Nanoformulation and Biological Evaluation of Kaempferol Nanoparticles for Enhanced Antibacterial Activity

INTRODUCTION
Specific chemicals and/or plant extracts obtained from natural sources (containing various secondary metabolites) have been proven to have antioxidant action. Plant metabolites have long acts as a means of inspiration for medicinal chemists and a platform for drug development They can play a series of ecological roles in the plant, including protecting it against herbivores, fungus, bacteria, and viruses, as well as assisting it in competing for water, light, and nutrients.[1] Kaempferol (3,4′,5,7-tetrahydroxyflavone) is a flavonoid found in kale, beans, tea, spinach, and broccoli, among other plants and plant derived foods. Kaempferol is a yellow crystalline substance that melts at 276–278 degrees Celsius (529–532 degrees Fahrenheit). It is soluble in hot ethanol, ethers, and DMSO but very slightly in water [2]. Because kaempferol’s structure is similar to estrogen’s, it could have both estrogenic and antiestrogenic effects depending on its concentration, and it could be used to treat hormonal cancers like ovarian, breast, and cervical cancers, as well as hepatocellular carcinoma, acute promyelocytic leukemia, and glioma. [3-4] There are four key stages in the production of kaempferol. In the first phase, phenylalanine is transformed into 4-coumaroylCoA via the phenylpropanoid metabolic pathway. Through the activity of chalcone synthase, 4-coumaroyl-CoA is joined with three molecules of malonyl coA to create naringenin chalcone (a tetrahydroxychalcone). The hydroxyl group of naringenin chalcone is implicated in the production of dihydrokaempferol in the third stage, when it is swapped with naringenin. Finally, dihydrokaempferol is transformed to kaempferol, which possesses a double bond [5-6].
About biomedical applications, the nanoparticles are almost surface modified. Furthermore, the modification can increase the chemical stability of the nanoparticles. An assortment of materials coating to modify the metal nanoparticles, like silica or biopolymers or until carbon [7-9]. This surface coating is important to avoid oxidation and the nanoparticles agglomerate, which increases their biocompatibility [10] in biomedical applications Fe3O4 NPs can be used in drug delivery magnetic resonance imaging (MRI), contrast agents and therapy [11-12]. There are several methods of synthesizing magnetite nanoparticles. The most common method is the co-precipitation of ferrous and ferric ions into an alkaline medium [13]. In addition, thermal decomposition [14], sol-gel synthesis [15], hydrothermal synthesis [16], electrochemical synthesis [17] and microwave assisted synthesis [18]. Recently, studies have shown that the incorporation of copper nanoparticles into chitosan significantly reinforce its antimicrobial and antifungal activity [19]. The choice of chitosan as a stabilizer for Cu NPs due to its ability to chelate metals, making it an ideal material for metal nanoparticles. Chitosan as a stabilizer and or reducing agent [20].

MATERIALS AND METHODS
Materials
Chitosan (code KB-002) (Merck) (Deacetylation content = 89 %), kaempferol locally. Propanaldyhede (Sigma-Aldrich) Succinic Anhydride (BDH).

Instrumentals
FTIR spectra were recorded in KBr discs on (FTIR model 8000) Testcan Shimadzu IR-Spectrometer under dry air at room temperature within the wave number range of 4000-600 cm-1.Samples were heated from 0 to 500 oC in a platinum pan with a heating rate 10 oC / min, in N2 atmosphere of flow rate 25 mL / min. X-ray diffraction technique Research Center, Baghdad (Shimadzu 6000 X-ray with 2θ angle covered from 20 to 80 degree) The roughness of the nanoparticles surface was characterized using AFM (type AT3000, Angstrom, Scanning Probe Microscope, Advanced Inc., USA). This technique provides two- and three-dimensional profile images. Scanning electron microscopy of nanoparticles were determined by (TESCAN-Czech Republic).
Extraction
The separation of medicinally active parts of plant or animal tissues from inactive or inert components using selected solvents in conventional extraction techniques is referred to as extraction in the pharmaceutical industry. Plant-derived products are impure liquids, semisolids, or powders intended for oral or external use alone. [21]. Aligi (kaempferol) , was weighed (100 g) and put in a beaker with (1000 ml) of 70 % ethanol. The solution is macerated for 48 h at room temperature with 360 rpm agitation until the soluble stuff has dissolved. The solution was purified by filtering under vacuum using filter paper after maceration, and then concentrated using a rotary evaporator (less than 70°C under decreased pressure). Finally, the extracted material was weighed and placed in the refrigerator to be analyzed later [22].

Methods
Preparation ofpoly Schiff base of chitosan (Ch-Pr) (1) [23]
A (1 gm) of chitosan in (50 ml) of glacial acetic acid (1%) was stirred at 30 °C for 1 h. A (0.009mol,0.52 g) of propanaldehyde mixed with chitosan The mixture was stirred and heated in a water bath at 70 °C for 6 h. 5% sodium hydroxide drops are added until precipitation of the desired compound. The Transparent white precipitate was collected and washed with (10 ml) diethyl ether and to remove any remaining materials. The products were filtered and dried in a vacuum oven at 60 °C overnight [24].

Synthesis of (chitosan– grafted - kaempferol) (2)
An amount of 0.007mol, 2 g of kaempferol was dissolved in absolute ethanol, added to 3 g of chitosan-g-succinic anhydride that had been dissolved in 10 ml of dimethyl sulfoxide (DMSO), and the mixture was refluxed for 6 hours at 70 °C. The black precipitate was collected, continuously washed with 10 ml of di-ethyl ether, and then dried in a vacuum oven at 50 °C [26].

Preparation of Schiff base–grafted-kaempferol-coated Cu NPs (3)
Copper-chitosan Schiff base nanoparticles synthesized by green method via one-step which is 1gr Schiff base – grafted - kaempferol dissolved in 50 ml of ethanol then add 25 ml of (0.3g) copper sulfate (CuSO4.5H2O) and stir in 70 °C for 13 hours until a reaction, is complete. After then, 13 hours a result product centrifuged in 5000 G to30 minutes till particles are separate from suspension. Resupended the precipitat in acetone (99% ᵛ/ᵥ) then the centrifugation has been reiterated five times to co-precipitation.

Preparation of Schiff base – grafted - kaempferol -coated Fe NPs (4)
Magnetite iron oxide (Fe3O4) nanoparticles are prepared via the co-precipitation, method which FeCl3.6H2O and, FeCl2.4H2O, which include (1.7g) and (4.7g) respectively of salts in 50 ml deionized water. Then ammonia solution (25%) has been added to the salts of iron solution meanwhile syringe pump beneath mechanical stirring until the pH of the solution arrive to ± 10. In the precedent process will generate the dark brown precipitant of Fe3O4 nanoparticles. The generated precipitant washing in deionized water to eliminate every alkali metals and until arrive the solution (pH±7). The resulting product dried via freeze process. To synthesize Fe3O4 nanoparticles coated chitosan –g-kamferol then added to it and was heated at 70 ºC for 2 hours to output a transparent solution which is known “sol”. The obtained sol has been heated to evaporate most of its solvent and getting an additional viscous gel [27,28].

Antimicrobial activity [29]
Antibacterial activity of the prepared compounds was evaluated against types of bacteria (Gram-positive and Gram-negative), Streptococcus, Bacillus, Escherichia coli, Klebsiella the testing was performed using Agar diffusion method. The inhibition zone is measured by using a metric ruler over the inhibition zone, at its widest diameter, and then measuring from one edge of the zone to the other. It may be helpful to hold the panel up to the light and then use measurements in millimeters. Most of the tested compounds showed clear antibacterial activity, especially compound 1,2 the results of these two compounds were nearly similar to those of Ampicillin, which was used as a reference. Compound 5 showed the greatest activity against bacteria Bacillus, Candida. While compound 3 showed the strongest effect against Candida and both compounds had a strong effect against other bacteria. While compound 4 showed the strongest effect against Candida and both compounds had a strong effect against other bacteria. 

RESULT AND DISCUSSION
This study includes synthesis and polymerization of new derivative compounds of nanoparticles of kaempferol and Schiff base through the reaction of amin groups in chitosan with propanaldehyde then Schiff base reaction with succinic anhydride by ring opening the carboxylic groups in succinate reaction with hydroxyl group in kaempferol.

FTIR
The infrared spectra of the compounds are presented in Figs.1-3. It was shown that a high intensity band at the range of (3379-3480) cm-1 which refer to the stretching vibrations of hydroxyl groups. Moreover, the high intensity of this band due to the presence of more than two hydroxyl groups in the structure of these compounds. The spectra of compounds (1-3) show two bands at (3000-3062) and (2924-2970) cm-1 due to (C-H)aliph and (=C-H)Ar, respectively. The stretching vibrations of (C=O) for these compounds appeared as a strong bands at (1693-1624) cm-1, while the stretching vibration of (C=C) appeared at (1620-1631) cm-1.The stretching vibration of (C=N) of these compounds appeared at the range of (1550-1631) cm-1 while the stretching vibrations of (C-N) group appeared at (1203-1207) cm-1. The (C-O) stretching vibrations appeared at the range of (1260-1319) cm-1. Finally, the spectra show in addition to the above-mentioned bands, the bands of the bending vibrations of (C-H)Ar and (C-H)Alph [29-30]. 
XRD is a swift analytical technique common in characterizing crystal structures and an atomic spacing. Firstly, it is used for stage coincidence of a crystalline material and supplying information on unit cell dimensions. The XRD peaks are formed via deductive confusion of a monochromatic x-rays beam scattered at specified angles from every set of network planes in a sample. XRD has good potential for nano-structure analysis due to the width and shape of the reflections provide information about the material substructure, to determine the structure and crystal phase compound 3, 4, and the intensity was determined by step scanning 2θ range of (10-80°). The X-ray diffraction analysis of compound 4 nanoparticles is presented in Fig. 4. The diffraction peaks are located at 2θ = 21°, 32° and 44° in Fig. 4. Also, the X-ray diffraction analysis of Characteristic peaks have been observed in the XRD pattern at 2θ = 29°, 41° and 53°.in Fig. 5.

The Scanning Electron Microscopy (SEM)
The SEM is a one of kind of electron microscope that produces images of a specimen via scanning the sample surface by using a concentered beam of the electrons. The interaction between electrons and atoms sample, leads to various signals which contain information on sample surface composition and topography. Fig. 6 shows the (SEM) images of Schiff base –g- kaempferol- coated Cu NPs, the SEM photographs that indicate the diameters of the particles with approximately (29 - 256 nm) diameters. Also, Fig. 7 shows the (SEM) images of Schiff base –g- kaempferol-coated Fe3O4NPs, that indicate the diameters of the particles approximately (35-46nm).

Atomic Force Microscopy (AFM)
This technique provides two and three-dimensional profiles of the surface at a nanoscale by measuring the forces between a sharp cantilever tip and a surface at a very short distance. Figs. 8 and 9 show the three-dimensional images of Schiff base –g- kaempferol coated of magnetite, copper and iron nanoparticles respectively, 10 and 11 show and display the granularity cumulation distribution charts for Schiff base –g- kaempferol -coated of magnetite, copper and iron nanoparticles respectively. Table 1 shows the particle size distribution of Schiff base –g- kaempferol -coated Cu NPs with a mean diameter range from 20-100 nm and an average diameter 62.99 nm. Table 2 shows the particle size distribution of Schiff base –g- kaempferol -coated Fe3O4 NPs with a mean diameter range from 65-100 nm and an average diameter 75.31 nm. 

Antimicrobial Activity
Biological activates of some prepared compounds 1, 2, and 3 is tested against bacterial strains, Rhizosporuim using agar well diffusion method. The results shown in Table 3
Antibacterial activity of the prepared compounds was evaluated against types of bacteria (Gram-positive and Gram-negative), Streptococcus, Bacillus, Escherichia coli, Klebsiella the testing was performed using Agar diffusion method. The inhibition zone is measured by using a metric ruler over the inhibition zone, at its widest diameter, and then measuring from one edge of the zone to the other. It may be helpful to hold the panel up to the light and then use measurements in millimeters. Most of the tested compounds showed clear antibacterial activity, especially compound 1,2 the results of these two compounds were nearly similar to those of Ampicillin, which was used as a reference. Compound 5 showed the greatest activity against bacteria Bacillus, Candida. While compound 3 showed the strongest effect against Candida and both compounds had a strong effect against other bacteria (Figs. 10 and 11).

CONCLUSION
1-The present study aims to achieving synthesis, and characterization of some new chitosan\ kaempferol polymer 
2- high molecular weights led to an increase in their biological activity
3-The prepared compounds proved to be resistant to some types of bacteria these compounds have medical application.
4- synthesis of new coated of chitosan -g- kaempferol nanoparticles of copper and iron.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.