INTRODUCTION:

One of endodontics' major goals is to sterilize the root canals. Persistent infection is one of the main causes of endodontic failure in teeth that have undergone root canal therapy^1,2. A complex root canal anatomy or bacteria like Enterococcus faecalis and C. albicans that are resistant to antibiotics may cause endodontic treatment to fail^3,4. Therefore, it is essential to effectively disinfect the root canal system.

Intra-canal medication is a critical component of endodontic therapy that depends on the effectiveness of antimicrobial medications to get rid of microorganisms².

The environmentally acceptable substance calcium hydroxide (Ca(OH)₂) has drawn a lot of interest due to its wide range of uses^5,6, including applications in hair care products, dental work, food manufacturing, leather production, and others. It is one of the most often used intracanal drugs during endodontic therapy. It has been demonstrated to be quite successful in the treatment of teeth with persistent periapical lesions⁷. Calcium hydroxide is the root canal treatment drug that is most frequently utilized. It has the ability to dissolve tissue and serves as a physical barrier. It also produces hydroxyl ions, which create a very alkaline environment ⁸.

A Gram-positive coccus called Dentinal tubules can be invaded by E. faecalis, and create biofilm, adhere to serum collagen, and decrease lymphocyte activity, which can keep them from being harmed⁹. Additionally, it contains a mechanism that inhibits the proton pump that enables it to tolerate a broad pH range, up to about pH 11.5^10.It may therefore resist the alkalinity of calcium hydroxide, persist after root canal treatment, and is usually discovered in recurrent infection^11,12. Also, another microbe linked to chronic post-treatment apical periodontitis is C. albicans¹³. Switching between hyphal forms and blastospore is one of C. albicans main pathogenicity traits. This allows it to enter host tissue while avoiding phagocytosis via macrophages^14.Deep dentinal tubules can be accessed by C. albicans due to thigmotropism¹⁵. Biofilm can be formed by C. albicans in 48 hours¹⁶. It can endure a wide pH range, an environment with a high alkalinity, and extreme ecological circumstances, allowing it to produce prolonged infection¹⁴.

On the other hand, deacetylation of chitin in crustacean shells produces the natural polysaccharide known as chitosan. Chitosan accelerates healing of wounds and possesses antifungal and antibacterial effects¹⁴. Chitosan may prevent the development of biofilms by interacting with the cytoplasm,cell membrane, and outer-layer components of microorganisms¹⁵. Additionally, E. faecalis and Candida albicans biofilm form and planktonic were inhibited by chitosan, which also has a low toxicity and high biocompatibility^16,17. As a result, chitosan may be useful as an antibacterial intracanal medicine. Certain chitosan derivatives have been proven to be efficient against common oral Candida species, such as C. albicans and E. faecalis, as reported in previous research^18,19. Particularly, it has been shown that 2100 kDa and 1700 kDachitosan are efficient against E. faecalis, but they need a lengthy contact period (over 1 h)¹⁹. As a result, we suggest that these chitosan derivatives show promise for use in the development of intracanal medications^19,20. Therefore, novel therapeutic drug release methods being developed, together with increased specificity, enhanced bioavailability, decreased pharmacological toxicity, and sensibilityhas all become very interesting due to the properties of chitosan micro-particles in biomedical engineering, nanomedicine, and other fields²¹.

Native to Asia, Africa, and America, Moringa oleifera is a well-known medicinal plant with a variety of traditional applications. In Africa and Eastern Nigeria, it is a typical vegetable. Several common nomenclatures are used such as the Miracle tree, Drumstick tree, Ben oil tree, Horseradish tree, and “Mother’s Best Friend”²². It has various tastes and flavors; its seeds and leaves are utilized as food supplements²³. Moringa oleifera has been shown to have antioxidant,anti-inflammatory antiviral,and antibacterial characteristics. Being widely known for treating a variety of diseases like colon cancer, myeloma, malnutrition, and malaria²⁴. As a result, M. oleifera leaf has the potential to be employed both in medicinal and food. Additionally, nanoparticles are known to be more reactive and hence more efficient in their antibacterial action due to the increased surface area of contact with the microbial membrane and the resultant aggregation on the surface of the microorganisms' cell wall^25,26,27. Therefore, inhibiting bacterial endotoxins and promoting apical and periapical healing are two of H₂CaO₂nanoparticles well-known properties²⁸.

In this study, chitosan and H₂CaO₂ nanoparticles assayedand GC-Mass of the Moringa oleifera of ethanol extractwere studied then the anti-inflammatory, antimicrobial, and cytotoxicity of chitosan-moringa composite and Calcium hydroxide nanoparticles as an intra-canal medicament in vitrowere also investigated.

MATERIALS AND METHODS:

1. Chitosan nanoparticles preparation:

Sigma-Aldrich was used as source of the chitosan. According to the method described in²⁹, nanoparticles were created. In order to create the solvent phase, acetic acid (1% and 2% v/v) was used to dissolve chitosan (0.05% w/v).

A peristaltic pump (Cole Parmer, Model Master flex C/L) was used to drop 5mL of this phase into the non-solvent phase (80mL of methanol), where it was kept under stirring. Once the nanoparticle solution had been created, the methanol was removed by spinning the mixture at 40^oC and 50rpm (Büchi Rotavapor R-114, Heating Bath B-491). It was then placed in a refrigerator set at 4°C.

2. Calcium hydroxidenanoparticles:

Calcium hydroxide nanoparticles from nanomaterial powder with purity: 99.95+%, and size: 10-70nm was used.

3. Characterization of Chitosan nanoparticles:

3.1. UV–Vis spectrophotometry:

Using a (T80+UV/VIS Spectrometer, PG Instrument Ltd., UK), the ultra violet evaluation of chitosan nanoparticles was carried out, and measurements were made to verify the absorbance of the particles over a range of absorbance between 200 and 600nm until no further absorbance changes were detected.

3.2. FTIR spectrum measurement:

The Nexus 670 FTIR spectrophotometer was used to perform Fourier transform infrared spectroscopy on a variety of samples containing chitosan nanoparticles (ATRS-FTIR). Sample spectra between 4000 cm^-1 and 400 cm^-1 in the middle infrared band were recorded with a resolution of 4 cm in the absorbance mode across ten scans at room temperature³⁰. By applying 1 mg of material to the instrument's sensor, FTIR spectra of chitosan nanoparticles were acquired.

3.3.Transmission electron microscopy (TEM):

TEM of obtained using (electron probe micro-analyzer JEOL-JXA 840A, Model Japan) offer specialist knowledge on morphological characteristics, such as size and shape. Onto a carbon-coated copper grid, the samples were produced by drop coating. Samples were vacuum dried before being put into a specimen holder. TEM micrographs were taken after the ready grids were analyzed.

3.4.X-Ray Diffraction (XDR):

For describing the structural characteristics of CNPs, XRD is one of the most important tools. At room temperature, measurements of X-ray diffraction were made using a diffractometer of the Bruker D2 Phaser second generation with Ni-filtered Cu K radiation (λ = 1.54A°). The generator was producing 30mA of electricity at 10kV. Data were gathered at a 2°/min scanning rate of 2°/min for 2θ between 0 and 40.

4. Extraction of Moringa oleifera leaves:

The leaves of Moringa oleifera were obtained from National Research Center, Giza, Egypt. After being air dried, the leaves were pounded into a coarse powder using a perfect mortar and pestle, sieved with a 1mm mesh thickness, and placed in a plastic container for storage. 50g of the powdered leaves were dissolved in 500mL of distilled water and left to dry for 72hours to create the aqueous extract. The ethanol extract, on the other hand, was made by steeping 45g of the leaves powder in 500mL of ethanol and letting it dry for 72 hours. Using Whatman filter paper, the ethanol mixture was filtered³¹, and the suspension was maintained at room temperature for 24hours while being continuously stirred. The extracted material was then collected and centrifuged for five minutes at 5000rpm after being filtered through two layers of muslin. The extract was concentrated for subsequent use in a vacuum evaporator and stored at -20°C. For the various studies, the extract was properly diluted³².

5. GC–MS of M. oleifera leaf extracts:

Ethanol extracts of Moringa oleifera leaf extracts was analyzed using gas chromatography-mass spectrometry QP2010PLUS system from Shimadzu Japan in 2010 with a fused GC column covered with polymethyl silicon at a size of 0.25nm by 50m under the following conditions:Temperature programming: from 80 - 200°C, for 1minute held at 80°C, at 200°C for 20 min, and rate 5°C/min. (FID) Field ionization detector at temperature 300°C, nitrogen at a flow rate of 1ml/min, injection temperature: 220°C, split ratio: 1:75. The column has a length of 30m, a diameter of 0.25mm, and a flow rate of 50ml/min. A mass spectrometer was used to collect the elute, the sample rate and the detector voltage were adjusted at 0.2 s and 1.5 kv, respectively.

The Hermlez 233 M-Z centrifuge and computer-fed mass spectra data weight after drying— weight before drying—weight before drying—100 bank was connected to the mass spectrum (Germany). By using Computer Wiley MS libraries to identify comparable peaks, the components of the extracts were first identified, and they were afterwards confirmed by comparison with peaks of the mass spectra in the published literature³³.

6. Biological activity:

6.1 Antimicrobialactivity:

The antimicrobial activity was carried out for Moringa (aqueous and ethanol) extracts, Chitosan, Ca (OH)₂, H₂CaO₂ nanoparticles, and Moringa-Chitosan composite (Chitosan+Moringa) in addition to control (Gentamycin for bacteria and Fluconazole for fungi) against dental pathogens Streptococcus mutans ATCC 25175, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 10541,Candida albicans ATCC 10221, and Escherichia coli ATCC 8739, were determined using agar well diffusion method³⁴.The density of the strain culture media was calculated using the McFarland standard turbidity as a reference. Using a photometer, each culture medium was adjusted to the McFarland turbidity standard No. 0.5 at 600 nm (absorbance 0.1, density of strain 10⁷ colony-forming units (CFU)/mL).Brain heart infusion and sabroad agar was used for bacteria and Candida albicans respectively, suspensions were spread on the plates using a sterile cotton swab, and then wells were made using a sterilized cork borer, and samples were added. Plates were incubated for 24h at 35^oC and finally the inhibition zones were measured.

6.2 Determination of MIC and MBC for Moringa - Chitosan composite:

The Chitosan-Moringa composite's MIC was determined using microtiter-plates, and 100µL of the 1% was then added to the first row of 96-well plates. Pipetting 100 µL of the material test from the first row to the other rows at serially decreasing concentrations (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and 1/128) was used to make serial dilutions. A bacterial solution (10µL) containing 1 x 10⁸ CFU/ml was applied to each well. They were incubated at 35°C for 18 to 24 hours, followed by the addition of 10µL of resazurin solution, and the plate was incubated for 24hours to check for any color change³⁵.

6.3 Anti-inflammatory activity:

According to Shinde et al., (1989) anti-inflammatory effects on HRBC hemolysis and membrane stability of Chitosan-Moringa composite, Moringa ethanol extract, Ca(OH)₂, and H₂CaO₂ nanoparticles have been done³⁶. Fresh whole blood drawn from healthy participants and placed into heparinized tubes was then for 10 minutes centrifuged at 3000rpm to prepare the erythrocyte suspension.The supernatant and the red blood pellets were dissolved in the equal volume of ordinary saline. An isotonic buffer solution and red blood pellets that had been dissolved were measured for volumewas used to reconstitute them as a 40% v/v suspension (pH 7.4, 10 mM sodium phosphate buffer). In 1 liter of distilled water, the buffer solution contained 1.15g of Na₂HPO₄, 0.2g of NaH₂PO₄, and 9 g of NaCl. Samples of the extract were dissolved in distilled water for this test (hypotonic solution). Duplicate pairs (per dose) of centrifuge tubes were filled with the calculated doses of the extracts (100, 200, 400, 600, 800, and 1000µg/ml) in the hypotonic solution (5 ml).

Additionally, duplicate pairs (per dose) of centrifuge tubes were filled with isotonic solution (5ml) containing graded doses of the extracts (100 - 1000µg/ml). The control tubes contained 5 ml of the drug (indomethacin 200g/ml) and 5ml of the vehicle (distilled water). To each of the tubes, erythrocyte suspension (0.1ml) was added and carefully combined. The solutions were first incubated for 1hour at 37^oC before being centrifuged for 3 minutes at 1300g. Using a Spectronic (Milton Roy) spectrophotometer, the supernatant's absorbance (OD) of the hemoglobin conc. was calculated at 540nm. Hemolysis in the presence of distilled water was assumed to be 100% in order to compute the percentage hemolysis³⁷.

The following estimate was made for the extract's percentage of hemolysis inhibition:

% Hemolysis Inhibition =

1-((OD2-OD1)/ (OD3-OD1)) x100

Where OD1 indicates the test sample's absorbance in an isotonic solution, OD2 indicates the test sample's absorbance in a hypotonic solution, and OD3 indicates the control sample's absorbance in a hypotonic solution.

6.4 MTT assay of cytotoxicity:

The International Center for Training and Advanced Researches, Cairo, Egypt, graciously contributed human oral epithelial cell line (OEC). Cells were grown in MEM-E media with 10% fetal bovine serum from Thermo Fisher Scientific, Waltham, Massachusetts, USA, at 37°C in a humidified 5% CO₂ atmosphere (Jouan SA, Saint-herblain, Pays de la Loire, France). Cells were kept in accordance with the manufacturing protocol, which included decanting the growth medium and washing the cells with phosphate buffer saline (Adwia Pharmaceuticals, Sharqia, Egypt). Cells were treated for 5 minutes at 37^oC with 0.05% (v/v) EDTA and 0.25% trypsin enzyme (GIBCO). Separated cells were spitted in accordance with need. Senthilraja and Kathiresan performed cytotoxicity on OEC cell lines that were grown in a 75cm cell culture (TPP-Swiss)³⁸. Cells were plated in 96-well cell culture plates at a density of 105cells/ml and incubated at 37°C for 24 hours before confluence was achieved. The growth medium was decanted and fresh medium containing serially diluted titanium samples was added to pre-cultured plates. After dead cells had been removed 24 hours later, each well received 50L of MTT stock solution (0.5mg/ml), which had been melt in phosphate buffer saline (PBS, pH = 7.2±0.2 (Adwia)). After 4 hours at 37^oC, the supernatant was removed, and the formazan precipitate was made soluble by adding 50 L of di-methyl sulfoxide to each well (DMSO). After the plates had been incubated at 37°C for 30 minutes in the dark, absorbance at 570nm was measured using a micro plate reader (ELx-800, Bio-Tek Instruments, Inc, Winooski, VT, USA).

The ratio of viable cells was calculated using the following formula:

Viability percentage (%) = Mean OD of test dilution × 100/Mean OD of control wells.

RESULTS:

1. Chitosan microparticles Characterization:

1.1 UV–visible spectrophotometer analysis:

The broad peak in the absorption spectra of the chitosan nanoparticles, which indicates a range in particle size, appears at 280- 300nm (Figure 1).

1.2 FTIR spectroscopy analysis :

The FTIR spectrum of chitosan revealed the intermolecular hydrogen bonding of the polysaccharide, the stretching vibration of OH, and the extension vibration of NH₂(Figure 2) as a broad and powerful band at 3442 cm¹. The amide I band's absorption peak, which is caused by the hydrogen-bonded -C=O-NHCOCH₃ group being stretched, occurs at about 1636 cm¹, indicating that N-deacetylation was successful.Also, at 1052 cm¹, stretching vibration (C-O) in the OH group was noticed.All the three band confirm the presence of the native chitosan.While, other bands confirm the presence of chitosan nanoparticles such as the asymmetric stretching vibration (CH₂) in the group CH₂OH was assigned to the absorption bands at 2067 cm¹. Both the symmetrical bending (CH₃) in the NHCOCH₃ group and the bending (CH₂) in the CH₂OH group were identified as the causes of the bands at 1417 cm¹respectively. Wavenumber at 1217 cm¹was connected to the complicated NHCO group vibrations (Amide III band).At a wavenumber of 1162 cm¹, the C-O-C symmetric stretching vibration in the glycosidic bond was investigated. In addition, at 1033 cm¹, the secondary OH group's stretching vibration (C-O) was seen. Finally, The C-H out of plane vibration was identified as the source of the wavenumber at 771 and 588 cm¹.

1.3 X-ray diffraction of chitosan nanoparticles:

Crystallographic structure of chitosan nanoparticles was detected by X-Ray Diffraction (XRD). The chitosan nanoparticles' XRD spectra showed characteristics of amorphous architectures. As illustrated in Figure 3, the diffractogram of chitosan there are three major unique peaks in the diffractogram of chitosan nanoparticles at 2 θ = 18.798°, 2 θ = 22.810°, and 2 θ 24.469^o indicating some degree of crystallinity of chitosan chains. Also, there are weak peaks in the XRD diffraction pattern of chitosan nanoparticles at 2 θ 61.702° and 2 θ 66.077° respectively.

1.4 TEM of chitosan nanoparticles:

We were able to determine the particle shape and size by TEM (transmission electron microscope) observation. In the present study, as illustrated at Figure 4 the TEM images showed physical aggregation of the chitosan nanoparticles. The chitosan nanoparticles size was between 76.61 – 126.91 nm under magnification of 200 nm. Also, the chitosan particles shape appears much less spherical with slightly wrinkled surface.

Figure 1: Chitosan nanoparticle UV-visible spectroscopy

Figure 2: Chitosan nanoparticles FTIR spectra analysis

Figure 3: Chitosan nanoparticles X-ray diffraction

Figure 4: Transmission electron microscope of chitosan nanoparticles

1. Extraction and characterization of Moringa oleifera leaf:

1.1. Extraction:

The yield of the Moringa oleifera leaves from the ethanol extract was higher than the yield from the aqueous extract, at 62.86% and 40.75%, respectively. According to Table 1, this indicates that ethanol performed better as an extracting solvent than aqueous extract.

Table 1: Physical characteristics and Percentage yield of the ethanol and aqueous extracts of Moringa oleifera leaf

Extracts

Powdered

leaf material

(g)

Yield in (g)

Yield in (%)

Physical

characteristics

Aqueous

18.34

36.68

Black

Ethanol

28.29

62.86

Light green

1.2. Characterization of Moringa leaf ethanol extract by GC-Mass:

The major chemical compounds in ethanol extract were found as illustrated in Figure 5 and Table 2 as follow: 6-Hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (2.45%), n-Hexadecanoic acid (11.91%) Cyclopropane butanoic acid, 2-[[2-[[2-[(2-pentylcyclopropy l)methyl] cyclopropyl]methyl]cy clopropyl]methyl]-, methyl ester (8.33%), Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (20.51%),Octadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester(25.95%), Phen-1,4-diol, 2,3-dimethyl-5-trifluoromethyl (6.65%), dl-à-Tocopherol (15.75%) and 24-Noroleana-3.12-diene (9.52%). Some other component couldn’t be identified and quantified, because they were obtained as minor components from the crude extract of ethanol.

Figure 5: GC-Mass of Moringa leaf ethanol extract

Biological activity:

1. Antimicrobial activity:

The antimicrobial activity of Moringa oleifera leaf (aqueous and ethanolextracts) in addition to control (Gentamycin for bacteria and Fluconazole for fungi) against dental pathogens Streptococcus mutans ATCC 25175, Staphylococcus aureus ATCC 6538, Escherichia coliATCC 8739, Enterococcus faecalis ATCC 10541, and Candida albicans ATCC 10221 are presented in Figure 6.The maximum zone of inhibition was seen with ethanol extract at 100µg/ml of 40±0.02mm for Enterococcus faecalis, 32±0.02 for Streptococcus mutans, 31±0.02mm for Candida albicans, 28±0.02mm for Escherichia coli,and 25±0.02mm for Staphylococcus aureus. While, foraqueous extract at 100mg/ml the results were not promising because it gave 17±0.02mm for Escherichia coli, 15±0.02 mm for Candida albicans, 12±0.02 mmfor Streptococcus mutans,11±0.02 mm for Staphylococcus aureus, and 10 ± 0.02 mm for Enterococcus faecalis.

Figure 6: Antimicrobial activity of ethanoland aqueous extracts of Moringa oleifera leaf

On the other hand, the antimicrobial activity was also carried out for chitosan nanoparticle , H₂CaO₂ nanoparticles, Ca (OH)₂, and Moringa-Chitosan composite (Mor.+Chit.) in addition to control; Gentamycin for bacteria and Fluconazole for fungi against dental pathogens Streptococcus mutans ATCC 25175, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 10541, and Candida albicans ATCC 10221 as illustrated in Figure 7.

Figure 7: Antimicrobial activity ofchitosan nanoparticle, H₂CaO₂ nanoparticles, Ca (OH)₂, and Chitosan-Moringacomposite

Moringa-chitosan composite had the highest zone of inhibition of 41±0.02mm for Escherichia coli, 39±0.02 mm for Staphylococcus aureus, 37±0.02mm for Enterococcus faecalis, 32±0.02mm for Streptococcus mutans, and 30±0.02mm for Candida albicans. Furthermore,by using H₂CaO₂ nanoparticlesthe results were 30±0.02mm for Candida albicans, 27±0.02mm for Escherichia coli, and 21±0.02mm for Staphylococcus aureus. While,by using Ca(OH)₂there is no antimicrobial activity against pathogenic microorganism except for Candida albicans at 22±0.02mm. Finally, chitosan has no antimicrobial activity against pathogenic microorganism under study.

On the other hand, Table 3 show the MIC (minimum inhibitory concentrations) and the MBC (minimum bactericidal concentration) of Moringa-Chitosan composite obtained for all the pathogenic microorganisms under test.The least MIC for Escherichia coli ATCC 8739 was at 1.97 μg/ml, MIC for Staphylococcus aureus ATCC 6538 was at 3.9μg/ml, MIC for Enterococcus faecalis ATCC 10541 and Streptococcus mutans ATCC 25175 was at 7.8μg/ml, and MIC for Candida albicans ATCC 10221 was at 15.62μg/ml. While, the minimum bactericidal concentration (MBC) for Escherichia coli ATCC 8739 was at 3.9μg/ml, MBC for Enterococcus faecalis ATCC 10541 and Staphylococcus aureus ATCC 6538 was at 7.8μg/ml, and MBC for Candidaalbicans ATCC 10221and Streptococcus mutans ATCC 25175 was at 15.62μg/ml

Table 3: The MIC and MBC of Chitosan-Moringa composite

Pathogenic microorganism	MIC Moringa-Chitosan composite (µg/ml)	MBC Moringa-Chitosan composite (µg/ml)
Staphylococcus aureus ATCC 6538	3.9	7.8
Streptococcus mutans ATCC 25175	7.8	15.62
Enterococcus faecalis ATCC 10541	7.8	7.8
Escherichia coli ATCC 8739	1.97	3.9
Candida albicans ATCC 10221	15.62	15.62

2. The Anti-inflammatory potential of Chitosan-Moringa composite, Moringa ethanol extract Ca (OH)₂, and H₂CaO₂ nanoparticles:

Inflammation is a potent response of the non-specific natural immune system that aids in the beginning of defensive responses against invading pathogens and the development of common immunity against invading factors. Chronic autoimmune disorders, however, might result from persistent inflammation. For chronic pathologies like metabolic disorders, medicinal plants have long been used as an effective source of treatment. In this manuscript the anti-inflammatory effects on HRBC hemolysis and membrane stability byChitosan-Moringa composite, Moringa ethanol extract, Ca(OH)₂, and H₂CaO₂ nanoparticles were depicted at Figure 8. At concentration 100 - 1000 μg/mL Ca (OH)₂gave the best lower production than the positive control Stander Indo meth followed by H₂CaO₂ nanoparticles, Moringa ethanol extract, and finally Chitosan-Moringa composite.

Figure 8: Anti-inflammatory of Chitosan-Moringa composite, Moringa ethanol extract, Ca (OH)₂, and H₂CaO₂ nanoparticles

3. Cytotoxicity:

The MTT assay for Chitosan-moringa composite, Moringa ethanol extract, Ca (OH)₂, and H₂CaO₂ nanoparticles against human oral epithelial cell line (OEC) have been studies. Figure 9 showed that, Ca (OH)₂ is the most cytotoxic compared to all the tested compounds with the lowest IC50 value 201.91±2.94 followed by Moringa ethanol extract with IC50 value of 202.46±1.83, then Chitosan-Moringa composite with IC50 of 222.05±0.79, and finally H₂CaO₂ nanoparticles with IC50 of 226.84±1.25. On the other hand, these outcomes show the impact of various compounds on human oral epithelial cell line (OEC) is based on the concentrations that were tested (31.25–1000μg/mL).Ca (OH)₂ exhibits high cytotoxic action at low concentrations (from 250µg/mL), whereupon the effect becomes very powerful and most cells die at about 31.25 g/mL, followed by Moringa ethanol extract, then H₂CaO₂ nanoparticles, and finally Chitosan-Moringa composite.

DISCUSSION:

The following headings serve as a simple summary of the results obtained: (A) preparation of chitosan nanoparticles by polymerization processand studies their properties; (B) Make extraction for Moringa oleifera leavesby ethanol and study their properties by FT-IR and GC-Mass; (C) Study the antimicrobial activity of ethanol and aqueous extracts of Moringa oleifera leaf, (D) Study the antimicrobial activity chitosan nanoparticle, H₂CaO₂ nanoparticles, Ca (OH)₂, and Chitosan-Moringa composite; (D) The MIC and MBC of Chitosan-Moringa composite; (E) and finally study the Cytotoxicity and Inflammation of all compounds under study.

To verify the accuracy of produced chitosan nanoparticles, whereas characterized by X ray, TEM, GC-Mass, FTIR,and UV Spectroscopy. The results of X ray diffraction of chitosan nanoparticle illustrated a peakat 2 θ 18.798°, 2 θ 22.810°,2 θ 24.469^o, 2 θ 61.702°, and 2 θ 66.077° and this confirm the formation of chitosan nanoparticles as proved by Kaur et al.; 2013³⁹.While the native chitosan's diffraction pattern exhibits two peaks at 2 θ 10^o and 2 θ 20^o which indicate the ordered crystalline structure and are in perfect agreement with the study that has already been publishedsuch as Matet et al.; 2013 who illustrated that,the peak at 20^o degrees is attributed to the crystal lattice of the chitosan orthorhombic unit cell and the peak at 10^o degrees comes from the incorporation of water molecules into the crystal structure of hydrated chitosan⁴⁰, Also, Rhim et al.; 2006 showed that that The size of the crystallite and an increase in the amorphous of the material are connected to the width of the X-ray diffraction peak⁴¹.

Also, A peak was visible at 280–300nm using a UV spectrophotometer. This might be because chitosan contains the amido group. In 2014, Krishnaveni and Priya discovered a peak for chitosan nanoparticles at 310nm⁴². Moreover, Chitosan peaked at 201nm was noticed by Liu et al in 2005⁴³.

Furthermore, Chitosan nanoparticle intermolecular interaction is revealed by FTIR characterization.The absence or presence of a wide range of functional groups in a molecule can be accurately determined using IR spectroscopy. FTIR examination of the chitosan spectrum revealed that, the peak of the OH group was visible at 3442 cm^-1, indicating that the H bonding is strengthened. The band1636 cm^-1 (amide I vibration, C=O stretching in amide group), Also, at 1052 cm^-1, stretching vibration (C-O) in the OH group was noticed.All the three band confirm the presence of the native chitosan. Similar results were detected by Lam et al.; 2006, Mohammad pour et al.; 2010, and Mohammad pour et al.; 2012, showed the peaks at 1636 cm⁻¹and 1650 cm⁻¹for amino group in chitosan^44,45,46.

On the other hand, throughout the dawn of civilization, people have turned to medicinal plants for their curative properties. An estimated 80% of people worldwide use traditional medicines as their first-line treatment option for a variety of illnesses⁴⁷. Numerous medicinal plant extracts have been utilized commercially, medically, and therapeutically⁴⁸. Moringa oleifera leaves are beneficial for human health⁴⁹. Several research have indicated that, Moringa leaves have anti-bacterial, anti-inflammatory, and cytotoxicity efficacy as illustrated on our study. Mangale et al.; 2012 showed that, the anticancer and antibacterial activities of fresh leaf extract⁵⁰. However, the impact of Moringa leaves on oral disorders is unknown.In this study the extraction of Moringa oleifera by aqueous and ethanol was done and the resulted proved that,compared to the water extract, the ethanol extract had a greater yield with 62.87% and 40.75%, respectively. Instead of the water extract, the ethanol extract produced a higher yield. This suggests that ethanol had superior extraction, quantitative, and solubility capabilities when it came to the active ingredients from Moringa oleifera leaves as compared to water. Studies conducted earlier found that, ethanol extracts of plants have higher antibacterial activity than water extracts of the same plants^47,51. Also, Ajayi and Fadeyi, 2015 and Mikore and Mulugeta 2017reported that the most effective extraction technique was with 70% ethanol^52,53.

Furthermore, this investigation was conducted to determine the phytochemical constituents of the ethanol leaf extracts of Moringa oleifera via chromatographic analysis using GC–MS. Ajayi and Fadeyi (2015) and Mikore and Mulugeta (2017), other investigations, found certain bioactive components in various parts of this plant^52,53.

According to Othman et al; 2019 publication, M. oleifera contained flavonoid and phenol contents that varied as a result of the various extraction techniques⁵⁴. A useful technology for the accurate identification of bioactive components in plant studies is gas chromatography-mass spectrometry (GC-MS)⁵⁵. Even though, some of the discovered chemicals matched those that Azwanida had previously described (2015): Cyclopropane butanoic acid, methyl ester, Octadecanoic acid, and Phen-1,4-diol, 2,3-dimethyl-5-trifluoromethyl³¹. The anti-cancer, antibacterial, and anti-inflammatory activities of certain chemical compounds might be due to them^56,57,58. However, Abd El- Hack et al. (2018) and Husni et al. (2021), who showed that ethanol extracts of sections of Moringa oleifera demonstrated potent in vitro and in vivo activities, supported the findings of this investigation ^58,59.

Also, the Moringa oleifera leaf's antimicrobial properties (aqueous and ethanol extracts) against dental pathogens Streptococcus mutans ATCC 25175, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 10541, and Candida albicans ATCC 10221 were studies and as illustrated in the results section the ethanol extract has strong antimicrobial activity compared to the aqueous extract. This probably infers that ethanol had better solubility, extracting and quantitative abilities of the active components from Moringa oleifera leaves than water.

This suggests that ethanol had superior quantitative, solubility capabilities, and extraction, when it came to the active ingredients from Moringa oleifera leaves as compared to water. This investigation indicated that, both leaf extracts (ethanol and water) demonstrated antibacterial activity on all the isolates. Even so, ethanol extracts outperformed aqueous extracts in terms of antibacterial activity. This suggested that alcohol concentrations had greater natural antibacterial properties than water solutions⁵². These findings support with Singa et al.; 2021 who conclusion that ethanol extracts of plants have higher antibacterial activity than aqueous extracts⁵¹. Moreover, Bukar et al.; 2010 research demonstrated that, the test bacterial isolates were most responsive to the ethanol extracts of Moringa oleifera leaf⁶⁰. Furthermore, Ajayi and Fadeyi, 2015 demonstrated that,plant aqueous extracts often have negligible to no antibacterial action⁵². Because aqueous extracts have access to a number of substances that may interact negatively with one another in their overall activities, Moyo et al. (2012) demonstrated how they differ from other extracting solvents⁶¹.

On the other hand, the antimicrobial activity of chitosan nanoparticle, H₂CaO₂nanoparticles, Ca (OH)₂, and Moringa-Chitosan composite (Mor. + Chit.) was studies and the results proved that Moringa-chitosan composite had the highest zone of inhibition, then H₂CaO₂ nanoparticles however, chitosan and Ca (OH)₂ have no antimicrobial activity against pathogenic microorganism except for Candida albicans. For chitosan and Calcium hydroxide (Ca (OH)₂) our results were disagreeing with many researchers such as Jia et al.; 2001, Chen et al.; 2002, Qi et al.; 2005, and Sanpui et al.; 2008 who demonstrated that,S. aureus and E. coli are two pathogens that are resistant to the antibacterial properties of chitosan^62,63,64,65. The concentration, source, molecular weight, and degree of deacetylation all affect chitosan's action⁶⁶. On the other hand, Siqueira and Lopes, 1999, Aguiar et al.; 2015, and Midena et al.; 2015 showed that, Calcium hydroxide (Ca (OH)₂) have antimicrobial activity because of its high pH, it encourages the breakdown of bacterial cytoplasmic membrane and microbial DNA^66,67,68,69.

Furthermore,according to our resultswhen chitosan companies with Moringa extract it have greatest effect on pathogenic microorganisms under study. These results in agreement with Adam et al.; 2013 who proved that, P. acnes was resistant to the antibacterial action of chitosan-alginate nanoparticles⁷⁰.

It has been demonstrated that, chitosan-alginate NPs cause bacterial cell wall membrane breakdown. The very large zone of inhibition of the chitosan composite with Moringa leaf powder demonstrated that it has considerable potential as a treatment for infections and disorders brought on by Salmonella typhi, Proteus bulgaris, Escherichia coli, Staphylococcus aureus, and Streptococcus pneumonia⁶⁶. Furthermore, as illustrated on our results for all of the experimental bacteria, calcium hydroxide nanoparticles were more efficient than regular calcium hydroxide.As reported by Darroudi et al.; 2016 and Khan et al.; 2018 Calcium hydroxide (CH) nanoparticle has gained extensive attentions as antimicrobial compound^71,72. Also, Pallotta et al.; 2007 revealed that, NCH (nanoparticles Calcium hydroxide) inhibited E. faecalis in lower doses than the conventional formof CH (Calcium hydroxide)⁷³.

Moreover,the minimum bactericidal concentration (MBC) and the minimum inhibitory concentrations (MIC) of Moringa-Chitosan composite obtained for all the pathogenic microorganisms under test. The least MIC for Escherichia coli at 1.97μg/ml, for Staphylococcus aureus at 3.9μg/ml, for Streptococcus mutans and Enterococcus faecalis at 7.8μg/ml, for Candida albicans at 15.62μg/ml. According to Olson and Fahey, 2011 and Martin et al.; 2013 the chemical molecule 4-(4'-O-acetyl—L-rhamnopyranosyloxy)-benzylisothiocyanate, whose mode of action involves inhibition of crucial cellular membrane enzymes, is what gives M. oleifera its antibacterial properties^74,75. The MIC of an aqueous extract of M. oleifera against S. aureus was also assessed by Muhammad et al. in 2016; it was discovered to be 6.25µg/ml⁷⁶.

On the other hand, the results of the minimum bactericidal concentration (MBC) for Escherichia coliwere at 3.9μg/ml, for Staphylococcus aureus and Enterococcus faecalis was at 7.8μg/ml, and for Streptococcus mutans and Candida albicans was at 15.62μg/ml.

Inflammation is the immune system's reaction to adverse stimuli including infections, damaged cells, poisonous substances, or radiation⁷⁷, which removes harmful stimuli and starts the healing process⁷⁸. The anti-inflammatory effects on HRBC hemolysis and membrane stability by Chitosan-Moringa composite, Moringa ethanol extract, Ca (OH)₂, and H₂CaO₂ nanoparticles was investigated and the results showed that, at concentration 100 – 1000μg/mL Ca (OH)₂ gave the best lower production followed by H₂CaO₂ nanoparticles, Moringa ethanol extract, and finally Chitosan-Moringa composite.There have been several anti-inflammatory effects of chitosan demonstrated.

A colorimetric technique for determining cell viability is the MTT assay (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyl tetrazolium bromide colorimetric assay), also known as Mosmann's Tetrazolium Toxicity assay⁷⁹. MTT, a yellow tetrazole, enters the mitochondria of living cells where succinate dehydrogenase converts it to purple formazan. To turn the insoluble purple formazan result into a colorful solution, an acidified solution is added^80,81. The results of MTT assay onhuman oral epithelial cell line (OEC) was proved that, Ca (OH)₂ is the most cytotoxic followed by Moringa Ethanol extract, then Chitosan-moringa composite and finally CaOH nanoparticles. The reason for the moringa leaf extracts the presence of phenolic and flavonoid compounds which are usually the most cytotoxic against any cells as observed in previous studies^82,83,84,85.

CONCLUSION:

Moringa-chitosan composite had great antimicrobial activity against common dental canal infection Staphylococcus aureus, Escherichia coli, Streptococcus mutans, Enterococcus faecalis, and Candida albicans. Ca(OH)₂ is the most cytotoxic compared to all the tested compounds followed by Moringa ethanol extract, then Chitosan-Moringa composite, and finally H₂CaO₂ nanoparticles.The anti-inflammatory effects of Chitosan-Moringa composite, Moringa ethanol extract, Ca (OH)₂, and H₂CaO₂ nanoparticles were supported the use of Moringa-chitosan composite as an intra-canal medicament.

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Received on 23.07.2023 Modified on 25.08.2023

Research J. Pharm. and Tech 2024; 17(2):776-788.

DOI: 10.52711/0974-360X.2024.00121

Retention Time (RT)	Name of compound	Structure	Chemical formula	Molecular weight (MW)	Peak Area (%)
43.97	6-Hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one		C11H16O3	196	2.45
52.91	n-Hexadecanoic acid		C16H32O2	256	11.91
58.03	Cyclopropane butanoic acid, 2-[[2-[[2-[(2-pentylcyclopropy l)methyl]cyclopropyl]methyl]cy clopropyl]methyl]-, methyl ester		C25H42O2	374	8.33
69.64	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester		C19H38O4	330	20.51
75.19	Octadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl este		C21H42O4	358	25.95
80.66	Phen-1,4-diol, 2,3-dimethyl-5-trifluoromethyl		C9H9F3O2	206	6.65
84.68	dl-à-Tocopherol		C29H50O2	430	15.75
88.91	24-Noroleana-3,12-diene		C29H46	394	9.52