Safflower yellow extract inhibits thrombus formation in mouse brain arteriole and exerts protective effects against hemorheology disorders in a rat model of blood stasis syndrome

Figures & data

Figure 1. UPLC-DAD (A) and ion chromatograms of SYE in positive (B) and negative (C) ionization models.

Table 1. Identification details of the components in SYE using UFLC-Q-TOF-MS/MS technique.

No.	Retention time (min)	Molecular formula	[M+H]^+ (△ppm)	[M-H]^− (△ppm)	Fragment ions (positive mode)	Fragment ions (negative mode)	Ingredients
1	1.200	C5H5N5	136.0615 (−2.2)		119.0342 [M+H–NH3]^+, 92.0231 [M+H–NH3–HCN]^+, 65.0149		Adenine
2	1.440	C5H7NO3	130.0497 (−1.4)	128.0371 (8.2)	84.0447 [M+H–HCOOH]^+, 56.0505 [M+H–HCOOH–CO]^+		Pyroglutamic acid
3	2.065	C10H13N5O4	268.1038 (−0.8)	266.0904 (3.3)	136.0601 [M+H–Ribose]^+, 119.0336 [M+H–Ribose–NH3]^+		Adenosine
4	2.320	C10H13N5O5	284.0981 (−3.0)	282.0845 (0.5)	152.0564[M+H–Ribose]^+, 135.0293[M+H–Ribose–NH3]^+, 110.0343[M+H–Ribose–CN2H2]^+	150.0375 [M–H–Ribose]^–, 133.0179 [M+H–Ribose–NH3]^–,	Guanosine hydrate
5	2.927	C9H11NO2	166.0858 (−2.7)	164.0726 (3.7)	120.0804 [M+H–HCOOH]^+, 103.0546 [M+H–HCOOH–NH3]^+, 77.0392 [C6H5]^+	147.0453 [M–H–NH3]^–, 103.0562 [M–H–CO2–NH3]^–	Phenylalanine
6	7.177	C10H7NO3	190.0491 (−2.6)	188.0361 (4.2)	172.0371 [M+H–H2O]^+, 144.0442 [M+H–H2O–CO]^+, 116.0483 [M+H–H2O–2CO]^+,		Kynurenic acid
7	9.134	C27H32O16	613.1773 (0.8)	611.1617 (0)	451.1221 [M+H–Glc]^+, 433.1137 [M+H–Glc–H2O]^+ 313.0655, 211.0224	491.1189 [M–H–C4H8O4]^–, 473.1091 [M–H–C4H8O4–H2O]^–, 325.0705 [M–H–2C5H10O4–H2O]^–, 283.0601	Hydroxysafflor yellow^a
8	9.988	C33H40O22	789.2098 (1.8)	787.1963 (2.9)	627.1551 [M+H–Glc]^+, 465.1024 [M+H–2Glc]^+, 303.0498 [M+H–3Glc]^+,	625.1442 [M–H–Glc]^–, 462.0903 [M–H–2Glc]^–, 301.0384 [M–H–3Glc]^–,	6-Hydroxykaempferol-3,6,7-tri-O-β-glucoside
9	11.621	C27H30O15	595.1668 (1.7)	593.1520 (1.3)	433.1111 [M+H–Glc]^+, 397.0925 [M+H–Glc–2H2O]^+, 301.0708, 181.0127	473.0935 [M–H–C4H8O4]^–, 447.1137 [M–H–C9H6O2]^–, 430.0902 [M–H–Glc]^–	Safflor yellow A
10	12.202	C9H8O3	165.0541 (−2.1)	163.0410 (4.8)	147.0436 [M+H–H2O]^+, 119.0497 [M+H–HCOOH]^+, 91.0545, 65.0403	119.0501 [M–H–CO2]^–, 93.0357	p-Coumaric acid
11	15.376	C27H30O17	627.1561 (0.9)	625.1415 (0.8)	465.1011 [M+H–Glc]^+, 303.0494 [M+H–2Glc]^+	463.0884 [M–H–Glc]^–, 301.0343 [M–H–2Glc]^–	6-Hydroxykaempferol-6,7-di-O-β-glucoside
12	15.835	C27H30O17	627.1562 (1.0)	625.1421 (1.8)	465.1023 [M+H–Glc]^+, 303.0500 [M+H–2Glc]^+	463.0901 [M–H–Glc]^–, 301.0351 [M–H–2Glc]^–	6-Hydroxykaempferol-3,6-di-O-β-glucoside
13	15.956	C33H40O21	773.2155 (1.7)	771.2014 (2.7)	627.1552 [M+H–Rha]^+, 465.1038 [M+H–Rut]^+, 303.0502 [M+H–Rut–Glc]^+	609.1514 [M–H–Glc]^–, 463.0911 [M–H–Rut]^–, 301.0352 [M–H–Rut–Glc]^–	6-Hydroxykaempferol-3-O-β-rutinoside-6-O-β-D-glucoside
14	18.216	C21H20O12	465.1032 (0.9)	463.0883 (0.2)	303.0501 [M+H–Glc]^+	301.0366 [M–H–Glc]^–,	6-Hydroxykaempferol-3-O-β-D-glucoside
15	18.229	C27H30O16	611.1607 (0)	609.1472 (1.5)	465.1033 [M+H–Rha]^+, 303.0505 [M+H–Rut]^+	301.0355 [M–H–Rut]^–	6-Hydroxykaempferol-3-O-β-rutinoside
16	18.727	C27H30O16	611.1611 (0.7)	609.1470 (1.4)	449.1066 [M+H–Glc]^+, 287.0551 [M+H–2Glc]^+	285.0407 [M–H–2Glc]^–	Kaempferol-3-O-β-sophorose
17	19.007	C27H31NO14	594.1821 (0.7)	592.1676 (0.7)	576.1708 [M+H–H2O]^+, 432.1288 [M+H–Glc]^+, 414.1174 [M+H–Glc–H2O]^+, 354.0972 [M+H–Glc–C2H4O2–H2O]^+, 312.0863 [M+H–Glc–C4H8O4]^+	472.1118 [M–H–C4H8O4]^–, 364.0800 [M–H–Glc–H2O–OCH2]^–, 339.0745, 244.0238	Hydroxycartormin
18	19.180	C27H30O16	611.1613 (1.1)	609.1475 (0.7)	465.1036[M+H–Rha]^+, 303.0496[M+H–Rut]^+	301.0359 [M–H–Rut]^–,	Quercetin-3-O-β-rutinoside (Rutin)^a
19	19.184	C15H10O7	303.0501 (0.4)	301.0340 (−4.5)	153.0164[M+H–RDA]^+		Quercetin^a
20	19.407	C21H20O12	465.1032 (0.9)	463.0885 (0.7)	303.0503[M+H–Glc]^+	301.0360 [M–H–Glc]^–,	Quercetin-3-O-β-glucoside^a
21	19.799	C48H54O27	1063.2947 (1.7)	1061.2816 (3.3)	901.2400 [M+H–Glc]^+, 613.1730 [M+H–C21H22O11]^+, 451.1239 [M+H–C21H22O11–Glc]^+, 433.1124 [M+H–C21H22O11–Glc–H2O]^+, 289.0705	611.1651 [M–H–C21H22O11]^–, 449.1112 [C21H21O11]^–	Safflor yellow B
22	20.360	C27H30O15	595.1663 (0.9)	593.1517 (0.8)	449.1073 [M+H–Rha]^+, 287.0553 [M+H–Rut]^+	285.0406 [M–H–Rut]^–	Kaempferol-3-O-β-rutinoside
23	20.388	C48H52O26	1045.2851 (3.0)	1043.2711 (3.4)	883.2274 [M+H–Glc]^+, 721.1766 [M+H–2Glc]^+, 433.1130,313.0704	923.2335 [M–H–C4H8O4]^–, 593.1551 [M–H–C21H22O11]^–, 449.1093 [C21H21O11]^–, 287.0566	Anhydrosafflor yellow B
24	20.626	C15H10O6	287.0553 (1.0)		153.0173 [M+H–RDA]^+,		Kaempferol^a
25	20.627	C21H20O11	449.1086 (1.3)	447.0935 (0.5)	287.0554 [M+H–Glc]^+,	285.0333 [M–H–Glc]^–	Kaempferol-3-O-β-glucoside^a
26	20.648	C21H20O11		447.0935 (0.2)		285.0413 [M–H–Glc]^–, 255.0297 [M–H–C7H10O5–H2O]^–, 227.0342	Quercetin-3-C-β-glucoside
27	20.953	C21H18O11	447.0930 (1.3)	445.0780 (0.9)	271.0601 [M+H–GlcUA]^+	269.0449 [M–H–GlcUA]^–	Apigenin-7-O-β-glucuronic acid
28	21.424	C27H29NO13	576.1715 (0.5)	574.1571 (0.8)	414.1178 [M+H–Glc]^+, 396.1100 [M+H–Glc–H2O]^+, 312.0866 [M+H–Glc–C4H6O3]^+, 276.0494 [M+H–Glc–C4H8O4–H2O]^+	454.0990 [M–H–C4H8O4]^–, 364.0828 [M–H–Glc–OCH2–H2O]^–, 411.0957, 231.0151	Cartormin
29	21.826	C30H30O14	615.1719 (1.7)	613.1573 (1.6)	453.1190 [M+H–Glc]^+, 289.0713 [M+H–Glc–C9H8O3]^+, 169.0132, 165.0545	551.1569 [M–H–CO2–H2O]^–, 287.0586 [M–H–C9H6O2–Glc–H2O]^–, 361.1093	Safflomin C

Note: Ribose: C₅H₈O₄; Rha: C₆H₁₀O₄; Rut: C₁₂H₂₀O₉; Glc: C₆H₁₀O₅; GlcUA: C₆H₈O₆.

^aIdentification compared to reference substances.

Figure 2. Chemical structural formulas of the identified constituents in SYE. Six constituents were confirmed by retention time and MS data of reference substances, and 23 constituents were characterized by MS data and related studies.

Figure 3. Time for arteriole occlusion of thrombus induced by laser irradiation in mice vessels. SYE was applied at three doses of 13, 26 and 52 mg/kg body weight by intraperitoneal injection on C57BL/6J mice. After 30 min, thrombus formation was induced in brain arterioles by laser irradiation and the time necessary for arteriole occlusion was measured. SYE was compared with heparin sodium injection (0.2 mL/kg). The graph depicts the mean values ± SD (n = 6); *p < 0.05 and **p < 0.01 as compared with the control.

Figure 4. Arteriole diameter measurement during laser irradiation in the control (A) and SYE (B) group with high dosage. A brain arteriole of C57BL/6J mice with diameter of 18 ± 3 μm was selected as the target vessel for occlusion and was zoomed in at 20× magnification under TPLSM.

Figure 5. Ratio of vessel diameter. SYE was applied at three doses of 13, 26 and 52 mg/kg body weight by intraperitoneal injection on C57BL/6J mice. After 30 min, thrombus formation in brain arteriole was induced by laser irradiation and the diameter changes were measured and calculated as the ratio of diameter change to the initial diameter. The graph depicts the mean values ±SD (n = 6); *p < 0.05 and **p < 0.01 as compared with the control.

Figure 6. Hemorheology parameters measurements: WBV (A) at different shear rates; EAI, ERI and RCEI (B); APTT and PT (C); MPAR (D). SYE was applied at three doses of 1.5, 3.0 and 4.5 mg/kg body weight by intramuscular injection on SD rats. The graph depicts mean values ± SD (n = 8); ^#p < 0.05 and ^##p < 0.01 as compared with the normal, *p < 0.05 and **p < 0.01 as compared with the model.