Studies On Anti-Ulcer Effect And Its Effective Substances Of The Rhizomes Of Alpinia Officinarum Hance

Peptic ulcer is a common disease and now triple therapy(antibiotics against Helicobacter pylori,drugs that inhibit the secretion of gastric acid and protective agents of gastric mucosal)is often used in clinic.However,the side effects of western medicine made many patients choose traditional Chinese medicines for treatments.Alpinia officiniarum Hance is the dry roots of the Alpinia family.Its property is acrid,and it tastes hot,which can warm stomach,dispel cold,promote digestion and relieve pains.It has been commonly used in clinic for the treatment of abdominal cold and pain,gastrofrigid vomiting,belching acid and regurgitation.A.officiniarum is also an edible medicine,with low side effects.So it is a good medicine in the treatment of gastrointestinal diseases.Many literature and documents have reported the chemical compositions and digestive disease research,but the effective substance of treatment of peptic ulcer and its mechanism were not discussed in depth.This paper first identified the anti-gastric ulcer effective parts of A.officiniarum.Then the chemical composition of the effective fractions were separated and identified.Third,preliminary exploration of the mechanism of treatment of gastric ulcer,which will provide an important theoretical basis for the development of A.officiniarum into a drug treatment of gastric ulcers.In this paper,the main research contents are as follows:Part 1,determination of the effective fractions with anti-ulcer activity.The anti-ulcer effect of four polar fractions from the rhizomes of A.officiniarum(30%ethanol elution fraction(A),80%ethanol elution fraction(B),95%ethanol elution fraction(C)from the 95%ethanol extract through macroporous resin,and the decoction of the residue after extraction by 95%ethanol(D))was investigated using gastric mucosal injury induced by ethanol in mice,the mice gastric ulcer induced by aspirin,gastric damage induced by indomethacin in rats of three pharmacological model and determine the effective fractions.The experiment of gastric ulcer induced by aspirin in mice shows that B and D fractions could reduce gastric mucosal injury caused by ethanol in mice and reduce the ulcer index(P<0.01,P<0.05),the three fractions A,B and D could significantly reduce the concentration of mouse plasma IL-1;B and D fractions could significantly increase plasma concentration of COX-2,reduce the TNF-? level in plasma and significantly increase PGE2 level in plasma.The result of gastric mucosal injury in mice induced by anhydrous ethanol showed that water extract,B and C fractions could reduce the gastric mucosal injury index(P<0.01,P<0.05);the four fractions A,B,C and D could reduce serum gastrin concentration.The pathological sections of gastric tissue in each group showed that multifocal coagulation necrosis could be seen in gastric antrum and accompanied with more severe inflammation,hemorrhage,edema in the model group while stomach inflammation and bleeding was evidently reduced in Group B.The results revealed that both the water extraction of residue after extraction by 95%ethanol and the 80%ethanol eluate from the 95%ethanol extract of the rhizomes of A.officinarum by macroporous resin column chromatography were the effective fractions in treating gastric ulcer.Therefore,the chemical constituents of the two fractions were studied in this paper.Part 2,separation and identification of chemical constituents from effective fractions.1.Isolation and identification of small molecule ingredients35 compounds were isolated from the two fractions by solvent extraction,silica gel column chromatography,Sephadex LH-20 column chromatography and ODS column chromatography and recrystallization,and 30 compounds were identified and their structures were elucidated by infrared spectroscopy(IR),mass spectrometry(ESI-MS)and nuclear magnetic resonance spectroscopy(1H-NMR,13C-NMR,DEPT,1H-'HCOSY,HSQC,HMBC and NOESY)and other methods.These compounds included 5 flavonoids:galangin(1),galangin3,5,7-trimethylether(2),kaempferol(3),quercetin(4),pinocembrin(5),8 diarylheptanoid compounds:officinarone A(6),E-1,3-diphenylpropene(7),7-(4"?hydroxy-3"-methoxyphenyl)-1-phenyl-3,5-dihydroxy heptane(8),5-hydroxy-7-(4"-hydroxy-3,"-methoxyphenyl)-phenyl-1-phenyl hept-5-phenyl hept-3-one(9),(4E)-7-(4"-hydroxy-3",methoxyphenyl)-1-phenyl hept-4-en-3-one(10),1,7-diphenyl-4E-en-3-heptanone(11),2,5-furan-1-(4"-hydroxy-3"-methoxy)-1-phenyl-heptane(12),yakuchinone A(13),7 glycosides:n-butyl-O-?-D-fructopyranoside(14),ethyl-?-D-glucopyranoside(15),uridine(16),2,6-dimethoxy-4-hydroxy-phenol-1-O-glucopyranoside(17),staphylionoside D(18),3,5-dimethoxy-4-hydroxy benzoic acid-7-O-?-D-glucopyranoside(19),isoquercitrin(20)and other compounds:?-sitosterol(21),dibuthyl phthalate(22),4-methoxyl-phenol(23),5-hydroxymethyl-2-furaldehyde(24),p-hydroxybenzaldehyde(25),vanillin(26),4-(4-hydroxy-3-methoxy phenyl)-2-butanone(27),3-methoxy-4-methyl benzaldehyde(28),protocatechuic aldehyde(29),protocatechuic acid(30).5 flavonoids,8 diarylheptanoid compounds and one sesquiterpene compound were isolated from the 80%ethanol eluate by macroporous resin,6 glycosides and 10 other types of compounds were from the aqueous extract.Compound 6 is a new compound,compound 7 is a new natural product,compounds 13,18,20,22,29,30 were isolated from this plant for the first time,compounds 14 to 17,19,and 23 to 28 were first isolated from Alpinia genus.2.Separation and structure characterization of polysaccharidesThe fraction of water extraction followed by alcohol precipitation was firstly undergone dialysis,concentration,freeze drying,reconstitution and centrifugation,then separated and purified by weaker anion tandem weaker cation ion-exchange resin,ion-exchange cellulose,ion-exchange agarose gel and various sephacryl(high resolution)column chromatography.Six homogeneous polysaccharides were isolated from A.officiniarum for the first time,including WN-A1,WN-A2,WN-B1,WN-B2,WN-B3 and WN-B4.The compositions of the isolated homogeneous polysaccharides were qualitatively studied by using spectral and chromatographic analysis and the monosaccharide compositions of the six homogeneous polysaccharides were also determined.The results are listed as follows:WN-A1 is an acidic polysaccharide,it consists of D-mannose,L-rhamnose,D-glucuronic acid,D-galacturonic acid,D-galactose and L-arabinose with the mole percentage of 4.2:4.5:1.7:6.1:45.8;37.8;WN-A2 is also an acidic polysaccharide,it is composed of L-rhamnose,D-galacturonic acid,D-galactose and L-arabinose with a mole percentage of 12.3:49.3:21.5:16.8;WN-B1 is still an acidic polysaccharide,which contains D-mannose,L-rhamnose,D-galacturonic acid,D-galactose and L-arabinose,and the mole percentage:is 15.6:7.5:42.2:14.4:20.3;WN-B2 is an acidic polysaccharide,which consists of D-mannose,L-rhamnose,D-glucuronic acid,D-galacturonic acid,D-galactose and L-arabinose,and their mole percentage is16.3:4.0:2.9:13.4:21.7:41.7.WN-B3 is an acidic polysaccharide which contains D-mannose,L-rhamnose,D-glucuronic acid,D-galacturonic acid,D-galactose and L-arabinose,and their mole percentage is 4.5:4.3:36.1:5.7:23.2:20.1.The results showed that the compositions of the homogeneous polysaccharides isolated from the rhizomes of A.officiniarum are all free of common monosaccharide(glucose),and all of them are acidic polysaccharides.Part 3,study on the pharmacological effects of A.officiniarum1.Evaluation of the cytotoxicity of small molecule compoundsMTT assay was used to investigate the cytotoxic activity of 30 compounds on five kinds of human cancer cells.The results showed that IC50 values of diarylheptanoid compounds on gastric cancer SGC-7901 cells and breast cancer MCF-7 cells were between 20 and 30 ?M,IC50 values of flavonoids on gastric cancer SGC-7901 cells werebetween 20 and 30 ?M,and IC50 values of other compounds on the five kinds of human cancer cells were higher than 100 ?M.The results showed that the cytotoxic activity of small molecule compounds were weaker,and when there will be no major toxicity on gastric mucosal cells when used for the treatment of gastric ulcer.2.Anti-ulcer and immunomodulatory effects of polysaccharidesThe results of polysaccharides in the rhizomes of A.officinarum on gastric injury in mice induced by anhydrous ethanol showed that the polysaccharide in the rhizomes of A.officinarum could alleviate the acute gastric mucosal injury in mice,reduce the bleeding of ulcer and significantly decrease the ulcer index of gastric ulcer mice induced by anhydrous ethanol,significantly decreased the serum MTT and GAS levels(p<0.05),whereas there was no significant effect on the PGE2 content in the serum(p>0.05).The polysaccharides in the rhizomes of A.officinarum may exert its anti-ulcer effect mainly by inhibiting the aggressive factors of gastric ulcer.The polysaccharide in the rhizomes of A.officinarum could significantly enhance the phagocytosis activity of peritoneal macrophages in normal mice,which indicated that the rhizomes of A.officinarum may have certain immunoregulation activity.The administration of high and medium dose of polysaccharide of A.officinarum can aggravate the ear swelling degree of mouse delayed type hypersensitivity(DTH)induced by 2,4-dinitroflurobenzene(DNFB)and there was no obvious effect on the immune organs.The polysaccharide of A.officinarum at a low dose had no evident effect on the ear swelling degree of mouse,while it can reduce the immune organs indexes of DTH mice.The above results indicated that the polysaccharide of A.officinarum could not improve the inflammation caused by immune mechanism.In summary,this paper explored the anti-ulcer mechanism and its effective substances through chemical and pharmacodynamic studies and determined the chemical compositions of anti-ulcer effective fraction,the role of the characteristics of the underlying mechanism of A.officiniarum for the first time,which made a foundation for the further study of the treatment of gastric ulcer and provided a scientific basis for the development and utilization of the rhizomes of A.officiniarum Hance.