INTRODUCTION:

Cancer is one of the major causes of morbidity and mortality globally. It is trigeered by a complex, poorly understood interplay of genetic and environmental factors. Unrelieved pain affects the overall life of the cancer patient. The pervasiveness of cancer pain is estimated as 25% of newly diagnosed, 33% for those undergoing active treatment and greater than 75% for those with advanced stages¹.Various therapies towards the treatment of cancer fail due to less target specificity, side effects, adverse drug reactions and its resistance^2,3. There is an rising interest in developing drugs that are target specific, lesser side effects and more effectiveness by using natural compounds. They act on multiple targets with reduced side effects and provide holistic treatment approach, which improves the quality of life of cancer patient.

Natural compounds offer a great opportunity and single most successful strategy for discovery of novel therapeutic candidates for the treatment of cancer⁴.Most (>60%) anticancer drugs that are in clinical use and have demonstrated significant efficacy for combatting cancer originate from natural products derived from plants, marine organisms, and microorganisms⁵. The research proposal is established to evaluate the antiproliferative effect of the threatened aromatic medicinal plant C.aromatica.

Curcuma aromatica Salisb. of Zingiberaceae family is commonly known as wild turmeric, vanaharidra or Kasturi manjal. And is the second most commonly used turmeric after C.longa. Kasturi arisina is a promising drug with high therapeutic potential due to its Antibacterial, Antifungal, Anti-Inflammatory, Anti-Carcinogenic, Anti-Oxidant, Anti-Ageing, Anti-Platelet, Anti-Nephrotoxic, Inhibitors of HIV-1, Wound Healing and Immunomodulatory Properties. Also, promotes blood circulation, removes blood stasis and in treating Alzheimer's disease⁶. In the present research study, the whole extracts and the essential oils (from rhizome and leaf) of Curcuma aromatica Salisb. was screenedfor its antiproliferative potential through the invitro cell viability assay method called MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on MDA-MB-231 cell lines.

MATERIALS AND METHODS:

1. Cell lines:The cell line under study was MDA-MB-231 (human breast carcinoma, ER-, tumorigenic) and Vero (ATCC- CCL-81 normal kidney epithelial cell line) were obtained from the National Centre for Cell Science (NCCS), Pune, India.

2. Viability study by the Trypan Blue Size Exclusion Assay:

The viability of the cell lines under study was evaluated by The Trypan Blue Size Exclusion Assay. To perform the assay, a cell suspension is mixed with an equal volume of dye (trypan blue solution) and incubated for a short period of time, typically 3-5 minutes. During this time, the trypan blue enters the cells that have lost membrane integrity, such as dead or dying cells, and stains them blue. Intact and viable cells, on the other hand, exclude the dye and remain transparent. The stained and unstained cells are then counted using a hemocytometer. The number of viable cells is determined by subtracting the number of stained (dead) cells from the total number of cells counted^7.

3. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay:

MTT assay is a commonly used colorimetric assay to measure cell viability and proliferation. The principle is that for most viable cells, mitochondrial activity is constant and thereby an increase or decrease in the number of viable cells is linearly related to mitochondrial activity. The assay works by measuring the ability of viable cells to convert MTT, a yellow tetrazolium salt, into a blue-colored formazan product, which can be quantified using a spectrophotometer.

To perform the MTT assay, cells are first seeded in a 96-well plate and allowed to attach and grow for 24 hours. The growth medium is replaced with the test samples, Viz., Curcuma aromatica Extract, Rhizome oil, leaf oil and incubate for the 24 hours. The drug concentration isadded to the wells from the lowest concentration to the highest concentration. A range of concentrations of formulation in the range of 3.906ug/ml, 7.8125ug/ml, 15.625ug/ml, 31.25ug/ml, 62.5ug/ml, 125ug/ml, 250ug/ml in methanol. The plate are then placed in the CO2 incubator and the data is collected for 24, 48 and 72hours time points.Then, MTT solution is added and incubated with the cells for a certain period of time, usually 2-4hours. During this time, the MTT is reduced by the enzyme succinate dehydrogenase (Mitochondrial enzyme) in viable cells to form formazan. After the incubation period, the formazan is solubilized using a DMSO (5mg/ml) solution. The absorbance is recorded at 535nm in a ELISA microtiter plate reader. The amount of formazan produced is directly proportional to the viable cells. Plot absorbance against number of cells/mL⁸.

RESULTS AND DISCUSSION:

Chemoprevention through phytopharmaceuticals form’s a vital role in reducing the occurrence of cancer and the same has been tested and proved in various invitro and invivo studies⁹. The present research study was taken up to study the effect of cell viability of the C.aromatica rhizome extract, rhizome oil and leaf oil respectively on the Breast cancer cell lines, MDA-MB-431 by MTT assay.

The results suggested the ethanolic extract, rhizome oil and leaf oil exhibited the antiproliferative activity against the MDA-MB-431cell line with the IC50 value of 33.38, 34.28 and 34.15ug/ml respectively (Table 1, 2, 3; Figures 1,2,3). The viability of the MDA-MB-431cell line, decreased linearly with increase in the concentration of the samples under study. The potentiality of the extracts and essential oils in decreasing the cell viability in a dose dependent manner and exhibiting the antiproliferative activity are attributed to Curcuminoids and Terpenoids of Curcuma aromatica.

The results are in accordance with the previous studies that reported curcuminoids (and terpenoids (monoterpenoids and sesquiterpenoids) exhibit the cytotoxic activity against various cancer cell lines^8,9,10. Curcuminoids are known to induce apoptosis, or decrease the cell viability by blocking the entry to cell cycle from Growth phase G2 to M phase. They are also known to trigger the expression of cd2/cyclin B, caspase-3 mediated cell death, elevates the expression of Bax, p53 and procaspases-3, 8 and 9 and decreases the expression of antiapoptotic genes (Bcl-XL)^9.

Terpenoids on the other hand, causes inhibition of cell proliferation and tumour growth based on various in vitro and in vivo studies in a variety of human cancers¹⁰. The mode of action involves, activation of apoptosis factors, inhibition of the ubiquitin-proteasome and NF-κB pathways, which is linked with up-regulation of proapoptotic protein Bax and down-regulation of anti-apoptotic protein Bcl-2, resulting in cytochrome c release, caspase activation and apoptotic cell death^11-15,Germacrone has demonstrated that it also possesses significant protective effects including anti-bacterial, antifungal, antifeedant, depressant, choleretic, antitussive and vasodilator activities. Germacrone has been reported to induce G0/G1 or G2/M phase cell cycle arrest in various cancer cell lines ^16. The volatile bioactives such as 1,8-Cineole (eucalyptol), α-lemenone, α-phellandrene, 2-carene and α-curcumene are also reported to act as chain breaking antioxidants and thus the leaf essential oils from different Curcuma spp. may also have significant health promoting effects. As per the report published by^17-22,the Curcuma rhizome volatile oil induces programmed cell death in liver and lung cancer cells and the cytotoxicity can be attributed to bioactive components such as α-phellandrene, camphor, curdione, eucalyptol, terpinolene, and α-pinene, which are known cause signaling interruption in cancer cell.

CONCLUSION:

C.aromaticarhizome extract and volatile bioactives of rhizome and Leaf essential oil clearly demonstrates the dose dependent decrease of Breast cancer cell lines, MDA-MD 231. The composition comprises of curcuminoid mixture consisting of curcumin, demethoxycurcumin and bisdemethoxycurcumin and essential oil with the major unique odour defining components such as cedrene, Xanthorrhizol, camphor, and germacrone isofuranogermacrone and 1,8-cineole. The results rationalize that the traditional ayurvedic medicine in optimum dosage can be considered as a effective antiproliferative agent, which gives maximum benefits with minimum or no side effects.

Table 1: MTT assay of C.aromatica Extract- % DEATH

Conc (ug/ml)	Set1	Set2	Set3	% Death	SD
3.906	1.8574	0.674821	2.620295	1.717505	0.980253
7.8125	5.392451	1.096584	7.003335	4.497456	3.05339
15.625	13.72079	12.52636	15.53121	13.926120	1.512909
31.25	44.997	40.95318	44.30681	43.419000	2.163164
62.5	97.60336	98.73471	97.47499	97.937680	0.693223
125	95.32654	96.28849	97.47499	96.363340	1.076177
250	98.5021	97.46942	97.04621	97.672580	0.748902
IC50	33.01	33.64	33.5	33.383730	0.327221

Figure 1:MTT assay of C.aromatica Extract

Table 2: MTT assay of C.aromatica Rhizome oil- % DEATH

Conc (ug/ml)	Set1	Set2	Set3	% Death	SD
3.906ug	3.880219	3.959525	2.654867	3.498204	0.731427
7.8125ug	5.6938	5.059393	5.718176	5.490456	0.373511
15.625ug	10.6706	10.8667	9.87066	10.46932	0.527644
31.25ug	39.81442	42.36692	39.55071	40.57735	1.555407
62.5ug	94.47491	94.54465	88.63172	92.55043	3.393877
125ug	95.57149	95.11659	93.66916	94.78575	0.993379
250ug	95.57149	97.00836	96.86862	96.48282	0.792324
IC50	34.1361	33.6804	35.0476	34.28803	0.696148

Figure 2: MTT assay of C.aromatica Rhizome oil

Table 3: MTT assay of C.aromatica Leaf oil- % DEATH

Conc (ug/ml)	Set1	Set2	Set3	% Death	SD
3.906ug	5.880077	3.471749	5.983282	5.111703	1.421178
7.8125ug	11.56673	12.8659	10.42675	11.61979	1.220439
15.625ug	15.47389	15.58884	11.83458	14.2991	2.135112
31.25ug	41.58607	42.75017	41.707	42.01441	0.640047
62.5ug	91.45068	89.4486	80.73031	87.20986	5.700046
125ug	93.9265	92.85228	85.17378	90.65085	4.773598
250ug	94.11992	94.69027	89.26529	92.69183	2.981139
IC50	34.6508	34.4075	33.9033	34.1554	0.356523

Figure3: MTT assay of C.aromaticaLeaf oil

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Received on 31.05.2023 Modified on 15.11.2023

Research J. Pharm. and Tech. 2024; 17(3):1352-1355.

DOI: 10.52711/0974-360X.2024.00213

Antiproliferative effects of C.aromatica Extracts and Essential oils in

MDA-MB231 as a Breast Cancer Cell Line