Green synthesis of zinc oxide nanoparticles from Phoenix dactylifera and their anti-microbial potentiality – An in vitro study

Introduction and Aim: The consumption of date palm (Phoenix dactylifera) fruit holds a widespread presence globally and holds notable importance in the diets of various Arabian nations. The adoption of green synthesis for nanoparticle production presents multiple advantages over conventional physio-chemical methods. Furthermore, these nanoparticles have found diverse applications within the medical field. In light of this, the synthesis of zinc oxide nanoparticles using extracts from Phoenix dactylifera (dates) was undertaken. The primary objective of this research was to employ a biosynthesis approach for the creation of zinc oxide nanoparticles utilizing extracts from Phoenix dactylifera (Mabroom dates), and subsequently assessing their potential antimicrobial efficacy. Materials and Methods: In the present study, the Dates extract and zinc sulphate solution was mixed and centrifuged to synthesise zinc oxide nanoparticles. Characterization was assessed using visual identification and UV visual spectrometry analysis. The anti-bacterial activity was analyzed in Mueller Hinton agar against the S. mutans pathogen, whereas anti-fungal activity was analyzed in Rose Bengal agar against the C. albicans pathogen to rule out the zone of inhibition. Results: UV-Visible spectrophotometric spectra acquired after 40 hours of incubation revealed an absorption peak at 275 nm, indicating the existence of zinc oxide nanoparticles. The assessment of antibacterial activity against S. mutans exhibited heightened efficacy with 100 µl of dates and ZnO nanoparticles, demonstrating stronger antibacterial effects. Similarly, the evaluation of antifungal activity against C. albicans displayed robust antifungal potency with 100 µl of dates and ZnO nanoparticles. Conclusion: Zinc oxide nanoparticles synthesized using dates exhibited potent anti-microbial activity and thus future clinical trials can use this for Oral potentially malignant disorders such as candidal leukoplakia, erosive lichen planus etc.


INTRODUCTION
he Phoenix dactylifera, commonly known as the date palm, belongs to the Arecaceae family of flowering plants and is predominantly cultivated for its edible fruit (1).The date palm (Phoenix dactylifera) holds a significant role in defining the agricultural landscape of arid regions in North Africa and the Middle East (2).Date fruits are abundant in phytochemicals, encompassing phenolics, anthocyanins, carotenoids, and dietary fibre.
These phytochemicals have been linked to a range of biological activities in date fruits, including antioxidant, antibacterial, anti-inflammatory, prebiotic, anticancer, and antitoxic properties.These attributes have led to their utilization in various industrial economies to prevent and manage diseases such as diabetes, cancer, gastrointestinal disorders, cardiovascular issues, and neurodegenerative conditions (3).In contemporary times, nanotechnology has gained recognition as a cuttingedge technology with diverse applications across fields such as chemistry, pharmaceuticals, mechanics, and food processing.Another role for nanotechnology functions in computing, electricity generation, and other interesting environmental sciences, medication delivery, and optics.Since the emergence of nanotechnology, several nanoscale devices have been created utilizing a variety of techniques, including physical, chemical, and environmentally friendly ways (4).Green nanoparticle synthesis, however, is a preferred method that is simple to manufacture and engineer.Green synthetic nanoparticles surround themselves with a variety of organic phytochemicals that aid in ligand-based complexation with different receptors on the surface of microorganisms, including proteins, lipids, phospholipids, and lipoteichoic acid.Bacterial biofilm development and growth are inhibited by this complexation of nanoparticles with bacteria (5,6).Conventional methods for the manufacture of nanoparticles have several disadvantages, including lengthy processing, high costs, time-consuming processes, and in particular the usage of hazardous substances (7,8).Almost all T pertinent study has focused on quick and environmentally friendly synthesis techniques for making nanoparticles because of these limitations.In this regard, green synthesis of NPs, particularly when employing plant extracts, is a developing trend that is regarded as straightforward, affordable, and nontoxic in green chemistry (9).The past ten years have seen a focus on the complete study of metal oxide nanoparticles because of their numerous uses in a variety of technical disciplines.Among these, with ZnO-NPs are an intriguing inorganic material with many advantages materials.ZnO-NPs can be utilized in a variety of fields, including electronics, healthcare, catalysis, energy efficiency chemical sensing, semiconductors, and cosmetics (10).
Semiconducting ZnO is an n-type metal oxide.These nanoparticles hold promise in various biological contexts, including drug administration, anti-cancer, anti-diabetic, antibacterial, antifungal properties, and agricultural benefits.Furthermore, their low cytotoxicity positions them as potential candidates for targeted drug delivery systems (11).Date fruits are recognized to contain substantial levels of dietary fiber (6.5% to 11.5%), proteins (1% to 2%), and ash (2%).They also boast a noteworthy concentration of phenolic antioxidants (12).Notably, the antibacterial activity of dates has shown remarkable potential against specific diseases (13).Date fruits are said to have a total of 6.5% to 11.5% dietary fibre (up to 90% of which is insoluble and 10% of which is soluble), 1% to 2% protein, and 2% ash.It also has a significant number of phenolic antioxidants (14).The data on antibacterial activity is fascinating since it shows that dates have potent disease-specific inhibitory effects (15).Al-Shwyeh et al., (2012) demonstrated that the phenolic compounds present in date palm fruits contribute to their antioxidant-rich properties and potent bioactivities against various bacterial pathogens ( 16).Here zinc oxide nanoparticles are added to substantially increase the potentiality of dates extract.Therefore, zinc oxide nanoparticles are created from date fruit due to their potential use in medicine due to their emerging features.
The primary objective was to synthesize the zinc oxide nanoparticles aqueous solution from Mabroom dates extract, characterize the synthesized zinc oxide nanoparticle solution in UV-Visible spectrophotometry and to analyse the anti-microbial potential of synthesized zinc oxide nanoparticle solution.

Fruit extract preparation
Mabroom dates were procured from retail outlets.The date fruits were finely diced, and their weight was measured.A quantity of 2 grams of these dates was introduced into 100 millilitres of distilled water (Fig. 1).Subsequently, the mixture was heated to 80 degrees Celsius and kept at this temperature for a duration of 30 mins.Following the boiling process, the solution underwent filtration using Whatman filter paper, and the resultant filtrate was utilized for subsequent experimental procedures.

Synthesis of ZnO NPs
A total of 0.287 grams of zinc sulphate was dissolved in 50 ml of distilled water and subjected to stirring for a period of 1 hour.Simultaneously, another 50 ml of the date extract was combined with 50 ml of the zinc sulphate solution (refer to Fig. 2).Following an incubation period of 1 hour, a noticeable alteration in the colour of the reaction mixture occurred.The solution was then left to undergo further stirring for an additional 3 hours, during which a yellow hue emerged, serving as confirmation for the successful synthesis of ZnO nanoparticles.The resulting precipitate was isolated from the reaction solution through centrifugation at 10,000 revolutions per minute for 5 minutes (Fig. 3).

Characterization of biosynthesized ZnO NPs
The UV absorption spectra were employed to analyse the optical attributes of ZnO nanoparticles, encompassing wavelengths between 300 and 500 nanometers.

Anti-bacterial activity
A myriad of mechanisms is used by nanoparticles to serve as antibacterial agents.One of the most significant mechanisms is the loss of phospholipid bilayer integrity, which results in a cell membrane's integrity being disrupted.Another significant role played by nanoparticles involves the induction of oxidative stress through the generation of Reactive Oxygen Species (ROS).Through the inhibition or modification of DNA replication, protein synthesis, food metabolism, or respiratory cycles, this ROS molecule also impacts to cell death.Anti-bacterial activity of ZnO nanoparticles was assessed against the strain streptococcus mutants.

Media preparation
A volume of 100 millilitres of Streptococcus mutansspecific Mueller Hinton agar was prepared, sterilized, and subsequently poured into Petri plates.Subsequently, the plates were permitted to undergo solidification.

Swabbing and well formation
Using a sterile 9 mm polystyrene tip, wells were fashioned, and the test organisms were subsequently swabbed onto the medium.Distinct concentrations of ZnO nanoparticles (25 µl, 50 µl, 100 µl) were introduced into designated wells, while the fourth well received the standard antibiotic amoxyrite.Following well preparation, the plates were subjected to a 24hour incubation period at 37°C.After the incubation duration, the extent of inhibition zones was measured.

Measurement of zone of inhibition
The measurement of the zones was performed using a ruler positioned against the rear side of the petri plate, with the test plates held in front of a desk lamp.The diameters of the growth-inhibited zones were then determined, accurate to the nearest whole millimeter.

Anti-fungal activity
Candida albicans is employed as the tested pathogen in the agar well diffusion assay.

Media preparation
The fungal medium was prepared using Rose Bengal Agar.Once prepared and sterilized, the medium was then swabbed with the test organism and varying concentrations of ZnO nanoparticles (25µl, 50µl, 100µl).

Swabbing and well formation
They were introduced into the wells, and in the fourth well, the standard fluconazole was introduced.Following this, the plates were subjected to incubation at 37 ºC.Upon completion of the incubation period, the measurement of the inhibition zones was performed.

Measurement of zone of inhibition
Using a ruler positioned against the rear side of the Petri plate, the measurement of the zones was conducted, with the test plates being held in front of a desk lamp.The diameters of the growth inhibition zones were then measured, rounded to the nearest whole ml.

RESULTS
Fig. 6 depicts the UV-Visible spectrophotometric spectra acquired after 40 hours of incubation display an absorption peak at 275 nm, indicating the existence of zinc oxide nanoparticles.Fig. 4 show the zone of inhibition in Mueller Hinton agar which reveals it is more in 100 µl of dates and ZnO nanoparticles when compared to other concentration of the solution.Fig. 7 reveals anti-bacterial activity against S. mutans which shows 100 µl of dates and ZnO NPs has more antibacterial activity against S. mutans.Fig. 5 shows rose Bengal agar revealing zone of inhibition is more in 100 µl dates and ZnO nanoparticles when compared to other concentrations of the solution against C. albicans.Fig. 8 shows antifungal activity against C. albicans which shows 100 µl of dates and ZnO NPs has more anti-fungal potential.

DISCUSSION
Due to growing drug use, microbes have developed a resistance to many antibiotics, which is reducing the effectiveness of conventional medicines.Therefore, the need to discover novel antimicrobial drugs has arisen.An emerging technology called nanotechnology has sparked a new scientific revolution in every discipline (17).Other environmentally friendly sources for the creation of nanoparticles include biocompatible compounds.In the nucleation and synthesis reaction of nanoparticles, it is a quick, cost-effective technique that eliminates the formation of any kind of side product.Due to its well-dispersed nature, it causes the creation of nanoparticles with regulated shape and size (18).The use of zinc oxide nanoparticles as antibacterial agents opens new possibilities for medical research.Due to its strong anti-bacterial properties, it is also used in cosmetics, wound dressing for quick healing, burn dressing, as a disinfectant, antiseptic ointments, or lotions, coating medical instruments to ensure their safety, and water treatment (19)(20)(21)(22).Previously a study by Gutierrez et al., showed excellent water solubility and photodynamic antibacterial activity of curcuminloaded nanoparticles (22,23).Samrot et al., proved with the use of polymeric nanoparticles, curcuminloaded chitosan/tetramethyl orthosilicate nanoparticles demonstrated a 60% reduction in bacterial growth (24).
The objective of this study was to produce zinc oxide nanoparticles utilizing Phoenix dactylifera (dates), a source known for its antimicrobial properties.The green synthesized ZnO NPs used in this study were characterized by visual identification and UV-vis spectroscopy.The visual colour change from brown to clear in response to time can be seen as evidence of zinc oxide nanoparticle synthesis in our study.There are several studies done which prove the antimicrobial activity of Phoenix dactylifera (25)(26)(27).

Future scope
It is a novel drug with can be used to treat certain oral mucosal lesions and potentially malignant disorders.Further in vivo studies can be done.

CONCLUSION
Green synthesis is an environmentally benign way of producing nanoparticles that do not require the use of hazardous chemicals, high temperatures, or expensive equipment required by conventional physical and chemical synthesis methods.The substantial antimicrobial effectiveness of nanoparticles stems from their remarkable surface area-to-volume ratio.This characteristic facilitates the attachment of numerous ligands to their surfaces, consequently enabling intricate interactions with receptors present on the surfaces of microorganisms.In the current study, zinc oxide nanoparticles (ZnO NPs) were made from Phoenix dactylifera using this eco-friendly method which were then evaluated for their antimicrobial effects.