Chemical constituents of the different parts of Colchicum baytopiorum and their cytotoxic activities on K562 and HL60 cell lines

Abstract

The plant chemistry and cytotoxic activity of Colchicum baytopiorum CD Brickell (Liliaceae/Colchicaceae), an endemic species growing in Turkey, has been studied for the first time. Nine known alkaloids were isolated and their structures were identified by spectral methods (UV, IR, ¹H-NMR, and ESI/MS), and the presence of three alkaloids, which could not be isolated from the plant, was also detected by LC/MS/MS spectrometry. Phenolic acids were elaborated using LC/MS and 11 phenolic acids were identified. The presence of two flavonoids appeared to be valuable for chemotaxonomic purposes. Guided by the brine shrimp lethality test (BSLT), methanol extracts were tested for cytotoxic activity by colorimetric MTT test on K562 and HL60 cells. Except the seed extract, all methanol extracts showed more cytotoxic activity on HL60 cells (IC₅₀: 6.5– < 0.1 μg/mL) than on K562 cells (IC₅₀: > 500–44 μg/mL).

Keywords::

• Colchicum baytopiorum
• cytotoxic activity
• phenolic acids
• tropolonic alkaloids

Introduction

The genus Colchicum (Liliaceae) is represented by 90 species (Vassiliades & Persson, 2002) in the world; 45 of these species grow in Turkey. Turkey is a major center of diversity and speciation (for Colchicum species) when the endemism rate (35%) is considered (Akan & Eker, 2005; Düşen Dinç & Sümbül, 2007). Colchicum baytopiorum CD Brickell is one of the endemic species (Brickell, 1984; Persson, 2000).

The medicinal importance of the genus Colchicum is attributed to the presence of tropolonic alkaloids, mainly colchicine. This type of alkaloid occurs only in the genera Colchicum L., Merendera Ram., Gloriosa L., Androcymbium Willd., Kreysigia Rchb., Iphigenia Kunth, etc., belonging to subfamily Wurmbaeoideae (Willaman & Hui-lin, 1970; Santavy et al., 1983).

Colchicine has been found to possess antitumoral and antiinflammatory properties and has still kept its importance in the treatment of gout and many other diseases, such as familial Mediterranean fever (FMF), Behçet’s syndrome, chirosis, and psoriasis (Le Hello, 2000). Unfortunately, due to its low therapeutic index, this alkaloid cannot be used as an antineoplastic agent. It is also used in biological and breeding studies to produce polyploids and in the tubulin binding assay as a positive control because of its affinity for tubulin (Al-Fayyad et al., 2002; Evans, 2002). Demecolcine has lower toxicity than colchicine and has been successfully used in treatment, especially for myeloid leukemia and Hodgkin’s syndrome (Le Hello, 2000).

Colchicine and its derivatives are known to be sensitive to light and high temperature. The main degradation products described are β- and γ-lumi colchicines and colchiceines (Capraro & Brossi, 1984; Körner & Kohn, 2005).

Previous studies have indicated that the composition of tropolone alkaloids differs in different parts of Colchicum species and varies during the different growth stages (Sütlüpınar et al., 1988; Husek et al., 1990; Alali et al., 2006). The aim of the present work was to determine the chemical constituents of the different parts (perigon, perigon tube, leaf, seed, and corm) of C. baytopiorum, an unexplored Turkish species, as a continuation of our studies on Colchicum species (Baytop & Özcöbek, 1970; Baytop et al., 1980; Sütlüpınar, 2002). After determining the chemical constituents, this species was evaluated for chemotaxonomic purposes. In addition, we aimed to investigate the cytotoxic activities of methanol extracts obtained from different parts of the plant using K562 and HL60 cell lines. All of the results were compared with each other.

Material and methods

General experimental procedures

Ultraviolet (UV) spectra were recorded on a Jasco 530V-vis spectrophotometer (UK). ¹H-Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity Inova 500 MHz spectrometer (USA). Mass spectra were obtained on Thermo Finnigan^™ LCQ^™ (USA) and Masse ZQ 2000 Waters (source: Z spray) (USA) electrospray ionization (ESI) (+) instruments. Infrared (IR) spectra were run using KBr disks on a PerkinElmer 1600 Series Fourier transform (FTIR) spectrophotometer (USA). Liquid chromatography (LC) separation was performed on a Waters Cap LC XE System (Milford, USA) using the reverse phase microcolumn Geminy, Phenomenex C-18 (150 mm × 300 μm I.D.). Mass spectrometry (MS) was done using a quadrupole time-of-flight (Q-ToF) instrument (Waters Micromass Q-ToF Premier; Milford, USA). MS/MS analysis of detected alkaloids was carried out using a Waters MassLynx v.4.0 spectrometer (USA).

Plant material

Colchicum baytopiorum was collected from Antalya-Termessos at 900 m altitude with fruits, July 10, 2003, and with flowers, October 20, 2003. Voucher specimens have been identified by Professor N. Sütlüpınar and deposited in the Herbarium of the Pharmacy Faculty of Istanbul University (ISTE 81438, ISTE 81439). All parts of the plant were separated and then left to dry at room temparature, except the corms which were separated and sliced then left to dry in a drying oven at 60°C.

Extraction and isolation

All dried plant material (perigon = P (11.71 g), perigon tube = Pt (16.60 g), leaf = L (60.0 g), seed = S (30 g), corm (collected in October) = CO (100.0 g) and corm (collected in June) = CJ (30 g)) was extracted separately with methanol using Soxhlet apparatus, apart from the seeds which were extracted first with petroleum ether then with methanol. The obtained methanol extracts were evaporated in a rotavapor.

Extraction, isolation, and determination of alkaloids

The methanol extract was dissolved in water using an ultrasonic bath, and filtered. The filtrate was made acidic with 3% H₂SO₄ to pH 3–4 and extracted with chloroform. The combined chloroform extracts were dried over anhydrous Na₂SO₄, filtered, and concentrated to dryness under vacuum to yield neutral-phenolic extract (extract A). Later, the acidic aqueous layer was made alkaline with 10% NH₄OH to pH 8–9 and extracted with chloroform. The combined chloroform extracts were dried over anhydrous Na₂SO₄, filtered, and concentrated to dryness under vacuum to yield basic extract (extract B). Extractions were carried out with 100 mL of solvent for each portion until negative to SbCl₃. Alkaloid extraction was carried out for all methanol extracts. The evaporation was done at 35°C, and during the study, direct light protection was provided (Malichova et al., 1979).

Isolation of alkaloids P-A (perigon, neutral- phenolic extract) (110.0 mg) was purified by preparative thin layer chromatography (TLC) on silica gel (Merck 0.5554) using a benzene–ethylacetate–diethylamine–methanol (50:40:10:8) solvent system to afford the pure alkaloids. Pt-A (150.0 mg) was purified by an aluminum oxide, activity I (Merck 1078) column, eluted with gradient mixtures of diethylether–chloroform–methanol (140 × 20 mL). Similar fractions were combined according to their TLC profiles, which were carried out with many different solvent systems. The pure alkaloids were isolated from fractions 1–38 (13.4 mg) and fractions 38–140 (48.4 mg), and were eluted with diethylether–chloroform (100% to 1:1) and chloroform–methanol (99:1 to 10:90). L-A (125.0 mg) and S-A (215.0 mg) were purified with preparative TLC on silica gel using a chloroform– acetone–diethylamine (7:2:1) solvent system. For CJ-A (50.0 mg) purification, silica gel and a chloroform–acetone–25% NH₄OH (50:50:1) solvent system were used. CO methanol extract (800.0 mg) was applied to a Chromatotron^® plate covered with Al₂O₃ (Merck 1092). A gradient mixture of diethylether, chloroform, and methanol was used as a solvent system (60 × 20 mL). Fractions 1–5 (37 mg) and fractions 22–38 (21.8 mg) were purified with preparative TLC to afford the pure alkaloids which were eluted with chloroform–methanol (9:2) and chloroform–methanol (70:30). Then, they were purified with preparative TLC using a benzene–ethylacetate–diethylamine–methanol (50:40:10:8) solvent system.

Pt-B (32.5 mg) was purified by TLC on silica gel using a benzene–ethylacetate–diethylamine–methanol (50:40:10:8) solvent system.

Determination of alkaloids by LC/MS/MS analysis The mobile phase was a mixture of two components: component A, containing 5.7 mmol/L acetic acid in 5% acetonitrile, and component B, which was 100% acetonitrile. The following gradient program was used: 0–5 min, isocratic at 10% B; 5–25 min, jump to 30% B; 25–40 min, jump to 90% B; 40–60 min, isocratic 90% B. Equilibration on the column was reconstituted during a 15 min delay with isocratic flow of 10% B. The mobile phase flow rate was 5 μL/min and the injection volume was 5 μL. Optimal parameters of ESI-MS were the following: capillary voltage +2.5 kV, sampling cone 30 V, source temperature 100°C, desolvation temperature 250°C, cone gas flow 50 L/h, and desolvation gas flow 250 L/h. Data were obtained in single V mode.

Extraction and isolation of flavonoids

Pt (methanol extract) (3.0 g) was dissolved in water (20 mL). It was made acidic with 3% H₂SO₄ to pH 3–4 then was extracted with diethylether (6 × 50 mL) in a separating funnel. Pt-E (perigon tube, diethylether extract) (150.0 mg) was purified with TLC on silica gel using a chloroform–acetone–formic acid (9:2:1) solvent system. Two known flavonoids were isolated (F1: luteolin, 8.2 mg; F2: apigenin, 11.7 mg). The diethylether extracts were compared with each other and authentic samples on TLC. NA (Naturstoffreagenz A: diphenyl boric acid β-amino ethyl ester) reagent was sprayed on a TLC plate for the determination of flavonoids.

Determination of phenolic acids by LC/MS

All methanol extracts were compared with phenolic acid standards using LC/MS. The retention times and MS spectra were determined and evaluated. The same composition of mobile phase that was used for determination of alkaloids was also effectively used for basic separation of the phenolic acid authentic sample. The optimal conditions for ESI-MS were: capillary voltage–2.1 kV, sampling cone 35 V, source temperature 100°C, desolvation temperature 200°C, cone gas flow 50 L/h, and desolvation gas flow 450 L/h. Data were also obtained in single V mode.

Spectrophotometric total alkaloid assay

Materials obtained from the different parts of the plant were weighed accurately and they were extracted in a Soxhlet apparatus for 6 h. The obtained extracts were evaporated under vacuum to dryness. The Sütlüpınar (1998) procedure was performed. UV values of the solutions at 352 nm were measured. A graph of colchicine as standard was drawn and the formula of the graph was calculated as y = 0.0475x – 0.0256 (R² = 0.9996) (y = average of solution absorbance, x = concentration). The value of the absorbance was determined with an average of five measurements for each sample.

Cytotoxic activity

The cytotoxicity tests were performed on the methanol extracts (P, Pt, L, S, CO, CJ) prepared from the separated parts of the plant.

Brine shrimp lethality test (BSLT)

The Meyer et al. (1982) procedure was performed for the BSLT test. Colchicine standard was used as a positive control. The activity is reported as LC₅₀ value (μg/mL) with 95% confidence interval as determined by a Finney PC computer program. LC₅₀ values > 1000 μg/mL are considered inactive while LC₅₀ values < 200 μg/mL are considered as highly active for extracts (Al-Mahmoud et al., 2006)

Cytotoxicity assay against human leukemia cell lines by MTT test

The cytotoxicities were measured using a system based on the tetrazolium compound MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma M-5655), which is reduced by living cells to yield a soluble formazan product that can be assayed colorimetrically (Mosmann, 1983). The cytotoxic activities of the extracts were evaluated in K562 (chronic myeloid leukemia) and HL60 (promyeloid leukemia) cell lines. K562 and HL60 cells were purchased from the American Type Culture Collection (ATCC, MD, USA). The cell lines were grown and maintained in a humidified incubator at 37°C and in a 5% CO₂ atmosphere. Leukemic cells were suspended at 1 × 10⁵ cells/mL in Iscove’s modified Dulbecco’s medium (IMDM; Sigma I 2510) that contained 10% fetal calf serum (FCS; Sigma F 4135), gentamycin (Genta^®), l-glutamine (Sigma G 3226), and sodium bicarbonate. This suspension (90 μL) was dispensed into 96-well round-bottom plates containing 10 μL of extract dilutions. Medium (10 μL) without extract was used as a positive control, and only medium which did not contain any cells and extract was used as a negative control. Stock colchicine and demecolcine solutions 0.5% were prepared and used as standard. Stock solutions of the extracts were prepared in dimethylsulfoxide (DMSO; Sigma D 5879), 5 mg/mL. Six aqueous concentrations (500, 100, 50, 10, 1, 0.1 μg/mL) were prepared from each solution. The cytotoxicity assay was repeated six times for each concentration of all methanol extracts. Colorimetric analysis was performed by enzyme-linked immunosorbent assay (ELISA) multiwell spectrophotometry, and optical density (OD) was measured with a 540 nm test wavelength and a 620 nm reference wavelength.

A cytotoxicity index (cytotoxicity %) was calculated using the following formula:

Also, IC₅₀ was calculated from the data obtained using GraphPad Prism software.

Statistical analysis

Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software. Results are expressed as the mean ± standard deviation (SD). Statistical differences were assessed by Student’s unpaired t-test, with p < 0.05 as statistically significant.

Results and discussion

The total alkaloid content ranges of the different parts of C. baytopiorum were determined according to the spectrophotometric method (Table 1). These results indicate that perigon (5.27%) has the richest alkaloid content and the corm collected in October (0.50%) has the lowest alkaloid content. Nine known alkaloids, namely, colchicine (1), 2-demethylcolchicine (2), 3-demethylcolchicine (3), 3-demethyl-N-formyl-N-deacetylcolchicine (4), colchifoline (5), 2-demethylcolchifoline (6), demecolcine (7), 2-demethyldemecolcine (8), and 2-demethyl-γ-lumicolchicine (12), were isolated from the all separated parts of C. baytopiorum (Figure 1). The structures of the isolated compounds were elucidated using a series of spectrophotometric (UV, IR; ¹H-NMR, ESI-MS) methods by comparison of these spectral data with the literature (Fabian et al., 1955; Cross et al., 1965; Freyer et al., 1987; Husek et al., 1990; Abu-Zarga et al., 1991; Al-Tel et al., 1991) and TLC by comparison with authentic samples. The structures of three other alkaloids, namely, cornigerine (9), N-formyl-N-deacetylcolchicine (10), and 3-demethyldemecolcine (11) (Figure 1), were only determined by LC/MS/MS analysis (m/z values = 358, 386, 358, respectively). The presence of colchicoside (glucose-containing derivative of colchicine) has been reported in a previous study from the corms and seeds of C. baytopiorum (Sütlüpınar, 1998). These results indicate that the presence of one lumi derivative and 13 tropolonic type alkaloids have been determined in this species. The alkaloid composition of each part of the plant is presented in Table 2. The yields of the alkaloids were evaluated, and colchicine and demecolcine were found to be major alkaloids in all plant parts. A published report on the colchicine, demecolcine, and colchicoside quantification of Turkish Colchicum species by high performance liquid chromatography (HPLC) showed that the major alkaloid of the seeds of C. baytopiorum is demecolcine (Sütlüpınar, 1998). In accordance with this study, demecolcine has been isolated as a major alkaloid from the seed extract.

Table 1.  Total alkaloid content of the different parts of C. baytopiorum.

Plant part	Total alkaloids (%)
P	5.27
Pt	2.96
L	1.96
S	0.85
CJ	0.69
CO	0.50

P, perigon; Pt, perigon tube; L, leaf; S, seed; CJ, corm (June); CO, corm (October).

Table 2.  Tropolonic alkaloids determined in the parts of C. baytopiorum and their yields.

P	Pt	L	S	CJ	CO
Colchicine (11.3 mg)	Colchicine (2.3 mg)	Colchicine (2 mg)	Colchicine (4.8 mg)^+	Colchicine (6.1 mg)	Colchicine (3.4 mg)
3-Demethylcolchicine	N-Deacetyl-N-formylcolchicine*	3-Demethylcolchicine (3.8 mg)^+	2-Demethylcolchicine (1.3 mg)	Cornigerine*	2-Demethylcolchicine (1.4 mg)^+
Colchifoline (5.2 mg)	Colchifoline (3.1 mg)	N-Formyl-N-deacetyl-3-demethylcolchicine(1.5 mg)^+	Colchifoline (2.2 mg)	Demecolcine (5.1 mg)	Colchifoline (2.9 mg)^+
2-Demethylcolchicine (2.5 mg)	Cornigerine*	2-Demethyl-γ-lumicolchicine (2.5 mg)^+	Cornigerine*	2-Demethyldemecolcine	Cornigerine*
Demecolcine (1.3 mg)	Demecolcine (3 mg)	Cornigerine*	Demecolcine (7.1 mg)^+	3-Demethyldemecolcine*	Demecolcine (2 mg)
2-Demethyldemecolcine	2-Demethyldemecolcine (9.5 mg)^+	2-Demethylcolchifoline (5.5 mg)^+	2-Demethyldemecolcine		2-Demethyldemecolcine
3-Demethyldemecolcine*	3-Demethyldemecolcine*	Demecolcine (0.7 mg)	3-Demethyldemecolcine*		3-Demethyldemecolcine*
		2-Demethyldemecolcine
		3-Demethyldemecolcine*

*Analyzed by LC/MS/MS.

⁺ Obtained and structures identified.

Identification of other substances was carried out by TLC. The substances were compared with standards using at least five different solvent systems. See Table 1 for definitions of plant parts.

Figure 1.  Structures of the alkaloids found in C. baytopiorum.

Eleven phenolic acids, namely, benzoic, coumaric, vanillic, cinnamic, ferulic, caffeic acids, 2-hydroxy-6-methoxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 2,5-dihydroxybenzoic acid, 2-hydroxybenzoic acid, and 4-hydroxybenzoic acid were detected in the methanol extracts prepared from all plant parts using LC/MS (Table 3). The phenolic acid composition of the plant is summarized in Table 4. In addition to phenolic acids, apigenin and luteolin were isolated from the perigon tube, and their structures were identified using UV, IR, and TLC, comparing with authentic samples. The presence of apigenin and luteolin was determined in perigon tube and leaf extracts, although only apigenin was confirmed in the perigon extract (Table 4).

Table 3.  Retention times and m/z values of phenolic acids from C. baytopiorum.

Phenolic acid	m/z	Retention time (Rt, min)
		P	Pt	L	S	CO	CJ
Vanillic acid	167	—	—	—	24.71	—	—
Coumaric acid	163	—	22.87	—	23.14	21.20	—
Ferulic acid	193	—	—	—	18.69	—	—
Caffeic acid	179	—	22.04	—	—	21.20	23.64
Cinnamic acid	147	—	—	—	21.25	—	—
Benzoic acid	121	—	18.65	21.51	21.76	20.21	21.79
2-Hydroxybenzoic acid	137	33.73	33.19	—	38.12	35.73	35.42
4-Hydroxybenzoic acid	137	33.73	33.19	—	38.12	35.73	35.42
2,5-Dihydroxybenzoic acid	153	—	—	20.31	19.37	19.00	23.33
2-Hydroxy-6-methoxybenzoic acid	167	—	—	—	24.71	—	—
4-Hydroxy-3-methoxybenzoic acid	167	—	—	—	24.71	—	—

Table 4.  Phenolic acids and flavonoids in C. baytopiorum.

Phenolic acid	P	Pt	L	S	CO	CJ
Vanillic acid	—	—	?	?*	—	—
Coumaric acid	—	+	—	+	+	?
Ferulic acid	—	?	—	+	—	?
Caffeic acid	—	+	?	—	+	+
Cinnamic acid	?	?	?	?	?	?
Benzoic acid	—	+	+	+	+	+
2-Hydroxybenzoic acid	?*	?*	?*	?*	?*	?*
4-Hydroxybenzoic acid	?*	?*	?*	?*	?*	?*
2,5-Dihydroxybenzoic acid	—	—	+	+	+	+
2-Hydroxy-6-methoxybenzoic acid	—	—	?	?*	—	—
4-Hydroxy-3-methoxybenzoic acid	—	—	?	?*	—	—
Apigenin	+	+	+	—	—	—
Luteolin	—	+	+	—	—	—

+, present; —, not available; ?, not confirmed; ?*, one or more isomers.

For determination of cytotoxic activity, the extracts prepared from all plant parts were tested for toxicity against the BSLT (Table 5). All of the extracts showed markedly high cytotoxic activity (LC₅₀: > 100–23.20 μg/mL). Guided by the BSLT, we decided to test the extracts for cytotoxic activity against human myeloid leukemia cell lines. K562 (chronic myeloid leukemia) and HL60 (promyeloid leukemia) cell lines were selected, and the cytotoxicity assay was performed six times for each concentration (500, 100, 50, 5, 1, and 0.1 μg/mL) of all methanol extracts prepared from the different parts of C. baytopiorum (Table 6). IC₅₀ values were calculated (Table 7) and the results were compared with each other and those for colchicine and demecolcine standards (Figure 2). Except for the seed extract, all extracts were found to be more cytotoxic on the HL60 cell line (IC₅₀: 6.5– < 0.1 μg/mL) than on the K562 cell line (IC₅₀: > 500–44 μg/mL). All extracts were found to be more active than the demecolcine standard, and they also showed similar activity to colchicine (Figure 2). Different cytotoxic responses of K562 and HL60 cells can be attributed to cell origin, receptors, and genetic mutations. Furthermore, the results also showed that the methanol extract of perigon, which had the richest alkaloid content (5.27%), was the most active extract (IC₅₀: < 0.1 μg/mL) on HL60 cells (Figure 2).

Table 5.  LC₅₀ values of standard and extracts from C. baytopiorum in the brine shrimp lethality test (BSLT).

Standard and extracts	LD50 value (μg/mL) (K562)
C	0.1406
P	57.9792
Pt	24.8582
L	> 100
S	36.2251
CO	65.5610
CJ	23.1961

C, colchicine.

Table 6.  Cytotoxicity results of the extracts obtained from C. baytopiorum for two different cell lines in the MTT test.

Cell line	Extract	500 μg/mL	100 μg/mL	50 μg/mL	5 μg/mL	1 μg/mL	0.1 μg/mL
K562	P	52.02 ± 22.03	48.57 ± 15.00	37.53 ± 18.63^d	40.56 ± 21.59^d	35.73 ± 21.46	32.41 ± 26.96
	Pt	52.83 ± 26.65	49.82 ± 15.95	50.90 ± 19.99^d	44.01 ± 22.93^d	40.01 ± 14.32^d	36.56 ± 13.38
	L	55.25 ± 2.51	44.81 ± 15.19	38.14 ± 17.78^d	30.58 ± 26.01^d	8.95 ± 19.45^c	–9.46 ± 22.87^c,d
	S	53.92 ± 17.42	54.83 ± 18.08	47.35 ± 26.62^d	38.75 ± 21.68^d	33.75 ± 14.47	24.03 ± 20.45
	CO	59.19 ± 15.01	52.27 ± 9.55^d	43.79 ± 8.62^d	32.52 ± 14.03^d	30.47 ± 11.98	9.92 ± 19.76
	CJ	41.96 ± 23.63	39.81 ± 25.11	34.72 ± 17.05^d	35.27 ± 8.05^d	32.59 ± 14.13	21.49 ± 10.59
	C	59.57 ± 9.39	43.84 ± 16.74	40.42 ± 15.35	38.66 ± 14.78	38.66 ± 15.00	32.02 ± 20.00
	D	57.14 ± 0.00	44.40 ± 18.08^c	16.60 ± 0.00^c	7.34 ± 0.00^c	22.39 ± 0.00	27.03 ± 0.00
HL60	P	87.14 ± 2.97	90.56 ± 4.01^c,d	84.93 ± 6.12^c,d	73.21 ± 5.14^c,d	75.74 ± 5.29	65.65 ± 10.18
	Pt	79.69 ± 9.69	77.45 ± 5.43^c,d	69.75 ± 4.81^d	59.67 ± 4.81	70.00 ± 6.72^c,d	32.69 ± 5.27^c,d
	L	83.29 ± 5.49^d	75.38 ± 6.34^d	73.12 ± 3.11^d	48.52 ± 4.27^c	5.68 ± 8.93^c,d	10.77 ± 10.86^c
	S	37.21 ± 18.60^d	31.06 ± 10.11^c	43.85 ± 6.20^c	30.90 ± 8.31^c	34.88 ± 13.82^c,d	28.41 ± 4.37^c,d
	CO	74.63 ± 4.46	66.84 ± 5.06^d	65.40 ± 5.82^d	64.42 ± 1.43^d	68.62 ± 4.27^d	57.18 ± 16.59
	CJ	71.39 ± 2.31^d	70.53 ± 2.41^d	69.55 ± 5.41^d	63.57 ± 3.25^d	64.24 ± 5.63^d	55.88 ± 6.12^c
	C	68.21 ± 14.49	67.10 ± 5.04	73.44 ± 3.99	63.78 ± 2.41	71.33 ± 3.92	77.67 ± 2.35
	D	77.71 ± 2.55^c	44.04 ± 6.56^c	46.01 ± 4.29	45.40 ± 17.38^c	47.85 ± 1.43^c	53.03 ± 2.10

^c,dMean differences are significant (p < 0.05) between extracts and colchicine (c), demecolcine (d) standards and between standards according to Student’s t-test.

C, colchicine; D, demecolcine.

Table 7.  IC₅₀ values of standards and C. baytopiorum extracts in MTT test.

Standards and extracts	IC50 value (μg/mL)
	K562	HL60
C	247	< 0.1
D	286	180.l
P	233.3	< 0.1
Pt	44	0.6
L	300	6.5
S	70	394
CO	76.6	< 0.1
CJ	> 500	< 0.1

C, colchicine; D, demecolcine.

Figure 2.  Cytotoxicity % graphs of C. baytopiorum extracts (in five different concentrations) and standards (C, D) on K562 and HL60. P, perigon; Pt, perigon tube; L, leaf; S, seed; CJ, corm (June); CO, corm (October).

When the results were evaluated for chemotaxonomic purposes, only tropolonic alkaloids and flavonoids (apigenin and luteolin) were isolated, and the presence of phenolic acids including caffeic acid and 2-hydroxy-6-methoxybenzoic acid, which have an importance for plants belonging to the subfamily Wurmbaeoideae (Potesilova et al., 1976; Ondra et al., 1995), were identified. 2-Demethyl-γ-lumicolchicine can be formed as an artifact during the extraction and has no chemotaxonomic importance.

This is the first report of the chemical composition and cytotoxic activity of C. baytopiorum CD Brickell. 3-Demethyl-N-formyl-N-deacetylcolchicine, 2-demethyl-γ-lumicolchicine, and 2-demethylcolchifoline were determined for the first time in Turkish Colchicum species. This study showed that C. baytopiorum is one of the richest Colchicum species, having a high quantity of alkaloids, among the studied species growing in Turkey, and is an important species with its variety of alkaloids and phenolic compounds.

Acknowledgements

This work was supported by The Scientific Research Projects of Istanbul University (Project No: T-435/08032004)

Declaration of interest: The authors report no conflicts of interest.