Research Article | Open Access
Hamed A. Ghramh, Essam H. Ibrahim, Mona Kilnay, Zubair Ahmad, Sadeq K. Alhag, Khalid Ali Khan, Ramadan Taha, Fawziah M. Asiri, "Silver Nanoparticle Production by Ruta graveolens and Testing Its Safety, Bioactivity, Immune Modulation, Anticancer, and Insecticidal Potentials", Bioinorganic Chemistry and Applications, vol. 2020, Article ID 5626382, 11 pages, 2020. https://doi.org/10.1155/2020/5626382
Silver Nanoparticle Production by Ruta graveolens and Testing Its Safety, Bioactivity, Immune Modulation, Anticancer, and Insecticidal Potentials
Ruta graveolens, a plant belonging to the family Rutaceae, is traditionally used as a medicinal plant and a flavoring agent in food. This work aimed to prepare silver nanoparticles (AgNPs) using the ethanol extract from R. graveolens leaves and test different biological activities as well as insecticidal potentials in the extract and extract prepared AgNPs. Dried and powdered R. graveolens leaves were subjected to extraction using ethanol, and this extract was used to synthesize AgNPs. AgNP synthesis was monitored by the change in color, UV spectrophotometry, and electron microscopy (scanning). Fourier transform infrared (FT-IR) spectroscopy was used to monitor the functional groups in the extracts. Immunological, physiological, anticancer, antibacterial, and insecticidal potentials of the extract and its prepared AgNPs were tested. Results showed the ability of the leaf extract to synthesize. SEM examination revealed a spherical shape of AgNPs with a size of 40–45 nm. The extract contained many functional groups as indicated by FT-IR. The extract alone inhibited the growth of normal rat splenic cells, while the extract containing AgNPs stimulated its growth. Extract alone stimulated HeLa cell proliferation and inhibited HepG2 growth, while both cell line growth was inhibited by the extract containing AgNPs. Both the extract and extract with AgNPs were safe on RBCs and did not cause any severe elevation in liver enzymes. The extract alone and with AgNPs showed insecticidal activity against Culex pipiens. Our findings suggest that the R. graveolens leaf extract, alone or with AgNPs, is biologically safe on animal cells and has antibacterial, insecticidal, and immunomodulation potentials.
Ruta graveolens L. (Rutaceae), known in some countries as Sazab and has a common name as rue, is widely distributed in various geographical regions of Afro Asian countries . This plant is cultivated as a decorative plant [4, 5] and is used in traditional medicine for treating many disorders such as hypertension, cramps to hysteria, edema, helminthes, skin conditions, gastrointestinal disorders, and womb diseases [3, 6, 7]. It is also used as a toxin antidote and insect repellent ; as a diuretic, antipyretic , anticancer , antifungal, antibacterial, purgative, antiparasitic, hepatoprotective, antioxidant, and hypotensive agent; and against epilepsy. Ancient Egyptians and early Greeks used rue to improve eyesight and was also used as an anti-inflammatory agent and immune stimulant and for the treatment of skin afflictions and malaria [9, 10]. Ruta has been demonstrated to be advantageous for multiple sclerosis treatment  as well as hypotensive action .
Phytochemical investigations have demonstrated the presence of at least 120 chemical compounds found in all of the different parts of the plant. These include oils, fats, flavonoids, alkaloids, furoquinolines, glycosides, terpenoids, essential oils, steroids, coumarins, sterols, tannins, saponins, phenols, carbohydrates, pyranocoumarins, cardioglycosides, proteins, and amino acids. All parts of the plant contain the active compound, but they are mostly found in leaves [13, 14].
Nobel metal nanoparticles such as silver and gold have obtained increased interest because of their multipurpose applications in several fields such as biology, medicine, and industry . The physiochemical properties of silver nanoparticles (AgNPs) made them get special attention . Nanoparticles can be prepared by chemical and physical methods [17–19], but green synthesis using plants [20, 21], yeast, bacteria, and fungi  got more attention because it is nontoxic, clean, and ecofriendly. AgNPs have many biological properties such as anticancer, antimicrobial, antifungal, antiviral, anti-inflammatory [23–26], antiparasite , and insecticidal potentials . In addition, silver nanoparticles have been used in the industries such as in paint, detergent , clothing , and pharmaceutical preparations . Preparation of nanoparticles using a plant extract is valuable due to the ease of preparation methods with low biohazardous contents.
Silver nanoparticles produced by Ruta graveolens were shown to have many characteristics that include antibacterial, antifungal , dye degradation , antiviral , and sun protection factor .
This study was designed to prepare the Ruta graveolens leaf ethanol extract and prepare silver nanoparticles (AgNPs) using this extract. Both the extract and the extract containing AgNPs were tested for their biological safety and as an insecticide.
2. Materials and Methods
2.1. Preparation of Plant Extracts
Ruta graveolens leaves were gathered during the rainy season at Abha, Aseer, Kingdom of Saudi Arabia (KSA), in the month of February 2018. Identification of the plant was kindly done by a taxonomist at the Biology Department, King Abdul-Aziz University, Saudi Arabia. The collected leaves were rinsed with distilled water after extensive washing using tap water. After that, R. graveolens leaves were dried in shade at room temperature. Following the method described in , the active ingredients in the leaves were extracted. In brief, powder of the dried leaves was prepared by grinding, and 100 g of the obtained powder was mixed with 350 mL of 70% ethanol. The mixture was agitated for 40 h at room temperature and then filtered through a filter paper (Whatman No. 5). Using a rotary evaporator, the obtained flow-throw was dried at 40°C for 3 h. The obtained dried material was about 4 g (semisolid crude extract). From this dried material, 1% acetone and 0.5% dimethyl sulfoxide (DMSO) stock solutions were prepared.
2.2. Synthesis of AgNPs Using R. graveolens Extract
To an Erlenmeyer flask, 98.5 mL 1 mM AgNO3, 0.5 mL Triton X-100 (both are from Sigma-Aldrich), and 1 mL acetone plant extract solution were added to prepare silver nanoparticles (AgNPs) . The flask was incubated at room temperature until the color of the mixture changed (about 24 h).
2.3. Characterization of R. graveolens-Produced AgNPs
Using a UV-3600 Shimadzu spectrophotometer, the production of AgNPs was monitored at the wavelengths from 200 to 600 nm at 1 nm resolution . The shape and size of the produced AgNPs were characterized using a scanning electron microscope (SEM, JEM-1011, JEOL, Tokyo, Japan) at an accelerating voltage of 90 kV. Fourier transform infrared (FT-IR) spectroscopy (PerkinElmer Spectrum 2000, USA) within the range 600–4000 cm−1 at a rate of 16 times, and a clarity of 4 cm−1 was used to explore the functional groups in the plant extract alone or with AgNPs.
2.4. Antibacterial Activities
2.4.1. Antibacterial Activity Assay
Bacterial strains: Gram-negative (Escherichia coli, Proteus mirabilis, and Shigella flexneri) and Gram-positive (Staphylococcus aureus) bacteria were used. These bacteria were kindly supplied by the Microbiology Lab, Biology Department, Faculty of Sciences and Arts, King Khalid University, Dhahran Al Janoub, Saudi Arabia, and were maintained at 4°C on nutrient agar slants. Nutrient agar and nutrient broth (Sigma-Aldrich) were prepared according to the manufacturer’s instructions.
2.4.2. Well Diffusion Assay for Antibacterial Activity
The antibacterial potential of the Ruta graveolens leaf extract and the extract prepared AgNPs was tested using the agar well diffusion method. Bacterial strains (Gram-negative Escherichia coli, Proteus mirabilis, and Shigella flexneri and Gram-positive Staphylococcus aureus) were inoculated into 15 mL nutrient broth and shaken (225 rpm) overnight at 37°C. Nutrient agar plates were prepared, and 6 mm wells in each plate were punched out. Separate plates were covered with one of the overnight bacterial suspension (50 μL) by spreading using a sterile cotton swap. Plant extract in DMSO and its prepared silver nanoparticles (30 μL each) were aseptically pipetted into the wells of agar plates separately. The positive control (penicillin/streptomycin at 20 units/20 μg, respectively) solution and the negative control (nutrient broth) solution, 30 μL each, were included. Different plates were incubated for 24 h at 37°C, and zones (mm diameter) of inhibition around each well were measured in triplicate .
2.5. In Vitro Effects of Ruta graveolens Leaf Extract and Extract Prepared AgNPs on Normal Splenic Cell Proliferation
2.5.1. Splenic Cell Culture Preparation
The test was done according to the procedures described in  with little modifications. Splenic single-cell suspension was prepared from healthy adult male Sprague Dawley rats weighing about 280 g. The rat was kindly supplied by the animal house found at King Khalid University. The cells were suspended at a density of 0.05 × 106/mL in a complete RPMI-1640 medium consisting of 10% fetal calf serum (Gibco), penicillin/streptomycin 100 U/100 μg/ml (Gibco), 2 mM L-glutamine (Gibco), 2 mM sodium pyruvate (Seromed), 2% sodium bicarbonate (Seromed), pH 7.2, and HEPES (N-2-hydroxyethylpiperazine-N-2-ehtanesulfonic acid) buffer (Sigma). The cell culture was performed on a 96-microwell tissue culture plate (Coaster) by adding 100 μL of cell suspension (5000 cells/well) and 100 μL Ruta graveolens leaf extract or extract prepared AgNPs at 200, 100, and 50 μg/mL separately.
2.5.2. Study of Cytotoxic/Proliferative Effects
Potentiality of the R. graveolens leaf extract and extract prepared AgNPs to kill splenic cells (cytotoxicity) or to induce normal cell division was tested by adding different concentrations of the R. graveolens leaf extract and extract prepared AgNPs at final concentrations of 200, 100, and 50 μg/mL to wells containing 5000 cells/well rat splenic cells prepared as mentioned above separately in triplicate. The control culture was prepared by adding 5000 splenic cells/well in 200 μL of the culture medium. All plates were incubated in a CO2 incubator (Memmert, GmbH) at 37°C for 72 h. The viability of the cells in all plates was assessed using the Vybrant® MTT cell proliferation assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
The percentage increase or decrease in cell number was calculated according to the formula given in .
2.6. Lytic Effects of Ruta graveolens Leaf Extract and Extract Prepared AgNPs on Red Blood Cells (RBCs)
The hemolytic activity which may be found in the R. graveolens leaf acetone extract and its biosynthesized AgNPs was tested separately at a final concentration of 200 μg/mL according to the method described in  using 10% hematocrit fresh cow red blood cells (RBCs) suspended in the phosphate-buffered saline (PBS, pH 7.4). To 900 μL of prepared RBCs, 100 μL of the acetone R. graveolens leaf extract and R. graveolens leaf extract prepared AgNPs were added in 1.5 mL tubes separately and incubated for 60 min at 37°C. The positive control was prepared by adding 100 μL of 1.5% Triton X-100 to 900 μL of prepared RBCs (positive control), and the negative control was prepared by adding 100 μL PBS to 900 μL of prepared RBCs. At the end of the incubation, all tubes were centrifuged for 10 min at 2,000 rpm; supernatants were taken from the tubes, and the absorbance of each test was measured at 576 nm. The percentage of hemolysis was determined using the following equation:
2.7. Acute Cytotoxicity Study of Ruta graveolens Leaf Extract and Extract Prepared AgNPs
To test the hepatic toxicity which may be found in the R. graveolens leaf extract or extract prepared AgNPs, 5 adult healthy rats (200–250 g) were injected with a single dose regimen of 100 μg/mL of the R. graveolens leaf extract or extract prepared AgNPs . The rats were left for 24 h and then sacrificed, and sera were obtained from their blood. Liver function was tested by assaying the level of serum aspartate aminotransferase (AST) colorimetrically according to the Reitman and Frankel method using the Randox kit (UK).
2.8. Mosquito Larvicidal Activity
The larval susceptibility test was conducted according the method given by the WHO . Early 4th instar larvae of Culex pipiens were treated with various concentrations of R. graveolens (1000–5000 ppm) and its prepared AgNPs (50–250 ppm) for 24 h in groups of glass beakers containing 100 mL of tap water. Five replicates of 20 larvae each per concentration, and so in the control, were set up and kept in an environmental chamber at 27°C with a photoperiod of 16 : 8 h light/dark regimen. The larvae were given the usual larval food during these experiments. Larval mortalities were recorded at 24 h after treatment. The dead larvae were identified when they failed to move after being probed by a needle in the siphon or cervical region.
2.9. Anticancer Activity Test
The cell lines HepG2 and HeLa were used to test the anticancer potential of the R. graveolens leaf extract. The cells were maintained and grown in the supplemented minimal essential medium (MEM), containing fetal calf serum (10%), penicillin/streptomycin (100 U/mL/100 μg/mL), and L-glutamine (2 mM) at 37°C and 5% CO2 (CO2 incubator). After reaching confluency, the cells were trypsinized (2% trypsin-EDTA) to prepare single-cell suspension. Single-cell suspension was adjusted to 1 × 105/mL, and then 100 μL (104 cells) were plated into each well of the 96-well plate and incubated overnight in a CO2 incubator. The medium in the plate was decanted, and 200 μL media containing the R. graveolens leaf extract at the concentration of 200 μg/well was added. The plate was incubated for an additional 24 hours in a CO2 incubator. The media in wells were replaced with a fresh 100 μL/well culture medium. The viability of the cells was monitored using the Vybrant® MTT cell proliferation assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions .
3. Statistical Analysis
The study was conducted in a completely randomized design (CRD) in a factorial experiment. All data were statistically analyzed using the analysis of variance (ANOVA), and means were compared by LSD at using the SAS software program, SAS Institute (2006) version 9.3. LC50, IC50, and IC95 were calculated according to the probit analysis program . Computerized log-probit analysis was used to analyze values, regression equations, 95% confidence intervals, and degrees of freedom of the χ2 goodness of fit tests. Using Abbot’s formula , the mortality percent was corrected for control mortality.
4. Results and Discussion
4.1. Characterization of AgNPs
The plant extract was mixed with silver nitrate to synthesize AgNPs. The change in the color of the mixture was an indication of AgNP synthesis where the color of the solution changed from yellow to brown and continued to dark brown (Figures 1(a) and 1(b)). The degree of color change was time dependent that enabled the visual monitoring by observation. This change in color may be assigned to the excitation of the surface plasmon response of AgNPs . Color changes of the solutions are due to some chemical compounds such as alkaloids, flavonoids, saponins, and steroids, and the plant extract may act as a reducing agent that reduced silver ions (Ag+) to a silver atom (Ag0) by active biomolecules that are found in the plant extract through an enzyme like nitrate reductase. Many enzymes released into the solution have the capability to reduce the silver ions in the form of nanoparticles through agents working as capping molecules such as proteins .
The nanoparticle synthesis was accomplished via one-pot reaction, including the reduction of silver ions using the ethanol extract of R. graveolens. Using the UV-Vis absorption, the spectrum of the solution indicated that the surface plasmon resonance that is derived from the silver nanoparticles was around 412–488 nm, which is the specific area of silver nanoparticles (Figures 1(c) and 1(d)). SEM analysis revealed that the prepared AgNPs are almost uniform spherical in shape with a size of 40–45 nm (Figure 2).
The function of the leaf extract to act as a reducing and capping agent may be due to the domination of several functional groups which were confirmed by FT-IR analysis of the extract containing AgNPs. FT-IR spectroscopy analyses were carried out to recognize the biological entities responsible for capping and stabilizing of AgNPs synthesized using R. graveolens. The FT-IR spectra of AgNPs prepared by the leaf extract of R. graveolens (Figure 3) showed a vibrational band at 3750 cm−1 that was assigned to O-H arising due to alcohols and phenols. The band at 3300 cm−1 corresponds to N-H, indicating the presence of primary and secondary amines. The strong stretching vibrational band at 2120 cm−1 may be assigned to N=N=N and N=C=N related to azide and carbamide. A weak band at 2000 cm−1 may be due to C=C=C and C=C=N, corresponding to allene and ketenimine. The sharp peak at 1630 cm−1 may be assigned to C=C, corresponding to alkene. A weak bending band at 1380 cm−1 may be assigned to C-H, corresponding to aldehyde and alkane. A weak stretching band at 1250 cm−1 indicates C-O and C-N, corresponding to aromatic ester and amine. A stretching vibrational band at 500 cm−1 assigned to C-I corresponds to the halo compound .
Some works showed that the ethanol extract of the R. graveolens plant contained more than 120 compounds such as flavonoids and furoquinolines, coumarins, acridone alkaloids, and essential oils . The components of the R. graveolens species are of medical importance because they have a broad range of biological activities which led to some of them being used in medication. Alcoholic extracts of R. graveolens have been examined for their antiproliferative effect, using many types of cancer cell lines to test their potentiality as a therapeutic in oncology [47–49].
4.2. Antibacterial Activity
The results of testing the antibacterial activity of the R. graveolens leaf extract and extract prepared AgNPs against Gram-positive and -negative bacteria are shown in Table 1. Both the plant leaf extract and the extract prepared AgNPs inhibited the bacterial pathogens. Average diameters of inhibition zones produced by plant extract + AgNPs against both Gram-negative and -positive bacteria were bigger than those produced by the plant extract alone.
Inhibition zones are presented as an average of triplicate ± SD. Means with the same superscript letters indicate nonsignificant differences.
Previously, many types and sizes of nanoparticles were effectively used to induce the delivery of therapeutic agents [50, 51] to cure bacterial infections in the skin . Also, nanoparticles were used to prevent colonization of bacteria on the surface of medical devices and as an antibacterial agent in food and clothing industries . Because of their unique and nearly known mode of action and antimicrobial properties against Gram-positive and -negative bacteria and the need for developing a new generation of antibiotics, nanoparticles are on the spot as an alternative to traditional antibiotics to bypass the problem of drug resistance.
4.3. Cytotoxic/Proliferative Effects
The cytotoxic or stimulatory properties that may be found in R. graveolens were studied at different concentrations. The results showed that there were growth inhibitor/cytotoxic effects of the R. graveolens leaf extract on normal rat splenic cells, while the extract containing AgNPs showed stimulatory effects on normal splenic cells (Figure 4 and Table 2).
% of splenic cell inhibition is expressed as the average of triplicate ± SD.
Many constituents of Ruta graveolens extracts such as alkaloids, quinolone alkaloids, graveolens, flavonoids, undecyl acetates, and mananones may contribute to its cytotoxic activity. The cytotoxic effects of Ruta extracts may be attributed to the induction of DNA damage, causing chromosomal aberrations. They are also reported to be phototoxic, mutagenic, and capable of binding to DNA to induce apoptosis . Flavonoids are reported to be genotoxic and possess oxidant activities. This pro-oxidant activity has been attributed to the apoptotic-inducing property of these flavonoids, and hence implicated in cancer chemoprevention . The study  on Ruta graveolens found that it produced ROS (reactive oxygen species) and also induced apoptosis, and hence the cytotoxicity activity. Alkaloids were obtained from leaves and stem extracts of rue. Alkaloids are common constituents of other Rutaceae plants . Alkaloids are known to have cytotoxic effects . This explains why the cytotoxic effects decreased with the decrease in the plant concentration added to the normal rat splenic cells. Terpenoids are the largest class of naturally occurring compounds having mainly cytotoxic properties. A large number of terpenoids exhibit cytotoxicity against a variety of cells [56, 57].
The inhibitory effects of the extract decreased with the decrease in the extract concentration. Also, the stimulatory effects decreased with the decrease in the nanoparticle containing extract.
The cytotoxic effects of R. graveolens have been demonstrated by other studies . In addition, R. graveolens was found to have antitumor activity , indicating its toxic behaviors. The stimulatory effects of the extract in the presence of AgNPs may be due to the antagonistic effects of the nanoparticles with the active toxic compounds found in the extract.
4.4. Lytic Effects of Ruta graveolens Leaf Extract and Extract with AgNP Treatment on RBCs
The degree of RBC lysis was calculated by comparing the absorbance of the target sample to the positive and negative controls (Table 3). The positive control demonstrated 100% RBC lysis, while the negative control demonstrated 0% RBC lysis. The acetone plant extract containing AgNPs showed 100% RBC lysis while that without AgNPs showed 9.58% RBC lysis (Figure 5 and Table 3). The lysis effect of the extract containing AgNPs may be due to the direct effect of the nanoparticles on the membranes of the RBCs. Other studies referred the hemolytic effects as due to the induced release of oxidative stress products following exposure .
4.5. Acute Cytotoxicity Study of R. graveolens Leaf Acetone Extract and Its Biosynthesized AgNPs
The hepatic toxicity which may be found in prepared extracts was tested in adult healthy rats. The levels of serum aspartate aminotransferase (AST) showed a nonsignificant increase (1.16 fold increase) in the extract alone and the same (1.09 fold increase) in the extract with AgNPs. These results indicate the plant is safe and can be used without any systemic toxic effects. Many authors demonstrated the cytotoxic effects of AgNPs on the liver either in vivo  or in vitro . In our study, the extract containing AgNPs showed no toxic effects on the liver in vivo. This may be due to the bioactive compounds found in the extract that abrogated the toxic effects of the nanoparticles on the liver.
4.6. Larvicidal Effects of R. graveolens Extract and Its Silver Nanoparticles on Cx. pipiens
Susceptibility levels of Cx. pipiens larvae following treatments with different concentrations of R. graveolens and R. graveolens synthesized AgNPs against 4th larval instars of Cx. pipiens are described in Table 4.
In general, 31.633–92.857% and 48.98–99.898% larval mortalities were obtained when the 4th instar larvae of Cx. pipiens were treated with the effective concentrations of R. graveolens alone (1000–5000 ppm) and R. graveolens with silver nanoparticles (50–250 ppm), respectively. The results showed a significant positive correlation between the tested concentrations and the percentage of larval death for both the extract alone and the extract containing nanoparticles. Taking the concentration which kills 50% of larvae (LC50) and LC90 values into consideration, the records showed that the R. graveolens with silver nanoparticles caused LC50 at 56.002 ppm and LC90 at 151.377 ppm, proving that the extract with nanoparticles is more effective than the extract of R. graveolens alone that showed LC50 at 1673.804 ppm and LC90 at 5460.374 ppm, meaning that the extract with nanoparticles has a potency exceeding the extract alone by about 29.888 folds (Figure 6 and Table 5).
The results obtained from this study showed that the effect of the extract of R. graveolens and the nanoparticles against the larvae of the Cx. pipiens are in accordance with those reported by other works [62, 63].
Kumar et al.  reported that 43% mortality was noted for fourth instar larvae of Culex quinquefasciatus treated with S. xanthocarpum, LC50 = 155.29, 198.32, 271.12, 377.44, and 448.41 ppm; LC90 = 687.14, 913.10, 1011.89, 1058.85, and 1141.65 ppm, respectively. The bioactivity of the plant as well as AgNPs against the larval instars of Ae. aegypti and An. stephensi mosquito larvae was determined, and the results of AgNPs showed excellent larvicidal activity of the first, second, third, and fourth instar larvae, which exhibit noticeable effects after 24 or 48 h of exposure at their LC50 and LC90 values .
4.7. Effects on Cancer Cells
Effects of the R. graveolens extract and extract-generated AgNPs on HeLa and HepG2 cancer cell lines were studied at one concentration. Results showed the ability of the extract alone to diminish the cell growth of the HepG2 cell line but not of the HeLa line. The extract containing AgNPs could lower the growth rate of both cell lines (Figure 7). Similar results were obtained by other studies  as they showed that the extract of R. graveolens has anticancer effects on some cancer cell lines but not on the HeLa cell line.
Alcoholic extracts of R. graveolens have been tested for its antiproliferative power against many types of cancer cells [47–49] and was found to inhibit the growth of these cell lines by many mechanisms. In our study, the R. graveolens leaf extract, alone or with AgNPs, inhibited the growth of HepG2.
Generally, the mixing of a plant extract with silver nitrate has led to potentiation. This may be due to the synergy that occurs between the extract and the silver particles during the reduction process, as the compounds present in the extract are connected to the surface of the particles, increasing the strength of their effectiveness ; or the strength of the effect may in turn be due to their small size, which make them easy to pass through the wall of the body into the cells where they interfere with the process of dissociation and other physiological processes, and this is consistent with that stated in .
From the above results, we can conclude that the leaf extract of R. graveolens could synthesize AgNPs with a size of 40–45 nm. The extract contained functional groups that inhibited the growth of splenic and HepG2 cells and stimulated HeLa cell proliferation. The extract and extract containing AgNPs are safe on RBCs and vital organs. The extract alone and with AgNPs showed insecticidal activity against Culex pipiens.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
All authors state that they do not have any financial/commercial conflicts of interest regarding this work.
The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through the Research Project (RCAMS/KKU/003/20).
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