INTRODUCTION:

Cancer is a state of abnormal cell growth and is one of the leading causes of mortality worldwide. Most of the cytotoxic agents used in chemotherapy of cancer are reported to exhibit toxicity towards normal tissues, accompanied by undesirable side effects. Natural substances of plant origin are reported to be biologically active, endowed with antioxidant and anticancer properties^1,2. Therefore, research efforts have been intensified on the development of anticancer drugs from natural sources³.

Curcuma caesia Roxb. (black turmeric) is a lesser known medicinal plant and rhizomes are used as folklore medicine for the treatment of wounds, cold, cough, inflammation, leucoderma, tumors, asthma, rheumatic pains etc⁴. Pharmacological attributes of black turmeric includes antifungal activity of essential oil⁵, smooth muscle relaxant effect⁶, neuropharmacological activity⁷, bronchodilator activity⁸, antioxidant activity^9,10, and analgesic and anti-inflammatory activity¹¹.

A study conducted to evaluate the antitumor activity of methanol extract of C. caesia rhizome on Ehrlich's ascites carcinoma (EAC) showed cytotoxicity with IC₅₀valueof90.70μg/ml¹². Hexane and methanol extracts of C. caesia and their bioactive compounds were assayed for tumor cell growth inhibition against MCF-7 (breast), SF-268 (central nervous system), NCI-H460 (lung), HCT-116 (colon), AGS (gastric), MIA PaCa-2 and BxPc-3 (pancreatic), and LNCaP and DU-145 (prostate) human tumor cells. The extracts inhibited the growth of tumor cells whereas their bioactive compounds did not inhibit the growth of these cancer cell lines¹³. In spite of its strong medicinal properties, there are only few reports on the cytotoxic properties of different extracts of black turmeric rhizome and identification of bioactive compounds. Therefore, the present study is an evaluation of the in vitro cytotoxic potential of sequential extracts of fresh and dry C. caesia rhizome against A375, HCT116 and A549 cell lines by the MTT assay and characterization of phenolic acids and flavonoids in the active extract (s).

MATERIALS AND METHODS:

Chemicals and reagents:

Dulbecco’s Modified Eagle’s Medium (DMEM), Antibiotic-Antimycotic (100X) and Fetal bovine serum (FBS) were obtained from Life Technologies (India). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO) and Doxorubicin from Sigma-Aldrich (India). All solvents were purchased from Merck Millipore (India) and were of analytical grade. Phenolic acid standards- caffeic acid, 2,4 dihydroxy benzoic acid, chlorogenic acid, ferulic acid, gallic acid, gentisic acid, o-coumaric acid, p-coumaric acid, p-hydroxy benzoic acid, protocatechuic acid, salicylic acid, syringic acid, t-cinnamic acid, vanillic acid; and flavanoid standards- apigenin, catechin, hesperitin, luteolin, myrcetin, naringenin, quercetin, rutin, umbelliferone were procured from Sigma (USA). The organic solvents used for the analysis were of chromatographic/MS grade.

Preparation of plant extracts:

C. caesia rhizomes, obtained from the experimental farm of ICAR-Indian Institute of Spices Research at Peruvannamuzhi, Kozhikode, were processed to produce solvent extracts. The fresh and dry forms of C. caesia rhizomes (50g of each) were sequentially extracted with 500ml each of solvents, in the following order of increasing polarity: hexane, petroleum ether, benzene, chloroform, ethyl acetate, methanol and water. The extracts were filtered with Whatmann filter paper no. 1 and evaporated to dryness using a rotary flash evaporator (Buchi Rotavapor R-205, Switzerland), except the water extract which was lyophilized (Heto Drywinner DW 1.0-110, Denmark). A methanolic solution of the different extracts (10mg/ml) was maintained as stock, and samples were stored at 4°C until further analysis. Each extract was tested in triplicates for the cytotoxicity study and mean values calculated.

Cancer cell lines:

Human cancer cell lines A375 (skin melanoma), HCT116 (colon carcinoma) and A549 (lung adenocarcinoma) were obtained from National Centre for Cell Science, Pune, Maharashtra, India. The cells were maintained in DMEM supplemented with 10% FBS and 10ml 100X antibiotic-antimycotic. The cells were cultured in T-25 culture flasks at 37°C in a humidified incubator containing 5% CO₂and 95% air (NuAire DH Autoflow Automatic CO₂ Air-Jacketed Incubator, USA). The cells were sub-cultured for every two-three days and routinely checked for any contamination under an inverted microscope (Olympus IX51, New York); confluent (80-90%) cell cultures were used for experimentation¹⁴.

Cytotoxicity assay:

Cytotoxicity of solvent extracts was evaluated by MTT assay^15,16. This assay was based on the cleavage of tetrazolium salt by mitochondrial dehydrogenases in viable cells. Briefly, cells were seeded in 96-well culture plates at 5x10³ cells/well density and incubated at 37°C in a humidified incubator containing 5% CO₂and 95% air to allow the cells to adhere. After 24 h, the cells were treated with extracts at five different concentrations, i.e. 1, 10, 100, 500 and 1000µg/ml and incubated for 24, 48 and 72h. Following incubation, 100µl of the MTT dye in DMEM media (5mg/ml) was added and incubated for 2 h. The insoluble formazan crystals formed were solubilized by the addition of MTT lysis buffer (100µl) followed by an incubation of 4 h and the absorbance was measured at 570nm using a microplate spectrophotometer (BioTek Power Wave XS, USA). The inhibitory rate was calculated as Inhibitory rate (Ir) % = [1-Abs sample/Abs control] × 100. The cytotoxicity of each extract was expressed as IC₅₀, the concentration of extract that causes 50% inhibition. DMSO was used to dilute the extracts (stock: 10mg/ml) and the final concentration of DMSO in each well did not exceed 1.0% (v/v). Doxorubicin served as the positive control, while 1.0% DMSO was the negative control. The IC₅₀ values were obtained using EasyPlot software.

Phenolic acids and flavonoids analysis in the active extract(s) by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS):

The standard curve for individual phenolic acids and flavonoids were made using six different concentrations of phenolic acids (2-20µg/ml) and five different concentrations of flavonoids (4-20µg/ml) which was identified and quantified by their molecular weight (parent-mass m/z) and most abundant fragmented daughters.

Sample preparation:

Active extracts, chloroform and ethyl acetate, were dissolved in 3ml of the mobile phase - 0.2% formic acid in methanol, centrifuged, filtered through 0.2µm nylon filter prior to injection.

Instrument:

LC-MS/MS analysis was performed on an ACQUITY UPLC-H class coupled with TQD-MS/MS with ESI source (Waters, USA) equipped with a degasser, quaternary pump, automatic injection system (0-10μl), a diode array detector and a thermostated column compartment. Instrument control, data acquisition and processing were performed using MassLynx software. The mass spectra obtained using negative ionization mode (ES^‒) for the most abundant forms of de-protonated [M-H]^‒ molecules of phenolic acids and flavonoids was found by direct sample infusion. These de-protonated molecules were respectively confirmed as precursor ions of the corresponding phenolic acids and flavonoids for the following collision induced decomposition (CID) fragmentation by their respective collision energy (CE) to develop the MRM methods, for further analysis (Table 1).

Table 1: LC-MS/MS characteristics of standard compounds in the negative mode

Compound	Molar Mass (M)	*Parent ion (m/z)*	*Daughters ion (m/z)*	Cone Voltage (V)	Collision Energy (V)
Phenolic acids
Caffeic acid	180	178.90	135.05	30	16
2,4-Dihydroxybenzoic acid	154	152.90	65.02	28	18
Chlorogenic acid	354	352.97	191.90	22	18
Ferulic acid	194	192.90	134.02	26	14
Gallic acid	170	168.90	125.03	28	12
Gentisic acid	154	152.90	108.98	24	12
o-Coumaric acid	164	162.90	119.06	22	12
p-Coumaric acid	164	162.90	119.05	24	14
p-Hydroxybenzoic acid	138	136.90	93.01	26	12
Protocatechuic acid	154	152.90	109.05	26	16
Salicylic acid	138	136.90	93.10	28	14
Syringic acid	198	196.97	182.07	26	10
t-Cinnamic acid	148	146.90	103.05	26	10
Vanillic acid	168	166.97	108.01	26	20
Flavonoids
Apigenin	270	268.97	107.04	46	30
Catechin	290	289.03	245.15	38	12
Hesperetin	302	300.97	286.15	42	16
Luteolin	286	284.97	150.99	54	26
Myricetin	318	317.03	151.06	42	28
Naringenin	272	271.03	151.00	34	16
Quercetin	302	301.03	151.12	36	20
Rutin	610	609.10	300.20	60	42
Umbelliferone	162	161.04	133.07	42	18

Table 3: Cytotoxicity (IC₅₀ µg/ml) of C. caesia sequential extracts

A375
Extract	24h		48h		72h
Extract	Fresh	Dry	Fresh	Dry	Fresh	Dry
HE	387.32^b	692.68^b	207.81^b	400.00^b	98.54^b	148.29^a
PEE	911.22^a	769.76^a	520.98^a	414.63^a	175.61^a	149.27^a
BE	364.88^c	299.51^c	119.02^d	98.54^c	64.39^d	42.93^c
CE	153.17^g	93.66^g	41.95^g	30.24^g	11.71^g	9.76 ^f
EAE	184.39^f	117.07^f	91.71^f	55.61^f	49.76^e	20.49^e
ME	251.71^e	180.49^e	98.54^e	77.07^e	45.85^f	33.17^d
WE	356.10^d	187.32^d	152.20^c	80.98^d	69.27^c	46.83^b
CV%	0.64	0.59	0.59	0.86	1.15	1.87
Dox*	67.32		6.83		<1

Continue………….

HCT116
Extract	24h		48h		72h
Extract	Fresh	Dry	Fresh	Dry	Fresh	Dry
HE	340.49^e	279.02^c	172.68^c	122.93^c	74.15^c	65.37^c
PEE	>1000^a	>1000^a	>1000^a	>1000^a	959.02^a	803.90^a
BE	388.29^c	239.02^d	132.68^d	91.71^d	54.63^e	44.88^d
CE	217.56^g	193.17^e	89.76^g	72.20^g	35.12^g	30.24^e
EAE	298.54^f	138.54^g	95.61^f	76.10^f	41.95^f	35.12^e
ME	376.59^d	173.66^f	107.32^e	85.85^e	68.29^d	47.80^d
WE	761.95^b	471.22^b	454.63^b	250.73^b	118.05^b	99.51^b
CV%	0.22	0.33	0.43	0.61	1.36	1.43
Dox*	52.68		7.80		<1

Continue……..

A549
Extract	24h		48h		72h
Extract	Fresh	Dry	Fresh	Dry	Fresh	Dry
HE	>1000^b	>1000^b	962.93^a	898.53^b	565.85^a	462.44^b
PEE	>1000^a	>1000^a	900.48^b	902.44^a	497.56^b	622.44^a
BE	452.68^c	383.42^c	169.76^d	155.12^c	76.10^d	50.73^e
CE	210.73^g	163.90^f	70.24^g	65.37^g	27.32^g	25.37^g
EAE	265.37^f	110.24^g	98.54^f	73.17^f	65.37^f	48.78^f
ME	300.49^e	253.66^e	115.12^e	100.49^e	70.24^e	62.44^d
WE	388.29^d	286.83^d	245.85^c	135.61^d	98.54^c	86.83^c
CV%	0.22	0.28	0.61	0.49	0.42	0.53
Dox*	72.20		33.17		<1

*Doxorubicin was used as the reference compound; (HE-hexane extract; PEE-petroleum ether extract; BE-benzene extract; CE-chloroform extract; EAE-ethyl acetate extract; ME-methanol extract; WE-water extract); Values with the different superscript are significantly different (P<0.05)

Generally, the fresh rhizome extracts showed weaker cytotoxicity profile against the human cancer cell lines tested compared to the dry rhizome extracts in a time- and dose-dependent manner. Among the extracts, chloroform and ethyl acetate extracts showed better cytotoxic activity compared to other extracts. The hexane extract of fresh C. caesia rhizomes showed low cytotoxic effect with IC₅₀ value 98.54μg/ml at 72h against skin cancer cell line (A375) and still lower activity at 24 and 48h. The IC₅₀ values hexane extract of dry rhizomes were even less impressive at 24, 48 and 72 h. The hexane extract of fresh and dry rhizomes displayed low cytotoxicity in HCT116 cells with IC₅₀ values of 74.15 and 65.37μg/ml respectively at 72h of incubation. Additionally, the hexane extract did not exhibit any cytotoxic effect against A549 cells at 24h. The petroleum ether extract of C. caesia rhizomes showed less impressive cytotoxicity than hexane extracts towards the three cell lines. In A375 cells, the petroleum ether extract from fresh and dry rhizomes displayed low cytotoxic effect at 24, 48 and 72h of exposure. It exhibited low cytotoxicity towards HCT116 cells at 72 h. Furthermore, there was no significant cytotoxic effect due to fresh and dry extracts in A549 cells except the IC₅₀ values of 900.48 and 497.56μg/ml; 902.44 and 622.44μg/ml at 48 and 72h respectively.

In A375 cells, the benzene extract displayed low cytotoxic effect for fresh rhizomes and dry rhizomes at 24 and 48h of exposure. However, the extracts from fresh rhizomes showed moderate cytotoxicity with an IC₅₀ value of 64.39μg/ml and greater effect (IC₅₀ at 42.93μg/ml) for dry rhizomes at 72h. Benzene extract did not have cytotoxicity effect on HCT116 cells at 24 and 48h but exhibited moderate cytotoxicity at 72h in fresh and dry rhizomes. However, the benzene extract displayed very low cytotoxic effect in A549 cells at 24 and 48h; at 72h, there was some activity with IC₅₀ values of 76.1 and 50.7μg/ml for fresh and dry extracts respectively.

The chloroform extract of C. caesia rhizomes displayed highest cytotoxic activity at 72h of exposure against A375 cells and A549 cells in fresh and dry rhizomes, compared to the remaining extracts. The fresh and dry rhizome extracts showed moderate cytotoxic effect in HCT116 cells at 72h and A375 cells for 48h, and very low cytotoxic effect in A375 at 24h and HCT116 and A549 cells at 24 and 48 h.

The ethyl acetate extract of C. caesia viz., dry rhizomes showed good cytotoxic effect with an IC₅₀ value of 20.49 μg/ml against A375 cells at 72 h. In addition, this ethyl acetate extract from fresh and dry rhizomes displayed moderate cytotoxic effect against HCT116 at 72h.

The methanol extract from fresh and dry rhizomes of C. caesia exhibited moderate cytotoxic effect against A375 cells at 72h (IC₅₀ values of 45.85 and 33.17μg/ml respectively). In addition, the extract from dry rhizomes exhibited moderate cytotoxicity in HCT116 with an IC₅₀ value of 47.80μg/ml at 72h. However, the water extract showed very low cytotoxicity in all investigated cancer cell lines at 24, 48 and 72h of exposure, except a moderate cytotoxicity of 46.83μg/ml by dry rhizomes on A375 cells at 72 h of incubation.

Characterization of phenolic acids and flavonoids:

Pharmacological properties of an extract are often associated with its bioactive components such as phenolic acids, flavonoids and tannins²¹. Chloroform and ethyl acetate extracts, identified as active extracts against the cancer cell lines considered in this study, were analyzed by LC-MS/MS in order to characterize the phenolic acids and flavonoids present in it. This was performed by comparison of retention times and mass fragmentation pattern obtained for commercial standards. From the LC-MS/MS results, it could be deduced that the cytotoxic activity of active extracts from C. caesia rhizomes observed in this study was contributed by the presence of phenolic acids and flavonoids. Phenolic acids and flavonoids are said to have protective effect in carcinogenesis, inflammation and have high antioxidant capacity, hence, 14 phenolic acids and 9 flavonoids have been detected in these active extracts (Table 4 and 5). The major phenolic acids found in C. caesia rhizomes belonged to the hydroxybenzoic acid derivatives (Table 4). Hydroxybenzoic acids have a general structure C6-C1. The content of gallic acid and vanillic acid were the highest in chloroform extracts, 106.1 µg/g fw and 701.6µg/g dw. Moreover, vanillic acid was a predominant compound of fresh ethyl acetate extract (414.6µg/g fw).

Table 4: Quantification of phenolic acids in chloroform and ethyl acetate extracts of C. caesia rhizome

Phenolic acids (µg/g fresh or dry rhizome extract)	Chloroform extract		Ethyl acetate extract
Phenolic acids (µg/g fresh or dry rhizome extract)	Fresh	Dry	Fresh	Dry
Caffeic acid	0.00^j	0.00ⁱ	124.11^e	28.14ⁱ
2,4-Dihydroxybenzoic acid	12.02^g	1.34^h	0.00^j	0.00^k
Chlorogenic acid	0.00^j	0.00ⁱ	0.00^j	0.00^k
Ferulic acid	7.09^h	23.14^c	119.41^f	257.13^b
Gallic acid	106.14^a	7.83^g	0.00^j	2.61^j
Gentisic acid	1.33^ij	2.22^h	0.00^j	0.00^k
o-Coumaric acid	2.96ⁱ	3.19^h	0.00^j	1.14j^k
p-Coumaric acid	0.00^j	2.78^h	13.64ⁱ	77.12^f
p-Hydroxybenzoic acid	27.48^e	14.38^e	397.27^c	218.54^d
Protocatechuic acid	17.93^f	14.57^e	407.97^b	354.17^a
Salicylic acid	1.51^ij	12.92^f	140.96^d	71.12^g
Syringic acid	65.28^b	26.11^b	104.45^g	241.54^c
t-Cinnamic acid	50.35^d	22.38^d	27.97^h	52.21^h
Vanillic acid	53.15^c	701.55^a	414.55^a	148.81^e

Values with the different superscript are significantly different (P<0.05)

But ethyl acetate extract from dry rhizome was dominant in protocatechuic acid (354.2 µg/g dw). The amount of syringic acid in ethyl acetate extract (241.5µg/g) was nine times higher than chloroform extract (26.1µg/g) of dried rhizome. Coumaric acids (o-coumaric: 0-3.2µg/g and p-coumaric: 0-77.1µg/g) in chloroform and ethyl actetate extracts were found at varied level. Gentisic acid was the lowest among identified phenolic acids in C. caesia rhizome.

Flavonoids include compounds with general structure having a C6-C3-C6 flavone skeleton²². Catechin was the predominant flavonoid in the active extracts: 66.4µg/g fw and 701.4µg/g dw in chloroform extracts and 436.0 µg/g fw and 369.7µg/g dw in ethyl acetate extracts (Table5). Luteolin and hesperetin followed the predominant flavonoid in chloroform extracts with concentrations 53.7 and 109.3µg/g in fresh and dried rhizomes respectively. But in ethyl acetate extracts where naringenin was the second most abundant flavonoid (37.9µg/g fw and 95.5µg/g dw). The active extracts had very low contents of apigenin, which ranged from 0.99-1.98µg/g. Except ethyl acetate extract from dried rhizome, which was deficient in rutin, a number of other flavonoids were identified and quantified in the chloroform and ethyl acetate extracts from fresh and dried rhizomes.

Table 5: Quantification of flavonoids in chloroform and ethyl acetate extracts of C. caesia rhizome

Flavonoids (µg/g fresh or dry rhizome extract)	Chloroform extract		Ethyl acetate extract
Flavonoids (µg/g fresh or dry rhizome extract)	Fresh	Dry	Fresh	Dry
Apigenin	0.99ⁱ	1.48^h	1.98^f	1.48^g
Catechin	66.35^a	701.43^a	436.02^a	369.67^a
Hesperetin	19.62^e	109.32^b	25.23^c	53.26^c
Luteolin	53.66^b	7.22^g	7.22^d	4.13^f
Myricetin	41.20^c	10.30^f	25.75^c	33.48^d
Naringenin	14.70^f	37.30^d	37.87^b	95.52^b
Quercetin	23.09^d	42.33^c	5.13^de	5.13^f
Rutin	13.67^g	16.82^e	7.36^d	0.00^h
Umbelliferone	5.23^h	11.51^f	4.19^e	6.80^e

Values with the different superscript are significantly different (P<0.05)

DISCUSSION:

Cytotoxicity tests use a series of increasing concentrations of the drug to determine the IC₅₀ value, concentration resulting in the death of 50% of cancer cells. Liu et al. assayed the extracts and pure isolates of C. caesia for tumor cell growth inhibition at single concentration i.e. 100µg/ml and 5µg/ml respectively¹³. According to the United States National Cancer Institute Plant Screening Program, a plant extract is generally considered to have active cytotoxic effect if the IC₅₀ value is 30µg/ml or less, following incubation of 72 h²³. It has been reported that methanol extract of C. caesia rhizome exhibited cytotoxic effect on Ehrlich's ascites carcinoma (EAC) with an IC₅₀ of90.70μg/ml¹². In our study, three cell lines were treated with different concentrations of different extracts for 24, 48 and 72h. The chloroform, ethyl acetate, benzene and methanol extracts were found to inhibit A375, HCT116 and A549 cell proliferation in a time- and dose-dependent manner, among which chloroform extract was highly toxic towards A375 cell lines (IC₅₀ values of 11.71 and 9.76 μg/ml for fresh and dry respectively) followed by A549 cell (IC₅₀ values of 27.32 and 25.37μg/ml for fresh and dry respectively) at 72h exposure. Although the cytotoxicity of C. caesia extracts is not as impressive as doxorubicin, the results suggest that bioactive components play an important role in the cytotoxicity exhibited by the extracts. Further studies are needed to evaluate the chemopreventive potentials of the black turmeric rhizome extracts when used alone or in combination with doxorubicin to mitigate the toxic side-effects of the latter.

These observations revealed that C. caesia possess potent anticancer activity in chloroform and ethyl acetate extracts, which could possibly be derived from polyphenols such as flavonoids, phenols and sterols. Extractant solvents of different polarity have pronounced effect on the phytochemicals extracted and therefore the cytotoxic effect, among other therapeutic properties. To the best of our knowledge, this is the first report on the cytotoxic properties of C. caesia rhizomes employing successive extraction of solvents differing in polarity, at different concentrations.

The principal phenolic acids of the present study, gallic acid, vanillic acid and protocatechuic acid, are well-known antioxidant compounds. Gallic acid, a naturally occurring polyphenol, has been reported as a free radical scavenger and showed significant inhibitory effects on cell proliferation, induced apoptosis in a series of cancer cell lines including skin, lung and colon adenocarcinoma cell lines^24-26. Vanillic acid has been shown to suppress the metastatic potential of human cancer cells by inhibiting the enzymatic activity of P13K and also decreased angiogenesis in vivo²⁷. Protocatechuic acid is shown to be a potent anticancer agent to cause apoptosis and metastasis in human breast (MCF7), lung (A549), liver (HepG2), cervix (HeLa) and prostate (LNCaP) cancer lines²⁸.

Flavonoids have remarkable anticancer activity and catechin, the major flavonoid of the active extracts in the present study, is proven to be effective for suppressing the activation of heptocyte growth factor receptor in human colon cancer cells²⁹. Catechin in combination with curcumin can inhibit the proliferation of human colon adenocarcinoma (HCT15 and HCT116) cells by inducing apoptosis³⁰. Luteolin and hesperetin, the second abundant flavonoid in chloroform extract from fresh and dried rhizomes, can be considered as a potent candidate for the treatment of cancer^31,32.

There have been no previous reports on the characterization of polyphenols in C. caesia. For the first time, we have shown that this species has the potential to slow down the growth of colon and lung adenocarcinoma and skin melanoma cells using immortalized cell lines. Previous studies have demonstrated that gallic acid, vanillic acid, protocatechuic acid, catechin, luteolin and hesperetin are potent anticancer agents. These phenolics are present in significant amounts in C. caesia and their presence can be correlated with the anticancer properties exhibited by these extracts. Accordingly, C. caesia could be regarded as potential anticancer agent and possible source of new therpaeutics.

CONCLUSION:

The current study have provided preliminary data proving that C. caesia extracts have potent cytotoxic activity against A375, HCT116 and A549 cells. Chloroform extract was found to be the most effective treatment against the cancer cell lines under study; A375 cell lines (IC₅₀ values of 11.71 and 9.76 μg/ml for fresh and dry respectively), followed by A549 cell (IC₅₀ values of 27.32 and 25.37μg/ml for fresh and dry respectively) and HCT116 ((IC₅₀ values of 35.12 and 30.24μg/ml for fresh and dry respectively) at 72h exposure The cytotoxicity could be attributed to high phenolic and flavonoid contents, viz. gallic acid, vanillic acid, syringic acid, catechin, luteolin and hesperetin, identified using LC-MS/MS. Therefore, C. caesia could be an excellent source for the possible development as promising anticancer drugs.

CONFLICT OF INTEREST:

The authors declare that they have no competing interests.

ACKNOWLEDGEMENTS:

The authors are thankful to the Directors of Indian Institute of Spices Research, Regional Cancer Center and Indian Institute of Horticultural Research for providing laboratory facilities for the sequential extraction, cell line studies and LC-MS/MS analysis respectively. We gratefully acknowledge KSCSTE for the fellowship awarded to the first author. We are also thankful to Head and members of Division of Crop Production and PHT, Indian Institute of Spices Research for their support. Authors are also thankful to Dr. Sreelekha T.T. (Regional Cancer Centre, Thiruvananthapuram) and Mr. T.K. Roy (Indian Institute of Horticultural Research, Bengaluru) for providing technical support in cell line studies and LC-MS/MS analysis respectively.

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Received on 06.02.2023 Modified on 08.04.2023

Asian J. Pharm. Res. 2023; 13(4):219-226.

DOI: 10.52711/2231-5691.2023.00041

Extracts	Yield
Extracts	Fresh	Dry
Hexane	6.7^b	17.5^c
Petroleum ether	1.2^d	1.5^f
Benzene	3.2^cd	6.6^e
Chloroform	4.3^c	9.5^d
Ethyl acetate	2.9^cd	6.6^e
Methanol	8.7^b	25.8^b
Water	40.0^a	80.0^a