In-vitro Characterization and Evaluation of Beta Vulgaris L. Extract Loaded on Chitosan Nanoparticles as Anticandidal and Antibiofilm Activity

Introduction
Nanotechnology is a field of applied science that studies materials on a scale between 1-100 nm. Recently, it has submitted submissions in biology, electronics, and medicine abroad. The extended development of nanotechnology and its science led to the motivation to develop industries (Hejazy & Koohi, 2017; Zahin et al., 2020). The small size of nanoparticles (NPs) means that they can easily enter the body and cross a variety of biological obstructions to reach the most vulnerable organs (Kaboutari et al., 2023; Othman et al., 2020). A wide range of plants are cultivated globally for food and medicinal purposes, with several traditional medicinal plants displaying significant potential in the management and treatment of various modern health problems. These include beetroot (Beta vulgaris L. extract [BVE]), known in the Middle East as Shamandar, a plant of the family Amaranthaceae (Albasher et al., 2020). B. vulgaris L. contains two major betalain pigments, red betanin, and yellow violaxanthin I, which have been considered for a long time as the unique source of betalains. Red beet is cultivated all over the world and is commonly and frequently consumed. The world production of beetroot was estimated to be about 275 million metric tons in 2018 (Fu et al., 2020). Betacyanins constitute approximately 75% to 95% of beetroot pigments, and the remaining 5% to 25% are betaxanthins (Sadowska-Bartosz & Bartosz, 2021). Fungal infections caused by Candida species and the increasing prevalence of Azole-resistant strains in immunocompromised patients are critical (Fetouh et al., 2023; Habib et al., 2007; Shokri et al., 2016). Recently, in addition to trying to make effective chemical treatments and probiotic therapy for the treatment of candidiasis, many metal NPs have been studied in the treatment of this disease and have received significant results (Yousefi et al., 2019). NPs prepared with chitosan and derivatives of these NPs typically possess a positive surface charge and mucoadhesive properties, such that can adhere to mucus membranes and release the drug payload in a sustained release manner. Chitosan-based NP has various applications in NPs as antimicrobial drugs (Al Sahib & Awad, 2022). Among the new antimicrobial agents, special attention has been paid to NPs. NPs have a higher level than other antifungal agents, and in addition, they have a better penetration in tissues and cells (Kumar & Anthony, 2016).

Materials and Methods

Microorganisms in the study
Candida spp. including (C. parapsilosis, C. glabrata, C. lusitaniae, and C. auris) were isolated from different clinical sources and identified using the CHROMagar Candida culture medium (C. auris is not included in this medium), ViteK-2 system, and molecular diagnostics. The isolates were obtained from the College of Science for Women laboratories.

Biofilm formation test
For the study of biofilm formation, the microtiter plate assay for studying biofilm formation is a method that allows for the observation of Candida adherence to an abiotic surface. In this assay, Candida was incubated in vinyl “u”-bottom or other types of 96-well microtiter plates. Following incubation, planktonic yeast is rinsed away, and the remaining adherent Candida (biofilms) are stained with crystal violet dye, thus allowing visualization of the biofilm. If quantitation is desired, the stained biofilms are solubilized and transferred to a 96-well optically clear flat-bottom plate for measurement by spectrophotometry.

Preparation of Beta vulgaris L. extract
The BV was purchased from a local market in Baghdad, Iraq, and identified at the herbarium in the Department of Biology, College of Science, University of Baghdad. Aqueous and ethanol extracts of the B. vulgaris L. were prepared according to (Sultana et al., 2009). The air-dried beetroot (150 g) was extracted using aqueous methanol (500 mL) as a solvent (methanol: water, 80% V/V) for 8 h, in the Soxhlet apparatus. The extract was concentrated using a rotary evaporator and then the remained extracts were dried. The extract yield was calculated by the dried concentrated crude extracts.

Biosynthesis of chitosan NPs loaded BVE 
Chitosan 5% (200 mg of CS +100 mL acetic acid) and BVE 1% (1 g of BVE + 100 mL distal water) were combined in equimolar ratios, and the condensation process was carried out in the presence of xylene using the Dean-Stark (Clevenger) apparatus until the theoretical quantity of water was separated. Chitosan amide product was separated by filtration, washed several times with methanol, hot distilled water, and ethanol, and then dried in an electric oven at 50 °C and weighed (Abd El-Ghaffar & Hashem, 2010).
Characterization (morphological and structural aspects) of chitosan NPs loaded BVE
Various approaches were used to characterize to identify chitosan NPs (CSNPs) loaded BVE in this study, namely, ultraviolet-visible absorption spectroscopy (Agarwal et al., 2018)., atomic force microscopy (AFM) (Du et al., 2008)., scanning electron microscopy (SEM) analysis (Atangana et al., 2019)., x-ray diffraction (XRD) (Anand et al., 2018)., energy dispersive x-ray (EDX) (Hodoroaba, 2020)., and high-performance liquid chromatography (HPLC) analysis for the determination of phenolic compounds (Dahl-Lassen et al., 2018). 

Determination of minimum inhibitory concentration and sub-minimum inhibitory concentration

Spread plate method (SPM) 
The spread plate method (SPM method is used as described by Wise, 2006. Sabouraud dextrose agar is put onto several sterile petri plates and allowed to solidify. Before using the plates, we must wait for a while to dry. As with the pour plate procedure, Candida suspension of moderate turbidity was prepared by picking 1-2 isolated colonies of candida from the original culture and introducing them into a test tube containing 4 mL of normal saline. It was compared to the standard turbidity solution 0.5 McFarland which approximately equals 1.5×108 colony-forming unit/milliliters (CFU/mL). The treatments were with different concentrations, namely 50%, 25%, 12.5%, 6.25%, and 3.12% of CSNPs loaded BVE solution mix with Candida suspension, in addition to Candida control (without treatment), pipetted with 100 μL quantities of each dilution over the surface of each of the three plates. An ethanol-flamed glass spreader was then used to disseminate the sample throughout the plate’s surface. The plates were then incubated for 24 h at 37 °C in aerobic conditions and the number of colonies that form is counted in the same way as the pour plate method.

Determination of inhibition efficacy against biofilm production
The microtiter plate assay is a qualitative technique that uses a microplate reader to determine an agent’s effectiveness against biofilm formation. The sub-minimum inhibition concentrations (sub-MIC) obtained from the previous experiment were used to study the effect of the test materials on the formation or inhibition of biofilm of the studied Candida spp. isolates that produce strong biofilm. The test materials were BVE, CSNPs loaded BVE solution, positive control (Candida suspension only), and negative control (brain heart infusion broth [BHIB] only). However, 100 μL of test compounds was added and the plate was incubated at 37 °C for 24 h. After that, all wells were washed, stained, and read at 600 nm wavelength using a microplate reader percent of biofilm inhibition was calculated by equation (Shinde et al., 2021). 

Results and Discussion

Detection of the fungal ability for biofilm formation
In this study, the determination of the ability of Candida spp. isolates for adherence and producing a slime layer (biofilm formation) was experienced by using a microtiter plate and read by a microplate spectrophotometer as in (Figure 2). We noticed that all isolates produced biofilm in varying proportions.
Our study’s findings are in line with the results of Marak and Dhanashree, (2018). Meanwhile, C. albicans (45.5%) was the most prevalent species among the 90 Candida spp. that were isolated, followed by C. tropicalis (28.88%), C. krusei (20%), C. glabrata (3.33%), and C. parasilosis (2.22%). Also, Candida spp. was found in the following samples: Pus, bile aspirate, deep tissue, high vaginal swabs, suction tips, blood, wound swabs, and urine.

Biosynthesis of chitosan NPs loaded BVE 
Chitosan and BVE adduct were prepared and then the tripolyphosphate (TPP) was added to form CSNPs loaded BVE. When chitosan reacts with TPP, the amine groups of the chitosan can cross-link with the phosphine groups of TPP to create NPs. During the action, the chitosan’s molecular structure will be altered, resulting in a change in solubility in an acid solution as well as a gel-like solution or a liquid form (Kahdestani et al., 2021). The appearance of a clear color indicates the formation of NPs loaded with the plant extract as shown in Figure 1.

Characterization of chitosan NPs loaded BVE
The CSNPs loaded BVE were characterized by employing ultraviolet-visible spectroscopy, SEM, EDX, AFM, and XRD, analyses through examining the morphological, structural, and optical properties as shown below.

Ultraviolet-visible spectroscopy
Ultraviolet-visible spectroscopy is a primary step in confirmation of the synthesis of CSNPs loaded BVE. The absorbances of BVE and CSNPs loaded BVE were measured and the results are shown in Figure 2 for BVE and CSNPs loaded BVE as shown in Figure 3.

In the CSNPs loaded BVE, the highest absorbance value was 0.087, while the lowest absorbance value was 0.006 at wavelengths 279 nm and 542 nm, respectively. On the other hand, for BVE the highest absorbance value was 3.135 and the lowest absorbance value was 1.063 at wavelengths 212 nm, and 282 nm, respectively. However, for chitosan the absorbance value was 2.85 at wavelengths 212 nm, The decrease in the absorbance value at wavelength 282 nm in the BVE to 212 nm in CSNPs loaded BVE, as well as the highest absorbance value at the wavelength 254 nm with a value of 0.006 in CSNPs loaded BVE material after it was the highest value in BVE at a wavelength of 282 nm with a value of 1.063, in addition to the disappearance of the wavelength 282 nm in the BVE and the appearance of a new wavelength with high absorbance at 542.00 in CSNPs loaded BVE all, indicate the formation of the nanomaterial and the successful loading of the BVE on chitosan NPs.

XRD 
The XRD patterns of chitosan show the main peak of 2θ value at 20.53° and an intensity level close to 1200 count; on the other hand, as shown in Figure 4, the CSNPs loaded BVE peak, which shows its main peak of 2θ value at 22.8596° and an intensity level at 736.5503 count, this change indicates the difference in the crystal structure between these two materials were CSNPs loaded BVE was more crystalline than CS, as well as the CS diffraction peak, which was previously found at 20.53, has shifted to a higher value 22.8596° in this study, which may be related to the interaction of CS loaded with BVE to form CSNPs loaded BVE.

The XRD results suggested that the particle size effects are generally responsible for the widening of peaks in crystalline XRD patterns. Wider peaks indicate lower particle sizes and reflect the influence of experimental circumstances on particle structures. Small crystals have a limited number of levels of reflection with low intensity, while large crystals have a large number of these levels with high intensity; therefore, XRD peaks are formed due to reflection from crystal levels and the decrease in intensity is visible in the pattern of CSNPs loaded BVE, which exhibits suppressed peaks (Table 1). 

These results were in line with the study of Anand et al. (Anand et al., 2018) who found the pure chitosan diffraction peak, which was previously found at 20.20 °C, has migrated to a lower value 19.85 °C, which may be attributed to the interaction of CSNPs with TPP and the crystalline structure of CSNPs. Kahdestani et al. (2021) found that the variations among the patterns might be traced to molecular arrangement changes in the crystal lattice, and the pattern of teicoplanin-loaded chitosan NPs displays reduced peaks at 2θ 22 °C and 25 °C, indicating an intensity decrease.

Atomic force microscopy 
AFM images were used to measure particle size and the topography of the surface of CSNPs loaded BVE, the particle size ranged from 26.74 to 53.96 nm, and the 3D image of CSNPs loaded BVE revealed a population of homogeneous particles with a regular surface shape (Figures 5 and 6).

The images of AFM demonstrated smart interaction among CSNPs loaded BVE, leading to the formation of well discrete aggregates, From the information included with the image, the evolution values of root mean square, surface roughness average, and average grain size were calculated and listed that exhibited information about coverage area minimum, maximum, and mean particle size. These results are in line with Zheng et al., 2021 who found that the resulting morphological-mediated chitosan was exhibited with uniform particles, and their average size was 40–96 nm.

Scanning electron microscope 
The morphology of CSNPs loaded BVE was investigated using a scanning electron microscope (SEM), the result was presented in Figure 7, CSNPs loaded BVE has a spherical appearance with a diameter range of 37.99-56.28 nm, and have a relatively homogeneous morphology, the larger particles may be a result of the unwanted aggregation that occurs throughout the drying process (Table 2). Agglomeration occurs as soon as the liquid evaporates leading to rising particle concentration. The increase in dissolved ion concentration caused by liquid evaporation can reduce the electrostatic repulsive force, facilitating agglomeration (Shrestha et al., 2020).

Energy dispersive x-ray 
The elemental analysis of CSNPs loaded BVE via SEM-EDX revealed the presence of C, O, Mg, Na, and Ca. Figure 8 shows that EDX examination reveals a significant signal at 1.5 keV and 2.3 keV due to the presence of carbon and oxygen respectively, confirming the presence of chitosan. The other elements, such as Ca and Mg are due to the presence of BVE components. The results are in line with Raza and Anwar (Raza & Anwar, 2017) who used scanning electron microscopy equipped with EDX to confirm the presence of CSNPs on the treated fabric. Also, Omidi and Kakanejadifard (Omidi & Kakanejadifard, 2019) reported that the EDX spectrum of CS10 contains four kinds of elements C, O, N, and Br. Also, CSNP10 consists of C, O, N, P, and Br elements.

Quantification of bioactive compounds by high-performance liquid chromatography (HPLC) mass spectrometry/mass spectrometry system
The chromatographic analysis of BVE showed the existence of flavonoids (ferulic acid, quercetin, and rutin), phenols (caffeic acid, gallic acid, and chlorogenic acid), lysine, and vitamin B12; however those were higher in the nano sample than the extracted sample and the highest concentration recorded in caffeic acid in nano sample was 865.5 parts per million as shown in Table 3.

The detection results of active compounds in the extract proved that it contained most of these compounds in good proportions, which are responsible for the biological activity of medicinal plants which indicates that the BVE possesses a good therapeutic property according to the function of each group.

Determination of the minimum inhibition concentration (MIC)
There were differences in MIC and sub-MIC of CSNPs loaded BVE and only BVE against the isolates; however, C. auris recorded the highest value in CSNPs loaded BVE which MIC of CSNPs loaded BVE was 52.3 and sub-MIC was 39.23 while the other isolates showed same MIC and Sub-MIC of CSNPs loaded BVE and only BVE as shown in Figure 9.

The findings are in line with Lin et al., 2023, who showed that berberine-loaded chitosan NPs released from berberine (BBR)-CSNPs could inhibit the growth of C. albicans and destroy the integrity of the cell wall and cell membrane of C. albicans. 

Estimation of antibiofilm activity of chitosan NPs loaded BVE
The inhibition rate of CSNPs loaded BVE was higher in 1:5 than in 1:4; however, the antibiofilm activity showed less against C. auris compared to other isolates as shown in Figure 10.
The findings are in line with Lin et al., 2023. that showed berberine-loaded chitosan NPs (the free BBR and BBR-CSNPs) had a significant inhibitory effect on C. albicans biofilm, and the inhibitory effect on biofilm was concentration-dependent. Free BBR even at the lowest concentration (64 μg/mL), about 40% of the C. albicans biofilm formation was inhibited. When free BBR concentration was higher than 512 μg/mL, almost no biofilm formation was observed. In the same concentration range, BBR-CSNPs showed lower inhibitory activity on the biofilm of C. albicans compared with free BBR. Also, the results are in line with Tan et al., 2022 who found that CSNPs loaded with amphotericin B inhibited planktonic cell growth and biofilm formation effectively and exhibited the highest efficacy on the removal of a mature biofilm than free amphotericin B or CSNPs- amphotericin B On Candida biofilm-related infections. 

The nanostructures and positive charge of CSNPs make them have good permeability and easy to combine with the cells on the surface and inside the biofilm, causing the change of cell surface charge, increasing the sensitivity of cells to drugs, and then affecting the formation of biofilm (Li et al., 2021). Moreover, the nanostructure and positive charge of BBR-CSNPs had good permeability and were easy to combine with microbial cells. Secondly, CS can also form NPs with polyanion TPP through electrostatic interaction in an aqueous solution. CSNPs have small particle sizes and large specific surface area (Tan et al., 2022). The results also indicated that CSNPs loaded BVE was easier to penetrate the biofilm and kill the cells in the biofilm of Candida spp. 

CSNP prepared using low molecular weight and high molecular weight of chitosan have been evaluated for their antifungal activity against C. albicans, Fusarium solani, and Aspergillus niger. The NPs prepared with different concentrations of chitosan showed an inhibitory effect against the three fungal species. While A. niger exhibited a strong resistance to CNPs which was fabricated with low concentrations of chitosan (Ing et al., 2012). The CSNPs were also evaluated as a controlling agent for various plant diseases caused by Rhizoctonia solani, Fusarium oxysporum, Collectotrichum acutatum, and Phytophthora infestans.

Moreover, CNP shave has been shown as an ideal coating agent for coated vegetables by improving the shelf life of tomato, chilly, and brinjal. CSNPs exhibited significant antifungal activity against all fungal species. Vegetable samples treated with different concentrations that ranged from 1% to 5% of CSNPs prevented weight loss compared to uncoated vegetable samples (Divya et al., 2018). 

Conclusion
The prepared NPS compound was exceeded on BVE alone and highly sensitive antibiotic as antifungal and antibiofilm activity against extensively drug-resistant (XDR) Candida spp. including C. auris isolate. CSNPs loaded BVE showed antibiofilm activity more than BVE against some resistance Candida spp.

Ethical Considerations

Compliance with ethical guidelines
The research did not involve any ethical considerations.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
All authors contributed equally to the conception and design of the study, data collection and analysis, interpretation of the results, and drafting of the manuscript. Each author approved the final version of the manuscript for submission.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors would like to express their sincere gratitude to all those who contributed to the successful completion of this research.

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