Study On Separation And Molecular Modification Of Capsaicinoids

Capsaicin, the major pungent component in Capsicum annuum L., has been used as amedicine in clinic for long time because of its extensive pharmacological activities and is gettingmore and more attention in recent years because it had been reported that it has thepharmacological activity such as promotion adrenal catecholamine secretion so as to enhance theoxidation of fatty acids and to inhibit obesity. But its usage as a food additive or a drug isconsiderably limited since capsaicin has a strong pungency and nociceptive activity and theusage in other fields is also badly restricted because it is very difficult to gain pure capsaicinoids.Capsinoids,a novel group of compounds, separated from a sweet-pepper of Japan andhaving a structure of ester of aliphatic hydroxyl group in vanillyl alcohol with a fatty acid whichis resemble to that of capsaicinoids that is a fatty acid amide of vanillylamine, may be used asfood additive or pharmaceutical ingredients alternated the capsaicin because of its similarphysiological activities of capsaicin without pungent and cytotoxicity. But its study for using ashealthy food or medicine cann't be gotten on because of its limited resource.In this thesis, the aim is marly focused on the method of separating capsaicinoids from hotpepper, of molecular modification of capsaicinoids, of synthesis of capsinoids fromcapsaicinoids by using biocatalysis technology and on screening their pharmacological activitiessuch as anti-obesity and regulating glucose and lipid metabolism by High ThroughputScreening(HTS) method.The resuts were as follows:1. Studying on method of effective extracting capsaicinoids from capsicum annuum L.(1) A method of simultaneous extracting capsaicinoids and pigment was established. Thecondition, which is 95% ethanol at 50℃and the solid to liquid ratio 1:11 with 3 times ofextraction in 1h pertime, was determined by studying single factor and orthogonal experimentmethod. The extraction rate of capsaicinoids got 95.9% with 2 times extration and that ofpigment got 79.3% and 96.4% with 2 and 3 times extraction, respectively.(2) A new method extracted capsaicinoids from capsicum annuum L. by using cellulaseenzymolysizing pepper peel was established. Some influence factors, such as enzymatichydrolysis temperature, pH of enzymatic hydrolysis solution, the time of enzymatic hydrolysisand the amount of cellulase, were optimized. The optimum parameters were that the pH ofenzymatic hydrolysis solution is 5.3, enzymatic hydrolysis temperature is 40℃, the amount ofcellulase is 10mg/g and the time of enzymatic hydrolysis is 3 hours.The extractive efficiency was7% more than that of traditional extract with ethanol comparatively. It is more important that theextract of water and 30% ethanol, in which the extracting rate of capsaicinoids gets 45.7% in all,could be purifyed by macroporous adsorption resin column chromatography with ethanol as eluent.2. Studying on method of separating capsaicinoids from pigment(1) A kind of macroporous adsorption resin was selected as the medium for purification ofcapsaicinoids on comparing adsorption and separation performance of 11 kinds of macroporousadsorption resin. The investigation of the adsorption behavior indicated that resinâ… has capabilityof selective adsorption of capsaicinoids from extract of capsicum annuum L. with a static anddynamic adsorption capacity of 26.1mg/g and 16.0 mg/g, respectively. The capsaicinoids,byextraction with 40% ethanol from the extract of capsicum annuum L. and then purification bymacroporous adsorption resin column chromatography using 70% ethanol as elutent,wasenriched from 1.5% to 81.7% determinated by HPLC,with 78.3～87.6% of recovery rate.(2) The method separated capsaicinoids from pigment was explored by adsorpting theextract with macroporouse resin directly and then eluting with ethanol solution. The resultshowed that the content of capsaicinids was enriched from 1.2% in extract to 28.1% in product(determination by HPLC) with 23 times enriched and the colour value of separated pigment got93.1 with 17.7 times enriched.(3) Pure capsaicinoids with the content of capsaicinoids at 99.9% (in which the content ofcapsaicin at 48.9%, dihydrocapsaicin at 42.4% and nordihydrocapsaicin at 8.7%) was gained bysilica gel column chromatography with petroleum ether-acetone (8:2) as eluent. The yield is0.13% from the peper peel.(4) Capsaicin, dihydrocapsaicin and nordihydrocapsaicin was obtained by separative HPLC,with a purity of 99.9%, 100.7% and 98.1% compared that with standard sample,respectively.(5) The preparative HSCCC procedure for separation capsaicin and dihydrocapsaicin fromcapsaicinoids was studied and capsaicin and dihydrocapsaicin was obtained with a purity 99.8%and 97.5% (by HPLC), respectively.3. Studing on the analysis methods of capsaicinoids(1) A reversed-phase high performance liquid chromatographic method was established forsimultaneous determination of capsaicin and dehydrocapsaicin in samples of purificatedcapsaicinoid or unpurificated capsaicinoid.The HPLC system was consisted of Vp-OSD C₁₈column (150mm×4.6mm, 5μm). The column temperature was 35℃and the moble phase wasV(CH₃OH):V(H₂O)=65:35 with flow rate of 0.8 mL / min. UV detecter was set at 280 nm. Thelinear equation was y=813237 x +21490 and correlation coefficients was 0.9997 (n=8) in0.0115～2.30μg for capsaicin. The RSD was 1.33% (n=5) or 0.49% (n=5) for standard orsample of capsaicin.The detection limit was 0.61ng and the average recovery was 99.88%. Thelinear equation was y=696861 x +2908 and correlation coefficients was 0.9997 (n=8) in0.006～1.20μg for dihydrocapsaicin. The RSD was 3.45 % (n=5) or 1.82% (n=5) forstandard or sample of dihydrocapsaicin.The detection limit was 0.72 and the average recovery was 99.29%.(2) A fluorescence spectrophotometer method was established for determination ofcapsaicinoid in samples of purificated capsaicinoid or unpurificated capsaicinoid. UnderEx=278nm and Em=312nm, The linear equation of the flouresence intensity(â… ) and theconcentration(c,μg/mL) of capsaicinoid was c=1.0377x10^-3 I-0.3667 and correlationcoefficients was r= 0.9994 (n=5) in 0.58～5.8μg/mL for capsaicin. The RSD was 0.08% (n=5).The average recovery was 95.39%. The result of determination by this method is as fine as thatby HPLC method, meanwhile it is very fast and simple.4. Molecular modification of capsaicinoids by chemistry method(1) Since capsaicin has a strong pungency, its usage as medecine or healthy food isconsiderably limited. In this study,β-D-tetraacetylglucose of capsaicinoids, a intermediate, wassynthesized by using pentaacetylglucose reacting with capsaicinoids with BF₃·Et₂O as acatalysis and characterized by IR,¹HNMR, MS and HPLC-MS. And then the glucoside ofcapsaicinoids was obtained by hydrolyzing the intermetiate and characterized by FT-IR.(2) Vanillyl nonanoate is the most similar to nature capsinoids in structure. In this paper,vanillyl nonanoate, i.e. (4-hydroxy-3-methoxyphenyl)methyl nonanoate, was synthesizedusing acetone as solvent and NaHCO₃ capturing the acid by-product instead of pyridine with theyield of 35.8% and characterized by the UV, FT-IR, ¹HNMR, ¹³CNMR and MS.5. Studing on synthesis of capsinoids by lipase-catalysis(1) The condition of synthesis of vanillyl nonanoate by lipase-catalysis, including theinfluences of sort of enzyme, organic solvent, amount of enzyme, molar ratio of the substracts,addtion of water, and reaction tempetature, was studied, Lipase Novozyme 435 was selected asthe catalysis for the reaction.The optimal reaction conditions are:50mmol/L vanillyl alcohol and75 mmol/L methyl nonanoate in 1mL of acetone which contain 0.1-0.3% of water(V/V), using20rag lipase(Novo 435). In the conditions, at 30℃, more than 60% of conversion was achievedafter 9h.Several capsinoid homologues having various acyl chain lengths(C2-C18) were gained bylipase-catalysis in 100mL scale under the condition with 18%～93% yield, respectively. Theywere purified by silica gel column chromatograph and characterized by IR,¹HNMR,¹³CNMR andMS spectra.(2) The natural capsinoids was preparated by conversing natural capsaicinoids with chemo-enzymic technology.The methyl fatty acid ester was got by capsaicinoids reacting with methanoland the capsinoids was gained from vanillyl alcohol reacting with the methyl fatty acid ester bylipase-catalysis under the enzymatic condition. The product was purified by silica gel columnchromatography and contains capsiate, dihydrocapsiate, nordihydrocapsiate andhomodihydrocapsiate characterized by GC-MS. Finally, capsiate and dihydrocapsiate was obtained by preparative HPLC and the chemical structure was characterized by ¹H NMR andMS.(3) The natural long chain capsinoids with acyl of colza oil or arachis oil was preparated bylipase-catalysis. Finally, vanillyl hexadecanoate,vanillyl octadecanoate,vanillyl oleate,vanillyllinoleate and vanillyl linolenate was obtained by preparative HPLC, respectively, and thechemical structure was characterized by ¹H NMR.(4) The dynamic model and kinetic equation was established by studing initial velocity ofvanillyl alcohol reacting methyl nonanoate using Novo 435 as biocatalysis.(5) The method of fermentation production of lipase was studied. The lipase made byourselves had the characteration of transesterification in organic medium and was resembl to thatof commercial lipase such as Novozym 435.6. Studing on the pharmacological activity of capsaicinoids and capsinoids by HighThroughput Screening (HTS) methodHigh Throughput Screen (HTS) method was developed since 1980s and is used widely bymany foreign research institutes because of the characteristic of micro-quantity, speediness andhigh efficiency. In this paper, the PPARγmodel and 3T3-L1 model of HTS was used to screenthe pharmacological activity of capsaicinoids isolated from Capsicum annuum L. and capsinoidssynthesized by lipase-catalysis. Results were as follows.(1) The MTS result showed that the cytotoxicity of capsaicinoids and capsinoids is above10μg/mL in all, but that of capsaicinoids is below 50μg/mL and that of capsinoids is above 50μg/mL.And sample N1-N12, i.e., capsinoids (N10 is the natural capisnoids), has littlecytotoxicity compared with the sample N13 (natural capsaicinoids) and sample N14 (capsaicin).(2) Capsaicinoids and capsaicin has the activity as a PPARγagonist with above 2 timesactivity at the concentration of 10μg/mL, but the capsinoids has no activity as PPARγagonist atthis concentration. However, vanillyl nonanoate, vanillyl decanoate, natural capsinoids andcapsinoids with acyl of colza oil has the activity as a PPARγagonist with the activity times at10.7, 9.2,9.9 and 1.5 at 50μg/mL, respectively. But capsaicinoids has no activity for PPARγatthis concentration because of its high cytotoxicity.(3) All sample shows no effect on 3T3-L1 at 10μg/mL.(4) Capsaicinoids isolated from Capsicum annuum L. and capsinoids preparated bylipase-catalysis possess the activity to inhibite MCF-7 and HepG2 under high concentration.