Discovery Of Novel Phthalides On Relaxation The Thoracic Aorta For "Treatment Of Dizziness" Of The Stem And Leaf Of Ligusticum Chuanxiong Hort.

The stems and leaves of Ligusticum chuanxiong Hort.,also known as Mi Wu,was the dried aerial parts of Ligusticum chuanxiong Hort.<Ming Yi Bie Lu>recorded that Mi Wu was able to treat dizziness.However,the rhizomes of Ligusticum chuanxiong Hort.were mainly used instead of the aerial parts in the clinic.These discarded parts from the aerial parts caused waste of resources and environmental pollution.It′s considered that the applications of Mi Wu and the association with hypertension,the pharmacological activities of phthalides and potential for the treatment of CCVDs,and the importance of vasodilators in hypertension.In this paper,we collated the records of Materia Medica between Mi Wu and Ligusticum chuanxiong Hort.We investigated the chemical composition of stems and leaves of Ligusticum chuanxiong Hort.Secondly,its vasorelaxant effect was investigated by isolated rat thoracic aortic rings.Finally,we explored the vasodilatory mechanisms of some active compounds.We aimed to illustrate the traditional efficacy and application value of Mi Wu.1.The herbal authentication of Mi Wu.The Mi Wu source before the Han Dynasty could not be determined,but could be identified as Umbelliferae through the literature and Materia Medica.After the Han Dynasty,the Mi Wu was identified as the stems and leaves of Ligusticum chuanxiong Hort from the<Ming Yi Bie Lu>.Herbs of past dynasties describeed the Mi Wu as a seedling leaf of Chuanxiong Hort and could treat dizziness.2.Isolation of phthalides from stems and leaves of Ligusticum chuanxiong Hort.The compounds were isolated by extensive chromatographic techniques including MCI,silica gel,Sephadex LH-20,and semi-preparative HPLC,and the structure was identified by HRMS,NMR,IR,ECD,X-ray.34 phthalides were isolated and identified from the petroleum ether parts,including 15 monophthalides and 19 phthalide dimers.Among them,compounds 1 and 2 are novel skeletal compounds and compounds 3～9are new compounds.All compounds were isolated from the stems and leaves of Ligusticum Chuanxiong,which compounds 1～9,13,19,23,24,34 were isolated from Ligusticum Chuanxiong for the first time.3.Vasodilation and structure-activity analysis of phthalides.All tested phthalides（12μM）could relax blood vessels through KCl（60 m M）induced constriction model of isolated thoracic aortic ring in rats.Compounds 1,3,5,6,7,11,15,18 had significant vasodilatory effect with IC₅₀ were 9.2,8.0,5.9,7.1,6.2,8.8,8.2,7.8μM(EC₅₀<10μM),respectively.Furthermore,we also analyzed the structure-activity relationship of vasodilatory activity of phthalides.the activity of phthalide dimers was significantly better than that of monophthalides.The activity of monophthalide was closely related to the type and position of its substituents,while the activity of phthalide dimer was closely related to its unsaturation,polymerization position,polymeric monomer,substituents,enantiomerism,etc.4.Mechanisms of vasodilation.Compounds 3（20μM）and 7（20μM）could significantly reduce the intracellular calcium concentration in VSMCs,and fluorescence values（F）were 33,18 VS control group（F=148）.The maximal vasodilation rates of compound 3（18μM）after pre-treatment with the four potassium channel blockers were TEA（94%）,4-AP（67%）,Gly（100%）and Ba Cl₂（60%）;EC₅₀were 8.6,13.6,8.5 and 15.6μM,respectively.However,the maximal vasodilation rates of compound 7（12μM）were TEA（56%）,4-AP（41%）,Gly（50%）,Ba Cl₂（52%）;EC₅₀were greater than 10μM.In addition,the compounds 1,3,5a also significantly inhibited the current of Ca_v1.2 by patch clamp electrophysiological experiments.EC₅₀ were 3.3,16.8 and 7.5μM,respectively.It was showed that the vasodilatory activity of phthalides was mainly through VOCC,which decreased the intracellular calcium concentration of VSMCs,such as potassium channels and L-type voltage ion channels.This study was the first to investigate the phthalides of the stems and leaves of Ligusticum chuanxiong（Mi Wu）through herbal authentication and modern techniques such as chemistry,cell biology and electrophysiology.These results not only preliminarily explained the Mi Wu"treatment of dizziness",but also enriched phthalides components and discovered novel vasodilators.At the same time,it was also beneficial to the utilization of non-medicinal resources of chuanxiong,which promoted the comprehensive development of medicinal resources for the Genuine Crude Drugs in Sichuan,and provided references for similar studies.