15 µg/mL and 2.7 µg/mL, respectively. Upon reviewing comparable studies (Fig. 6), it's evident that the observed inhibitory concentrations are notably low, thus underscoring the promising potential of our synthesized Ag-AgCl NC as a formidable agent against biofilm formation in C. albicans.
, Claims:1. A process for the preparation of silver chloride (Ag-AgCl) nanocomposite comprising:
a. obtaining a curry leaf extract to obtain an extract;
b. heating the curry leaf extract;
c. obtaining a silver nitrate solution;
d. mixing the curry leaf extract with silver nitrate solution; and
e. centrifuging and washing to obtain Ag-AgCl nanocomposites.

2. The process as claimed in claim 1, wherein the curry leaf extract is obtained by boiling clean chopped curry leaves, cooling and filtering the curry leaves.

3. The process as claimed in claim 1, wherein the ratio of the curry leaves and water is 1:8-1:12.

4. The process as claimed in claim 1, wherein the curry leaves are boiled in water for 20-60 min and filtered with Whatman filter paper 1.

5. The process as claimed in claim 1, wherein the silver nitrate solution has a concentration of 1mM to 5mM silver nitrate and 5 to 15mL extract in 120 ml silver nitrate solution.

6. The process as claimed in claim 1, wherein the mixture of the curry leaf extract and the at room temperature for 30 minutes to 90 minutes.

7. The process as claimed in claim 1, wherein the curry leaf extract facilitate the reduction of silver ions to form Ag-AgCl NC.

8. The process as claimed in claim 1, wherein the colloidal solution containing the synthesized Ag-AgCl NC is subjected to centrifugation at 9000 to 12000 rpm for 30 to 90 minutes.

9. The process as claimed in claim 1, wherein the antifungal activity of Ag-AgCl NC of bio film formation in C. albicans ranges from 15 µg/mL - 1.79 µg/mL respectively at which a 50% reduction in adhesion (MIC50).

10. The process as claimed in claim 1, wherein the Ag-AgCl nanocomposite has a diameter ranging from 10 to 80 nm.