Coumaroylspermidines From Carthamus Tinctorius L. And Their Biological Activities

Safflower, the dry tubular flower of Carthamus tinctorius L., is a member of the family Compositae. The flower of the plant is utilized for producing herbal medicines for over 2500 years, known to be effective of activating blood circulation and stimulating meridians, removing stasis and relieving pain, lowering blood pressure and anting-hyperlipidemia. Phytochemical studies by others have showed that more than 250 compounds have been found from safflower containing flavonoids, alkaloids, poly acetylenes, alkyldiols, organic acids, steroids, coumaroylspermidines and so on. Coumaroylspermidines are specific constitutes in safflower which containing H2N(CH2)3NH(CH2)4NH2 construction unit. They have been reported to be effective in many biological activities. However, the studies investigating biological activities of coumaroylspermidines all studied one configuration of the coumaroylspermidine, there are no study systematically investigating biological activities of different configuration of coumaroylspermidines and the connection between configuration and their biological activities. There is one study showed that N1, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine has inhibition effect on 5-HT reuptake. This article separated and purified 4 coumaroylspermidines and investigated the inhibition of 5-HT transportation of different configuration of coumaroylspermidines and preliminary discussed the connection between configuration and their inhibition activity. In addition, there have been many articles studying the separation and purification and biological activities of coumaroylspermidines, however, there are no article studying the quantitative determination of coumaroylspermidines. This paper first established the method for quantitative determination of coumaroylspermidines which can determine the content of 4 coumaroylspermidines at the same time. This provide scientific basis for quantitative determination of coumaroylspermidines. This paper is divided into the following areas:1. The separation and purification of coumaroylspermidines:The dry safflower was extracted by ethyl acetate, the ethyl acetate extracts were fractioned by medium pressure flash chromatography. HPLC-Q-Exactive -MS was used as a powerful tool to identify constitutes from ethyl acetate extracts of Carthamus tinctorius L.. The position which contains coumaroylspermidines was found and then the target compounds were isolated by sephadex LH-20 column chromatography and preparative HPLC. This paper isolated 4 coumaroylspermidines quikly, and also isolated one aromatic compound and one flavonoid glycoside, the structures were identified by ESI-MS, 1H-NMR spectral data as N1,N5,N10-(Z)-tri-p-coumaroylspermidine (1), N1,N5-(E)-N10-(Z)-tri-p-coumaroylspermidine (2), N1(E)-N5-(Z)-N10-(E)-tri-p-coumaroyl-spermidine (3), N1,N5,N10-(E)-tri-p-coumaroylspermidine (4), syringin (5), kaempferol-3-O-rutinoside (6).2. Photoisomerization phenomenon of coumaroylspermidines:There are studies showed that the coumaroylspermidines of E configuration undergo (E)→(Z) isomerization under UV light or normal daylight conditions, so the photoisomerization phenomenon of the 4 coumaroylspermidines were investigated in this paper. The photoisomerization phenomenon of coumaroylspermidines was observed by HPLC. The results showed that coumaroylspermidine could transform into each other when irradiated by light and the four coumaroylspermidines reached a same equilibrium state eventually. However, the equilibrium state under daylight was different from the equilibrium state under 254 nm UV light. However, the coumaroylspermidines were stable and keeping high pure when placed in dark place.3. Biological activities of coumaroylspermidines:The inhibition evaluation of coumaroylspermidines on 5-HT reuptake was performed by rat synaptosomes. This paper primarily discussed the connection between configuration and their inhibition activitied of coumaroylspermidines. The results showed that the four coumaroylspermidines all having inhibition activity on 5-HT reuptake. However, the inhibition effect was different for different isomers, coumaroylspermidines 2 and 3 were better than Z,Z,Z-isomer coumaroylspermidine 1.This paper also investigated the prothrombin time (PT) of the 6 compounds by semi-automated blood coagulation analyzer which were separated and purified from Carthamus tinctorius L.The results showed that the six compounds all could not extend prothrombin time (PT) of blood plasma.4. The quantitative determination of coumaroylspermidines:This paper first established the method for quantitative determination of coumaroylspermidines using HPLC method and the content of coumaroylspermidines 1,2,3,4 as indicator. This method can determine the content of 4 coumaroylspermidines at the same time. The methodological evaluation showed that this method is simple and convenient, accurate and reliable. This paper also determined the content of the 4 coumaroylspermidines in Carthamus tinctorius L. This paper provide scientific basis for quantitative determination of coumaroylspermidines.