Study On The Chemicalconstituents In Vivo Of Acanthopanax Fruit And The Immunomodulatory Effects

The Acanthopanax belongs to Araliaceae that is is dried roots and rhizome.The plant have good effect to the function of skeleton,heart,spleen,kidney and anti-aging using Qi deficiency of spleen and lung,inappetency,deficiency of yin of both lung and kidney,chronic cough,aching lumbus and knees,deficiency of both heart and spleen,insomnia.The dried roots and rhizome was excessive used leading to the destruction with wild resources and plantation resources.The Acanthopanax fruit as a reproductive organ has a lot of nutrients.Using the resources will didn't effect the sustainable development of Acanthopanax resources.Study the chemical composition and the chemical composition in vivo of Acanthopanax fruit and detected the immunomodulatory effects will be propitious to sustainable development of Acanthopanax resources.Purpose:To analysis the chemical composition and the absorbed into blood of Acanthopanax senticosus fruit.To knows the immunomodulatory effects of Acanthopanax fruitMethods:1 The chemical composition analysis of acanthopanax senticosus fruit.:The 100g Acanthopanax fruit was extracted two times under relux in the 10 times amount 30%alcohol,every time for 1 hour,recovering alcohol under reduced pressure,until no alcohol flavor,freeze-dried.The freeze-dried powder was extracted one times under ultrasonic in the I times amount 50%methanol for 10 mins,centrifuged and the supernatant was got.Using mass spectrometry for the detection of the nuclear ratio will screened possible element.According to the fragments of the secondary ion of the chromatographic peaks determined the possible chemical structure.We compared with literature that determine the chemical composition of the corresponding chromatographic peak.2The chemical composition analysis of Absorbed into Blood of acanthopanax fruit extract:The freeze-dried powder of acanthopanax fruit was dissoved into the distilled water to make the solution with the concentration of 0.2mg/ml.The rat of Wistar was given orally distilled water and the solution of acanthopanax fruit after fasting at dose of 10ml/kg.We take the vein blood of hilar afer 60 minutes with anesthesia.The blood sample was separated,purified,gathered with the method of SPE.Finally,we used the UPLC-HD/MS method that has established to analyzing and screening the potential bioactive compounds from the blood sample,using a comparison methods of intuitive,multivariate data processing approach and the MetaboLynx tool.3The immunomodulatory effects of Acanthopanax fruitAccording to Inspection and Assessment Standard for Health Food issued that we use the test method.The clean Kunming mice were respectively given Acanthopanax fruit decoction with single dose of 12.5 g/kg,25 g/kg and 50g/kg every days,altogether 7days.After 30 days,we determined the thymus index,spleen index,the hemolytic plaque count,serum hemolysin level,carbon clearance index,delayed type hypersensitivity(DTH),natural killer cell activity,transformation of spleen lymphocyte and so onResults:1 We used the methods of analysis of Acanthopanax fruit with Ultra-high perfor mance liquid chromatographycoupled with time-of-fight mass finding 104 compounds of Acanthopanax fruit in negative ion mode including flavonoids,triterpenoid saponin,and lignans etc.at 24min.The Chemical composition is 4-O-Galactopyranuronosyl-galactopyranuronate?Sucrose?Melezitose?3-Flydroxy-2-butanyl6-O-[3,4-dihydroxy-4-(hydroxymethyl)tetrahydro-2-furanyl]-glucopyranosid?Protocatechuic acid-glucoside?Methyl5-({3,4-dihydroxy-4-(hydroxymethyl)tetralydro-2-furanyl]-glucopyranosyl}oxy)-2-hydroxybenzoate?caffeoylquinic.acid diglucoside?isopropyl-apiofuranosyl-(1->6)-glucopyranoside?Buty 4/6-O-galactopyranosyl-?-D-glucopyranoside?caffeoylquinic aci d glucosid?3-Carboxy-4-hydroxy-phenoxy glucoside?6-O-(4-Hydroxy-3-methoxy benzoyl)-glucopyranose?Scopolin?Protocatechuic acid?caffeoylquinic.acid glucoside?N eochlorogenic acid?Ferulyl quinic acid glucoside?3,4-Dihydroxyallylbenzene?4-O-[rhamnopyranosyl-(1>6)]-glucopyranoside?1-Caffeoylglucose?4-Formyl-2,6-dimethoxyp henyl ?-D-glucopyranoside?caffeoylquinic acid?Magnoliosid?10-Deoxygeniposidic acid?1-O-sinapoyl-?-D-glucose/Methyl-1-(?-D-glucopyranosyloxy)-7-(hydroxy methyl)-1,7a-dihydrocyclopenta[c]pyran-4-carboxylat?trans-4,4'-Dihydroxy-3,3'-dimethoxystilben e?Chlorogenic acid?Caffeic acid?Syringin?Sinapyl alcohlo?p-Coumaroylquinic acid?{(1 R,2R)-2-[(2Z)-5-(?-D-glucopyranosyloxy)pent-2-en-1-yl]-3-oxocyclopentyl}ace tic acid?Benzyl 6-O-(6-deoxy-?-L-mannopyranosyl)-?-D-glucopyranoside?Dicaffeoyl quinic acid glucoside?(2E)-3-[4-({(1S,2S)-1-[4-(?-D-Glucopyranosyloxy)-3-methoxyph enyl]-1,3-dihydroxy,2-propanyl}oxy)-3-methoxyphenyl]acrylicacid?2Hydroxy-5-isoprop enyl-2-methylcyclohexyl?Astilbin?Methyl(1 R,4aS,6S?7R,7aS)-6-({[(2S,3R,4S)-2-(?-D-glucopyranosy loxy)-4-(2-hydroxyethyl)-3-vinyl-3,4-dihydro-2H-pyran-5-yl]carbonyl}oxy)-1-hydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylat?6-O-[3,4-dihydroxy-4-(hydroxymethyl)tetrahydro-2-furanyl]-?-D-glucopyranosid?4-{(1S.3aR.4S.6aR)-4-[4-(?-D-Glucopyranosyloxy)-3-mehoxyphenyl]tetrahydro-1H,3H-furo[3,4-c]furan-l-yl}-2-methoxyphenyl ?-D-glucopyranoside?Orientin?Hyperin?4-{4-[4-(?-D-Glucop yranosyloxy)-3,5-dimethoxy phenyl]tetrahydro-1H,3H-furo[3,4-c]furan-1-yl}-2-methoxyphenyl?Eleutheroside E?Hesperetin 7-O-glucosid?4-(1,3-Dihydroxy-2-{4-[(1E)-3-hydr oxy-1-propen-1-yl]-2-methoxyphenoxy}propyl)-2-methoxyphenyl hexopyranoside?Niduloic acid?Quercetin-3-arabinoglucoside?Rutin?4-{(1S,2R)-1,3-Dihydroxy-2-[4-(3-hydr oxypropyl)-2-methoxyplienoxy]propyl}-2-methoxyphenyl ?-D-glucopyranoside?3-O-caf feoyl-FeruloylquinicAcid?(1S,2S)-3-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-h ydroxy-propyl)-2-methoxy phenoxy]propyl ?-D-glucopyranoside?Methyl2-O-?-D-glucosy loxy-4-methoxybenzenepropanoate?matairesinol?7-(Acetoxymethyl)-4-[(?-D-glucopyra nosyloxy)methyl]-6,7-dihydroxy-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-1-yl 3-meth ylbutanoate?Medioresinol 4'-O-?-D-glucopyranoside?Azelaic acid?4-{[(3R,4R,5S)-5-(4-Hydroxy-3,5-dimethoxyphenyl)-4-(hydroxymethyl)tetrahydro-3-furanyl]methyl}-2,6-di methoxy phenyl ?-D-glucopyranosid?3,4-Dicaffeoyl-quinic acid?4-(1,3-Dihydroxy-2-{4-[(1E)-3-hydroxy-1-propen-1-yl]-2,6-dim ethoxy phenoxy} propyl)-2-m ethoxy phenyl ?-D-al lopyranoside?3,5-Dicaffeoyl-quinic acid?2-O-Caf-feoyl arbutin?7-Hydroxy-1-(4-hydr oxy-3-methoxyphenyl)-3-(hydroxy in ethyl)-6-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl]methyl?-D-glucopyranoside?Quercitrin?1-Methoxyoxaly 1-3,5-dicaffeoylquinic acid?(2 E)-3-Phenyl-2-propen-1-yl-6-O-(6-deoxy-?-L-mannopyranosyl)(?-D-glucopyranoside?4-[4-(4-Hydroxy-3-methoxyphenyl)tetrahydro-1H,3H-furo[3,4-c]furan-1-yl]-2-methoxyphen yl ?-D-glucopyranoside?4,5-Dicaffeoyl-quinic acid?Secoisolariciresinol?804/804-de hydrotriferulic acid?Acanthopanaxoside E?Acanjaposide G/Acanjaposide H?Trihydr oxyoctadecanoic acid?trihydroxy-10(11)-octadecenoic acid?28-Hydroxy-28-oxoolean-1 2-en-3-yl-3-O-xylopyranosyl-glue opyra nos id uronic acid?3-{[2,6-Dideoxy-3-0-methyl-ribo-hexopyranosy 1-(1->4)-2,6-dideoxy-3-O-methy l-arabino-hexopyranosyl-(1->4)-2,6-di deoxy-3-O-methyl-?-D-arabino-hexopyranosyl-(1->4)-2,6-dideoxy-3-O-methyl-?-D-ribo-hexopyranosy 1-(1->4)-2,6-dideoxy-3-O-methyl-?-D-ribo-hexopyranosyl]oxy}-8,14,17-trih ydroxy-20-oxopregn-5-en-12-ylbenzoate??-HEDERIN?Hederagenin 3-O-arabinoside?3-O-?-D-Glucuronopyanosyl-gypsogenin-28-O-?-D-glucopyranoside?Silphioside G?hedeagenin-3-o-glucuronopyraoside?Ciwujianoside E?3-{[2-O-(Lyxopyranosyl)-galactop yranosyl]oxy}olean-12-en-28-oic acid?Oleanoicacid-3-O-glucuronide?Eleutheroside K?13-Hydroxy-9,11-octadecadienoic acid?3-Hydroxyhexadecanoic acid2We used the methods of analysis of Acanthopanax fruit with Ultra-high performance liquid chromatographycoupled with time-of-fight mass anlysis the potential bioactive compounds of Acanthopanax fruit in vivo,using multivariate data processingapproach and the MetaboLynx tool founding33 components come from the acanthopanax fruit in the blood of rats including 24 prototype,9metabolites.Those components metabolism including glucose metabolism,glucuronic acid metabolism,methylation metabolism.The Chemical composition:Methy5-({3,4 dihydrox y-4-(hydroxy methyl)tetrahydro-2-furanyl-glucopyranosyl}oxy)-2-hydroxyb-enzoate 6-O-(4-Hydroxy-3-methoxy-Benzoyl)-glucopyranose?Phenyl-2-O-(6-deoxy-?-L-galactopyranosyl)-?-D-galactopyranoside?(1s?4aS,5R,7aS)-l-(?-D-gluc opyranosyloxy)-5-{[2(E)-3-(4-hydroxyphenyl)-2-propenoy-L]oxy}-7-(methoxy methyl)-1,4a,5,7a-tetrahydrocyclopcnta[c]pyran-4-carboxylate?2-Hy-Droxy-5-isopropenyl-2-methy Icyclohexyl-glucopyranoside?Acanthopanaxoside E?Hederagenin 3-O-arabinoside?hederagenin-3-O-glucuronopyraoside?Rutin?Astilbin?hyperin?3-O-caffeoylFeruloylquinic Acid?Niduolic acid?(2E)-3[4-({1S,2S)-1-[4-(?-D-Glucopyranos-yloxy)-3-methoxypenyl]-1,3-dihydroxy-2-propanyl}oxy)-3-methoxypenyl]acrylic aci?Protocatechuic acid?Chlorogenic acid?1-Caffeoylglucose?Magnolioside?10-Deoxygeniposidic acid?Caffeic acid?Dicaffeoylquinic acid glucoside?Benzyl6-O-pentopyranosylhexopyanoside?3(1)Cellular immune experiment:There was significant difference in the low dose group with the control at the delayed type hypersensitivity(P<0.05),There was very significant difference in the high dose group with the control group at the transformation of spleen lymphocyte test(P<0.01).There was significant difference in the middle dose group with the control at thet hymus index(P<0.05).There was very significant difference in the high dose group with the control group at the spleen index.(P<0.01)These results suggest that Acanthopanax fruits could enhance the level of cellular immunity.The immunomodulatory effects of Acanthopanax fruit certified on the regulation of cellular immunity in mice was certified that increased the wight of immune organs.the level of DTH,and the prompted the lymphocyte proliteration.(2)The humoral immune experiment:There was the significant difference in the each dose group with the control group at the test of the hemolytic plaque count(P<0.01).There was the very significant difference in the high-dose group with the control group at the test of serum hemolysin level(P<0.05);There was the significant difference in the middle-dose group with the control group at the test of serum hemolysin level(P<0.01).The immunomodulatory effects of Acanthopanax fruit certified on the regulation of humoral immunity in mice was certified that increased the serum hemolysin level.(3)The text of natural killer cell activity:The low and high dose of acanthopanax fruit extract can improve the activity of NK cells in mice.There was the significant difference in the middle-dose group with the control group at the test of natural killer cell activity.(P<0.01)There was the significant difference in the high-dose group with the control group at the test of natural killer cell activity(P<0.05).The immunomodulatory effects of Acanthopanax fruit certified on the regulation of natural killer cell activity in mice that increased the natural killer cell activity.(4)The macrophage immune experiment:Three doses of acanthopanax fruit extract didn't improve the phagocytosis of macrophage in mice.These results suggest that the effect of enhance immunity from Acanthopanax fruits is not achieved through this way.Conclusion:The chemical composition of acanthopanax senticosus fruit and absorbed into blood of acanthopanax fruit was identified.The regulation of immune function was increased by increasing the weight of immune organ,promoting the proliferation of lymphatic T cell,regulating the lymphatic B cell function and killing ability of NK cells.The experiment will illuminate the pharmacodynamic material basis,develop new chinese medicine,provide the chemical information for sustainable development of Acanthopanax resources.