INTRODUCTION:

Cancer is a condition characterised by uncontrollable proliferation of cells. The homeostatic conditions favour the death of old or damaged cells and replace them with new cells. A malfunction in the cell division proteins or tumour suppressor protein leads to cancer^{1, 2}. Among various cancers, cervical cancer is one of the common cancers found in women worldwide. It was classified into two major types namely adeno carcinoma and squamous cell cancer. Other types of cervical cancers have also been found, but they are not as frequent as the aforementioned types.

The majority of the cervical cancers are due to human papilloma viral (HPV) infection. Till date, more than 100 different strains of HPV have been reported. Among the 100, about 35 have been found to have high carcinogenic potential whereas 40 others have been reported to possess low carcinogenic potential³. Tumour suppressor proteins p53 and pRb have been reported to be disrupted by HPV proteins E6 and E7 respectively^{4, 5}. Various proteins found insignaling pathways such as ERK/MAPK, PI3K/Akt/mTOR, Wnt/β-catenin and anti-apoptotic proteins have been found to be over expressed and pharmaceuticals are actively involved in exploiting them as drug targets for chemotherapy^6-9. The incidence and mortality of cervical cancer are higher in the economically under developed countries when compared to developed countries. Cervical cancer takes a long time to develop and a Pap smear test could detect cancer at an earlier stage. Developed countries have already included the Pap smear test in the basic annual medical tests which considerably decreases the number of cervical cancer cases. However, people in the developing countries with a poor economic condition not able to do the Pap smear tests onanannual basis and hence the incidence is more¹⁰. On comparison, it was found that 1 in 53 women in India was affected by cervical cancer whereas in developed countries the ratio stands at 1 in 100. In India, 70% of the cases detected for cervical cancer were found to be of stage 3 or stage 4. Therefore effective chemotherapeutic drugs are very much required to tackle the cervical cancer menace¹¹.

Natural products obtained from plants and other sources serve as an important therapeutic source for treating various diseases. C. sebestena also was known as Geiger tree belongs to the family Boraginaceae was chosen for this study based on its traditional use as a medicinal plant. It is found in the tropical and sub-tropical regions growing up to a height of 25 feet^12,13. It was not been fully explored for its medicinal properties. It was already been proved that various parts of the tree possess anti-diabetic activity, anti-bacterial activity, anti-oxidant activity, anti-ulcer activity and anti-hypolipidemic activity^14-17. But there is no report on the anti-cancer activity of phytoconstituents these plant. Hence, an attempt was made to study the anticancer cytotoxic effect of phytochemicals obtained from the leaf extract of the tree.

MATERIALS AND METHODS:

Preparation of leaf extract:

The leaves of Cordiasebestena tree was collected, washed with water and shade dried for 48 hours. The moisture level was observed periodically. After shade drying was done, the leaves were placed in a hot air oven and the temperature was set to 50ºC for 10min. The completely dried leaves were crushed into a fine powder using a mortar and pestle. 500 ml of crude extract was prepared in a flask using ethyl acetate, petroleum ether, and water by adding 2 g of leaf powder. Then the flasks were sealed with para-film and placed in an orbitary shaker for 48 hours at an RPM of 120. The extract was filtered using a muslin cloth and the filtrate was concentrated using rotary vacuum evaporator and the concentrate was lyophilized.

GC-MS analysis of crude extracts:

All the crude extracts prepared were subjected to GC-MS analysis. The extracts were dissolved in their respective solvents and fed into the system with the injector. The analysis was carried out on a Perkin-Elmer work station, with model Clarus 600 GC coupled to a mass spectrometer. Elite-5MS (30mx0.25mm width film depth of 250μm capillary tube was used under the following condition. The instrument has an oven with aninitial temperature of 55°C for 3 minutes and a ramp program which elevates from 6°C/minutes up to 310°C, further 3 minutes isothermal hold. The Helium (He) carrier gas was used, with flow rate split ratios of 10:1. The sample (2µl) volume was vaccinated and temperature of the injector was maintained to 250°C. The spectrum obtained was compared with spectra available in the National Institute of Standards and Technology (NIST-LIB 0.5) library for matching using the Inbuilt software of the GCMS system (Wiley GC-MS-2007) as per the literature data available.

Assay of cytotoxic activity by MTT method:

The anti-cancer cytotoxic activity of ethyl acetate, petroleum ether, and aqueous crude extracts was tested on cervical cancer cells (HeLa) by MTT method. The MTT assay was carried out in a 96-well plate. To the 96 well plate,1×10⁵HeLa cells were added. The plate was incubated for three days in 5% CO₂ incubator. Then the crude extracts of various concentrations were added to the 96 well plate. To each well5 mg/ml of 0.5% MTT was added.1ml of DMSO was added to each well after four hours of incubation. The viable cells were determined by observing the absorbance at 540nm. The following formula was used to calculate the cell viability:

Absorbance at 540 of treated cells/Absorbance at 540 of control cells×100%

The concentrations of crude extracts were taken along the X axis and the corresponding viable cells (%) were taken along the Y axis. From the graph, the IC₅₀value of the crude extracts against HeLa cell lines was obtained¹⁸.

Assay of antioxidant activity of C. sebestena crude extract:

The (DPPH) is a purple colored crystalline powder composed of stable free-radical molecules. The free radical,2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging ability was carried out as described earlier¹⁹. Different concentrations of petroleum ether crude extract (5-25mg/ml) was mixed with 2 ml of DPPH (0.002% w/v in methanol) and was incubated for minutes in the dark. After incubation, the absorbance was measured at 517 nm. The DPPH scavenging activity was calculated using the following equation. Ascorbic acid was used as a standard.

Ac - At

DPPH Scavenging activity (%) =-------------- X 100

Extraction and identification of the compound:

The crude extract was subjected to thin layer chromatographic separations. Various solvents based on polarity at varying proportions were tested to find the optimal solvent system for TLC. Petroleum ether:Acetone (7:3 v/v) was used as the optimal solvent system. The separated fractions assessed for anticancer cytotoxic activity on HeLa cells. The purity of the active fraction was analysed further using preparative TLC.

In Silico docking studies:

Autodock 4.2 was used to carry out the in-Silico docking studies. Pentane2,4-dione was used as the ligand. Autodock 4.2 was used to carry out the in-Silico docking studies. The ligand was drawn in Marvin Sketch Tool and saved in pdb format. 3D structures of the proteins; (Kras, Raf1, ERK1, ERK2, MEK1, MEK2, Pi3K Alpha, Pi3K Gamma, PDK1, AKT1, AKT2, mTOR, Beta-catenin, LRP6, EGFR Kinase domain, VEGFR-1 Kinase domain, BCL-2, BCL-XL, IAP,MDM2) with their respective PDB ID (4OBE, 4OMV, 4QTB, 4XJ0, 3VVH, 1S9I, 3ZIM, 5EDS, 3RWP, 3O96, 2UW9, 4JSV, 1JDH, 3S94, 5FED, 3HNG, 4MAN, 4TUH, 5COK, 4ZFI) were downloaded from RCSB PDB website (www.rcsb.org).

The water molecules found in the proteins were removed using Pymol software. For each docking, the proteins were initially imported into the Autodock working file folder and appropriate editing was done. The editing includes the addition of polar hydrogen bonds, merging of non-polar hydrogen bonds and the addition of Kolman charges. Then the editing performed was saved by overwriting the existing protein file. The ligand was introduced and saved in PDBqt format. Then grid parameter file was created by assigning macro molecules and ligands. Then the grid box was set up in the region for the docking to be performed. The autogrid was run with the created grid parameter file in .gpf format. After autogrid was done, docking was performed by creating a dock parameter file. The macromolecule and ligand were assigned and the Lamarckian genetic algorithm was chosen for Autodock operation. Autodock was performed with the dock parameter file saved in .dpfformat. After docking was done Pymol software was used to find the number of hydrogen bonds formed²⁰.

Nitric oxide inhibition assay:

The RAW 264.7 cells were seeded at a density of 5*10⁵cells/well in 24 well plates and incubated for 12h at 37ºCand 5% CO₂. Then media of each well were aspirated and fresh FBS-free DMEM media were replaced. Pentane-2,4-dione (10µg/ml) was prepared in FBS-free DMEM. After 24h of drug treatment, cells were stimulated with 1µg/mL ofLPS for 24h. The presence of nitric oxide was estimated using Griess reagent assay. 100µL of cellculture medium with an equal volume of Griess reagent in a96-well plate was incubated at room temperature for 10 min.Then the absorbance was measured at 540 nm in a micro titre plate reader. The amount of nitrite inthe media was calculated from sodium nitrite (NaNO2) standardcurve²¹.

RESULTS:

In vitro anticancer activity of crude extracts:

Among the three different extracts obtained from C. sebestena leaves, the petroleum ether extract demonstrated cytotoxic activity on HeLa cell lines with an IC₅₀ value of 250µg/ml. The viability of HeLa cells treated with different concentrations of the crude extracts of C. sebestenais shown in Figure 1.

Figure1: Cytotoxic activity of crude extracts prepared from Cordiasebestena against HeLa cell lines

Antioxidant activity of C. sebestena crude extract:

Crude petroleum ether extract of C. sebestena leaves was tested for its antioxidant potential using the DPPH assay. Crude extract demonstrated promising antioxidant activity, higher than that of the standard drug ascorbic acid. The antioxidant activity of petroleum ether extract of C. sebestena leaves and standard ascorbic acidis shown in Figure 2.

Figure2: DPPH assay result of petroleum ether crude extract

Separation of phytochemicals by preparative TLC:

Separation of petroleum ether crude extract on silica gel TLC sheet using petroleum ether:acetone (7:3 v/v) asthesolventsystemis given in Figure 3A.The crude extract was subjected to preparative TLC to separate the individual phytochemicals using the same solvent system. The lemon yellow or light green coloured single band obtained at R_f of 2.32 appeared to contribute the major percentage of the crude extract (Figure 3B). The separatedband was further purified using preparativeTLC using the same solventsystem. In the preparative TLC separation, the lead compound was separated as a single band (Figure 3 C).

Figure 3: Thin layer chromatography of C. sebestena. A)TLC of crude extract,

B) Preparative TLC of crude extract and C) TLC of pure compound

Structure elucidation of the compound:

The purified compound was subjected for FT-IR, GC-MS, ¹H NMR and ¹³C NMR to identify the structure of the compound. The FT-IRspectrum of the pure compound is given in Figure 4.The peak at 1737 cm^-1represents C=O (Carbonyl),and peak at 3020 cm^-1 corresponds to C-R (Alkenes) and peak at 1215cm^-1indicates C-H (Alkanes) groupsin thestructure.GC-MS analysis and the mass spectrum obtained is shown in Figure 5.The ¹H NMR shifts are given in Figure 6 and ¹³C NMR shifts are given in Figure 7.The structure of the pure compound was identified by elucidating the spectral data obtained from FT-IR, GC-MS, ¹H NMR and ¹³C NMR and the compound was identified as pentane-2,4-dione (Acetylacetone). It has a molecular weight of 100.12g/mol and a molecular formula of C₅H₈O₂.

Figure 4: FT-IR spectrum of the pure compound

Figure 5: Mass spectrum of the pure compound

Figure 6: ¹H NMR spectrum of the Pure Compound

Figure 7: ¹³C NMR spectrum of the Pure Compound

Figure 8: Structure of the pure compound (Pentane-2,4-dione)

MTT assay of the pure compound:

Pentane-2,4-dionedemonstrated a significant cytotoxic activityonHeLa cells, with the IC₅₀ value of 10µg/ml. The cytotoxic activity of pentane-2,4-dione at various concentrations against HeLa cells is shown in Figure 9. The effect of pentane-2,4-dione on HeLacells are shown in Figure 10. Pentane-2,4-dione (10µg/ml)treated cells showed apoptotic changes which include cell breakage, cell rounding up and membrane blabbing.

Figure 9: Cytotoxic activity of pentane-2,4-dione at varying concentrations on HeLa cells.

Figure 10: Effect of pentane-2,4-dione on HeLa Cells; A) Control Cells and B) Cells treated with pentane-2,4-dione (10µg/ml).

In Silicoprotein-ligand docking studies:

The interactions of pentane-2,4-dione, with the selected anticancer protein targets, are given in Table 1. Pentane-2,4-dione showedthehighest affinity towardsK-Rasprotein with the least binding energy of -5.15kcal/mol and124.37µM as inhibition constant and formed 5 hydrogen bonds. Theinteractionis very strong polarinteraction. This indicates that the ligand pentane-2,4-dionewould probably exert its cytotoxic activity, by inhibiting Kras protein, which is an important kinase protein in the ERK/MAPKpathway.

Table 1: Autodock results of pentane-2,4-dione on cancer protein targets

Protein Name	Inhibition constant	Binding energy (Kcal/Mol)	No. of. Hydrogen bonds
AKT1	852.59 µM	-4.19	2
AKT2	259.92 µM	-4.89	2
BCL-2	3.68 mM	-3.32	0
BCLXL	730.09 µM	-4.28	2
Beta catenin	1.91 mM	-3.71	2
EGFR Kinase domain	2.6 mM	-3.53	1
ERK1	777.11 µM	-4.24	2
ERK2	498.35 µM	-4.51	2
IAP	4.29 mM	-3.23	2
Kras	124.37 µM	-5.15	5
LRP6	978.57 µM	-4.11	3
MDM2	7.12 mM	-2.93	0
MEK1	904.61 µM	-4.15	2
MEK2	530.07 µM	-4.47	3
mTOR	1.2 mM	-3.99	2
PDK1	1.76 mM	-3.76	2
PI3K Alpha	884.63 µM	-4.17	3
PI3K Gamma	985.93 µM	-4.1	2
RAF1	1.51 mM	-3.85	0
VEGFR-1 kinase domain	2.48 mM	-3.55	1

Nitric oxide inhibition assay:

Pentane-2,4-dione reduced the nitric oxide production by 47% in LPS induced RAW 264.7 cells. This shows that the compound also contains anti-inflammatory activity on cells.

DISCUSSION:

Secondary metabolites from plants possess significant chemical diversity which could be responsible fortheunique biological activity.With the rediscovery of many known phytochemicals more often, currentlythefocus has shifted towards the unexplored medicinal plants. There was no report onthecytotoxic activity of phytochemicals obtained from the C.sebestena tree. It was reported that the presence of tannins, cardenolides, and alkaloids in methanol fraction of C.sebestena leaves. The extracted fraction had inhibited Mycobacterium fortuitum and Mycobacterium smegmatis with an IC₅₀ value e of 200 mg/ml²².Ethyl acetate extract obtained from C.sebestena leaves possessed significant anti-bacterial activities against B. cereus and S. aureus²³ and also reported that one of the fractions obtained from C.sebestena possessed low toxicity to rat liver cells while having significant anti-bacterial activity.

The emphasis of our work was to identify the anticancer cytotoxic phytochemical from three different extracts from C. sebestena leaves. Each of the extracts was assessed for their cytotoxic effects on cervical cancer cell lines by MTT assay. The petroleum ether extract was found to be effective with an IC₅₀ value of 250 µg/ml.It was chosen for further studies to purify and to identify the active phytochemical compounds. Using preparatory TLC, the lead compound was separated and the structure of the lead phytochemical was identified by GC-MS, NMR, and FT-IR studies. The lead phytochemical was identified as pentane-2,4-dione (acetyl acetone). Extraction of pentane-2,4-dione from the ethyl acetate fraction of C.sebestena flowers was already been reported¹⁶. So far there have been no reports on the anticancer cytotoxic activity of pentane-2,4-dione. It showed the highly significant IC₅₀ value of 10µg/ml in HeLa cells. In order to predict the mechanism of cytotoxic activity of a lead compound on cancer cells, in-Silico protein-ligand docking study was performed using Autodock-4.2. Proteins involved in various signaling pathways such as Wnt/β-catenin, ERK/MAPK, PI3K/Akt and anti-apoptotic proteins responsible for cervical cancer were chosen based on literature review^6-9. The pentane-2,4-dionelig and was docked with the proteins to find the binding energy and inhibition constant. Pentane-2,4-dione was found to be inhibiting Kras protein with least binding energy and therefore it is proposed that pentane-2,4-dione exerts its anti-cancer activity by inhibiting Kras protein which is involved in ERK/MAPK pathway. The lead compound also reduced the nitric oxide production in LPS induced RAW 264.7 cells. It further confirmed the antioxidant activity of the lead compound. However further in vitro and in vivo studies are needed to confirm the afore mentioned mechanism of action.

CONCLUSION:

Cervical cancer is currently one of the major concerns for women in the developing countries. With the increasing population rate and incidence of cancer, there is always a strong need to find a novel chemotherapeutic drug. Natural products are unique for their chemical complexity, diversity and the associated biological activity. The phytochemical constituent of Cordiasebestena, Pentane-2,4-Dione exhibited significant anticancer cytotoxic activity on cervical cancer cells. Molecular docking study suggests that pentane-2,4-dione could exert its anticancer activity by inhibiting Kras protein.

ACKNOWLEDGMENTS:

Necessary facilities provided by the management of VIT University to carry out this study is gratefully acknowledged.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 28.06.2017 Modified on 17.07.2017

Research J. Pharm. and Tech 2018; 11(6): 2191-2196.

DOI: 10.5958/0974-360X.2018.00405.5