Study On Chemical Constituents And Anti-AD Effect Of Hemp Seed Water Extract

Hemp seed(Fructus cannabis)is the dried and mature fruit of Cannabis sativa L.Many studies have shown that hemp seed extract has antioxidant,anti-aging,antiinflammatory,neuroprotective,and pharmacological effects such as improving the learning and memory abilities of chemical-induced Alzheimer’s disease(AD)model mice.Although many studies have mentioned the anti-AD effect of hemp seed,little is known about the chemical constituents of the water extract of hemp seed,and the therapeutic material of hemp seed are not clear.In recent years,the relationship between AD and intestinal flora has been reported,and it is worth exploring the potential of traditional Chinese medicine to exert anti-AD effects through flora regulation.In this paper,the water extracts of hemp seed were chemically separated by a variety of column chromatography methods,and the structures of the obtained compounds were identified by nuclear magnetic resonance,mass spectrometry and other data,as well as literature comparison methods.A total of 27 compounds were isolated and identified,including seven nucleosides:2’-O-Methyluridine(1),Adenosine(2),Thymidine(3),2’-O-Methyladenosine(4),2’-O-Methylguanosine(5),Guanosine(6),N,N-dimethylguanosine(7);five phenylpropanamides:4-[(E)-pcoumaroylamino]butan-1-ol(8),N-trans-coumaroyltyramine(9),N-transcaffeoyltyramine(10),N-trans-feryroyltyramine(11),((E)-N-(2-hydroxybutyl)-3-(4hydroxyphenyl)prop-2-enamide)(24);three organic acids:Dihyroferulic acid(12),Vanillc acid(13),Phenylalanine(14);three alkaloids:1-Methyl-1,2,3,4-tetrahydro-βcarboline-3-carboxylic acid(15),Orychophragines-A(16),Perlolyrine(26);two cyclic dipeptides:Cyclo(Phe-Ser)(17),Cyclo(Tyr-IIe)(25);and Uracil(19),Tryptophan(18),five others compounds:p-Hydroxybenzaldehyde(20),p-Cresol(21),Butyl 1Himidazole-4-carboxylate(22),1-(β-D-ribofuranosyl)-1H-1,2,4-triazone(23),Crotonine(27).Among them,20 compounds were isolated from hemp seed for the first time.Here nucleosides,cyclic dipeptides,pyrimidines and imidazoles are the first reported classes from hemp seed.In order to carry out anti-AD animal experiments,a quantitative study was conducted on the water extract of hemp seed after alcohol precipitation(Extract of Fructus Cannabis,EFC),with the two main chemical components Uracil and Adenosine as the reference substances,and their content in the extract was 1.30 mg/g and 2.04 mg/g,respectively.APP/PS1 model mice were selected for anti-AD and gut flora regulation animal experiments,and the EFC high-dose group(1.8 g/kg/d),medium-dose group(1.2 g/kg/d),and low-dose group(0.6 g/kg/d)were the administration groups,and the blank model group and the wild-type control groups were also set.The results of the novel object recognition experiment showed that EFC could enhance the novel object recognition ability of APP/PS1 mice;the Nissl staining results showed that EFC could increase the number of Nissl bodies in the hippocampus of APP/PS1 mice;the thioflavin-s staining showed that EFC can reduce the deposition of senile plaques in the hippocampus;16S rDNA amplicon of mouse fecal flora Sequencing results showed that EFC can reduce the relative abundance of Cyanobacteria,Proteobacteria,Desulfobacterota;increase the relative abundance of Actinobacteriota,and inhibit the rise of Prevotellaceae.These changes in the flora may be related to its anti-AD effect,which needs to be further explored.Chemical studies in this paper revealed the rich chemical composition of hemp seed water extract.Anti-AD animal experiments evaluated its efficacy on APP/PS1 model animals for the first time.The study of microflora will contribute to further explore the relationship between microflora changes and anti-AD effect.