Evaluation of the cytotoxic effects of the colchicine compound isolated from the leaves of Calotropis procera (Ait) against MCF-7 and SK-GT-4 cancer cell lines .

: Alkaloids are regarded as important nitrogen-containing chemical compounds that serve as a rich source for discovering and developing new drugs where most plant-origin alkaloids have antiproliferation effects on different kinds of cancers. Alkaloids’ continence of Calotropis procera leaves are detected by two biochemical alkaloid reagents. Also GC-MS analysis for leaf alkaloid extract was done that showed the existence of one type of alkaloid compound at retention time12.8min detected as colchicine (C 22 H 25 N0 6 ( by comparing it with colchicine standard reference (Sigma Aldrich) with M.wt 399g/mol and percentage area 7.1% . Furthermore, identification, separation, and purification for purified colchicine compound were conducted by HPLC technique that gave one main peak at RT reached 2.5min compared with the standard reference. Evaluation of the anticancer activity of purified colchicine on two (MCF-7 &SK-GT-4) cell lines revealed significant cytotoxicity on the MCF-7 cell line that was superior to its cytotoxicity on the SK-GT4 cell line. With calculated IC 50 reached 55.33µg/ml &522 µg/ml respectively.


Introduction:
More than 80% of the world's population still depends on traditional medicines for many diseases, and natural products have been used worldwide for medicinal purposes for along years 0 .As a result, this is a new era of natural product discovery.Thus, there is an urgent need to discover modern therapeutic phytochemical agents with different chemical structures and also rare mechanisms of actions are needed for new and emerging infectious diseases [2][3] .Alkaloids can be found in Flowers, roots, fruits, leaves, seeds and non-flowering plants, such as paclitaxel, 4 .Calotropis procera (Family: Asclepiadaceae) is a cultivable wild xerophytes' shrub found across Africa, Asia, and South America 5 .Calotropis spp. is a small genus of shrubs or small trees that can be found in the tropical and subtropical regions of Asia, Africa, and central and southern America.This species was found in India in two species, C.procera and C.gigantean L, which resemble each other in structure and uses, that is, fiber used as fuel 6 .C.procera is used to reduce Filarial symptoms alleviated by attaching a red thread to the afflicted area 7 .There is a plethora of literature available that demonstrates the blossoms of this plant have hepatoprotective properties when taken in conjunction with paracetamol to protect the liver.Different plant parts, particularly the latex, are tested against various cancer cell lines 8,9 .alkaloid compounds founded in C. procera leaf will be isolated ,purified, and tested their anticancer effect on MCF-7 and SK-GT-4 cell lines.

Preparation of selection plants extraction
After plant's classification, the leaves of plant were thoroughly washed by using tap water to remove any contaminates and then shade dried at 40 o Cand the dried leaves were ground to a fine powder through a mechanical grinder and then stored in tight plastic bags labeled for study.

Preliminary Biochemical test for alkaloid compounds detection
Extraction for leaves of C. procera was done by using methanol (Hot continuous extraction) method for detection of alkaloid compounds 10 .
Dragendorffs reagent: 1ml of a reddish-brown or orange precipitate formed when the reagent was added drop by drop to the extract shows the presence of alkaloids.
Mayer reagent: 1 ml of drop-by-drop reagent is applied to the extract and a creamy precipitate is formed as a result.

Extraction of total Alkaloids
Alkaloid compounds were extracted by method of Harborne 10 .After 24 hrs. of continuous extraction with 80 percent Ethanol, the soxhlet apparatus 250 ml volume was filtered and the filtrate was concentrated under vacuum at 45C⁰ until the solution reached 10ml and transferred to a separate funnel where 2 N HCl was gradually added to make it reach (pH=2), then the extract was washed with 10 ml chloroform three times, the pH value of the extract was increased to reach (pH=10) using NH4 OH that partitioned with 10 ml chloroform 3times.The chloroform portion dried to obtain the total alkaloid extract, that kept in a clean container at 4⁰C for later research.

Screening for alkaloid compounds by using Gas chromatography-mass spectrum (GC-Mass) analysis
GC-MS analyses were done in Nihranbinomar-Laboratory-Basra oil company.Leaf Methanolic alkaloid extract of C.procera for detecting their alkaloid types and structure was done by using a modified method by GC-.MS analysis 11 .Spectrometer Agilent gas chromatograph equipped and coupled to a mass detector Agilent 5977A spectrometer with an HP-5MS (5% Phenyl methyl siloxane), 30m ×0.25mm × 0.25 mm ID of the capillary column.The temperature of the injector was 40 C maintained for 5 min then raised gradually to 300 ⁰C at a rate of increment 10\min.Helium gas 99.99% used as mobile phase at a flow rate of 1ml\min.an injection volume of 1 µL.Mass spectra were taken at 70 ev; a scan-interval of 4 min.and fragments from 45 to 450 Da.The solvent delay was 4min.and the total GC-MS running time was 45min.The samples were injected in (Split mode) 50:1 Mass spectral scan range was set at 45 to 650 m/z .

Identification , Separation & Purification of alkaloid compounds by using the High Performance Liquid Chromatography (HPLC) technique
HPLC analysis was carried out using a modified method 12 , LC-W100A HPLC (USA) system connected to LC-UV100 plus UV detector with manual injectors.Data interfered using PC with (les (x86) \HPLC SYSTEM) separation was performed through Exformma technologies Column Arcus EPC18 5um, 4.6 x 250mm, isocratic mobile phase used for the analysis of leaf methanolic alkaloid extract consist of (acetonitrile75: water:25), with 1ml/min flow rate at 25 ⁰C, and pressure of 100 p.s.i, injection volume was 10 µL.0.05 mg\ml sample of extract that was filtered by 125mm filter paper before injection, the run time was for…delete for 10 min for each run, and detection was conducted at 300 nm wavelength.Thus, a modified method was used to separate and purify the 1592 colchicine compound by HPLC [12][13] .The eluted mobile phase during the appearance of the identified peak of colchicine with recorded retention time by HPLC at R.T reached 2.5min.under the same conditions.Isolated mobile phase portions then undergo HPLC analysis to confirm the purity of the isolated.

In vitro anticancer activityMTT assay against the (SK-GT-4 and MCF-7) cell cultures Maintenance of cell cultures
To maintain the cancer cell lines,of MCF-7 (breast adenocarcinoma derived from metastatic site: pleural effusion) and SK-GT-4 (esophagus adenocarcinoma derived from metastatic site: pleural effusion) ( the IRAQ Biotech Cell Bank Unit in Basrah) provided cancer cell lines, and RPMI-1640 supplemented with 10% Fetal bovine serum, 100 units/mL penicillin, and 100 g/mL streptomycin was used.Reseeded at 70 percent confluence twice a week, the cells were kept at 37 C⁰ and 5% CO2 for three weeks 14 .

Cytotoxicity Assays
C. procera's colchicine was used in the MTT cell viability assay on 96-well plates to investigate the cytotoxic effect of purified colchicine.10 4 cells/well were used to seed cell lines.Cells were treated with (colchicine) at varying concentrations 100, 250, 500, 750, 1000 of µg/µl after 24 hours or after a confluent monolayer had formed.A solution of 2 mg/mL MTT (after incubating the cells for 2 hours at 37°C) was used to evaluate cell viability after 72 hours of treatment.To dissolve the residual crystals, 10 µL of DMSO (Dimethyl Sulphoxide) was added to the wells and incubated for 15 minutes at 37 °C 15 , The assay was carried out in triplicate and the absorbency was measured using a microplate reader at a test wavelength of 620 nm.The proportion of cytotoxicity (cell growth inhibition) was estimated as follows: If A is the mean optical density in untreated wells and B is the mean optical density in treated ones, then PR = B/A*100 and IR = 100 -PR [16][17][18][19] .

Acridine Orange/Ethidium Bromide (AO/EB) staining
Ethidium bromide and Acridine Orange 100µg/ml both were added to the cells and kept in dark at room temperature.The morphological changes were observed using a fluorescence microscope 20 .

Results and discussion: Biochemical tests for alkaloid compounds' detection
A positive test appeared for methanolic leaf extract of C. procera as orange and white precipitate for Dragendorff and Mayer's reagent respectively, as shown in Table 1.Methanol was used as a strong solvent for bioactive extraction for the leaf of C.procera.An alkaloid compounds found in large amounts in the leaf of C. procera 15 .

GC-MS analysis for the leaf methanolic alkaloid extracts
As shown in Table 2 and Fig. 2 the results of GC-MS analysis for leaf's methanolic extract proved the existence of one type of alkaloid compound that is colchicine by comparison with its actual retention time to the retention time of colchicine standard reference (Sigma Aldrich) at R.T(12.8)min with area percentage reached 7.1% with molecular weight 399g/mol, that was in line with the study of Naser et al. 15 , that revealed the presence of colchicine compound in leaf methanolic extract of C. procera by GC-MS analysis in Iraq that, C.procera appeared to produce many bioactive compounds, such as alkaloids [21][22][23][24][25] .7] .In vitro anticancer activity of studied alkaloid compounds Cytotoxicity study of the colchicine compound against (MCF-7) and (SK-GT-4) cell lines.

Name of alkaloid compound
The cytotoxic effect of the purified alkaloid compound colchicine was tested by studying their ability as anti-proliferative against human cancer cells (MCF-7&SK-GT-4) which showed that the colchicine compound has significant cytotoxic activity on the MCF-7 cell line that is superior to that against the SK-GT-4 cell line.Assessment of anti-cancer activity using MTT assay is a colorimetric assay that correlates between cell activity and the number of viable cells by measuring the absorbance of a specific wavelength to determine the cytotoxic effect of the drugs or substances 16 .Study of Krishnasamy et al 19 proved anticancer activity of colchicine compound that purified from Indigofera aspalathoids against another type of cell lines that was ( Hep-B ) cell line with a value of IC50% reaching 344 µg/ml.Also as seen in study of Lu et al. 28 and Zhang et al. 29 colchicine has antiproliferative effects on both two human gastric cancer cell lines (i.e., AGS and NCI-N87) by induced apoptosis,Thus , colchicine that purified from C.procera could be a good suggestion for the treatment of breast cancer cell line MCF-7 and esophagus cancer cell line SK-GT-4.

Calculation of the IC50 value of the colchicine compound against SK-GT-4 &MCF-7 cell lines.
As shown in Figs. 5, 6, the concentrations of colchicine tested plotted against viable cell percentage for both the MCF-7&SK-GT-4 cell lines and the IC50 value was calculated and found to be 55.33 µg/ml &522 µg/ml respectively and that differences of IC50 Value for colchicine towards two different cell lines Because every cell line has its own "cell specific response" where , Each cell line has a completely unique system, with its individual biological characteristics even when cell lines were established from the same tissue 30,31 .

Conclusion:
It is clear from the results of the current study that the leaves of a plant C. procera contain a chemical compound namely (Colchicine) with high efficacy against some cancerous lines, and that it shows high purity and distinctive anti-cancer efficacy that enables it to be a promising treatment after completing other studies on it to know its side effects.Also the MTT assay which is used to study the cytotoxic effect of the Colchicine compound showed significant anti-cancer activity against both MCF-7 and SK-GT-4 cell lines and The plant C. procera, especially its leaves, is an important source of this compound, which has anti-carcinogenic activity In turn, it is considered a promising plant in the treatment of this disease, after extensive studies of this compound, and the study is the first of its kind for purification this compoundby HPLC technique with high purity compared with standard fromthe leaves of this important plant.

Figure 2 .
Figure 2. GC-MS Chromatogram for leaf Methanolic alkaloid compoundIsolation, Identification, and Purification of colchicine compound by HPLC techniqueHPLC analysis for leaf methanolic alkaloid extract of C procera as shown in Fig.3, resulted in the identification, separation, and purification of colchicine that was identified by comparison with

Figure 3 .
Figure 3. HPLC chromatogram for purified colchicine founded in leaf methanolic alkaloid extract in C. procera.