Authenticity Evaluation Of Traditional Chinese Medicine Puffballs And Comparative Study On Their Hemostatic Activities

The Chinese medicinal puffballs are commonly collectively known as"Lasiosphaera seu Calvatia"（Latin name for this kind of Chinese herbal medicine）in Chinese pharmacopoeia.In order to understand the current market situation and application types of the traditional Chinese medicinal puffballs and provide scientific basis for their clinical application.On the basis of literature review,this paper carried out market investigation,authenticity identification and inspection,resource investigation,infrared and gas chromatography analysis and comparative study on hemostatic activity of traditional Chinese medicinal puffballs.The details are as follows:Literature review:Herbal textual research on Chinese medicinal puffballs were conducted,and the chemical compositions and pharmacological effects of Chinese medicinal puffballs were summarized.Experimental part:（1）Market investigation:The survey of the traditional Chinese medicinal puffballs were conducted in several major medicinal markets in China using representative and random methods.And the samples were examined for moisture,ash moisture,total ash,acid-insoluble ash and extract.Results:The market survey showed that five species were identified from the 47 samples randomly selected from the representative medicinal markets of Hehuachi,Anguo,Bozhou,and Sankeshu,among which 13 were Calvatia gigantea,1 was Calvatia lilacina,and only 14 samples（accounting for 30%）were“Lasiosphaera seu Calvatia”as the Chinese medicinal in China Pharmacopoeia;The remaining 33 samples were Lycoperdon perlatum,Mycenastrum corium,and Bovistella utriformis,of which Bovistella utriformis was the species with the largest number of samples in the survey（64%）.（2）Resource investigation:From 2021 to 2022,more than 50 samples of puffballs were collected in Jilin Province and Inner Mongolia Autonomous Region,and 11 species were identified,belonging to 7 genera and 2 families.They are Bovista aestivalis,Bovista promontorii,Lycoperdon perlatum,Vascellum pretense,Calvatia gigantean,Lycoperdon cau datum,Lycoperdon mole,Bovistella sinensis,Mycenastrum corium,Scleroderma Bovista,Scleroderma cepa.（3）Infrared and temperament analysis:Infrared spectroscopy and GC-MS analysis were performed on the fruiting bodies of five species of Chinese medicinal puffballs,such as,Calvatia gigantean,Calvatia lilacina,Bovistella utriformis,Lycoperdon perlatum and Mycenastrum corium.The results showed that the five Chinese medicinal puffballs shared common peaks at 3370 cm^-1,2925 cm^-1,2855 cm^-1,1640–1550 cm^-1,1380 cm^-1,1075 cm^-1and870 cm^-1.The chemical components of the fruiting bodies of five different puffballs have both common components and differences.All the five puffballs contain a large amount of alkane chemical components.The ether chemical components in the fruitingbodies of C.gigantean、C.lilacina and B.utriformis are more,while the ester chemicalcomponents in the fruiting body of M.corium is relatively more,and the aldehyde chemical components in the fruiting body of L.perlatum is more.In which only that C.gigantean contain nonyl tetradecyl ether,1,2,3,4-tetramethylbenzene and the like;Only C.lilacina containsβ-caryophyllene and toluene,etc;Only B.utriformis contains D-ismenthol,P-Xylene,etc;Only M.corium contains N-octacosane,ALPHA-pinene and the like;Only L.perlatum contains isobutyric acid,decyl undecyl carbonate,2,6-di-tert-butylbenzoquinone,etc.（4）Comparative study of hemostatic activity:Comparative study on the hemostatic activity of C.gigantea,C.lilacina,and B.utriformis.Pharmacological experiments included determination of bleeding time and coagulation time of mice by tail-cutting method and capillary method.The four coagulation indexes were measured by blood coagulation analyzer,and the coagulation factors in mouse plasma were determined by ELISA kit.The platelet count（PLT）and mean platelet volume（MPV）in whole blood of mice were measured.The relative protein expression of PI3K/Akt/GSK3βsignaling pathway in platelets was detected by Western-Blot.The comparison of hemostatic activities showed that compared with the blank group,all three kinds of Chinese medicinal puffballs showed hemostatic activities.But the aqueous extracts of C.gigantea and C.lilacina had more significant hemostatic effects.The water extract group of C.gigantea could significantly enhance the coagulation function,and the hemostatic effect of the high dose group was extremely significant.This study provided a scientific basis for regulating the market of puffballs medicinal materials and rational utilization of puffballs resources in clinical practice of traditional Chinese medicine.