IN-VITRO ANTICANCER ACTIVITY OF FLOWERS OF COUROUPITA GUIANENSIS AGAINST SKIN CANCER CELL LINE
HTML Full TextIN-VITRO ANTICANCER ACTIVITY OF FLOWERS OF COUROUPITA GUIANENSIS AGAINST SKIN CANCER CELL LINE
K. S. Nisana *, Alkka Anna Sabu, Ansa Elezabeth Mathew, Chinchu K. Moncy, B. Gopika, V. Jiju and Elessy Abraham
Nazareth College of Pharmacy, Othera, Thiruvalla, Kerala, India.
ABSTRACT: Objective: This study aim’s to deal with the in vitro anticancer activity of flowers of Couroupita guianensis (family: Lecythidaceae) against B16F10 (skin cancer cell line). Materials and Methods: MTT assay for flowers of Couroupita guianensis against B16F10 (Skin Cancer cell line) is conducted. Results: At the Concentration 44.44 µg/ml, the methanolic flower extract of Couroupita guianensis showed good percent inhibition of B16F10 (Skin Cancer cell line) as compared to the standard drug. Conclusion: The present attempt provides information that may generate researchers' interest in exploring such natural resources.
Keywords: Couroupita guianensis (CgF), Lecythidaceae, MTT Assay, Skin cancer cell line
INTRODUCTION: Skin cancer is one of the most common types. Every year, about a million cases of skin cancer are discovered. Squamous and basal cell carcinoma, which are various types of non-melanoma skin cancers, are not likely to spread and may need little more than minor surgery or topical care. Melanoma, which accounts for about 1 % of all skin cancers but is the main cause of most skin cancer deaths, may spread (metastasize) through the bloodstream to other body parts and the lymphatic system. Couroupita guianensis (CgF), also known as Cannonball tree (family: Lecythidaceae), Nagalinga pushpam. The native of Couroupita guianensis is in tropical forests of Central and South America, and it is cultivated in many other tropical areas throughout the world. Isatin (1H-indole-2,3-dione) is one of the active constituent in Couroupita guianensis. It is an endogenous compound that has cytotoxic activity against human cancer cell lines. Isatin has been isolated from the flower of Couroupita guianensis to study anticancer activity in various cell lines.
FIG. 1: FLOWERS OF COUROUPITA GUIANENSIS
MATERIALS AND METHODS
Plant Collection: The Flowers of Couroupita guianensis were collected from St. Thomas College, Kozhencherry, Kerala. The flowers were dried using shade drying method, powdered uniformly, and stored. 100g coarsely powdered flowers of Couroupita guianensis were subjected to maceration separately and successively with 70% methanol and distilled water. The flower extract was used to conduct MTT assay.
In-vitro Anticancer Activity: Cell proliferation and viability are analyzed by the most widely used MTT assay method. The cell's metabolic activity is measured by MTT assay, a colorimetric assay.
It is based on the capacity of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its purple color insoluble formazan. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol -2 -yl)-2, 5-diphenyltetrazolium bromide or MTT) by metabolically active cells to purple formazan crystals.
The viable cells contain NAD (P)H- dependent oxidoreductase enzymes which reduce the MTT to formazan. The insoluble formazan were dissolved by solubilizing solution and the resulting colored solution is quantified by measuring absorbance at 500-600 nanometers using a multi-well spectrophotometer. The darker the solution, the greater the number of viable or metabolically active cells.
In-vitro Anticancer Activity of Flowers of Couroupita guianensis:
Cell Line: B16F10 (Skin Cancer cell line).
Media: DMEM with high glucose (Cat No-11965-092), FBS (Gibco, Invitrogen) Cat No -10270106 Antibiotic – Antimycotic 100X solution (Thermo fisher Scientific)-Cat No-15240062
Experimental Procedure:
MTT Assay: The cells were incubated at a concentration of 1 × 104 cells/ml at 5% CO2 and 37°C for 24 hours. Cells were seeded in 100 µl culture medium at a concentration (70µl) 104 cells/well and 100µl sample of flowers of Couroupita guianensis (10,40,100µg/ml) into micro plates respectively (96 wells and tissue culture grade).
The Control wells were incubated with DMSO (0.2% in PBS) and cell line. All specimens were incubated in triplicate. Controls were maintained and it was used to determine the control cell survival and the percentage of live cells after culture. Then the cell cultures were incubated for 24 hours at 5% CO2 and 37°C in CO2 incubator (Thermo scientific BB150).
FIG. 2: CO2 INCUBATOR
FIG. 3: ELISA PLATE READER
After incubation, the medium was completely removed and added 20 µl of MTT reagent (5mg/min PBS), after the addition of MTT, cells are incubated for 4 hours at 37oC in CO2 incubator, then observed the wells for formazan crystal formation under a microscope. The yellowish MTT was decreased to dark-colored formazan by viable cells only. After removing the medium completely, add 200 µl of DMSO (kept for 10 min) and incubate at 37°C (wrapped with aluminium foil). Triplicate samples were analyzed by measuring the absorbance of each sample with a microplate reader (Benesphera E21) at a wavelength of 550 nm.
RESULTS: In-vitro Anticancer activity of flowers of Couroupita guianensis (CgF)
TABLE 1: EFFECTS OF COMPOUND AGAINST B16F10 CELL LINE (SKIN CANCER CELL LINE) BY MTT ASSAY
Sr. no. | Sample | Concentration (µg/ml) | OD | % inhibition | IC 50 (µg/ml)) |
1 | Control | 0.914 | 30.25 | ||
2 | Std. 5 FU | 10 | 0.551 | 39.71 | |
30 | 0.431 | 52.84 | |||
100 | 0.409 | 55.25 | |||
3 | CgF | 10 | 0.713 | 22.10 | 44.44 |
30 | 0.646 | 29.32 | |||
100 | 0.549 | 39.93 |
FIG. 4: IN-VITRO ANTICANCER ACTIVITY OF FLOWERS OF COUROUPITA GUIANENSIS
Result: The antiproliferation effect is the first indication to be assessed when investigating antitumor agents; thus, the cell growth inhibitory activity of CgF was assessed on B16F10 Cell line (Skin cancer cell line) at different concentrations. A dose-dependence decrease in cell viability was observed at an IC 50 value of 44.44µg/ml.
DISCUSSION: The anticancer activity of methanolic extract of flowers was studied against Skin cancer cell line (B16F10) by MTT assay and observed that at the concentration 44.44µg/ml, methanolic extract of Flowers of Couroupita guianensis showed good percent inhibition B16F10 cell line as compared to standard drug (5-FU).
Cytotoxicity against B16F10 (skin cancer cell line) was determined by (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay (MTT assay). The vital compound present in C. guianensis is isatin which shows solid inhibitory activity with IC50 value of 44.44µg/ml in a dose-dependent manner aganist standards 5 FU. The flower of Couroupita guianensis consists of an isatin compound that has cytotoxicity against human carcinoma cell lines.
It has the potential to be used as a chemotherapeutic agent against cancer. Isatin isolated from floral parts exhibited cytotoxicity against various cell line.
CONCLUSION: Cancer remains a leading cause of death in nowadays. A plan for the diagnosis and treatment of cancer is a key component of any overall cancer control plan. Anticancer activity of methanolic extract of flowers of Couroupita guianensis against cell line B16F10 (Skin cancer cell line) by MTT assay was found to have a good percent inhibition at the concentration of 44.44 µg/ml as compared to the standard. The present attempt provides information that may generate researchers' interest in exploring such natural resources. Outcomes of the study suggest that the flower extract of Couroupita guianensis is intoxicating chemopreventive and supplemental in-vivo animal studies are necessary to demonstrate extract as safe molecules for cancer management.
ACKNOWLEDGMENT: We are thankful to the management of Nazareth College of Pharmacy, Othera, Thiruvalla, Kerala, for the help and support and Dr. P. Selvam, Aravindh Herbal Labs Pvt. Ltd, Tamil Nadu.
CONFLICT OF INTEREST: Nil
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How to cite this article:
Nisana KS, Sabu AA, Mathew AE, Moncy CK, Gopika B, Jiju V and Abraham E: In-vitro anticancer activity of flowers of Couroupita guianensis against skin cancer cell line. Int J Pharmacognosy 2022; 9(12): 202-05. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.9(12).202-05.
This Journal licensed under a Creative Commons Attribution-Non-commercial-Share Alike 3.0 Unported License.
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IJP
K. S. Nisana *, Alkka Anna Sabu, Ansa Elezabeth Mathew, Chinchu K. Moncy, B. Gopika, V. Jiju and Elessy Abraham
Nazareth College of Pharmacy, Othera, Thiruvalla, Kerala, India.
nisanaks7790@gmail.com
01 August 2022
10 December 2022
14 December 2022
10.13040/IJPSR.0975-8232.IJP.9(12).202-05
31 December 2022