Cytotoxicity Assay of Biosynthesized Gold Nanoparticles for Synephrine Extracted from Citrus Sinesins Peels

In this study synephrine extracted from C. sinesins peels. Detected by TLC, HPLC and biosynthesized gold nanoparticles from it, then characterization of synthesize of gold nanoparticles (AuNps) by UV-visible spectroscopy Fourier Transform Infrared (FTIR), which confirm presence of AuNps in diameter size range to (9-31) nm and determined their toxicity assay on lymphoid human cell in 24, 48 and 72 hours. The finding obtained from applying gold nanoparticles at different concentrations for 24, 48, and 72 hours showed that’s the least cytotoxicity value was in 2.351 in 10 mg/ml for Citrus sinensis extract in 24 hours. which gave indicate that synephrine extracted have not toxic effect inhibition rate of this maximum 5.511 in AuNp biosynthesized in 20 mg/ml concentration in 72 hours.


Introduction
Nanotechnology is a brilliant field of study in modern science.The use of nanoparticles (NPs) and nanomaterials is rapidly expanding due to the development of new applications.The manufacturing of nanoparticles has greatly assisted the development of nanomaterials [1].
Research on AuNPs has lately risen Because of their promising potential in cancer treatment.Their unique physicochemical properties associated with their size.The augmentation of the surface area to volume ratio, within the range of 1-100 nm, results in adverse consequences in biological barriers.The integration of nanomaterials with other compounds has facilitated [2].Additionally, by more precisely targeting the drug delivery system, AuNPs can be employed as drug carriers to enhance drug delivery and minimize.Adverse effect Recent developments in the creation of AuNPs have demonstrated that they may also be excellent candidates for viral targeting and diagnostics [3,4] and that they may be utilized as standalone substances or as carriers for the administration of other medications or vaccines.In addition to cytotoxicity, the exposure to AuNPs has been found to induce cell cycle arrest in several cell phases [5,6].According to [7] AuNPs blocked the S phase, causing oxidative stress, G0/G1 cell cycle arrest, and apoptotic cell death.Using modified AuNPs, similar outcomes have also been documented [8].According to some theories, the size, shape, surface charge, composition, concentrations, and the toxicity of AuNPs is influenced by the specific cell type.Citrus fruits are extensively grown globally and mostly utilised in the production of various citrus-based food products such as jams, canned fruits, and beverages [9,10].The nutritional significance of citrus fruit is widely acknowledged.Multiple studies have shown the positive effects of citrus-based diets on degenerative disorders.like hypertension, cancer, diabetes, and cardio-vascular disease [11].Synephrine is alkaloid secondary metabolites chemically formula ‫,)2ܱܰ31ܪ9ܥ(‬ synephrine the main component of various citrus species, including Citrus sinesins and Citrus aurantium (often known as bitter orange), and it exhibits analogous structural characteristics to ephedrine and adrenaline.Synephrine is becoming more revalent in food supplements designed for weight reduction and athletic performance, namely those that aim to provide energy and enhance muscle growth utilized as a substitute for ephedrine, frequently in combination with caffeine among other chemicals Ortho o-, para p-, and meta m are the three positional isomers of synephrine) [12].

Collection of Plant Samples
This study included the use peels of C. sinesins was collected from Diyala province, Iraq.The plant was identified in herbarium, The plant peels dried and grinded in to powder from by mechanical grinder, it kept at 4 C° until further investigation.extraction of synephrine according to [13] .100g of fruit peels from Iraqi C. Sinesins were subjected to defatting using n-hexane for a duration of 24 hours.Subsequently, the peels were allowed to dry at room temperature.The Soxhlet extractor was loaded with the defatted plant material, which was enclosed in a thimble.One-liter round bottom flask, fitted with a Soxhlet extractor, contained 500 ml of 80% methanol as the solvent.The extraction process was extended for an additional 12 hours.The sample was initially agitated and dissolved in 2N hydrochloric acid over a heated water bath.It was then filtered and concentrated under reduced pressure until it completely dried using a rotary evaporator, maintaining a temperature below 40°C.The resulting filtrate was then shaken with chloroform to remove unwanted components.Alkaloidal bases were released from the acidic aqueous layer by raising its pH with ammonia.

Using TLC to Identify Plant Components
The extract was investigated using TLC on premade silica gel GF254 (20 x 20 cm) plates with a 0.25 mm thickness from MERCK 2% ninhydrin in n-butanol spraying reagent was used for detection.The standard for synephrine was bought from Chengdu Biopurify Phytochemicals [13].

Synephrine Identification Using HPLC
Synephrine was estimated both qualitatively and quantitatively using HPLC.Using (Knauer/Germany), HPLC analysis was performed.By comparing the retention durations obtained from examined materials with genuine standards under identical chromatographic conditions, identifications were made.[13].The column employed was a C18 column with dimensions of 150mm length, 4.6mm diameter, and 5um particle size.The mobile phase consisted of a mixture of acetonitrile, water, and trifluoroacetic acid at a ratio of 5:95:0.01.The flow rate used was 0.6 ml/min, and the detection of the analyte was performed using a UV detector set at a wavelength of 220 nm.Gold Nanoparticle Biosynthesis: An Environmentally Friendly Method in order to create gold nanoparticles, [13] employed C. sinesins peel extract in the bio reduction of chloroauric acid.This extract contains amino, alkane, amide, and alcohols that helped to stabilize and bio reduce the gold nanoparticles.

Analysis of Gold Nanoparticles Produced via Biosynthesis
The characterisation of particles is a crucial step in the biosynthesis of gold nanoparticles.The synthesis of gold nanoparticles is often characterised by their stability, shape, size, surface area, and dispersion.Fourier Transform Infrared (FTIR) Spectroscopy and UV-visible spectroscopy are two of the various techniques employed for nanoparticle characterization.[14].

Testing of Toxicity of Synephrine and Gold Nanoparticles
are tested on lymphoid blood cell lines After we created lymphocytes, we looked at them under a microscope to make sure they were growing normally.After being distributed in a 96-hole multi-hole plate, the cells were exposed and treated with three concentrations (10,15,20) mg/ml of produced AuNPs using a micropipette absorbent [15,16].

TLC detection
The existence of synephrine in the extract of Iraqi Citrus Sinensis fruit peels was confirmed by the appearance of a single round compact spot-on TLC plates in five various developing solvent systems, sharing the same color and Rf values as the reference standard, as shown in figure 1.

The HPLC detection
Synephrine was further confirmed to be present in Iraqi C. Sinensis fruit peels using HPLC.In this way, the sample ingredients are separated.Synephrine was further confirmed to be present in Iraqi C. sinensis fruit peels using HPLC.retention period (6.2 minute) for the synephrine extracted from Iraqi C. sinensis fruit peels, and (6.5 min) reference to the standard was in the chromatogram indicating that the peak was most likely synephrine, as shown in figure 2 and 3. Appearance of single peak indicate the purity of the stander.In figure 3 the peaks that appeared in HPLC presence of There are five other biogenic amines found in sweet orange related to synephrine likely to be: Octopamine, phenylephrine (m-synephrine), tyramine, N Methyltyramine, and hordenine are the substances mentioned according to [17].

Analysis of the Finite Element Structural Model (FESM)
The surface properties of nanoparticles (NPs) were analysed using field emission scanning electron microscopy (FESEM).Microscopic investigation revealed that the Au NPs were stabilised, indicating that they did not make direct contact with each other, even when they formed aggregates.The morphology and dimensions of the aggregations were analysed, revealing that the majority of the biosynthesized Au NPs exhibited spherical shapes.The analysis indicates that the surface continues to undergo a process of decrease.The histogram depicting the distribution of particle sizes of the gold nanoparticles (Au NPs) was acquired following a reaction period of 10 minutes at a pH of 7.0.The particle size range, as depicted in figure 5, was calculated to be (9-31) nm based on a representative FESEM micrograph.The study revealed that the formation of new nanoparticles through nucleation and the merging of these particles to form larger ones happened simultaneously.This was due to the presence of gold nanoparticles of varying sizes, which were produced during both the initial and later stages of the process.The Au NPs spot-profile EDX.

The Fourier transform infrared (FTIR)
Approach refers to a spectroscopic technique that can identify alterations in the entire makeup of biomolecules by detecting modifications in functional groups.FTIR is used to measure the vibration and rotation of molecules that are affected by an infrared wavelength.The detection of molecular interactions can be achieved by analysing changes in the structure of molecule binding.Transmittance FTIR, attenuated total reflectance (ATR-FTIR), and micro-spectroscopy are techniques used in spectroscopic analysis FTIR are three widely used methods in FTIR analysis for material characterizations [18].6 demonstrated the presence of an aliphatic peak at (2813), a broad peak for the hydroxyl groups at (3170-3394), and a single peak for the secondary amine group at (3029).The presence of meta compensation on the ring, which creates a peak that is regarded as the compound's fingerprint between (1667-2000), is the most significant group in this molecule.

Figure 7. FTIR spectrum of synthesized Au NPs
Figure 7 FTIR study of the production of AuNp in C. Sinensis.In figure 7, aliphatic aggregates were visible at (2812), hydroxyl groups were visible as a broad peak at (3170-3384), and secondary amine groups were visible as a single peak at (3049).The presence of meta compensation on the ring, which creates a peak that is regarded as the compound's fingerprint between (1631-1764), is the most significant group in this compound.The presence of gold appearance near the peak 890.Another benefit of biologically synthesised nanoparticles is their ability to streamline the process by eliminating the need for attaching certain functional groups to the surface of gold nanoparticles in order to make them biologically active.This step is often necessary in chemical synthesis.[19] inhibition rate of this maximum 5.511 in AuNp biosynthesized in 20 mg/ml concentration in 72 h.increased with an increase in its concentration and exposure time (20).Research conducted by [16] indicates that nanoparticles disrupt cellular components and the process of cell death by enhancing the permeability of the cell membrane and reactivating the oxygen atom in an interactive manner.
In the absence of a health-based reference value, a potential upper limit for p-synephrine in dietary supplements might be determined by considering the quantity of p-synephrine typically taken through the regular diet, as p-synephrine is commonly found in citrus fruits.

Conclusion
The usage of gold nanoparticles in many applications is limited in comparison to biologically synthesised gold nanoparticles due to the absence of harmful contaminants that are typically associated with nanoparticles produced through chemical synthesis.biological compatibility is a necessary requirement for gold nanoparticles used in biomedical applications.The biological synthesis of gold nanoparticles has several advantages, such as the creation of biocompatible, ecologically friendly, costefficient, and straightforward nanoparticles in a single procedure.The process of synthesising gold nanoparticles is time-efficient.compared to the chemical synthesis of gold nanoparticles.There is a scarcity of toxicological information regarding synephrine, especially when it comes to chronic toxicity.

Figure 4 .
Figure 4.The UV visible spectra of the synthesised gold nanoparticles exhibited a peak at a wavelength of 470 nm

Figure 5 .
Figure 5. FESM images of the synthesized Au NPs Figure below demonstrates.

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Table 1 .
Cell death percentage (cytotoxicity assay) measured on human blood lymphoid exposed to 10,15,20 mg/ml of plant extract and biosynthesis AU Nps for 24 ,48,72 hours.The cytoxicity assay for gold nanoparticles (AgNPs) and Plant Extracts on Blood Lymphoid Cells The toxicity assay for gold nanoparticles (AgNPs) and plant extracts; Although some information has been given regarding their impact on human blood cells, there is still a lack of knowledge about the underlying mechanisms.The experiments were measured the death of human blood lymphoid cells percentage after exposing to different solutions gold nanoparticles at different concentrations for 24 ,48 and 72 hours.As the table shows study that's the least cytotoxicity value was in 2.351 in 10 mg/ml for C. Sinensis extract in 24 h.which give indicate that synephrine extracted have not toxic effect