Secondary metabolites of Centaurea calolepis and evaluation of cnicin for anti-inflammatory, antioxidant, and cytotoxic activities

Figures & data

Table 1.  ¹H-NMR data obtained with compounds 1–4 (δ ppm).

Position of proton	1^a (J)	2^b (J)	3^c (J)	4^a(J)
1	5.09^†	–	–
2	2.62 ddd (2, 5.2, 8.4)	–	–	2.0–2.16 m
	2.33 ddd
3	2.36 ddd 2.01^†	6.44 s	6.6 s	5.58 m
4	–	–	–	3.70 m
5	4.94 d (9.6)	–	–	4.16 m
6	5.2 dd (8.4, 9.6)	–	–	2.0–2.16 m
7	3.3 m	–	–	–
8	5.11^†			7.62 d (12)
9	2.59 dd 2.21			6.36 d (12)
10	–	–	–	–
11	–	–	–	–
12	–	–	–	–
13	6.18 d (3.2) 5.84 d (3.2)	–	–	–
14	1.53 s	–	–	–
15	4.01 d (13.6) 4.26 d (13.6)	–	–	–
16	–	–	–	–
17	–	–	–	–
18	4.55 dd (3.6, 6)	–	–	–
19	3.71 dd (3.6, 11.6) 3.48 dd (6, 11.6)	–	–	–
20	6.38 s 6.07 s	–	–	–
1′	–	–	–	–
2′	–	7.40 d (2.0)	7.92 d (8.8)	7.16 s
3′	–	–	6.91 d (8.8)	–
4′	–	–	–	–
5′	–	6.82 d (8.2)	6.91 d (8.8)	7.05 d (8.4)
6′	–	7.43 dd (2.0, 8.2)	7.92 d (8.8)	6.79 d (8.4)
3′-OMe	–	–	–	3.87 s
6-glucose
1′′	–	4.70 d (9.8)	4.75 d (10)	–
2′′	–	3.88 bs	3.08–3.61^†	–
3′′	–	3.27^†	3.08–3.61^†	–
4′′	–	3.27^†	3.08–3.61^†	–
5′′	–	3.24^†	3.08–3.61^†	–
6′′	–	3.67 dd (2.1, 11.8) 3.54 dd (4.7, 11.8)	3.58 m 3.63^†	–
8-glucose
1′′′		4.84 d (9.7)
2′′′		3.82 t (9.7)
3′′′		3.34^†
4′′′		3.38^†
5′′′		3.32^†
6′′′		3.75 d (11.2) 3.58 dd (5.7, 11.2)
8-arabinose
1′′′			4.95 brs
2′′′			3.64–3.87^†
3′′′			3.64–3.87^†
4′′′			3.64–3.87^†
5′′′			3.64–3.87^†

^aIn CD₃OD at 400 MHz.

^bIn DMSO-d₆ 600 MHz.

^cIn DMSO-d₆ 400 MHz.

^†Signal pattern was unclear due to overlapping.

Table 2.  ¹³C-NMR data obtained with compounds 1–4 (δ ppm).

Position of carbon	1	2	3	4
1	129.6			75.8
2	26.8	163.5	165.3	31.5
3	35.01	102.1	102.5	73.4
4	142.3		183.0	73.9
5	130.1	159.7	161.5	73.9
6	78.6	108.8	104.5	33.0
7	53.2	159.7	162.4	169.8
8	71.9	105.1	104.5	117.2
9	48.9	155.3	155.1	147.4
10	133.2		104.0
11	137.4
12	166.5
13	125.2
14	171.1
15	66.6
16	171.2
17	145.4
18	74.4
19	60.8
20	127.2
1′		121.9	122.1	128.6
2′		113.6	128.9	115.7
3′		150.6	115.9	151.3
4′		146.3	162.4	150.4
5′		116.0	115.9	112.5
6′		119.4	128.5	124.7
3′-OMe				57.2
COOH				180.5
6-Glucose
1′′		74.4	71.1
2′′		71.6	70.1
3′′		79.0	79.1
4′′		70.4	69.7
5′′		81.2	81.4
6′′		61.3	62
8-Glucose
1′′′		74.8
2′′′		72.0
3′′′		79.2
4′′′		70.8
5′′′		82.0
6′′′		61.7
8-Arabinose
1′′′			74.7
2′′′			69.5
3′′′			77.7
4′′′			69.5
5′′′			70.4

^aIn CD₃OD at 100 MHz.

^bIn DMSO-d₆ 150 MHz.

^cIn DMSO-d₆ 100 MHz.

Figure 1.  Chemical structures of 1-4 isolated from Centaurea calolepis.

Figure 2.  Chemical structures of compounds of Centaurea calolepis determined by direct thin–layer chromatography and high performance liquid chromatography comparisons with authentic samples.

Table 3.  Inhibition of NF-κB, Sp-1, iNOS and toxicity to macrophages by cnicin (values are IC₅₀ in µM).

Compound	NF-κB	SP-1	iNOS	Toxicity to macrophages
Cnicin	1.8 ± 0.1	16.0 ± 1.3	6.5 ± 0.8	7.9 ± 1.0
Parthenolide^a	6.4 ± 0.8	49.3 ± 1.8	1.7 ± 0.4	41.3 ± 6.2

Inhibition of NF-kB and SP-1 was determined in human chondrosarcoma (SW1353) cells induced by phorbol myristate acetate and inhibition of iNOS activity was determined in mouse macrophages (RAW264.7 cells) induced by lipopolysaccharide as described in materials and methods. Data is represented as mean ± SEM of three determinations.

iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor κB.

^aPositive control.

Table 4.  Cytotoxic activity of cnicin (values are IC₅₀ in µM).

	Cell lines
Compound	SK-MEL	KB	BT-549	SK-OV-3	Vero	LLC-PK11
Cnicin	14.0 ± 0.7	NC	18.3 ± 2.4	NC	NC	23.3 ± 2.2
Doxorubicin^a	1.1 ± 0.1	1.8 ± 0.2	1.7 ± 0.3	2.2 ± 0.1	>10	1.2 ± 0.1

NC, no cytotoxicity up to 25 µM; SK-MEL, malignant melanoma; KB, oral epidermal carcinoma; BT-549, breast ductal carcinoma; SK-OV-3, ovary carcinoma; Vero, monkey kidney fibroblast; LLC-PK₁₁, pig kidney epithelial cells.

Data is represented as mean ± SEM of three determinations.

^aPositive control.