Int J Curr Pharm Res, Vol 9, Issue 4, 42-46Original Article


COMPARATIVE STUDY OF IN SILICO AND IN VITRO ANTICANCER ACTIVITY OF TRADITIONAL INDIAN MEDICINAL PLANTS-A REVERSE PHARMACOLOGICAL APPROACH

RADHIKA RAMASWAMY1*, J. SRIKANTH2, C. UMAMAHESWARA REDDY2

1SRM College of Pharmacy, SRM University, Chennai, 2Faculty of Pharmacy, Sri Ramachandra University, Chennai
Email: radhikar1326@gmail.com

Received: 26 Jan 2017, Revised and Accepted: 20 Apr 2017

ABSTRACT

Objective: Cancer is one of the major deaths occurring worldwide and its prophylaxis demands the daily consumption of extracts or dietary supplements of traditional medicinal plants which possess anticancer activities. This study focuses on the evaluation of the chemo preventive and antiproliferative effects of the active constituents of Indian medicinal plants such as Withaniasomnifera, Phyllanthusemblica and Zingiberofficinale by in silico and in vitro studies.

Methods: In silico docking analysis is performed using Molegro Virtual Docker choosing the targets as p-glycoprotein and thymidylate synthase for the identified phytoconstituents. In vitro colorimetric cell metabolic activity assay is performed for the standardized extracts of these plants in various cell lines using the standards.

Results: The phytoconstituents in the plants, Withaniasomnifera and Phyllanthusemblica revealed good binding affinity towards thymidylate synthase and p-glycoprotein respectively as compared to that of the standards.

Conclusion: Phyllanthusemblica showed a maximal antiproliferative effect on breast cancer cell lines (MCF-7) when compared to the other plant extracts. Zingiber officinalis was found to inhibit HT-29 cell lines to a greater extent and Withaniasomniferum resulted in highest A549 cell death. A combination of these extracts in any dosage form could be used in the therapeutic efficacy in cancer.

Keywords: Molegro Virtual docker, MCF-7, HT-29, A549, MTT assay

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2017v9i4.20761

INTRODUCTION

India is rightly called as the “home” to all the medicinal plants. Colon, lung, breast, liver and stomach cancers are the most cause of deaths reported every year and the estimate is said to be around 7.9 million [1]. Use of tobacco and other drug abuses are the commonly occurring deaths worldwide [2]. Death due to cancer is projected to rise continually with an estimate of 13.1 million deaths in 2030 [3]. The consumption of these medicinal plants will promote the resistance to the host against any infection by conditioning the body tissues and by re-stabilizing body equilibrium [1]. Novel cancer drug discovery is mainly focussing on some of the better strategies for treatment of cancer by minimizing the side effects and without impairing the drug resistance [4].

This study aims at the evaluation of the cytotoxic and antiproliferative effects of the active constituents of medicinal plants such as Withaniasomnifera, Phyllanthusemblica and Zingiber-officinale by in silico and in vitro studies on the targets p-glycoprotein and thymidylate synthase [5-9].

MATERIALS AND METHODS

In silico docking analysis

The major active constituents were identified from the selected medicinal plants namely and are as follows:

These constituents were found to possess anti-cancer properties according to traditional claims. In silico docking, analysis is performed using Molegro Virtual Docker choosing the targets as p-glycoprotein and thymidylate synthase for the identified phytoconstituents in the plants and the results are compared against the standards raltitrexed, tamoxifen, vinblastine and fluorouracil [10-13]. Docking analysis is done by initially selecting the target for the disease and followed by obtaining the 3D structure of thymidylate synthase (1HVY) and p-glycoprotein (3G61) protein data bank in pdb format [14, 15]. Then the 3D structures of the various active constituents (ligands) are retrieved from PubChem chemical databases and saved in. mol format. The ligands and proteins are imported to the workspace and prepared for docking. The results thus obtained are compared against the standards [12, 13].

In vitro MTT assay

Cell lines were obtained from National Centre for Cell Sciences (NCCS), Pune. The cells were maintained in Minimal Essential Media supplemented with 10% Fetal Bovine Serum, penicillin (100U/ml) and streptomycin (100μg/ml) in a humidified atmosphere of 50μg/ml CO 2 at 37˚C [16, 17].

Reagents–MEM (Minimal Essential Media) was purchased from Hi-Media Laboratories, Fetal Bovine Serum (FBS) was purchased from Cistron laboratories, MTT (3-(4,5 Dimethyl thiazole-2-yl)-2,5 Diphenyltetrazolium bromide) reagent and DMSO (Dimethyl sulfoxide) were purchased from Sisco research laboratory chemicals, Mumbai. Other chemicals were obtained from Sigma-Aldrich, Mumbai.

In vitro MTT assay was performed for the plant’s standardized extracts in cell lines namely, A-549 (lung cancer cell line), HT-29 (colorectal cancer), MCF-7 (breast cancer cell line)using standards such as tamoxifen for MCF-7 and A-549 cell lines and 5-Florouracil for HT-29 cell line) [18].

The sensitivity of MCF-7, HT-29 and A549 cells to Withaniasomnifera, Phyllanthusemblica, Zingiberofficinale was determined individually by the MTT colorimetric assay. Cells were seeded in a flat bottomed 96-well plate and were incubated for 24 h at 37 °C and 50% CO 2. Both the cell lines were exposed to all three plant extracts mentioned above. The solvent (Dimethyl sulfoxide) treated cells served as control. The cells were then treated with MTT reagent (20μl/well) for 4 h at 37◦C and then DMSO (200μl) was added to each well to dissolve the formazan crystals. The optical density was recorded at 540 nm in a microplate reader. The percentage of cell inhibition was determined as [1-(OD of treated cells/OD of control cells]*100 [19-21].

RESULTS

In silico docking analysis

The ability of the phytoconstituents to bind with the targets is given in terms of Mol Dock Score. The Mol Dock Score is used as the parameter for analyzing the docking results. The phytoconstituents are ranked according to their Mol Dock Score. The ligand possessing the highest Mol Dock Score shows a strong affinity towards its target.

The top 5 ligands for the target p-glycoprotein are ellagic acid (-60.7406); gallic acid (-57.7957); Curcumene (-57.1762); Phyllemblin (-54.874); alpha-farnesene (-49.0781). The constituents of Phyllanthusemblica was found to have a moderate affinity to p-glycoprotein when compared to that of the standard, raltitrexed (-141.817) and tamoxifen (-115.666). Refer table 1.

The top 5 ligands which were found to have a greater affinity to thymidylate synthase were withaferin A (-140.681); curcumene (-140.656); withanolide A (-109.302); withanoloide E (-106.49); withanoloide B (-102.595). The constituents of

Withaniasomnifera were found to have a maximum affinity to thymidylate synthase when compared with standards, raltitrexed (-151.264) and tamoxifen (-129.451). Refer table 2.

Table 1: P-GLYCOPROTEIN-3G61-ranking based on mol dock score

Mol dock score Ligand Name
-141.817 raltitrexed [00] raltitrexed
-115.666 tamoxifen [00] tamoxifen
-101.516 vinblastine [00] vinblastine
-60.7406 ellagic acid [02] ellagic acid
-57.7957 gallic acid [02] gallic acid
-57.1762 cucurmene [00] curcurmene
-54.874 phyllemblin [02] phyllemblin
-54.6759 flourouracil [00] flourouracil
-49.0781 alpha farnesene [01] alpha farnesene
-44.6231 phyllantidine [01] phyllantidine
-44.5979 withaferinA [01] withaferinA
-37.4563 withanoloide A [01] withanoloide A
-33.4113 withanoloide E [01] withanoloide E
-32.6451 withanone [00] withanone
-21.8885 ascorbic acid [02] ascorbic acid
-15.5545 sesquiphellandrene [02] sesquiphellandrene
10.5933 withanoloideB [02] withanoloideB
54.6561 gingerol [00] gingerol
71.3498 alpha zingiberin [00] alpha zingiberin

In vitro MTT assay

Screening of extracts of Withaniasomnifera, Phyllanthusemblica, Zingiberofficinale resulted in impotent anticancer activities against A-549, MCF-7 and HT-29 cell lines. The inhibitory properties of these extracts are compared with the standards, tamoxifen for MCF-7 and A549 cell lines and 5-flourouracil for the HT-29 cell. The percentage cancer cell inhibition profiles were found to be concentration dependent. The maximum concentration (μg/ml) used in the study was 1000μg/ml.

The inhibitory properties of plant extracts are compared with standard 5-Fluorouracil for HT-29 cell line and tamoxifen for A-549 and MCF-7 cell lines and are represented in the tables 3, 4 and 5 respectively.

Fig. 1: 3D View of 3G61 docked with raltitrexed

HT-29 cancer cell line, when subjected to various concentrations of fluorouracil, resulted in 87.2% of cell death. A maximum cell inhibition of 76.1% was observed with Zingiberofficinale at a concentration of 1000 μg/ml. Withaniasomnifera and Phyllanthusemblica extracts resulted in 71.1% and 64.7% of HT-29 cell inhibition respectively. Refer fig. 5 and table 3.

Fig. 1 and 2 represent the 3d view of the protein 3G61 docked with raltitrexed and the ligand having the highest affinity respectively.

Fig. 2: 3D view of 3G61 docked with the ligand having the highest affinity, vinblastine



Table 2: Thymidylate synthase-1HVY- Ranking based on MolDock Score

MolDockScore Ligand Name
-151.264 ralitrexed [01]ralitrexed
-140.681 withaferin A [00]withaferin A
-140.656 curcumene [00]curcumene
-129.451 tamoxifen [00]tamoxifen
-109.302 withanoloide A [00]withanoloide A
-106.49 withanoloide E [01]withanoloide E
-102.595 withanoloide B [00]withanoloide B
-101.426 gingerol [00]gingerol
-95.4284 vinblastine [00]vinblastine
-95.0899 ellagic acid [00]ellagic acid
-88.4583 withanone [00]withanone
-88.0655 alpha farnasene [00]alpha farnasene
-86.0758 phyllemblin [00]phyllemblin
-85.5623 alpha zingiberene [00]alpha zingiberene
-84.6785 sesquiphellandrene [00]sesquiphellandrene
-77.1248 phyllantidine [00]phyllantidine
-75.5379 ascorbic acid [00]ascorbic acid
-70.5988 gallic acid [02]gallic acid
-59.0146 florouracil [00]florouracil

Fig. 3 and 4 shows the 3d view of the protein 1HVY docked with raltitrexed and the ligand having the highest affinity respectively

Fig. 3: 3D view of 1HVY docked with raltitrexed


Fig. 4-3D: View of 1HVY docked with the ligand having the highest affinity, withaferin a


Fig. 5:


Fig. 6:


Table 3: Percentage cell inhibition of plant extracts on HT-29 cell lines

Concentration (µg/ml) % cell inhibition
Withaniasomnifera
7.8 4
15.6 12.8
31.2 21.7
62.5 45.5
125 34.4
250 53.3
500 60.9
1000 71.1

A-549 cell lines, when subjected to different concentrations of Withaniasomnifera extract resulted in 87.3% inhibition at a concentration of 1000μg/ml. Similarly, Zingiberofficinale and Phyllanthusemblica resulted in 85.5% and 80% of A-549 cell death respectively. Comparison with tamoxifen showed 96.4% of cell inhibition at the maximum concentration. Refer fig. 6 and table 4.

Table 4: Percentage cell inhibition of plant extracts on A-549 cell lines

Concentration (µg/ml) % cell inhibition
Withaniasomnifera
7.8 38.7
15.6 48.8
31.2 54.3
62.5 59.8
125 60.9
250 71.7
500 80.9
1000 87.3

MCF-7 cell lines, when subjected to different concentrations of Phyllanthusemblica extract resulted in 87% inhibition at a concentration of 1000μg/ml. Similarly, Zingiberofficinale and Withaniasomnifera resulted in 83.9% and 82.4% of MCF-7 cell death respectively. On the other hand, comparison with tamoxifen showed that 95.6% MCF-7 cell line inhibition at the same tested dose (1000 μg/ml).

Refer fig. 7 and table 5.

Table 5: Percentage cell inhibition of plant extracts on MCF-7 cell lines

Concentration (µg/ml) % cell inhibition
Withania somnifera
7.8 14.7
15.6 22.4
31.2 27
62.5 36.2
125 57.7
250 67
500 77.7
1000 82.4

Fig. 7:

DISCUSSION

The present work aimed towards the evaluation of the cytotoxic and antiproliferative effects of the phytoconstituents in Withania-somnifera, Phyllanthusemblica, Zingiberofficinale by docking analysis and MTT assay.

The phytoconstituents in the plant, Withaniasomnifera and Phyllanthusemblica revealed good binding affinity towards thymidylate synthase and p-glycoprotein respectively as compared to that of the standards.

From the results of MTT analysis, it is concluded that Phyllanthusemblica showed a maximal antiproliferative effect on breast cancer cell lines (MCF-7) when compared to the other plant extracts. Zingiber officinalis was found to inhibit HT-29 cell lines to a greater extent and Withaniasomnifera resulted in highest A549 cell death. Almost all the extracts were found to produce an excellent anticancer activity. The activity can be attributed either to the expression of the molecular targets having a maximal affinity to the chemical constituents present in these plants or might also be due to the higher penetration power of the active principles which might have resulted in cell inhibitions. Overall, it can be stated that all the extracts contain potential compounds or active principles which might render the plants with anticancer proliferative activities against the various cell lines.

The results of the present study support the anticancer properties of medicinal plants used in the traditional Indian medicine system. It can also be recommended that daily consumption of some of the medicinal herbs in the form of extracts or dietary supplements are a promising therapy for the prophylaxis of cancer. The rate at which cancer is progressing seems to have an urgent and effective effort for improving the health of humans and animals as well.

ACKNOWLEDGEMENT

I would like to extend my heartfelt thanks to my guide, Mr. J. Srikanth for providing constant encouragement and timely help in the completion of this project. I would also like to thank my HOD, Dr. C. Uma Maheswara Reddy for his support. I am highly indebted to my institution, Sri Ramachandra University, Porur, Chennai. I am also grateful to the Tamil Nadu Pharmaceutical Sciences Welfare Trust for providing the financial aid and for the scholarship given for the successful completion of my project without any hindrance. I would like to thank the International Pharmaceutical Federation for accepting this research for the presentation during the PSWC, 2014 held in Melbourne. Lastly I would like to thank my parents and other staff in my department for supporting me.

CONFLICT OF INTERESTS

Declare none

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