Isolation and Identification of Flavonoid Compounds from Euphorbia Milii Plant Cultivated in Iraq and Evaluation of its Genetic Effects on Two Types of Cancer Cell Line

The "crown of thorns" or Euphorbia milii , a decorative and therapeutic plant species, is regarded as belonging to the genus Euphorbia. E. milii is abundant in phenolic compounds, terpenoids, steroids and alkaloids. The main objective of this study was to investigate the extracted flavonoid and nano-flavonoids against two types of cell lines. Nano-flavonoids were synthesized via chitosan-maleic complex reaction. The structure of nano-flavonoids was analyzed by UV-sp -8001 spectrophotometer with a 200–1000 nm scanning wavelength and the produced compounds were examined. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are used to determine the morphological properties. A strong and cutting-edge anti-proliferative drug against breast cancer cells (MCF-7) and prostate cancer cells (PC3) is the flavonoid and nano-flavonoid. The anti-cancer efficacy of flavonoids and nano-flavonoids against two different cancer cell lines as well as healthy cell lines was examined using the MTT assay. DNA fragmentation and the AO/ EtBr double staining technique were used to examine apoptotic markers. To determine the dispersion of the cell cycle, flow cytometry was used. The recent findings showed that the quantities of flavonoid and nano-flavonoid considerably improved the cytotoxic effect against breast and prostate cancer cell lines. Additionally, the cell cycle arrest in the G0/G1 phase was linked to the apoptosis of the cell lines induced by flavonoids and nano-flavonoids. According to these results, flavonoids and nano-flavonoids prevent cancer cell lines from proliferating, which causes the cell cycle to be arrested and apoptosis to be induced. The available findings suggested that the flavonoid and nano-flavonoid complex would represent a promising therapeutic approach for treating cancer cells of other types.


Introduction
The worldwide search for new medications and drugs is a continuous exploration.Improvements, modifications, and new drug detection are constant processes worldwide that seek to find a remedy for numerous diseases, especially cancer.Among many rich sources for curing cancer are medicinal plants that evermore represent a fruitful exporter of modern, unique drug detection and historical folk medicine -3 Published Online First: February, 2024 https://doi.org/10.21123/bsj.2024.9276P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal China, Nepal, Brazil, and other equatorial districts as a treatment for cancer, warts, hepatitis, and eyesores 5 .Modern research states numerous pharmacological properties of E. milii such as analgesic, anti-inflammatory, antimicrobial, antitumor as well as many others 6 .Different phytochemical studies exposed that E. milii is rich in many phytochemical compounds namely phenolic compounds and terpenoids; nevertheless, other studies noticed cardiac glycosides, alkaloids, steroids, and anthocyanins 7 .As a disease, cancer is described as a condition where some bodily cells grow uncontrollably and spread to the body's other organs.The trillions of cells that make up the human body are home to cancer, which can spread to any location 8 .Without the person's knowledge, many malignancies such as breast, lung, stomach, and esophagus exist in all sections of the body.These types of cancer may mainly disappear owing to the immune response of the body.If the body doesn't resist cancer cells, the tumor progresses and leads to harm the body, which in turn, causes cancer.Several symptoms can be noticed during cancer infection, like weight inconsistency like unexpected weight loss or weight increase; different colors of the skin (yellowing, darkening, or redness), uncured wounds, changes to present moles representing marks of skin variations.A persistent cough and respiratory issues are some potential symptoms 9 .The most common malignancies all over the world are women's breast cancer and men's prostate cancer, respectively 10 .Moreover, gastric cancer is considered to be one of the most universal cancers around the world as well as being the fourth leading source of cancer-related deaths all over the world 11 .With roughly 9000 different compounds, flavonoids are one of the classes of bioactive particles with the greatest diversity 12 .The most diverse group of polyphenols are flavonoids, which have a wide variety of structural and functional properties 13 .The name "flavonoid" refers to phenyl-substituted propylbenzene compounds and flavonolignans, which are formed by combining these compounds with C6-C3 flavonoid substrates with a C15 backbone 14 .Flavonoids are divided into 10 different species based on their chemical makeup, with flavones, flavanones, anthocyanidins, flavonols, isoflavones, and catechins being the most prevalent in foods consumed by humans.In both in vitro and in vivo experiments, it has been demonstrated that several of these flavonoids have anti-tumor effects 15 .The aim of this study was to isolate flavonoids from E. milii, then loaded them on chitosan nanoparticles to develop their anticancer activity against breast and prostate cancer cell lines.The results proved that the load nanoparticles have cytotoxic influence against cancer cell lines higher than the flavonoids alone.

Fractionation and extraction of several active compounds
The different plant components were separated according to the scheme shown in Fig. 1, [16][17][18] .

Qualitative analysis of active compounds in Euphorbia milii
The medicinal plants under research were subjected to phytochemical analysis using the standard techniques outlined by Harborne 19,20 .

Analyze for phenolic compounds
Extracts (10 mg) were diluted in 10 ml of distilled water, filtered, and the filtrate was then combined with 3 ml of a 5% w/v FeCl3 solution.It was thought that the production of a blue-black or dark-green precipitate.

Analyze for Flavonoids compounds
A few milligrams of extracts were dissolved in ethanol, followed by the addition of a few drops of 5% ethanolic KOH and a few drops of 5% HCl.The variations in color were noted. 21ed HPLC equipment to measure the presence of flavonoids.

Chitosan-Maleic-Flavonoid Synthesis
Step 1: 400 mg/ml of maleic anhydride in THF was added after 600 mg/ml of nano chitosan in THF was agitated at room temperature.The combination Chitosan-Maleic was produced by refluxing this mixture for two hours at 70 o C.
Step 2: Using an ultrasonic probe (150 W) for 5 minutes, 600 mg/ml of steroid or maleic was distributed in THF.This mixture was added to the chitosan-maleic complex made in step 1 and refluxed for three hours at 50 o C. To remove THF, the resulting product was centrifuged for 10 minutes at 10,000 rpm.The same process was used to separate the nanocomposite chitosan-Maleic-Flavonoid after it had been dissolved in 300 ml of deionized water.This substance was dissolved in water for five minutes with an ultrasonic probe before being 22, 23 .

Maintenance of Cell Line Cultures
The effects of flavonoids and nano-flavonoids were examed on MCF-7 and PC3 cancer cell lines as well as MCF-10 normal cells used 24 .

Anticancer activity of compounds
The cytotoxic activity was conducted using established procedures 25, 26, , for Flavonoids and nanoflavonoids on MCF-7 ,PC3 and MCF-10.

Acridine Orange-Ethidium Bromide Staining
AO/ EtBr was used to test the flavonoids and nanoflavonoids capacity to cause apoptosis.Briefly stated, cells were exposed to the IC50 preparations of the flavonoids, Nano flavonoids, for 24 hours after being seeded on 12-well plates for 24 hours.After two PBS washes, two fluorescent dyes were put into each well.A fluorescent microscope was utilized to view the cells 27 .

Flavonoids, Nano flavonoids and their impact on DNA content
In order to study the cell cycle in cancer cell lines that had been exposed to flavonoids and nano flavonoids, flow cytometry was used.Briefly stated, cells (5104 cells mL -1 ) were displayed for 24 hours to the nano flavonoids' IC50 concentration.Following cell fixation, extra ethanol was flushed away with PBS washing.The exposed cells were stained with propidium iodide (PI; 10 mg mL -1 ) for 1 hour at 37 °C.To prevent DNA molecules from interacting with the PI stain, RNase A (10 mg mL -1 ) was used.Using flow cytometry, the DNA content of the treated cells was examined 28 .

Analytical Statistics
The resulting data were statistically processed using an unpaired t-test (GraphPad Prism 6), and they were presented as the mean and standard error of the three replicates per experiment 29 .

Qualitative analysis of active compounds in Euphorbia milii:
The results of the phytochemical screening are shown in Table 1.+, -represent presence and absence of phytoconstituents respectively.

Quantitative analysis of flavonoids in Euphorbia milii
By using a hyper clone ODCC C18 V-25cm column and a mixture of ethyl acetate: water (7:30 ratio) as the mobile phase with a flow rate of 0.5 ml/min, the active constituents were estimated qualitatively and quantitatively using HPLC.Identifications were made by based on retention times obtained at equal chromatographic circumstances of analyzed samples and authentic criteria, and detected at 280 nm.

UV-Vis spectrum
This compound's UV-visible spectrum revealed the existence of a single high-pitched peak with an absorbance value of 1.028 at 206 nm, which is attributable to π-π* electronic transitions 30 .This peak's location, which differs from the primary peak of nano-chitosan, which typically appears at about 250 nm 31 , is proof of the success of the reactivity between flavonoids and nano-chitosan, as shown in Fig. 3.

SEM
The produced chitosan-alkaloid nanoparticles' size and shape were determined using this measurement.The measurement revealed that regular sphere-like particles, as shown in Fig. 4, were present.Additionally, as shown in Fig. 5, the measurement demonstrated that the prepared particles' size was within the nanoscale range, which spans 1 to 100 nanometers.The prepared particles' size ranged from 22 to 97 nanometers.It was noted that the average particle size as obtained by SEM and the particle size as determined by Scherer's equation are quite close.

3-TEM
The characterization of the flavonoids-coated chitosan nanoparticles was studied using TEM.In accordance with the findings displayed in

Anticancer activity of compounds
When doing colorimetric experiments to determine cytotoxicity or cell viability, it is clear that MTT (3-(4,dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) is one of the chemicals that are most frequently used 33 .This assay identified the cell viability of the examined cell lines in response to treatment with flavonoids, Nano flavonoids.(Table 3,4,5 /Fig.6,7,8).When MTT enters metabolically active cells, it is transformed into insoluble purple MTT-formazan crystals, which are then dissolved in a solvent and spectrophotometrically assessed 34 .
Flavonoids used in the current study compared to MCF10, nano flavonoids showed more effectiveness against MCF-7 and PC3.Fig. 1-6 illustrates that when cells were exposed to nano flavonoids, there was dose-dependent cellular damage.The cytotoxicity of flavonoids against MCF-7 was the highest (71.66%) and the highest for nano-flavonoids was (90.67%).Flavonoids and nano-flavonoids had the maximum cytotoxicity against PC3 at 67% and 84.33%, respectively.The results against the typical cell line (MCF-10 cells) revealed a negligible cytotoxic effect.This effect of flavonoids and nanoflavonoids on NCF-7 and PC-3 cells may be indicative of a mechanism that inhibits proliferation by triggering apoptosis 35 .In contrast, MCF-10 showed no signs of such action.When compared to cancer cells, normal cells rate of proliferation showed less evidence of harmful effects.this study is consistent with [36][37][38]

Morphological changes in cancer cells
Morphological changes were used to investigate the cytotoxicity effect of the tested cells, as shown (Figure 9,10,11).The findings showed that nanoflavonoids and flavonoids have less cytotoxicity against MCF-7 and PC3 cells.Normal cells (MCF-10) did not exhibit any such modifications.When compared to cancer cells, normal cells proliferated at a lower rate and showed fewer cytotoxic effects.The basis of the crystal violet test is the dye's affinity for the outside of the DNA double helix.It is feasible to determine the estimated number of viable cells by correlating the dye absorption and the culture's overall DNA content 40 .Other alterations caused by flavonoids and nano flavonoids, according to the research, were modifications to cell structure, clumping of treated cells with few cellular extensions, and cell communication inhibition.Cells going through cell death create less crystal violet staining in culture because they lose their adherence and are subsequently eliminated from the population of cells.Cells were treated with nano flavonoids, although there was less change than what was shown in the control cells that weren't treated.Numerous studies suggest that the fatal effects of flavonoids and nano-flavonoids on cancer cells may be caused by the membrane-disrupting, accumulation of lactic acid, reactive oxygen species (ROS), DNA damage, and apoptosis-inducing activities of these substances 41 .our results in consistent with 42 When treated with flavonoids and nano-flavonoids, the  membrane integrity has been disrupted.Previously, it was shown that the treated cells were recorded as dead (red color), indicating that it had a cytotoxic impact, whereas the live cells (green color) had a uniform distribution and huge nuclei 43 .By causing cell cycle arrest or apoptosis in oral squamous cancer cells, human esophagus, lung, liver, colon, and prostate cancer cells, flavonoid, nano-flavinoid can have anti-cancer effects.By simultaneously activating anti-apoptotic proteins and decreasing pro-apoptotic proteins and caspases, flavonoids can trigger cell death signaling pathways in cancer cells 44,45 .this study is consistent with 46 After the treatment cellS (MCF-7) with flavonoids, we noticed fragmented and pyknotic nuclei with condensed chromatin, which became more noticeable after a longer incubation period.The outcomes showed that treatment with flavonoids, nano flavonoids, or both raised the proportion of cancer cells in the sub-G1 phase in MCF-7 cells from 2.11% to 34.74% (Fig. 15).Generally, the findings of this study showed that manufactured flavonoids, or nano flavonoids, had an anti-proliferative effect via inducing apoptotic cell death 47,48 .

Conclusion
In the past two decades, there has been a lot of research on the potential of using natural components as chemo preventive agents against cancer.According to studies, using anti-cancer phytochemicals with various mechanisms or modes of action may be more efficient in treating diseases and reducing side effects.Flavonoids could have an important role in the prevention and treatment of diseases like cancer, as well as nutritional supplements.The outcomes demonstrated that flavonoids and nano-flavonoid are effective chemotherapeutic agents that can be employed alone or in combination with other agents to treat various cancer cell types.

Figure 2 .
Figure 2. HPLC chromatogram of isolated compound and standard compound.
Means with different big letters in the same column and small letters in the same row are significantly different.** (P≤0.01).

Published
Online First: February, 2024 https://doi.org/10.21123/bsj.2024.9276P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal noticeable morphological alterations in the cancer cells section were indicated by a decrease in the number of darkly pigmented cells, cell disintegration, irregular form, cell lysis, and necrotic cells because of the onset of programmed cell death.

Figure 9 .Figure 10 .
Figure 9. MCF-7 cell morphological alterations as shown.A, untreated control cells.B, Nanofalvo was used to treat cells; C, Flav was used to treat cells

Figure 12 .
Figure 12.Apoptosis markers in MCF-7 cells using AO/EtBr staining.A, untreated control cells.B, Nanofalvo was used to treat cells; C, Flav was used to treat cells.

Figure 13 .
Figure 13.Apoptosis markers in PC3 cells using AO/EtBr staining.A, untreated control cells.B, Nanofalvo was used to treat cells; C, Flav was used to treat cells.

Figure 14 .
Figure 14 .AO/EtBr labeling of MCF-10 cells to identify apoptosis markers.A, untreated control cells.B, Nanofalvo was used to treat cells; C, Flav was used to treat cells.Flavonoids, Nano flavonoids and their impact on DNA contentIn the current investigation, an additional experiment was conducted to look into the potential inductive

Figure 15 .
Figure 15.The DNA cycle in MCF-7 cells follow the recommended treatment.A, untreated control cells.B, Nanofalvo was used to treat cells; C, Flav was used to treat cells.

Table 3 . Anticancer activity of flavo. and nanoflavo. against MCF-7 cells. LSD value Cell line Concentration µg/ml
Flavonoids have a wide range of anticancer properties, including the ability to regulate ROS-scavenging enzyme activity, participate in cell cycle arrest, reduce the proliferation or invasiveness of cancer cells, and trigger apoptosis 39 .