INTRODUCTION:

The family Zingiberaceae has been always used as spice ingredient, medicinal having the properties of antimicrobial, anti-inflammatory, antioxidant and anticancer effects^1,2. There are earlier reports on Asian countries medicinal plants to be used in cancer treatment³. Aromatic medicinal plants and its large number of secondary metabolites are used all over the world to be effective in tumour treatment. The gingers and its essential oils are valuable sources to be used commonly by local people in treatment of stomach problems, sore throat, cough, liver complaints, rheumatism, fever, swelling, muscular pains and other various disorders. There are many natural compounds having medicinal effects but their biological properties are yet to be characterized. Cancer cells have uncontrolled growth characteristics for which effective inhibition of cancer proliferation is a method for cancer therapy⁴.

Curcuma longa or ‘turmeric’ is known worldwide for its medicinal use, in cosmetics, as food flavouring and in textile industries. It has various pharmacological activities as anti-inflammation, hepato-protection, antimicrobial, healing of wounds and anticancer properties due to the presence of curcumin in its rhizome⁵. Curcuma aromatica or ‘wild turmeric’ is another medicinal plant whose rhizomes are used for flavouring and colouring of foods all over the world. The rhizome essential oil is antimicrobial, anticarcinogenic due to the presence of curcumin and has been reported to be used in early stages of cervical cancertreatment^6,7. Kaempferia galanga or ‘sand ginger’ are used in ayurvedic drugs preparation, as spice ingredient, in perfumeries and cosmetic industries. It possesses antioxidant, antinociceptive and anti-inflammatory activities helpful in treatment of mouth ulcers, migranes, rheumatism and eye infections^8,9. Hedychium coronarium or ‘white ginger lily’ is another important plant due to presence of coronarin D in its rhizome possesses cytotoxic, inflammatory and antimalarial activities^10,11. The cost of essential oils varies in international market due to exploitation by local people and being used as raw material by pharmaceutical industries. The present study has been carried out to find out the cytotoxicity effect of rhizome essential oil from micropropagated C. longa, C. aromatica, K. galanga and H. coronarium (CL, CA, KG and HC) plantlets against Human cervical cancer (HeLa) cell line. There are reports on rhizome oil analysis of these medicinal species but yet no report on its in vitro rhizome oil cytotoxicity studies of these important medicinal species¹². MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity, based on reduction of the yellow colored water soluble tetrazolium dye MTT to formazan crystals^13,14. Thus there is a need to study the activity of essential oils to improve human health conditions with fewer side effects.

MATERIAL AND METHODS:

Plant material collection and GC-MS analysis:

The plants were collected from different areas of Eastern Ghats and maintained in medicinal plant garden of Centre for Biotechnology, Siksha ‘O’ Anusandhan University, Bhubaneswar. The steririlized explants were used for culture establishment of all four plant samples on basal Murashige and Skoog (MS) medium supplemented with various combination of growth regulators like kinetin (Kin), benzyl adenine (BA), indole- 3-acetic acid (IAA), naphthalene acetic acid (NAA) and adenine sulphate (Ads) with 30 gm/l of sucrose and 0.8 % of agar following our earlier reports^15,16,17. In vitro regenerated plants with well developed shoots and roots were studied for rhizome oil extraction. For this a round bottom flask containing 100gm of sliced rhizomes in 500ml of distilled water was heated for 6 hours and the condensed vapour separated in oil-water surface was collected using a Clevenger’s apparatus by hydro-distillation process using our earlier published reports^{15,16,18,19,20}. The component identification was achieved by the GC–MS analysis using HP 6890 series GC (Hewlett-Packard, USA) coupled with a mass selective detector (MSD), HP 5973 series (Hewlett-Packard). Helium was used as a carrier gas and the sample was injected in split less mode in a column HP5 phenyl methyl siloxane [25μl film thickness × 320μm internal diameter × 30 m length of the column]. Mass spectra were acquired over a 40–400 atomic mass unit range. Compounds were identified by comparing the mass spectral data with those in the NIST library provided with software and with commercially available data. Temperature programming was: initial temperature 60⁰C, ramping rate 3⁰C, final temperature 243⁰C, run time 61 min.

Culturing of cell lines, treatment groups and MTT cell viability assays:

Human cervical cancer (HeLa) cell line was procured from National Centre for Cell Science, Pune, India. The cells were subcultured in dulbecco modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine in tissue culture flasks and incubated in a CO₂ incubator in a 5% CO and 95% humidity atmosphere. After trypsinization the cell count was done and the cell viability was tested by trypan blue dye exclusion method using haemocytometer. 200μl cell suspension was seeded into 96-well plate at required cell density without the test agent and the cells were allowed to grow for about 24 hours. Then appropriate 5 concentrations of the test agent were added i.e. 6.25, 12.5, 25, 50 and 100uL/mL and plates were incubated for 24 hours at 37°C in a 5% CO2 atmosphere. After the incubation period the plates were taken out from the incubator, spent media were removed and MTT reagent was added to a final concentration of 0.5mg/mL of total volume. Then the plates were wrapped with aluminium foil to avoid exposure to light and incubated for 3 hours. After incubation the MTT reagent were removed and 100 μl of solubilisation solution (DMSO) was added. Finally the absorbance of the 4 test samples as well as the control was taken on an ELISA reader at 630nm used as reference wavelength. The IC50 value was calculated by using linear regression equation i.e. Y =Mx+C where Y = 50, M and C values were derived from the viability graph^21,22.

RESULTS AND DISCUSSIONS:

The rhizome oil yield in all four samples i.e. CL, CA, KG and HC were 0.4%, 0.5%, 0.7% and 0.4% respectively. The compound ar-tumerone was the major compound in C. longa, ar-tumerone in C. aromatica, ethyl p-methoxycinnamate in K. galanga and eucalyptol in H. coronarium respectively. The percentage inhibition at different drug concentrations (6, 12.5, 25, 50 and 100 mg/mL) of all samples against cancer cell lines along with the percentage inhibition of positive control (camphothecin) at 10 mM concentration as shown (Fig. 1). The MTT assay expressed the effective concentration required for 50% of HeLa reduction against different concentrations of rhizome oil. The study shows the best activity in HC followed by CA, KG and CL respectively (Table 1, Fig. 2). There are reports on essential oils of Curcuma longa and other Curcuma species that screened cytotoxic activity against the HeLa cell line²³. Earlier scientists have reported on the traditional use of C. longa having ar-tumerone as the major compound beneficial in gastrointestinal disorders, inflammatory conditions and cancer prevention tried both in in vitro and in vivo cancer models^{24, 25, 26, 27}. But to the best of our knowledge there is no report on micropropagated Curcuma species rhizome oil cytotoxicity activity. Other studies on C. aromatica rhizome oil to be effective against against melanoma cells (B16) and prostate cancer cells (LNCaP)²⁸. Very few reports on GC-MS analysis of Kaempferia species and Hedychium species are available where we have reported earlier on GC-MS of micropropagated C. aromatica, K. galanga and H. coronarium rhizome oil analysis^15,16,19. Similar active cytotoxic effects of K. galanga extracts and essential oil on cervical cancer cell line has been done thus could be used as an anticancer agent²⁹^. There are other reports on Zingiber species essential oil composition alongwith its bioactivity having wide pharmacological effects. They revealed that chemical composition of the oil is influenced by their geographical region, extraction methods, freshness and dryness of rhizomes³⁰. Few study aims on Zingiber species to be used as a folk medicine to treat various diseases, including cancer like HeLa, SiHa, MCF-7 and HL-60 cells. Their results suggest the anti-proliferative effect of α-zingiberene present in gingers to be developed as cancer chemotherapeutics³¹. Other report from our lab has been done on H. coronarium extract arrests cell cycle progression in HeLa cervical cancer³². But till now no studies has been done on cytotoxicity activity of its micropropagated rhizome oil. The anticancer activity of 11 Zingiberaceae species was done using two different cell lines i.e. HT-29 and MCF-7 cancer cells which included C. longa that showed strong inhibitory effects on the growth of both cell lines but no effect in K. galanga was found³³. Other studies on cytotoxic potential of Hedychium species rhizome oils from different regions of India has been done against lung, colon, breast, head, neck and cervix cancer cell lines³⁴. In addition Lamiaceae family, the largest flowering plants essential oils are reported as bearing anticancer properties. It contains menthol, geraniol, eucalyptol, camphor and thymol as the active components in its essential oils³⁵. The essential oils mediated antiproliferative activity mechanisms involved in cell cycle arrest, apoptosis and DNA repair mechanisms. But in the present study among all four samples, HC was significantly most active against the HeLa cell. The aim was to assess the anticancer properties of essential oils obtained from different members of family Zingiberaceae to be considered as a source of anticancer drugs. In the present study, the major compound present in the essential oils might be responsible for the cytotoxic activity of the samples. Further studies should be carried out to evaluate the mechanism of these bioactive compounds for potential therapeutic application.

Fig. 1: Graph of IC50 concentrations of Curcuma longa (CL), Curcuma aromatica (CA), Kaempferia galanga (KG) and Hedychium coronarium (HC) against HeLa cell line observed in the MTT assay after the incubation period of 24hours

Fig. 2: (a) HeLa cell control (b) 10uM of Camptothecin as standard (positive control) (c) HeLa activity of rhizome essential oil in 12.5 uL/mL concentration of Curcuma longa (d) 50 uL/mL of Curcuma longa (e) 12.5 uL/mL of Curcuma aromatica (f) 50 uL/mL of Curcuma aromatica (g) 12.5 uL/mL of test sample of Kaempferia galanga (h) 50 uL/mL of test sample of Kaempferia galanga (i) 12.5 uL/mL of test sample of Hedychium coronarium (j) 50 uL/mL of test sample of Hedychium coronarium

Table 1: Percentage (%) of viability of HeLa cells against Curcuma longa (CL), Curcuma aromatica (CA), Kaempferia galanga (KG) and Hedychium coronarium (HC) rhizome oil in different concentrations at an incubation period of 24hours

Sl. No.	Species name	Viability %
		Untreated	Standard	Samples (in uL) treated in HeLa cell
		Untreated	Standard	6.25	12.5	25	50	100
1	C. longa	100	50.50	86.19	70.24	40.98	32.61	18.09
2	C. aromatica	100	50.50	99.21	89.27	74.67	59.37	40.27
3	K. galanga	100	50.50	97.92	82.18	73.74	41.05	28.11
4	H. coronarium	100	50.50	98.71	91.63	75.46	63.59	44.42

ACKNOWLEDGEMENTS:

The authors are grateful to Prof (Dr.) M.R. Nayak, President and Prof (Dr.) S.C. Si, Dean, Centre for Biotechnology, Siksha ‘O’ Anusandhan University for providing facilities and encouraging throughout.

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Received on 24.06.2020 Modified on 12.08.2020

Research J. Pharm. and Tech 2022; 15(1):325-328.

DOI: 10.52711/0974-360X.2022.00053