INTRODUCTION:

Cancer by definition is the uncontrolled cell growth^1,2. Colon cancer is a cancer of the gastro-intestinal system, particularly of the large intestine. According to cancer statistics, it is the second largest cause of fatalities and is also the third most common cancer among all^3,4. There is a need to screen for better drugs which can be used as a treatment modality⁵.

Plants possessing medicinal properties are in use in the traditional system of medicine and also the Ayurvedic medicine since a long time^6,7,8. Plants have biologically active substances in high concentrations⁹. Curcuma amada is such a medicinal plant with wide variety of active phytochemicals reported up to date¹⁰. The plant possess antioxidant, antimicrobial, cytotoxic and platelet aggregation inhibition properties¹¹.Use of this plant rhizome extracts in determining its cytotoxicity against various cancer cells has been under constant research and has yielded promising results. A novel compound isolated from this plant and named as amadaldehyde has shown potent cytotoxicity against A-549, which is the human small cell lung carcinoma cell line¹¹. Extracts have also shown cytotoxicity against BHK-21 cells, human cervical cancer cell line as well as Dalton lymphoma ascites cell line^12,13. Methanolic extracts of the rhizomes have shown cytotoxicity against breast cancer cell lines¹⁴. Therapeutic effects of supercritical CO₂ extracts have shown a significant cytotoxicity against human alveolar (SJRH30) and embryonal (RD) rhabdomyosarcoma cell lines¹⁵. Curcumin (diferuloylmethane), a yellow colored pigment found in some curcuma species has been screened for anti-colorectal cancer effects ^16,17,18. The current scope of this study was to evaluate and establish cytotoxic activity of chloroform-methanol, protein as well as ethanolic extracts and fractions of ethanolic extracts of Curcuma amada rhizome against colon cancer cell line which is a less approached area of study.

MATERIALS AND METHODS:

Plant Authentication:

C. amada rhizomes were purchased from Sangli, Maharashtra, India and were authenticated at Department of Botany, Mahatma Gandhi Memorial College Udupi, Karnataka by Professor Mrs. Usharani. S Suvarna, Associate Professor and Head of the Botany department.

Preparation of plant rhizome extract:

C. amada rhizomes were washed thoroughly and sun dried for 30 days to dry the rhizome completely and get rid of all the possible moisture. The dried rhizomes were cut into smaller parts and powdered finely into a grinder. 100g of finely powdered rhizome was added to 500ml of 90% ethanol and was kept in an amber bottle with sealed lid for cold extraction. The solution was shaken well and thoroughly mixed after every 2 hours for 7 days. After 7 days, the upper liquid layer was carefully separated from the settled powder of the rhizome to obtain a clear ethanolic extract. This extract was concentrated in a rotary evaporator (BUCHI) at 40°c and pressure to separate ethanol from the extract. The concentrated extract was kept in water bath (ThermoFisher) at 37°c for 4 hours to evaporate traces of ethanol. Similar procedure was carried out to obtain a chloroform-methanol (1:6 v/v) extract.

Fractionation of the ethanolic extract by different solvents:

The ethanolic extract (15g) was poured into a separating funnel. To this 50ml of petroleum ether was added and the solutions were mixed vigorously. The solution was allowed to stand still for 20 minutes. Two distinct layers were obtained of which the upper layer was of petroleum ether which was separated in a china dish and concentrated in water bath at 37°c for 4 hours. This yielded petroleum ether extract. The remaining components of the extract after petroleum ether extraction were added with 50ml of ethyl acetate and similar procedure was used to obtain ethyl acetate extract. The remainder components after ethyl acetate extraction were subjected to n-butanol extraction by similar procedure. This gave 3 different fractions of ethanolic extract.

Protein extraction:

After the solvent extraction, the remaining residue of the plant powder was dried overnight. It was then added with 1:6 ratio (wt./vol) of chilled acetone and was stirred and kept in the fridge at 4°C for 4 hours. The acetone was then completely removed, and the residue was completely dried. The dried residue was suspended in 1% solution of polyvinylpyrrolidone overnight for protein extraction. The next day supernatant was taken and centrifuged at 10000 rpm for 10 minutes to obtain crude protein extracts.

Total Protein estimation:

Total protein estimation was done by Lowry’s method¹⁹. 0.05ml of crude protein extract was made up to 1ml with distilled water. To this 4.5ml of Lowry reagent 1 (Prepared as per protocol) was added. It was incubated for 10 minutes at room temperature. After incubation, 0.5 ml Folin phenol reagent was added. It was incubated for 30 minutes at room temperature. Optical density of the final solution was measured at 660 nm in a spectrophotometer. A standard of BSA (0.25mg/ml) (bovine serum albumin) was run alongside the test sample and a standard curve of BSA was prepared. The amount of protein in the test sample was calculated from the standard curve of BSA.

Sulphorhodamine B assay for cytotoxicity determination of various extracts:

HCT 116 Cells were seeded in a 96 well plate at a density of 5000 cells per well. The plate was incubated for 24 hours. Concentrations of the test compounds were made in different concentrations from 500µg/ml to 7.5µg/ml and added to the wells in triplicates. The plate was incubated for 48 hours. After the incubation, the plate was removed from the incubator, 100 µL of 10% TCA was added to each well and incubated for an hour at 4°C. After 1 hour, TCA was removed, and the plate was washed thrice under running water. 100 µL of SRB reagent was added to each well and incubated for 30 minutes in dark at room temperature. The wells were then washed thrice with 1% acetic acid and 200 µL of Tris base was added to the wells and placed on a shaker for 10 minutes. The plate was then read at 540 nm using ELISA plate reader. The IC50 values were calculated using statistical analysis software.

RESULT:

Sulphorhodamine B Assay:

Table 1. SRB assay of Ethanolic extract:

Treatment	Concentration of Test Extract (µg/mL)	% Cell death	IC50 Value
Ethanolic Extract	500	97.68	14.46µg
	250	97.63
	125	97.61
	62.5	97.58
	31.25	87.68
	15.63	54.08
	7.81	18.56

Table 2. SRB assay of Ethyl acetate extract

Treatment	Concentrations of Test Extract (µg/mL)	% Cell death	IC50 Value
Ethyl Acetate Extract	500	9.17	1012 µg
	250	6.14
	125	-19.48
	62.5	-15.50
	31.25	-11.64
	15.63	-2.79
	7.81	15.57

Table 3. SRB assay of n-butanol extract.

Treatment	Concentrations of Test Extract (µg/mL)	% Cell death	IC50 Value
n-butanol Extract	500	86.37	43.19 µg
	250	83.44
	125	81.83
	62.5	72.35
	31.25	13.51
	15.63	20.19
	7.81	19.41

Table 4. SRB assay of Chloroform-methanol extract.

Treatment	Concentrations of Test Extract (µg/mL)	% Cell death	IC50 Value
Chloroform-methanol Extract	500	97.27	18.31µg
	250	97.37
	125	97.30
	62.5	97.01
	31.25	83.11
	15.63	36.83
	7.81	12.60

Table 5: SRB assay of Protein extract

Treatment	Concentrations of Test Extract (µg/mL)	% Cell death	IC50 Value
Protein Extract	500	97.20	43.1 µg
	250	97.34
	125	97.37
	62.5	90.21
	31.25	13.08
	15.63	-21.52
	7.81	-30.35

DISCUSSION:

100g of rhizome powder of Curcuma amada after ethanolic extraction gave 15g of extract. Similarly, 100g of rhizome powder of Curcuma amada after chloroform-methanol extraction gave 14g of extract. Petroleum ether yielded 4g, ethyl acetate 8.5g and n-butanol fraction yielded about 2.6g of extract fractions from the ethanolic extract. All solvent extracts had greasy, semi solid consistency except for ethyl acetate fraction which completely dried out with stretchy, rubbery consistency. The ethanolic whole extract showed a good cytotoxic activity with IC50 of 14.46µg. The ethyl acetate fraction showed very low cytotoxicity with an IC50 of 1040µg. The n-butanol fraction gave a good cytotoxicity with an IC50 of 43.19µg. The chloroform-methanol extract also showed potent cytotoxicity with an IC50 of 18.31µg. The crude protein extract of the rhizome of mangoginger also showed cytotoxicity with an IC50 value of 43.1µg. 4 out of 6 extracts showed promising cytotoxicity against colon cancer which reveals a great anticancer potential of this plant against colon cancer. The whole extracts of ethanol and chloroform-methanol showed better cytotoxicity than the fractionated once which indicates that more than one compound of the extract in combination with one another is responsible for the activity. This could be further proved by identifying all the compounds of the extract and testing them for cytotoxicity alone and in combination with one another. Total protein estimation of the crude protein extracts gave a concentration of 4.095mg/ml of extract. Protein extracts exhibited cytotoxicity which is a completely new area and dimension of study.

CONCLUSION:

Plant proteins have been used for various purposes and many of them have found medicinal importance too. Hence the proteins of mangoginger rhizomes exhibiting cytotoxic effect is an area which needs to be explored.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCES:

1. Saranya M, Punnagai K, Darling Chellathai David, Anusha D. Evaluation of Anticancer Effects of Vasopressin Receptor blocker in Colon Cancer – An In vitro Study. Research J. Pharm. and Tech. 2020; 13(1):77-80. doi: 10.5958/0974-360X.2020.00014.1

2. Burali Sanganna, A.R. Kulkarni. Antioxidant and Anti-colon cancer activity of fruit peel of Citrus reticulate essential oil on HT-29 cell line. Research J. Pharm. and Tech. 6(2): Feb. 2013; Page 216-219.

3. Siegel, R. L., Miller, K. D., Fedewa, S. A., Ahnen, D. J., Meester, R. G. S., Barzi, A., & Jemal, A.. Colorectal cancer statistics, 2014. CA: A Cancer Journal for Clinicians, 2014; 67(3), 177–193. https://doi.org/10.3322/caac.21395

4. Maheshwari Rajalekshmi, Chandrashekhara Shastri Shreedhara, Richard Lobo, Polu Picheshwara Rao. The Review on Genetics, Epigenetics, Risk Factors and Diagnosis of Colon Cancer. Research J. Pharm. and Tech 2018; 11(11): 5147-5151. doi: 10.5958/0974-360X.2018.00940.X

5. Keerthi Priya, M Manjunath Setty, K Sreedhara Ranganath Pai. In vitro and In vivo Evaluation of Anticancer Properties of Clerodendrum indicum (L.) Kuntze in Colon Cancer. Research J. Pharm. and Tech 2020; 13(5): 2321-2328. doi: 10.5958/0974-360X.2020.00418.7

6. Apeksha Saraf , Nidhi Dubey, Nitin Dubey, Mayank Sharma. Box Behnken Design Based Development of Curcumin Loaded Eudragit S100 Nanoparticles for Site-Specific Delivery in Colon Cancer. Research J. Pharm. and Tech 2019; 12(8):3672-3678. doi: 10.5958/0974-360X.2019.00627.9

7. Mrs Neeli Rose Beck, Pradeep Samal. Traditional Medicinal Plants used by the Tribes and Rural People of Bilaspur District, Chhattisgarh (India). Research J. Pharm. and Tech. 5(10): October 2012; Page 1281-1282.

8. Shrivastava Alankar, Jain R., Agrawal R.K., Ahirwar D.. Clinical Trial of Herbal Drugs and Products in India: Past and Current Status and Critical Issues. Research J. Pharm. and Tech. 1(2) April-June. 2008; Page 69-74.

9. Mohammad Abdul Motalib Momin, Mamunur Rashid, Kaniz Fatima Urmi, Sohel Rana. Phytochemical Screening and Investigation of Antioxidant and Cytotoxicity Potential of different extracts of selected Medicinal Plants of Bangladesh. Research J. Pharm. and Tech. 6(9): September 2013; Page 1042-1050

10. Samant, L. R. Curcuma amada Roxb.: A Phytopharmacological Review. J. Pharm. Res., 2012; 5(4), 1992–1993.

11. Policegoudra, R. S., Rehna, K., Rao, L. J., & Aradhya, S. M. Antimicrobial, antioxidant, cytotoxicity and platelet aggregation inhibitory activity of a novel molecule isolated and characterized from mango ginger (Curcuma amada Roxb.) rhizome. Journal of Biosciences, 2010; 35(2), 231–240. https://doi.org/10.1007/s12038-010-0027-1

12. Joshi, A., & Chauhan, R. S. Phytochemical Analysis and Cytotoxicity Studies of Curcuma amada rhizomes in BHK-21 Cells. International Journal of Scientific Research in Environmental Sciences; 2013;1(12), 365–371. https://doi.org/10.12983/ijsres-2013-p365-371

13. Prema, D., Kamaraj, M., Achiraman, S., & Udayakumar, R.In vitro antioxidant and cytotoxicity studies of Curcuma amada Roxb (Mango ginger). International Journal of Scientific and Research Publications, 2014; 4(4), 1–6.

14. Jambunathan, S., Bangarusamy, D., Padma, P. R., & Sundaravadivelu, S. Cytotoxic activity of the methanolic extract of leaves and rhizomes of Curcuma amada Roxb against breast cancer cell lines. Asian Pacific Journal of Tropical Medicine, 2014;7 (S1), S405–S409. https://doi.org/10.1016/S1995-7645(14)60266-2

15. Ramachandran, C., Quirin, K. W., Escalon, E. A., Lollett, I. V., & Melnick, S. J. Therapeutic Effect of Supercritical CO2 Extracts of Curcuma Species with Cancer Drugs in Rhabdomyosarcoma Cell Lines. Phytotherapy Research, 2015; 29(8), 1152–1160. https://doi.org/10.1002/ptr.5360

16. Chauhan, D. Chemotherapeutic Potential of Curcumin for Colorectal Cancer. Current Pharmaceutical Design, 2005;8 (19), 1695–1706. https://doi.org/10.2174/1381612023394016

17. Sri Vasavi Reddy A, J. Suresh, Hemant K.S. Yadav, Apurva Singh. A Review on Curcuma longa. Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 158-165.

18. Hemant Tomar, V. K. Shukla, Kunal Arora, Nitesh Tomar, Sumit Roy, Vikash kumar. Curcuma longa: Review of Advances in Pharmacology. Research J. Pharm. and Tech. 5(9): September 2012; Page 1141-1144.

19. Oliver H. Lowery, Nira J. Rosebrough, A. Lewis Farr, R. J. R. 195. Protein measurement with the Folin phenol reagent. http://www.jbc.org/.

Received on 05.07.2021 Modified on 15.12.2021

Research J. Pharm. and Tech 2022; 15(11):4955-4958.

DOI: 10.52711/0974-360X.2022.00833