Qualitative And Quantitative Study Of Chemical Constituents In Hemp Seed And Their Anti-AD Activity

Recent studies reported that hemp(Cannabis sativa L.)seed extract had antioxidant,anti-aging,anti-inflammatory,neuroprotection activities and the potential to improve the learning and memory damage induced by chemical drugs in Alzheimer's disease(AD)-like model mice.Neuroinflammation is an important pathological feature of AD.In our previous work,lignanamides from hemp seed showed anti-neuroinflammatory activity by preventing lipopolysaccharide(LPS)-induced activation of BV2 microglia cells.In order to further study the neuroprotective material basis of hemp seed,here,we continued studying the chemical constituents of hemp seed,and evaluated the protective effect of hemp seed extract on neuroinflammatory mice model.In this study,an ethanol extract of hemp seed was isolated by various column chromatography(macroporous resin,polyamide,ODS,sephadex LH-20),high-speed countercurrent chromatography(HSCCC)and high-performance liquid chromatography(HPLC);the isolated compounds were identified on the basis of nuclear magnetic resonance(NMR),MS,UV and IR analysis.Twenty compounds were isolated and identified from hemp seed,including fifteen phenylpropionamides:Cannabisin Q(1),coumaroylaminobutanol glucopyranoside(2),Cannabisin A(3),Cannabisin B(4),Cannabisin M(5),3,3'-demethyl-grossamide(6),Cannabisin F(7),Cannabisin G(8),N-trans-caffeoyloctopamine(9),N-trans-coumaroyloctopamine(10),N-trans-coumaroyltyramine(11),N-trans-feryroyltyramine(12),N-trans-caffeoyltyramine(13),(S)-N-(2-(4-hydroxyphenyl)-2-methoxyethyl)cinnamamide(14),4-[(E)-p-coumaroylamino]butan-1-ol(15);three glycosides:trans-ferulic acid-4-O-?-D-glucopyranoside(16),adenosine(17),sucrose(18);two monophenyl ring:p-hydroxybenzaldehyde(19),4-hydroxy-3-methoxybenzoic acid(20).Among them,1 and 2 are new compounds,compounds 9,10,14-18,20 were isolated from hemp seed for the first time.In view of the fact that the phenylpropionamides may play an important role in many effects of hemp seed,this paper optimized the extract process of phenylpropionamides,and qualitative and quantitative research were carried out on the phenylpropionamides in hemp seed extract by using HPLC-MS and HPLC methods.Fifteen phenylpropionamides were identified in hemp seed extract;the contents of Cannabisin A(CA),N-trans-caffeoyltyramine(NC)and total phenylpropionamides(TPA)in six batches of hemp seed collected from different places were determined using HPLC,and the content of CA ranged from 27.14 to 145.48 ?g/g,NC ranged from 23.93 to 113.13 ?g/g and TPA ranged from 129.39 to 641.90 ?g/g,respectively.The optimized extraction process was used to enrich the TPA from hemp seed and their neuroprotective effect on neuroinflammatory mice model was evaluated.In this work,eight-week-old male KM mouse were used.LPS(250?g/kg/d)was intraperitoneal injected for a week to make neuroinflammatory model.High-dose TPA(2 g/kg/d)group,low-dose TPA(1 g/kg/d)group and positive drug(piracetam 0.4g/kg/d)control group were set.The result of Morris water maze showed that TPA could significantly improve learning and memory impairment in LPS-induced neuroinflammation model mice;ELISA test revealed that low dose of TPA could significantly reduce the expression of inflammatory cytokines IL-1?,IL-6 and TNF-a in brain from LPS-induced mice;HE staining showed that TPA could improve the damage.of neurons in hippocampal CA3 region of mice induced by LPS;immunohistochemical staining showed that TPA had the effect of decreasing the expression of iNOS protein in hippocampus of LPS induced mice.This work further revealed the anti-AD material basis of hemp seed,together with our previous studies,providing theoretical basis for the utilization of hemp seed.