The Biological Synthesis of Silver Nanoparticles by Mango Plant Extract and Its Anti-Candida Effects

Among the main causes of infectious diseases with Candida fungal infections are a very important model of human pathogens. Candida albicans is a dwarf fungus and an important component of the natural flora of healthy people. In a situation where the immune system is weakened, Candida, as an opportunistic pathogen, can develop diseases after transferring from the symbiotic phase to the pathogenic phase. Different species of this yeast are able to form biofilms and are the leading cause of mortality in immunocompromised patients. Since they cause invasive candidiasis, it is difficult to eradicate them using antifungal therapy. In fighting against the fungal infections, 3 main flaws can be considered for some antifungal drugs: first, they operate in a limited range, second they can negatively react with different types of antifungal agents, and third they can make the microorganisms resistant. In addition, despite the advances in the treatment of fungal infections using antifungal compounds over the past thirty years, the antifungal resistance is still a major concern in clinical practice. In general, the advancement in the production of new antibiotics involves long and costly processes that can now be solved with the development of nanoscience science.1 Nano-biotechnology has been making progress in recent decades and will have widespread effects on various aspects of human life including pharmacy and medicine in the near future. Nowadays, the growing need for reliable methods for making nanoparticles which do not harm the environment has made the researchers extremely interested in this field. Many organisms, whether monocellular or multicellular, are known to produce inorganic nanoparticles acting as intracellular or extracellular. Plant extracts are among the biological systems which have been recently considered in this field. Mango is one of the most popular tropical fruits in most parts of the world which is cultivated abundantly in South Asian countries. Mangoes belong to the Mangifera The Biological Synthesis of Silver Nanoparticles by Mango Plant Extract and Its Anti-Candida Effects


Introduction
Among the main causes of infectious diseases with Candida fungal infections are a very important model of human pathogens.Candida albicans is a dwarf fungus and an important component of the natural flora of healthy people.In a situation where the immune system is weakened, Candida, as an opportunistic pathogen, can develop diseases after transferring from the symbiotic phase to the pathogenic phase.Different species of this yeast are able to form biofilms and are the leading cause of mortality in immunocompromised patients.Since they cause invasive candidiasis, it is difficult to eradicate them using antifungal therapy.In fighting against the fungal infections, 3 main flaws can be considered for some antifungal drugs: first, they operate in a limited range, second they can negatively react with different types of antifungal agents, and third they can make the microorganisms resistant.In addition, despite the advances in the treatment of fungal infections using antifungal compounds over the past thirty years, the antifungal resistance is still a major concern in clinical practice.In general, the advancement in the production of new antibiotics involves long and costly processes that can now be solved with the development of nanoscience science. 1 Nano-biotechnology has been making progress in recent decades and will have widespread effects on various aspects of human life including pharmacy and medicine in the near future.Nowadays, the growing need for reliable methods for making nanoparticles which do not harm the environment has made the researchers extremely interested in this field.Many organisms, whether monocellular or multicellular, are known to produce inorganic nanoparticles acting as intracellular or extracellular.Plant extracts are among the biological systems which have been recently considered in this field.Mango is one of the most popular tropical fruits in most parts of the world which is cultivated abundantly in South Asian countries.Mangoes belong to the Mangifera species from the Anacardiaceae family.Mangifera contains several species of edible fruit.The tree of fruits we know as mango belongs to the Mangifera indica species.Nowadays, the mango is considered to be one of the 3 or 4 optimal fruits of tropical regions.Its origin is northeastern India, northern Burma and probably Silane.Mango core contains active bioactive compounds with high antioxidant activity. 2Mango has anti-viral and antibacterial properties, due to the presence of compounds such as phenolic compounds. 3Ethanol extract of mango kernel has a good antimicrobial activity against a variety of pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, and Klebsiella pneumoniae. 4 Ethyl acetate and ethanol derived from mango peel extract showed strong antifungal activity against pathogenic fungi such as Rhizoctonia solani. 5Given that nanobiotechnology is seeking to discover the substances that are more specialized and effective in treating various diseases, including fungal diseases and based on the antimicrobial properties of green nanoparticles and the lack of precise knowledge of the specific effects of Candida species, it is necessary to conduct precise tests for studying the effects of these compounds on the above-mentioned yeast.The mango core contains bioactive compounds with high antioxidant activity.Researchers have shown that the ethanolic extract of mango core has antimicrobial activity against pathogenic bacteria such as E. coli, P. aeruginosa, S. aureus, S. pyogenes, and K. pneumoniae. 5

Materials and Methods
In this study, first the standard strains of C. albicans PTCC 5027and C. glabrata PTCC 5297 and Candida krusei PTCC 5295 were purchased from the Tehran Research Institute of Technology.Clinical isolates were comprised of 16 isolates from Shafa Laboratory, Isfahan, 3 isolates from Mahdieh Laboratory, and 2 isolates from Shahid Beheshti Hospital in Isfahan.All clinical isolates were identified by direct lam, Germ tube formation, clamidoconidia production in corn meal agar medium, and culture CHROMagar medium.In order to prepare the nanoparticles of mango kernel, first the mango core was removed from 2 lignin and cellulose shells.Then, they were washed several times with sterilized distilled water and crushed and autoclaved into ultra-small pieces by a meat grinder.After on, the pieces were dried in an oven at 250°C for 2 hours.In the next step, 500 g of powder was dried.The mango kernels were extracted with 50% cc of alcohol within the Soxhlet tank and then the extracts were passed through Whatman filter papers grade 4.
In order to synthesize the nanoparticles of mango kernels, first 1.69 g of silver nitrate powder was mixed with 100 mL sterile distilled water.Then, 2 g of mango extract powder were added to the above substance and were then placed in oven at 60°C for 30 minutes.The device was turned off after reaching the boiling point (100°C).In the next step, the solution was biphasic and some crystals appeared on the solution.Then, the supernatant was washed several times with sterilized deionized water to wash the excess salts and the obtained sediments were dried by using an oven at 37°C for 42-72 hours. 6For the final use, the dried powder obtained from the previous stage was re-exposed at 60°C for 48 hours and the samples were completely dried.After analyzing qualitative changes in the black color of the prepared samples, an amount of the resulting liquid was isolated to evaluate the absorption spectrum using a UV-VIS spectrophotometer.Then, the samples were sent to the Faculty of Materials Science, Isfahan University of Technology, to determine the morphological and physicochemical properties of the samples analyzed by the X-ray diffraction (XRD) device.
The samples were then sent to the Ferdowsi University of Mashhad's laboratory for the microscopic identification of the samples synthesized by the TEM device.In order to determine the sensitivity of yeasts to the nanoparticles of mango kernels, the micro dilution method was used.For this purpose, the serial dilution of 0.002, 0.004, 0.008, 0.016 and 0.032 mg/mL from mango bio-nanoparticles was prepared in the injectable distilled water solvent.With the help of this method, the minimum inhibitory concentration (MIC) and minimum fungal concentration (MFC) were determined.Fluconazole was used as a positive control and the physiologic serum was used as a negative control. 7n order to compare the mean diameter of non-growth yeast halves for each patient between different groups, oneway ANOVA test was used by SPSS software version 22.Moreover, Duncan test was used to examine the significant difference between different groups.

The Results of XRD Diagram for Mango Core Nanoparticles
The results obtained from Figure 1, illustrated by the XRD device, showed the peaks at 111, 200, and 220 levels being consistent with silver nano-crystal reference cards (JCDD04-0789).Figure 1 shows the scheme obtained from an XRD device for silver nanoparticles with mango core produced at 60°C.

The Results of UV-VIS Diagram for Mango Core Nanoparticles
A UV-VIS absorption spectrum of the mango core nanoparticles has been presented in Figure 2, in the presence of 1 mM silver nitrate at 500°C.As it is clear, it has a peak at 345 nm.
The Results of TEM Images for Mango Core Nanoparticles The analysis results of TEM images showed the production of nanoparticles produced by mango core extract in a size of 65 nm. Figure 3 shows the image prepared from the transmission electron microscope (TEM).

The Results of Isolates Collection
This study was conducted on 21 samples sent by several laboratories in Isfahan.
After the initial screening, 11 isolates of C. albicans, 7 isolates of C. krusei, 2 isolates of Candida tropicalis and 1 isolate of C. glabrata were among the isolates.The results of identifying these isolates are presented in Table 1.
Table 2 shows the MIC of fungi and also the MFC concentration in mg/mL against the various concentrations of mango core nanoparticles.

Discussion
The importance of opportunistic fungal diseases in patients with immune deficiency (diabetics, cancers, transplants, and certain diseases) and especially cellular immunity is not hidden to anyone.Candidiasis is undoubtedly one of the most prevalent opportunistic fungal diseases in humans which is acute and chronic in the skin, nail, mucosa, vagina, lung and gastrointestinal tract or is observed systemically with blood poisoning, endocarditis, and meningitis. 8The main problems in the treatment of patients with fungal infections, especially Candida, is resistance to antifungal agents, as well as an increased incidence of fungal infections or their recurrence, and also the use of different antifungal medicines. 9In addition, the infections associated with C. albicans, C. glabrata, and C. krusei are often treated with difficulty and most of the strains are less susceptible to antifungal drugs.Thus, it is necessary to adopt new therapies which are not only effective on these resistant strains, but also have no harmful or toxic effects for animal or human consumptions. 10Nowadays, many researchers investigate the antifungal effects of metallic nanoparticles alone or in combination with plant extracts which may be attributed to the different physico-chemical properties of these substances and antibacterial, antiviral, and anti-parasitic effects of these nanoparticles. 11The XRD analysis was performed to prove the nanocrystals of mango core.Based on the findings of Figure 1, the nanocrystals of mango core were consistent with silver nanocrystal reference card (JCDD04-0783) on 111, 200, 220 levels at 60°C. 12 In this project, the results of the UV-VIS graph had a specific absorption peak at 345 nm, and also showed a peak at 278 nm.The results of analyzing the TEM images according to Figure 3 indicated the production of mango core nanoparticles in spherical cubic and the size of 65 nm.Based on the findings, the changes created in the type of production, diameter, shape, and concentration of different nanoparticles have led to different results in the antifungal activity of these particles. 13,14im et al reported that the antifungal activity of silver nanoparticles in spherical shape against pathogenic fungi such as Trichophyton mentagrophytes and Trichosporon beigelii had a greater effect than the current antifungal agents such as fluconazole and amphotripsin B. 15 Based on Table 2 indicating the MIC and MFC results of mango core nanoparticles and Fluconazole standard and clinical samples of Candida by microdilution method, the highest MIC belonged to C. albicans 2, 5 and 9, C. krusei 1 and 2, and C. tropicalis 1 affected by 0.032 mg/mL concentration of mango kernel nanoparticles.However, the highest MFCs of these isolates was 0.064 mg/mL. 15The results of Bahrami Abdehgah et al showed that the highest MIC for C. glabrata belonged to copper and silver nanoparticles each with a concentration of about 31 ppm and MFC of 62.5 ppm. 16However, in the   same study, the lowest MIC for silver nanoparticles with amphotericin B was reported to be 8 ppm and MFC equivalent to 15.5 ppm.The results of this study are partly consistent with the present study where the lowest amount of MIC in standard C. glabrata 5297 was 0.002 mg/mL against silver nanoparticles along with mango kernel.In addition, its MFC level was reported to be 0.04 mg/mL.
In the present study, the effect of mango core nanoparticles had more appropriate antifungal property against C. glabrata than fluconazole.These findings are in line with the results of a study by Nafisa et al. which had been conducted on fungal pathogens of C. albicans, C. glabrata, and C. tropicalis. 17urthermore, in another study, Asghari et al investigated the antifungal effects of silver nanoparticles on Candida vulvovaginal factors in vitro.The results of this study showed that silver spherical nanoparticles with a diameter of 10 nm had antifungal activity against C. albicans to some degree.The MIC amount of the isolates was reported 25.31 to ppm and the MFC of the samples was 62.5 to 250 ppm. 18In 2016 Valenti et al evaluated the activity of two antifungal drugs (amphotericin B and voriconazole) simultaneously against 5 different candida species that were able to create biofilm.The reported MIC rate of this compound was 0.004 mg/mL and MFC was 0.008 mg/mL, which is comparable to some candida species in the present study. 19

Conclusions
The findings of this study revealed that the nanoparticles of the mango core had the most anti-candida property on the clinical strain of C. glabrata by the micro dilution method as well as on the standard strain of this yeast, C. glabrata 5297 which could be compared to the antifungal agent of fluconazole.However, the effect of flokonazole was more than the cubic mango core nanoparticles with a diameter of 65 nm in case of C. albicans, C. krusei, and C. tropicallis.

Figure 1 .
Figure 1.The Scheme Obtained From the X-Ray Diffraction Device of Mango Core Nanoparticles.

Figure 2 .
Figure 2. The UV-VIS Absorption Spectrum of Mango Core Nanoparticles.

Figure 3 .
Figure 3.The Image Prepared From Mango Core Nanoparticles Using the TEM Microscope

Table 1 .
The Results of Identification Tests on Different Candida Species

Table 2 .
MIC and MFC Rates in mg/mL for Nanoparticles of Mango Core and Fluconazole on Standard and Clinical Candidates of Candida