Hashim S. S, Mahmood Z. F, Abdulateef S. F, Dheeb B. I. Evaluation Cytotoxicity Effects of Centaurea Cineraria Extracts Against some of Cancer Cell Lines. Biomed Pharmacol J 2023;16(1).

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Manuscript received on :10-08-2022
Manuscript accepted on :12-12-2022
Published online on: 09-02-2023

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Plagiarism Check: Yes
Reviewed by: Dr. Ana Golez
Second Review by: Dr. Zena Sideeq Tawfeek
Final Approval by: Dr. Ian James Martin

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Sumayah Sami Hashim¹ , Zainab Farqad Mahmood¹, Safa M. Abdulateef ^2*, and  Batol Imran Dheeb ³

¹University of baghdad, College of Science, biotechnology department, Iraq.

²Dijlah University college, Medical laboratory techniques, Baghdad, Iraq.

³Samaraa University, College of applied science, pathological analysis, Iraq.

Corresponding Author E-mail: batoolomran@yahoo.com

DOI : https://dx.doi.org/10.13005/bpj/2606

Abstract

This research included the detection and determination of flavonoids from Centaurea Cineraria extract collected from public gardens using high-performance liquid chromatography technique. The compounds (Cirsilineol, Jaceosidin, Melitensin) were found, their concentrations were measured and found (36254,63719,89035 µg/ml), and qualitative chemical detections were performed. Study of the effect of the methanolic extract of Centaurea Cineraria containing the flavonoids mentioned above. The extraction process was carried out using an ordinary organic solvent ethanol and using a rotary evaporator device. The flavonoids present in the extract were detected, using standard flavonoids (Cirsilineol, Jaceosidin, Melitensin) and study the effect of the  extacts on some cancer cell line also study on utilizing stable free extreme compound 1, 1-Diphenyl-2-Picryl-hydrazil (DPPH). Results uncovered that the more expanding in centralization of the concentrate the seriously rummaging level of the free extremists. The fixations that were utilized gone from36.12 to 2000µg/ml, considered the Methanol concentrate of Centaurea Cineraria essentially showed high anticancer activity agent movement (96% at 2000µg/ml) connected with hexane extricates which were 94.61% at 2000µg/ml. This work additionally planned to concentrate on the cytotoxic and anticancer impacts of Centaurea Cineraria extricates The predominant decrease in attainable count cell was 500/ml in various cell lines, which was associated to methanolic isolates and appeared to cause cell loss in the cell lines L20B, PC-3, and HCT116 separately.

Keywords

Centaurea Cineraria; Cancer cell lines; Cytotoxicity; DPPH; HCT116; L20B; PC-3

Introduction

Malignant growth is perhaps the most widely recognized infections in both created and emerging nation. Plant items have been utilized over the entire course of time to treat and forestall illnesses as a result of their huge number of various phytochemicals with various organic activities ¹ indeed, the mixtures got from plants assume a significant part in the advancement of anticancer specialists to be utilized in clinical practice². Since significant proof has demonstrated that plant auxiliary metabolites are a likely wellspring of anticancer mixtures and disease cells might foster protection from existing medications, today broad examination is being done all around the world to find new plant species with anticancer properties ³.

The family Centaurea Cinerariahaving a place with the Asteraceae, is the third biggest class in Turkey ⁴. Some Centaurea species are utilized as cures against different infections in Turkish society medicine ⁵. Previousinvestigations inspected the pharmacological and organic properties of Centaurea Cinerariaand a few Centaurea animal groups showed cytotoxic impacts against some cell lines ⁶. The significant constituents of Centaurea species were flavonoids, and greasy acids ⁷. There were no sufficient reports  an publication about the anticancer impacts of C. Cineraria and activity of extracted flavonoid ⁸. Accordingly, the current study aimed to extract the flavonoid from C. Cinerariaand study its  antioxidant activity and anticancer activity against some cancer cell line.

Materials and Method

Plant collection and Extraction of Centaurea Cineraria flavonoid

leaves were collected from a Baghdad area garden. The Herbalist composed the plant depiction, and the new leaves were withdrawn and disinfected from dust with tissue paper prior to being set in the shade inside a by and large ventilated room until they displayed at an expected weight. The powder was made by crushing dried leaves into a fine powder and dealing with it at 4°C the flavonoids were extracted according to the following : determine the degree ofsolubility of flavonoids in organic solvents, adding (85% methanol and 15% water) (volume / volume) to (100g) of drypowder of C.. Cineraria, themixture is shaken for 12 (an hour) at a temperature of4 °C, after which the solution is filtered through aglass cotton, the first filtrate is kept at (4 °C), then theprecipitate is extracted again in the same way, butusing (50% methanol and50%water) (volume/volume).

We get the second filter. The two filters aremixed and then left for several hours, then filteredusing filter paper and then evaporated using a (RotaryEvaporator) device at a temperature of (40°C).  avolume of hexane is added to a volume of the extract, and it produces an organic part (hexaneextract) and waterpart. The organic extracts (organic parts) were subjected toan evaporation process to remove the organicsolvents, then dried and kept at a temperature of (20-C) ⁵.

Inferential tests were carried out included detection of neutral ferricchloride, base solution, Base leadacetated etection to detect flavonoids and Estimation, Quantitative, determination and characterization of C.Cineraria flavonoids using HPL Canalysis  using  number of standard flavonoids (Cirsilineol, Jaceosidin, Melitensin) , concentrates of C.Cineraria flavonoids leaves papered as (640,320,160, 80,40,20,10µg/ml) to zero in on its implications for disease presumption ace progress utilizing DPPH moderate glancing through measure as shown by [9]. ELISA tests was used to measure spectrophotometrically against a stable DPPH medium. When DPPH is lowered, the colorimetric changes (from fundamental violet to light-yellow) are learned at 517nm ⁹.

The cytotoxicity and anticancer activity  condition percent limitation of moderate: Percentage is equal to (Absorbance of -ve control – Absorbance of test/Absorbance of -ve control) multiplied by 100. obstacle The negative control was a mixture of (DMSO: Solvents (Methanol1:9 v: v), and tests were facilitated (concentration of C. Cineraria flavonoids leaves which was the positive control). The L20B, PC-3 prostate prison line, and HCT116 cell lines were gotten from the Improvements were made with 10% FBS (Sigma), 1% 5,000 units/mL penicillin, and 5,000 lg/mL streptomycin, and the cells were kept alive in RPMI (Gbico, Carlsbad, CA) (Sigma). In a 37°C, 5% CO2 atmosphere, the cells were transferred ¹⁰.

The 3-[4,5-dimethylthiazoyl]-2, 5-diphenyltetrazolium bromide (MTT) test is used to determine cytotoxicity[9]. This test was carried out by dissolving 3-[4,5-dimethylthiazoyl]-2, 5-diphenyltetrazolium bromide in phosphate-buffered saline (PBS) at 2 mg/ml, filtering the solution through a 0.22 lm millipore channel, and adding 50 l of the MTT tone to each of the microliter plate wells containing cell lines treated with various social events of concentrates for 24 hours.

The MTT-formazan huge stones were disengaged in 100 l Dimethyl sulphoxide (DMSO), and the optical thickness of each was generally pulled in using an ELISA peruser at a sending rehash of 620 nm  then using the following equation (Absorbancy of treated cell/Absorbancy of non-treated cell) 100] = toxicity ¹¹.

Statistical analysis

When compared to untreated controls, The centralization of the concentrate with a half loss of full metabolic movement is defined as the IC50 value, and are accounted for as mean S.D. GraphPad Prism was used to calculate IC50 values with 95% confidence limits.Programming level 3.3 (GraphPad Software, Inc., San Diego, CA). P values of less than 0.05 were considered enormous. All of the analyses followed a three-step process.

Result and Discussion

The results of  neutral ferricchloride, base solution, Baseleadacet at detection  test showed that the methanolic extract of C. Cineraria contains flavonoids through using the three detection. methanolic extract of C.Cineraria showed that it contained flavonoids, and this was confirmed by [10]  which recorded positive results of presence flavonoids in alcoholic extract of C. Cineraria contained flavonoids. HPLC analysis comparing with standard flavonoids(Cirsilineol, Jaceosidin, Melitensin,) were used in the chromatographic analysis shows the bundles of identical to the standard compounds approved by chromatography analysis peaks and retention time-Rt(1.233,2.048,3.448mints)  and the area was (107.6213, 118.7381, 62188) for each of(Cirsilineol, Jaceosidin, Melitensin)respectively as show in table (1) and figure (1) which shows the peaks and retention time of theisolated flavonoids their concentrations were measured and found (36254, 63719, 89035 µg/ml) .

Table 1: HPLC analysis  and Molecular formula Determination time and bandarea for standard flavonoids and methanolic extract of Centaurea Cineraria.

A. Standard B. Extract.

Figure 1: HPLC analysis for standard flavonoids(Cirsilineol, Jaceosidin, Melitensin,) and extract were show three sharp peaks and retention time-Rt(1.233,2.048,3.448mints)  and the area. A. standard B. extract.  Click here to view Figure

Antioxidant Activity of Centaurea Cineraria Leaves Methanolic extracts

The activity of  Centaurea Cineraria leaves Methanolic extracts was determine using the assay of free radical scavenging (stable DPPH) . results show that the antioxidant activity incrase with the concentration increase more scavenging percentage of the free radicals ranged from 10 to 640µg/ml, the highest activity of  antioxidant (69% at 640µg/ml) while the antioxidant activity (43,32, 21,17 ,11,9%)  at the concentrations  (320,160, 80,40,20,10µg/ml) respectively these results indicated the free radical scavenging activity DPPH was dose dependent that’s mean higher concentration lead to higher antioxidant activity ⁷.

The value ­ for each concentration determine in present study and display the activity of  inhibition effects of methanolic DPPH extracts was(189µg/ml) to high concentration. when compare with the  values it was show high significant difference between the applied concentration which lead to causing  scavenging activity 50%of the DPPH (35, 47,52, 66, 71, 102µg/ml) respectively.

The present results regarding Centaurea Cineraria antioxidant activity of the methanolic extract may be attributed mainly topresence of different type of flavonoid in the methanolic extracts (Cirsilineol, Jaceosidin, Melitensin,) which have the capacity  to reduce and remove  free radicals through  reactive oxygen species quenching and then trapping radicals thean reaching their cellular targets ¹².

Diffent research display the activity of Centaurea Cineraria flavonoid and show its role using DPPH extracts method in radical-scavenging activity of and the other active ingradiant of Centaurea Cineraria like alkaloid and phenols, using different concentration of Centaurea Cineraria leaves extracts ⁷ different study focus in the activity of Centaurea Cineraria extracts comparing  with prunica flavonoid its show stronger effects than that of butyl hydroxytoluene which lead to of lipid oxidation delay [2].The organic solvents  like methanol show very low activity DPPH scavenging ¹¹.

The solvent used in present analysis methanol is an amphiphilic compound and important in extract various activ groups from the  medical plant material. present results are agreed with previous researcher used methanolic extract in the study the activity of antioxidant and against DPPH ⁶ Free radicals responsiblefor health conditions development espatially cardiovascular disease ,cancer, and aging process acceleration and initiation finally  controlled of antioxidant substances ¹².

Cytotoxicity Effects of Centaurea Cineraria methanolic Extract 

Cell Line in vitro Using MTT Assay

The  cytotoxic test of Centaurea Cineraria methanolic Extract using (MTT) 3-(dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide  applaied to determine the significant activity of Methanolic extract of Centaurea Cineraria againistL20B, PC-3 and HCT116 cell lines. This experement done to determine the viability and inhibition rate  of L20B, PC-3 and HCT116 . Results show good indicators   regardind the activity of  studied extracts incubation of   L20Bcells with the(640,320,160, 80,40,20,10µg/ml)  concentrations of methanolic extract for 48 hours results indication that viability af cell  and inhibition were pattern dose-dependent the viability of L20Bdecreased with the  increase the concentration of the methanolic extract extract. The percentage of inhibition ranged from57.47% to 100%  with the concentrations(640,320,160, 80,40,20,10µg/ml) respectively  as display in  table (2)  and Figure (2), and the rate of cytotoxic activity calculated using the special equation listed in material and methods the our present results means that the extract have high activity in inhibition of  studied cancer cell line L20B.

At the same time , Figure (3) displays  the activity of Centaurea Cineraria Leaves Methanolic extracts againist the viability of PC-3 during 48 hrs the viability of the cell after exposure to Methanolic extract.

Results indicated that the cell PC-3  viability decrease gradually with the increase of extract concentrations and show high significant difference between studied concentrations  p value ≥ 0.05 and the cytotoxic effects increase with the increase of concentration  viability was not significantly affected by the application of extract concentrations  as show in (table 2),  these serial cocentrations (640,320,160, 80,40,20,10µg/ml) show high gradual percentage of inhibition (67.06, 72.46, 85.54, 90.32, 92.25, 97.98, 100 ), when compared with the viability of  HCT116 cancer cell line (49.43, 71. 87, 82.76, 82.43, 91.87, 95.06, 97.43%) , show a maximum cytotoxic rate (97.43%) of methanolic extract at 640µg/ml, Result showed that, the highest concentration at 400 appeared to effect significantly (p≤0.05) level on viability of  PC-32 comparing to other doses when the lowest concentration apeare 97% viability of the PC-3 cell line the results display in figure (4) and table (2).

Show high activity againist this type of cell line and the cytotoxic effects increase with the increase of methanolic extract concentrations, the potent cytotoxic effects significantly difference at the probability value(p≤0.05).

Table 2:  Cytotoxic Effects of Methanolic extracts on L20B, PC-3 and HCT116.

*(P<0.05) probability value.

In contrast, viability PC-3 cells with methanolic corrosive concentrate at fixations ranging from zero to 67.06 % for 48 hours revealed a gradual decrease in cell practicality in a portion subordinate example in which the cell suitability  gradual decreased with methanolic extract increasing. corrosive concentration had the lowest PC-3 cell reasonability (percentage) (86.57 %t) at fixation 500/ml, but it was 98.80% at 20/ml. With an IC50 of 28g/ml, the Ascorbic corrosive concentrate had a relatively strong cytotoxic activity.

Figure 2: Cytotoxicity effect of Centaurea Cineraria Methanolic extract on ,L20B after 48 hours  in cubation at 37ºC. Click here to view Figure

Figure 3: Cytotoxicity effect of Centaurea Cineraria Methanolic extract on ,PC-3 after 48 hours  in cubation at 37ºC. Click here to view Figure

Figure 4: Cytotoxicity effect of Centaurea Cineraria Methanolic extract on ,HCT116 after 48 hours  in cubation at 37ºC. Click here to view Figure

C. Cinerariais consistently used as solid flavors region of the world considering its inside and out expected activities like bactericidal, antifungal, and antiviral as well as cell support improvement ¹³ regardless, not a huge number analyzes were watched out for the antitumor headway of C. Cineraria kills. The cytotoxic effect on a remarkably basic level worked out precisely true to form by the Ascorbic terrible spotlight separate on MCF-7 The presence of polyphenol, a large ingredient with a wide range of standard development, can be attributed to the cells.

This finding is consistent with ¹⁴, who found that C. Cineraria concentrates had a strong cytotoxic impact against Human leukemic cell lines HL-60 and NB4, with LC50 values of up to 86.5g/ml. Similarly, polyphenol, one of the major ingredients of C. Cineraria, was found to be effective in preventing MIAPaCa2 pancreatic tragic advance cells (60-90 percent) ¹⁵ . The L20B, PC-3, and HCT116 cell lines were all affected by the C. Cineraria ascorbic stunning store, with the prostate dungeon line (PC-3) being the least affected. A few out of every odd one of the types of tumor cells demonstrated a general response for a The glycosides from Anemopsis californica leaves, for example, were used to assess their Bioactivity against a variety of advanced cell lines with anticancer properties.

It showed no progress against HePG2 and A549 cells, but was a proliferative antagonist against AN3CA and HeLA cells ¹⁶. Certain chemical components in Ascorbic dreadful concentrate could unmistakably impact the sensitivity of L20B, PC-3, and HCT116 cells, Ascorbic terrible concentrate was used for multi-limit cytotoxicity measure and MnSOD oxidative assertion test on PC-3 and HCT116, which According [1] and [7], specific anticancer medicines can affect distinct types of illness cells in different ways.

Conclusion

We concluded from the present results that the flavonoids from Centaurea Cineraria extract have higher activity againist  subjected cancer cell line using different concentration.

Conflict of Interest

There is no conflict of interest

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