Encapsulation of propolis extracted with methylal in the chitosan nanoparticles and its antibacterial and cell cytotoxicity studies

Drug delivery systems using nanocarriers provide an improved drug stability, effectiveness, and deep penetration [1, 2]. The choice of the nano carrier is critical as it may have an impact on the medication's or the natural active ingredient's protection, retention, and bioavailability of chosen drugs. Due to its non-toxic and non-immunogenic activities, chitosan biopolymer based nanocarrier is ideal for biomedical and pharmaceutical applications. As a nano-carrier, chitosan has the ability to encapsulate and protect drugs and other bioactive compounds, allowing for targeted delivery to specific cells or tissues. Chitosan offers the best nano carrier for natural products due to its ability for encapsulation of multi-component. Deacetylation, molecular weight, pH, ionic strength, and non-aqueous solvents all impact chitosan's antibacterial, biodegradability, and biocompatibility [3]. Microbial mortality results from interactions between the cationic chitosan and its amino group and the negatively charged cell surface of microorganisms. Therefore, chitosan combination with other antibacterial compounds, especially natural antibiotics like propolis, is an emerging concept for a successful therapy [4, 5]. The development of new antibiotics has slowed down in recent years, making it more challenging to treat infections caused by antibiotic-resistant bacteria. One interesting optionfight bacterial infections would be to use natural antibiotic solutions that have the capacity to inhibit biofilm development without bacterial resistance. Natural antibiotics have antimicrobial properties serving as potential alternatives to pharmaceuticals which prove ineffective against widespread antibiotic resistance in various industries. The resinous material such as propolis, which is gathered by honeybees, contains more than 300 distinct substances, including resins (50%), wax (30%), balms (10%), essential oils and aromatics (5%), pollen, polyphenols, flavonoids, and active ingredients (5%). Some of useful compounds in propolis are, cinamic, amino acids, benzoic acid derivatives, flavonoids (galangin, chrysin, pinocembrin), polyphenols, vitamins, carotenoids, sterols, and terpenes [6, 7]. Propolis color, smell, and chemical composition depend on the location, climate, time of collection and the type of bee species [8]. Propolis has proven with antibiotic, antioxidative, anti-inflammatory with eliminating IL-1 beta and IL-6, anti-prostatitis, anti-hepatotoxic, and anti-anemic properties. [7, 9], Besides propolis possesses antiviral activity against influenza virus type A and B, HSV-1, HSV-2, Adenovirus, HIV, infectious bursal disease virus, and avian reovirus, bovine rotavirus, bovine viral diarrhea virus, Newcastle virus, feline calicivirus, pseudorabies virus, and canine adenovirus type 2 [10], antifungal [11],as well as wound healing, and anti-cancer properties [12]. Propolis has antibacterial properties that can be used to reduce bacterial viability, interfere with bacterial growth, and prevent bacterial adhesion by inhibition of glucosyltransferase enzymes (GTFs) [9] and affect the gene expression [13]. Moreover, propolis has been used in certain instances to combat several murine and human melanoma cell lines, including B16 and A375 with reduction of NF-KB and JAK2 / STAT3, and blocking PAK1 [14]. Despite the extensive therapeutic potential of propolis, its formulation is often hindered due to its complex multi-component nature, resinous and adhesive consistency, limited solubility, and physical instability. On the other hand, due to propolis astringency and bitterness, is not as widely accepted as it could be by consumers. According to several studies, polyphenols cause those flavour characteristics and have a detrimental effect on consumer acceptability [15]. However, propolis can be encapsulated in nanoemulsions to reduce its effect on the organoleptic characteristics of the foods it is added to. Propolis's use in foods is supported by its nanoencapsulation, which improves its antimicrobial activity while lowering the impact on taste and allergy. Numerous studies have investigated the effects of different solvents on the extraction of phenolic and flavonoid compounds from propolis such as Ethyl acetate, Methanol, Dimethyl sulfoxide [16], Water [17], Chloroform, Acetone, Dichloromethane and Hexane [18]. For extracting biologically active compounds from propolis, ethanol is an appropriate solvent that is frequently utilized. [19]. Ethanol extracts of propolis have been shown to contain high level of phenolic and flavonoid compounds. Ethanol can extract these compounds effectively from propolis as it can dissolve a wide range of polar and nonpolar molecules. Phenolic compounds and their subclasses, flavonoids in propolis are known for their antioxidant properties. Chitosan-propolis nanoparticles tested against S. epidermidis [20]. Teik's group proposed that cationic nature in chitosan can potentially kill gram-positive bacteria biofilm with changing the zeta potential of the biofilm [20]. Dimethoxymethane, also known as Methylal, with the molecular formula C₃H₈O₂, is a commonly used organic solvent that classified as an acetal. Methylal is an essential starting material and a superior solvent in the chemical industry [21]. It is a colorless liquid with a mild, ether-like odor and is soluble in a wide range of organic solvents, including alcohols, ketones, and esters. Methylal is a polar aprotic solvent, which means it can dissolve a wide range of organic and inorganic compounds that we can find in the Ps. Methylal is commonly used as a solvent in the production of resins, coatings, and adhesives, as well as in the synthesis of pharmaceuticals, agrochemicals, and other special chemical compunds. It is also used as a fuel additive and as a solvent for oils and waxes. The most important step in extracting Ps extracts is to choose an effective and economical solvent. According to the published article the phenols in Ps can be protected using Methylal [22].This protection increases the properties of phenol in the long term. High concentrations of methyl can show some exhibited symptoms of eye and respiratory tract irritation [23]. Methylal can affect breathed in and may be absorbed through the skin. But like all chemicals, most chronic (long-term) effects result from repeated exposures to a chemical. But Methylal evaporates readily during the Ps extraction procedure. Bacteria can form biofilms on the surfaces of the devices, making them difficult to eradicate with antibiotics. It does not cause harm to its human host under normal circumstances. S. epidermidis is a species of Gram-positive bacteria that is part of the human skin microbiota. However, S. epidermidis can cause opportunistic infections in individuals with weakened immune systems or in medical settings, such as hospitals. S. epidermidis, as normal flora and resistance to β-lactam antibiotics that used in clinical settings, can adhere to both biotic and abiotic surfaces [24]. S. aureus is a species of Gram-positive bacteria that can be found on the skin and in the nasal passages of healthy individuals. S. aureus is also a significant human pathogen that can cause a wide range of infections. S. aureus infections can range from minor skin infections, such as boils and impetigo, to more serious infections, such as pneumonia, endocarditis, and sepsis. The bacterium is known to produce a number of virulence factors, including toxins, enzymes, and surface proteins, that contribute to its pathogenicity. In recent years, S. aureus has become a significant public health concern due to the emergence of antibiotic-resistant strains, such as methicillin-resistant S. aureus (MRSA). Prevention of S. aureus infections involves good hygiene practices, such as regular hand washing, and appropriate use of antibiotics [25]. The development of new antibiotics is also being explored, in combating antibiotic-resistant S. aureus [26]. E. coli is a gram-negative, facultative anaerobe, meaning it can grow in both aerobic and anaerobic environments. However, some strains of E. coli have developed resistance to antibiotics, making treatment more challenging [27]. The other gram-positive, rod-shaped, non-spore-forming bacteria L. monocytogenes is what causes listeriosis. Food is the usual source of contamination. P. aeruginosa, a kind of germ-negative bacterium, can infect the blood, the lungs (pneumonia), or other areas of the body. These bacteria are always coming up with novel strategies to evade the antibiotics that are used to treat the diseases they cause. These microorganisms may become multidrug-resistant if they acquire resistance to multiple different classes of antibiotics. Primary intestinal pathogen S. typhimurium can infect both people and animals. Salmonellae enter the intestinal epithelium after being consumed in tainted food or drink, which starts the infection process. Due to the slightly polar nature of propolis components (which typically contain multiple OH groups in their molecules), HPLC is the technique of choice for propolis component identification. In this study, we are following several objectives which include 1) for the first time extracting propolis by Methylal solvent (PM), 2) investigation of various compounds from Ps extracted with Methylal using the HPLC and GC-MAS, PE and PM encapsulation with different volumes of chitosan NPs, and analyzing the in vitro propolis release profile, 3) characterizing the morphological and physicochemical properties of Ps-NPs such as particle size, polydispersity index, zeta potential, and surface morphology, 4) analyzing the antibacterial effectiveness of chitosan-propolis NPs (PM-CH) on gram-positive and negative bacteria, and 5) disruption study of PM effect on the biofilm and pre-biofilm on the (S. aureus, S. epidermidis, L. monocytogenes) and three gram-negative (E. coli, P. aeruginosa, S. typhimurium) bacteria growth. Furthermore, maybe the antibacterial qualities, toxicity, and cell growth will be different with phenol and flavonoids extracted using various solvents. Methylal and chitosan NPs can shield to phenolic compounds and exhibit a delayed release of the drug, which may offer an improved feature of Ps at a reduced concentration. Therefore, the goal of this work is to investigate the antimicrobial and antioxidant activities as well as chemical components of propolis extracted using Metylal and create a nanoformulation for use as a natural antibiotic.

Propolis has different antimicrobial mechanisms, including inhibition of cell division, disruption of cell wall integrity, inhibition of bacterial motility, surface-exposed adhesions and polypeptides and cell membrane enzymes, bacteriolysis, blocking of ion channels, Inhibition of electron by capturing electrons and protein synthesis inhibition [55] change surface protein three dimensional (3D) structure [56]. With the latter mechanism, the microbial proteins and propolis polyphenols create ionic and hydrogen interactions change the proteins' 3D structure and functions [57]. In multiple studies it was found that Gram-positive bacteria were more susceptible than Gram-negative bacteria [58] which was attributed to specific structure of the outer membrane such as a multilayered membrane in Gram-negative than a single layered membrane in the Gram-positive bacteria [59]. Also, Gram-negative bacteria create hydrolytic enzymes that disintegrate the propolis' active components [49, 60]. There are many different reports on the characterization of propolis and evaluation of its anti-bacterial effect against many bacteria such as S. mutans [61], S. epidermidis [56], Enterococcus faecalis [13] P. aeruginosa, S. typhi, E.coli, S. aureus, B. subtilis, Enterococcus sp, and Candida spp [62]. Additionally, the antibacterial efficacy of propolis from various parts of the world was comapred. These reports mentioned that the mechanism of propolis activity depends on the synergy between compounds in propolis, the effect of extracting methods and their interference with microbes. The different ranges of MIC and MBC for various solvents, including alcohol, water, DMSO, DCM, Hexane, and supercritical fluid have been recorded [5]. We conducted a comparison between the composition and biological activities of propolis extracts and a nonalcoholic a substitute solvent mixture, such as Metylal, which we used as a green solvent. In the literature, there are references to other nano formulation ns that contain propolis extracts, but they have distinct formulations and uses [63‐65]. The size, shape, and surface charge of nanoparticles (NPs) as well as how they interact with biological systems can influence NP behavior and ultimately determine their toxicity. Also, encapsulation inside chitosan NPs offered a better dispersability, and prolonged release time of propolis. Drugs encapsulated in nano particles carriers with surface charges are resistant to enzymatic degradation [66]. We hypothesize that extracting propolis with Methylal and formulating it into nano-sized chitosan will improve its antibiotics effects and thus it’s cytotoxicity. We found that Metylal is a excellent solvent for the most active ingredients of propolis, including hydrophilic and lipophilic compounds. The PM-CH formolations revealed that a chitosan polymeric system does not have a negative impact on the stability, potency, or biological activity of flavonoids and phenols in propolis extract. Therefore, in this study, we have chosen 0.2% w/v as an ideal concentration of chitosan. Only physical interactions between the propolis extract, and the chitosan were observed; no significant chemical interactions were evident. Due to positive zeta potential of some molecules like chitosan, enhances drug delivery by facilitating adherence to the negatively charged cell membrane. The kinetics of drug release from NPs should therefore be a key component of their design and a trait that is checked for quality [67]. Chitosan with a positive surface charge, has interacted with negatively charged bacteria surface, thereby resulting in the cell surface permeability and then inhibiting of bacterial growth with bind to the anionic bacteria and increase their zeta potential [20]. The results of this investigation agree with that reported by Teik and co-worker which indicated that decreasing and increasing the chitosan concentration below 0.2% w/v resulted in clumping. These authors also confirmed that chitosan solution above 0.5% w/v lead to the formation of large, aggregated nanoparticles with aparticle size above 400 nm [13]. In general, propolis was released from PE-CH and PM-CH at a slower rate than from PE and PM. Consequently, chitosan NPs released the propolis continuously and under control, preventing changes in the release rate. As a consequence, both Gram-positive and Gram-negative strains showed significant antibacterial activity for Middle Eastern propolis [58]. In the present study, PE, PM, PE-CH and PM-CH inhibited the growth of Gram-negative bacteria such as E. coli, P. aeruginosa, and S. typhimurium with MIC values ranging from 12.8 to 230 μg/ml. Also, PE, PM, PE-CH and PM-CH inhibited the growth of gram-positive bacteria such as S. aureus, S. epidermidis, and L. monocytognes with MIC values ranging from 2 to 13 μg/ml. The outcomes were consistent with another study on Gram-positive bacteria, which had MIC values ranging from 3 to 100 g/ml [68]. The majority of Gram-positive bacteria create extracellular thin coatings of glucans, which, when present with sucrose, cause adhesion and biofilm development. The succeeding bacterial population produces biofilms after the initial bacterial adhesion to a particular surface [69]. Both pre-formed biofilm and biofilm assay are important methods for evaluating the effectiveness of antimicrobial agents against biofilms, which are known to be more difficult to eradicate than planktonic bacteria. Since propolis extracted with Methylal has a high antioxidant activity and the potential to suppress bacterial growth, it was used to create the nano emulsion. The antioxidant activity may be caused by phenolic substances [70]. Chemically, phenols are composed of an aromatic ring that is linked to one or more hydrogenated substituents, along with their derivatives. While all propolis extract methods lower the membrane potential of bacteria, our result shows that chitosan-propolis Methylal extracted NPs (PE-CH and PM-CH) have a great effect against suspension planktonic as well as biofilm of E. coli, S. aureus, S. epidermidis bacteria. The results of MIC and MBC for PE and PM activity demonstrate that compounds like phenolic acids, esters, flavonoids, and terpenes are likely to react with light, air, water, and other extrinsic elements, promoting rapid degradation or inactivation of biological activity. However encapsulation of propolis inside in chitosan NPs could prevent degradation and deactivation of the propolis extract. Our data shows that lower PE-CH and PM-CH concentrations are sufficient to kill planktonic bacteria compared to biofilm and pre-formed biofilm established by S. aureus and S. epidermidis due to small particle size and the positive zeta potential, has better antibacterial efficacy as compared to PM and PE extracts which enables them to penetrate in to the biofilm. Contrary to our results obtained with nanoparticles of propolis, although PM-CH could reduce E. coli in biofilms by 98–100% (at concentration of 300μg/ml), at the same concentration, only 65–80% of bacteria in pre-formed were eradicated. Similar results were obtained for the other positive bacteria. The DPPH assay displayed good antioxidant activity, with inhibition values of 43% for PM-CH and only 36% for the PE-CH sample. Our findings indicate that propolis's high potential for antioxidants in nature makes it a potential functional drug and food. There is a strong correlation between propolis's antioxidant capacity and its phenolic compounds, minor components, amino acids, flavonoids, terpenes, steroids, aldehydes, and ketones, which are the main sources of propolis's antioxidant capacity (Tables 2 and 3). Propolis's antioxidant properties are thought to be mediated by a variety of mechanisms, including metallic ion chelation, hydrogen donation, free radical sequestration, and serving as a substrate for radicals like hydroxyl and superoxide. Additionally, it has been proposed that the organic acids (Tables 2 and 3) in propolis enhance the effects of flavonoids and, via metal chelation, contribute to antioxidant activity. It should be noted that flavonoids are more easily oxidized the more hydroxyl groups they contain. For this reason, maybe nanoencapsulation of propolis can reduce the oxidation of flavonoids to have a lasting and more effect [40]. Natural-derived propolis has cytotoxic effects on several cell lines. For various cell lines, it was shown that the reported IC50 values of propolis varied greatly, which might be explained by the fact that the ingredients of propolis depend on its geographic origin, bee type, plant pollen, etc. Due to propolis's reliance on different solvents, nanocarriers, and compositions, the reported IC50 values of the compound varied greatly for different cell lines. The cytotoxice results showd a discernible difference between IC50 values of PE and PM with PE-CH and PM-CH, confirming the relative safety of pure propolis and encapsulation form. Similarly, great doses of PE-CH and PM-CH showed comparable cellular viability of healthy cells but compromised the cancer cells. Overall, the choice of solvent, extraction method, and choice of suitable nano carrier should be carefully considered based on the targeted properties of the propolis material and the biological effects of the extract.

In this study, we have substantiated that the formulation based on chitosan NPs encapsulated propolis, and the extract of propolis with Methylal have great therapeutic effecieces toward combating bacterial biofilms. Our MIC, MBC, and biofilm assay results like previous studies showed that Gram-negative bacteria are more resistant than Gram-positive bacteria. The synergistic effect of chitosan NPs and propolis has amplified the the antimicrobial effeciencies. Our MIC, MBC, and all biofilm assay results are in agreement to those from propolis-producing Middle Eastern nations like Turkey and Oman which indicates the same plants were the food source of bees use to make propolis. Our research has shown that the Methylal extract of the propolis has a high total flavonoid and phenolic contents. In comparison to free propolis, the release profiles of PE-CH and PM-CH demonstrated that propolis compound release from NPs is slower, which sustained the release of phenols and flavonoids against destruction. Considering these factors, MIC, MBC, and all biofilm assays reports reports on the successful extraction of propolis with metylal and successful encapsulation of propolis in chitosan, can be possible great substitutes for industrial antibiotics. Early indications that both PE-CH and PM-CH compounds have great effect against some bacteria that have resistance to the routine antibiotics. Both PE-CH and PM-CH had a favourable effect on the cell culture and increased the quantity of viable cells in terms of cell viability. Propolis and NP were found to be relatively safe, as evidenced by the significant difference in cytotoxicity between cancer cells and normal human fibroblasts. A final general protocol's development may be challenged by the fact that various experiments produce different results from each other. Furthermore, at higher concentrations than indicated in the manuscript, methylal can be dangerous for the researcher if safety precautions are not taken. This suggests that the extract may have potential antioxidant properties, which could be beneficial for various healthcare applications, including wound healing, natural antibiotics, cosmetic uses, and cancer therapy.