Cytotoxic Potential of 4-Hydroxypentan-2-Oneextracted from Jacaranda mimosifolia on Colorectal Cancer Cells

 

Adhithya Ragunathan, Lokesh Ravi, Kannabiran Krishna*

Department of Biomedical Sciences, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India.

 

ABSTRACT:

The incidence of colorectal cancer is comparatively higher in the industrialized countries. It has been estimated that the incidence, of colorectal cancer, would be increased significantly in the next twenty-fiveyearsworldwide. Therefore researchers are looking for an effective anti-colorectal cancer drug from natural sources. The aim of the present study was to isolate and to identify anticancer cytotoxic phytochemical compound from Jacaranda mimosifolia and to study its mechanism of action. The cytotoxic activity of the crude extracts (acetone, petroleum ether, and chloroform) prepared from J.mimosifolia on HCT-15 cell line was evaluated by MTT assay. Among the extracts, acetone extract showed the IC₅₀ value of 600µg/ml on theHCT-15 cells. The acetone extract was purified by silica gel column chromatography, and the lead compound obtained was characterized byFT-IR, ¹³C-NMR, and GC-MS. The structure of the pure compound was identified as 4-hydroxypentan-2-one and it showed the IC₅₀ value of 100µg/ml against HCT-15 cell line. The mechanism of action of 4-hydroxypentan-2-one on HCT-15 cancer cells drug target proteins was assessed by In Silicomolecular docking analysis using the bioinformatics tool AutoDock. The pure compound 4-hydroxypentan-2-one inhibited the K-Ras protein and showed the least binding energy of -5.22 Kcal/mol with the inhibition constant of 148.89 µMand  6 hydrogen bonds were formed during the interaction.

 

 

 

INTRODUCTION:

Colorectal cancer is a type of cancer which is characterised by the proliferation of abnormal cells in the large intestine and rectum¹.The large intestine comprises of four colon sections namely ascending, transverse, descending and sigmoid. The sigmoid colon is followed by rectum and anus. The ascending and transverse colon together make up the proximal colon whereas the descending and sigmoid colon are regarded as the distal colon. While women and older patients were reported to have tumors in the proximal colon more commonly, men and younger patients were reported to have tumors developed in the distal colon more frequently^2-4. The precursor for colorectal cancer is the development of polyps⁵.

 

Though polyps are generally considered as non-cancerous lesions, they progressively gain size and eventually manifest as colorectal cancer⁶. Amongst many different types of polyps, 95% of colorectal cancer cases are due to the development of adenoma polyp. The glandular cells which secrete mucus in the intestine give rise to adenoma polyps^7-9. The development of colorectal cancer takes place over a long period of time¹⁰. In the US, 90% of the incidence and 93% deaths due to colorectal cancer were reported to have been observed in individuals over the age of 50¹¹. It was also observed that men were 30-40% more likely to have colorectal cancer than women and the exact reason for this disparity is unknown¹².Lifestyle practices such as excessive alcohol consumption, smoking and lack of physical activity have been found to be a risk factor for colorectal cancer^13-15. In addition to these risk factors, individuals who had a diet rich in processed meat and saturated fats were more likely to develop colorectal cancer when compared to individuals who consumed the same in low or moderate scale¹⁶.Though the incidence of colorectal cancer is more in developed countries when compared to developing/underdeveloped countries. However, the mortality rate in developed countries is on the decline owing to better health care system when compared to developing and underdeveloped countries^{17, 18}. Hence, researchers around the world are actively looking for a drug that could be used to treat colorectal cancer. Many phytochemical compounds from plants served as a potential drug for the control of colorectal cancer.

 

Jacaranda mimosifolia belongs to the family of Bignoniaceae was originally found to be an indigenous tree in the river banks of Argentina. It was later imported into Africa from Brazil in the late 18^th century. It is a fast growing tree and would grow to a height of 15-22 m¹⁹. One of the prominent features of this tree is that it’s beautiful violet color flowers and for this reason, it is regarded as an ornamental tree all around the world²⁰. In addition to that root, stem, bark, and leaves ofJ.mimosifoliapossessseveral phytochemical compounds with various pharmacological properties. Traditionally various parts of the tree are used to treat diseases like hypertension, ulcer, and amoebiasis²¹.It is also an essential part of folklore medicine in the treatment of venereal diseases²². Previously researchers have reported the anti-candidal, anti-bacterial, antiplasmodial and anti-oxidant activity of various parts of the J.mimosifolia tree. Though there is a report on the cytotoxic activity ofJ.mimosifolia bark extract on HepG2 cell line,there has been no report on the cytotoxic effect of leavesextract^23-26. Hence a study wasundertaken to assess the cytotoxic effect of J. mimosifolia leaves on HCT-15 cell lines.

 

MATERIALS AND METHODS:

Preparation of crude extract from leaves:

The J. mimosifolia leaves were collected and thoroughly washed with tap water. After washing, the leaves were shade dried for a period of 48 hours with thetheperiodic observation of moisture content.The leaves were then placed in a hot air oven and exposed to a temperature of 50 degree Celsius. Using a mortar and pestle, the completely dried leaves were crushed into fine powder. Three leaf extracts were prepared usingthethree different solvents such as petroleum ether, chloroform, and acetone by soaking 5 g of leaf powder in 100 ml of the respective solvents. All the three flasks were then placed in an orbital shaker for 48 h at 140rpm.All the solvent extracts were filtered and concentrated using Rotaryevaporator as described earlier²⁷.

 

GC-MS analysis of crude extracts:

The crude extracts prepared were fed into a combination of Perkin Elmer workstation andClarus 600 GC coupled to a mass spectrometer. Elite-5MS (30m x 0.25mm) width film depth of 250μm capillary tube was used underthefollowing condition. Helium (He) was used as the carrier gas to carry the sample throughout the column. A library match was done usingtheWiley GC-MS-2007software and the spectral data obtained from the samples were compared to the data available inthe National Institute of Standards and Technology (NIST-LIB 0.5)²⁸.

 

In vitro cytotoxicity of crude extracts:

The cytotoxicityof acetone, chloroform, and petroleum ether crude extracts was evaluated on colorectal cancer lines (HCT-15) using MTT assay. 96 well plates were used to carry out the MTT assay. 1 × 10⁵ HCT-15 cells were added to the 96 well plates and incubated in5% CO₂ incubator for 72 hours. The crude extracts were added to the 96 well plate in varying concentrations and after that 5 mg/ml of 0.5%, MTT was added to each well.  DMSO (1 ml) was added to each well after four hours of incubation. Then the absorbance was measured at 540 nm. Cell viability was calculated using the following formula

 

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

The crude extract concentrations were plotted along the X axis and the corresponding viable cells (%) were taken along the Y axis. From the graph, the IC₅₀ value of the crude extracts on HCT-15 cell lines was obtained ²⁹.

 

Extraction and identification of the pure compound:

In order to isolate the pure compound from the crude extract, column chromatography was performed using pencil column.Acetone and petroleum ether (8:2  ratio) wereused as a mobilephase as optimised in TLC plates.The same solvent system was used as mobile phase in the column chromatography. A total of 50 fractions were collected and the fractions showing cytotoxicity were pooled and concentratedand evaluated for purity using TLC²⁵.The activefractions were subjected to C¹³ and H¹NMRanalysis to determine the position and number of carbon and hydrogen atoms. The function groups present in thecompound was evaluated by FT-IR analysis²⁵.

 

Assay of cytotoxic activity of the pure compounds:

The cytotoxicity of the pure compound against colorectal cancer (HCT-15) cell lines was evaluated using MTT assay. The cytotoxicity assessment was performed in 96 well plates. After the addition of 1 ×10⁵ HCT-15 cell lines to the 96 well plate, they were incubated in 5 % CO₂ incubator for 72 hours. Then in varying concentrations,thepure compound was added to the 96 well plate. To each, well 5 mg/ml of 0.5% MTT was added and then incubated for 4 hours.1ml of DMSO was added to each well after the incubation period. The absorbance was observed at 540 nm.The cell viability was calculated using the following formula:

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

 

The percentage of viable cells were plotted along the Y axis and the respective concentrations pure used were plotted along the X axis. The IC₅₀of the pure compound against HCT-15 cell lines were obtained from the graph²⁹.

 

In silico studies:

The in silico docking studies was performed using AutoDock 4.2. The 3D structure of 4-hydroxypentane-2-one was downloaded from PubChem compound database and used asaligand. The proteins with their PDB Ids were downloaded from the RCSB website(listed in Table1).The proteins downloaded from RCSB website usually contains water molecules and ligand which were removed using Pymol. Prior to each docking study, proteins were initially imported into the working file folder of AutoDock.Polar hydrogen bonds were added to the protein. Non-polar hydrogen bonds were merged and Kolman charges were added.All these modifications performed were saved. Then the ligand molecule was introduced into the systemand saved in pdbqt format. The grid parameter file was prepared and saved in .gpf format. After the completion of Autogrid program, dock file was prepared and saved in .dpf format and executed. After docking was done Pymol software was used to find the number of hydrogen bonds formed ³⁰.

Table1: List of proteins and their PDB IDs used forinsilicodockinganalysis

Protein	PDB Id
Kras	4OBE
Raf1	4OMV
ERK1	4QTB
ERK2	4XJ0
MEK1	3VVH
MEK2	1S9I
Pi3K Alpha	3ZIM
Pi3K Gamma	5EDS
PDK1	3RWP
AKT1	3O96
AKT2	2UW9
mTOR	4JSV
Beta-catenin	1JDH
LRP6	3S94
EGFR Kinase domain	5FED
VEGFR-1 Kinase domain	3HNG
BCL-2	4MAN
BCL-XL	4TUH
IAP	5COK
MDM2	4ZFI

 

RESULTS:

Assay of cytotoxicity of Jacaranda mimosifolia crude extracts:

Acetone, petroleum ether, and chloroform extracts prepared fromJacarandamimosifoliawereevaluated for their cytotoxic potential on HCT-15 cell lines by MTT assay. Acetone crude extract displayed an IC₅₀ value of 600 µg/ml, while petroleum ether and chloroform crude extracts possessed IC₅₀ values of 800 µg/ml. The cytotoxic activity of the crude extracts on HCT-15 cell lines are shown in Figure 1.

 

 

Figure1:Cytotoxic activity of crude extracts from J. mimosifolia on HCT-15 cell line

 

Extraction and identification of the Pure compound:

Since acetone extract displayed the highest cytotoxic potential, it was chosen for further studies. The pure compound from the acetone extract was separated by silica gel column chromatography using petroleum ether:acetone(8:2) as the mobile phase solvent system. The GC-MS data of the acetone crude extract and the pure compound is given as Figure 2. A and B. The mass spectrum of the pure compound is given n Figure 3. From the spectrum, the pure compound hasa retention time of 9.77 minutes and a molecular weight of 102.07 g/mol. The TLC separation of the acetone crude extract and the pure compound are shown in Figure 4. The FT-IR spectrum of different functional groups present in the pure compoundare shown in Figure 5. The FTIR peaks were identified as O-H (3396.64 cm^-1), C-H (2926.01 cm^-1), C=O (1689.64 cm^-1), C-H (1452.40 cm^-1) and C-O (1058.92 cm^-1). The C¹³ NMR spectrum of the pure compound is shown in Figure 6.From these data, the structure of the pure compound was elucidated and identified as 4-hydroxypentan-2-one.

 

 

Figure2: GC-MS spectrum of A)acetone crude extract  and B) the pure compound

 

 

Figure3: Mass spectrum of the pure compound

 

 

Figure 4: TLC separation of  A) the acetone crude extract and B)the pure compound

 

 

Figure5:FT-IR spectrum ofthepure compound

 

 

 

Figure6:¹³C NMR spectrum of the pure compound

 

Cytotoxic activity of the pure compound:

The cytotoxic activity of 4-hydroxypentan-2-one on HCT-15 cell line was assessed by MTT assay.  The pure compound 4-hydroxypentan-2-one displayed an IC₅₀ value of 100µg/mlonHCT-15 cells. The control cellsand the cells treated with 4-hydroxypentan-2-oneare shown in Figure 7. The cytotoxic activity of 4-hydroxypentan-2-one on HCT-15 cell line is shown in Figure 8.

 

 

Figure 7:Microscopic image ofHCT-15 cells  A) Control HCT-15 cells and B) 4-hydroxypentan-2-onetreated HCT-15 cells

 

Figure 8: Cytotoxic activity of 4-hydroxypentan-2-oneon HCT-15 cell line

 

In Silicodockingstudies:

The docking study was performed between 4-hydroxypentan-2-one and 20 proteins (drug targets) responsible for causing colorectal cancer to predict the mode of action of 4-hydroxypentan-2-one. The pure compound 4-hydroxypentan-2-one had a significant interaction with K-Ras protein with the least free binding energy ofbinding energy of -5.22 Kcal/moland inhibition constant of 148.89 µM and formed6 hydrogen bonds during the interaction. The interaction of 4-hydroxypentan-2-one with K-Ras protein is shown in Figure 9.The interaction of 4-hydroxypentan-2-one with the proteins responsible for causing colorectal cancer is given in Table2.

 

 

Figure 9: Interaction of 4-hydroxypentan-2-one with K-Ras protein

 

Table 2: Interaction of 4-hydroxypentan-2-onewith selected colorectal cancer drug targetproteins

Proteins	Inhibition constant	Binding energy (Kcal/mol)	No. of. H-Bond
Kras	148.89 µM	-5.22	6
AKT2	274.41 µM	-4.86	2
GSK3 Beta	672.91 µM	-4.33	3
Raf1	854.35 µM	-4.19	2
MEK2	928.1 µM	-4.14	4
ERK1	947.55 µM	-4.12	4
LRP6	1.01 mM	-4.09	5
Pi3k Gamma	1.04 mM	-4.07	3
mTOR	1.43 mM	-3.88	2
Pi3k Alpha	1.68 mM	-3.79	3
MEK1	1.79 mM	-3.75	3
IAP	2.05 mM	-3.67	2
ERK2	2.1 mM	-3.65	4
Beta catenin	2.25 mM	-3.61	3
Survivin	2.29 mM	-3.6	4
Bclxl	2.56 mM	-3.53	2
PDK1	3.08 mM	-3.43	3
BCl2	3.97 mM	-3.28	1
AKT1	4.64 mM	-3.18	4
MDM2	6.41 mM	-2.99	1

 

DISCUSSION:

Natural compounds obtained from plants are considered to be one of the novel sources for obtaining new drugs. There have been no reports on the cytotoxic potential of J.mimosifolia leaves and only very few reports are available on the cytotoxic potential of J.mimosifoliaand hence this plant waschosen for this study.Sidjui et al (2016), reported the cytotoxic potential of J.mimosifoliaroot and stem extracts onHEpG2 cell line. They reported that methylene chloride extract obtained from theroot and ethyl acetate extract obtained from stem displayed the highest cytotoxic potential with IC₅₀ values of 7.99µg/ml and 17.38 µg/ml respectively²⁶.In the present study, the acetone extract obtained from the leaves displayed the highest cytotoxic potential when compared to petroleum ether and chloroform extracts with an IC₅₀valueof600 µg/ml.There have been no reports on the isolation of 4-hydroxypentan-2-one from J.mimosifolia and therefore this is the first report on extraction and identification of  4-hydroxypentan-2-one isolation from J.mimosifolia.The pure compound 4-hydroxypentan-2-one was obtained from the acetone extract and displayed an IC₅₀value of100 µg/ml onHCT-15 cell lines.InSilico study suggest that4-hydroxypentan-2-one inhibit K-Ras protein which is part of the ERK/MAPK pathway. The results of the study suggest that 4-hydroxypentan-2-oneexerts its cytotoxicity by inhibiting K-Ras protein, however further studies are required to confirm the aforementioned hypothetical mode of action.

 

CONCLUSION:

Natural products based drug discovery have reaped numerous rewards in the past. The discovery of known compounds has been on the rise. Therefore it is of paramount importance to seek for phytochemicals from a medically underexploited plant and for this reason,Jacaranda mimosifolia was chosen. The pure compound 4-hydroxypentan-2-oneextracted from the leaves of Jacaranda mimosifoliashowed an IC₅₀ value of 100 µg/ml against the same cell line. In Silicodocking studiessuggested that the cytotoxic activity of 4-hydroxypentan-2-one is due to its inhibition ofK-Rasprotein.

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Accepted on 25.08.2017           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(6): 2251-2257.