辣椒素的制备及分子修饰方法研究
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摘要
辣椒素(capsaicin)具有广泛的药理作用和应用价值，在医药领域的应用有着悠久的历史，近些年报道具有降脂减肥作用后，引起人们的更加关注。但辣椒素强烈的辛辣味制约了它作为药品或食品添加剂的使用，由于高纯品制备的困难也限制了它的其他用途。
辣椒素酯类(capsinoids)物质是近年发现的结构类似辣椒素的物质，没有辣味且细胞毒性比辣椒素更低，但具有与辣椒素相似的药理作用及生理活性，认为是辣椒素在医药及保健食品行业的替代品。由于辣椒素酯类物质资源非常有限，因而严重限制它的应用研究。
本研究在辣椒素的制备方法，辣椒素糖苷化的分子修饰，辣椒素转化为辣椒素酯类化合物的生物催化方法，以及辣椒素与辣椒素酯类物质的糖脂代谢等生物活性的高通量筛选四个方面进行了研究，取得了以下实验研究结果。
1．辣椒素的提取方法研究
(1)建立了同时提取辣椒素与红色素的工艺：乙醇浓度95％，温度为50℃，提取时间为1h，固液比为1：11分三次提取(提取两次时辣椒素提取率为95.9％，色素提取率为79.3％；提取三次时色素提取率可达到96.4％)。
(2)纤维素酶预处理法提取辣椒素的研究：考察了用纤维素酶预处理辣椒粉后以30％和95％乙醇分步提取辣椒素的方法。首先，以单因素实验和4因素3水平的正交实验为基础，确定了辣椒粉的酶解条件为：酶解液初始pH值=5.3，酶解温度为40℃，酶用量为10mg／g，酶解时间为3h。辣椒粉酶解后用乙醇提取时，辣椒素的提取率与超声提取法接近，比乙醇直接提取时高出7％左右。更重要的是酶解液及30％乙醇提取液(占提取辣椒素总量的45.7％)可直接用吸附树脂进行分离纯化。
2．辣椒素的分离方法研究
(1)考察了11种大孔吸附树脂对辣椒素类物质的吸附分离性能，以乙醇溶液为洗脱剂，筛选了一种适合富集辣椒素的大孔吸附树脂Ⅰ。该树脂的静态饱和吸附容量及动态饱和吸附容量分别为26.1mg／g及16.0 mg／g。以40％乙醇溶解辣椒浸膏后上Ⅰ型大孔吸附树脂柱，以70％乙醇解吸，HPLC分析显示纯化样品的辣椒素从辣椒浸膏中的1.5％富集到81.7％，回收率为78.3％～87.6％。
(2)探索了吸附树脂拌样法分离辣椒素和色素的实验方法，选用的2号树脂对辣椒素、辣椒色素的分离效果较好，过柱后产品中辣椒素含量从提取物中1.2％提高到28.1％，辣椒素富集23倍；辣椒色素的色价为93.1，富集17.7倍。
(3)利用硅胶柱色谱法以石油醚-丙酮(8：2)洗脱，结合重结晶方法，实现了辣椒素纯化的目的，所得结晶辣素总含量达到99.9％(其中辣椒素含量为48.9％，二氢辣椒素含量为42.4％，降二氢辣椒素含量为8.7％)，相对于辣椒干果的得率为0.13％。
(4)用制备性HPLC色谱分离得到降二氢辣椒素、辣椒素及二氢辣椒素，纯度分别为98.1％、99.9％及100.7％(HPLC方法)。
(5)建立了制备性HSCCC色谱分离辣椒素与二氢辣椒素的方法，得到纯度为99.8％的capsaicin及97.5％的dihydrocapsaicin(HPLC方法)。
3．辣椒素的分析方法比较研究
(1)建立了以甲醇-水为流动相的同时测定样品中的两种主要辣椒素类物质含量的RP-HPLC分析方法。辣椒素进样量在0.0115～2.30μg范围内，峰面积与进样量之间呈线性关系，回归方程为y=813237x+21490，相关系数r=0.9997(n=8)，以3倍信噪比计算最低检出限为0.61ng；二氢辣椒素进样量在0.006～1.20μg范围内，峰面积与进样量之间呈线性关系，回归方程为y=696861x+2908，相关系数r=0.9997(n=8)，以3倍信噪比计算最低检出限为0.72ng。
(2)建立了辣椒素的荧光定量分析方法。在Ex为278nm，Em为312nm荧光条件下，辣椒素在0.58～5.8μg／mL浓度范围内其浓度C(μg／L)与荧光强度I具有良好线性关系，回归方程c=1.0377×10~(-3)I-0.3667，r=0.9994，精密度RSD=0.08％(n=5)。平均回收率95.39％。
该方法具有快速、操作简便等特点，特别适合大量样品的快速分析。
4．辣椒素分子结构的化学修饰方法研究
(1)辣椒素强烈的辛辣味制约它在医药及保健品方面的广泛应用。本研究利用分子修饰方法，采用Heferich法以BF_3乙醚液为催化剂将辣椒素糖苷化，合成了四乙酰辣椒素葡萄糖苷中间体，经IR、HNMR、MS及HPLC-MS表征结构；中间体经碱性水解+得到辣椒素葡萄糖苷，以IR表征结构。
(2)以丙酮为溶剂、NaHCO_3为缚酸剂替代有毒、恶臭的吡啶的方法，合成了壬酸(3-甲氧基-4-羟基)苄基酯(即壬酸香草醇酯)，以UV、IR、~1HNMR、~(13)CNMR、MS表征了结构。
5．辣椒素的生物催化转化为辣椒素酯的方法研究
(1)以香草醇和壬酸甲酯为底物，通过对脂肪酶、有机溶剂、反应温度、酶用量、底物摩尔比、反应时间、溶剂中微量水及酰基供体中的酯基结构等单因素考察，选择Novozyme 435作为催化用酶，丙酮为反应介质；并对反应温度、酶用量、底物摩尔比、反应时间进行了四因素三水平正交实验，优化其反应条件：在1mL反应体系中，底物VA∶MN=50∶75(mmol／L)、酶用量20mg、温度30℃、反应时间24h。在该反应条件下合成壬酸香草醇酯时，反应10 h产率达65.9％(二氧六环为反应介质反应21 h时产率可达81.4％)。
将体系扩大至100mL，合成了碳链长为C2-C18的一系列辣椒素酯类化合物，以硅胶柱色谱方法纯化得到目标产物；产物经UV、IR、~1HNMR、~(13)CNMR及MS表征结构。
(2)用化学-酶法实现了辣椒素向辣椒素酯的转化。辣椒素经甲醇分解转化为脂肪酸甲酯，然后在脂肪酶催化下与香草醇反应得到产物，经硅胶柱层析(石油醚-乙酸乙酯洗脱)纯化，HPLC分析表明不含原料辣椒素，两种主要辣椒素酯的含量之和达到97.4％；用制备性HPLC分离得到两个主要成分——capsiate及dihydrocapsiate，以~1HNMR及MS表征结构。
(3)以食用菜油及花生油为酰基供体，在有机介质中以脂肪酶催化合成了混合长链脂肪酸的辣椒素酯类物质，产品经硅胶柱层析纯化后，HPLC分析表明主要脂肪酸香草醇酯的含量分别达到96.6％及99.7％(面积归一法)；菜油酰基香草醇酯类物质以制备性HPLC分离得到5个主要成分，其中包括亚油酸香草醇酯、亚麻酸香草醇酯、芥酸香草醇酯，以~1HNMR表征结构。
(4)对脂肪酶Novozyme 435催化壬酸甲酯与香草醇转化为壬酸香草醇酯的反应机制进行了探讨，建立了转酯反应的动力学模型，根据反应机制推导并求解得到初速度方程。
(5)研究了发酵产脂肪酶的方法。自制的固定化酶与商品酶具有相近的催化性质，能进行有机相中的转酯反应。
6．辣椒素及辣椒素酯的高通量筛选研究
(1)MTS结果表明，辣椒素及辣椒素酯类物质的细胞毒性范围大于10μg／mL，但辣椒素的毒性范围小于50μg／mL，其中样品N1-N12为辣椒素酯类化合物(N10为天然辣椒素酯)，这些样品的细胞毒性较小；N13为天然辣椒素类物质(capsaicinoids)及N14为天然辣椒素(capsaicin，C)，细胞毒性较大。
(2)样品浓度为10μg／mL，天然辣椒素类物质(capsaicinoids)及天然辣椒素(capsaicin，C)两个样品激活PPARγ受体在2倍以上；辣椒素酯类物质则对PPARγ受体基本没有作用；在部分样品初筛浓度为50μg／mL的对比实验中，壬酸香草醇酯、癸酸香草醇酯、天然辣椒素酯类物质、菜油基香草醇酯类物质四个样品的MTS实验毒性范围大于50μg／mL，激活PPARγ受体分别达到10.7、9.2、9.9、1.5倍，显示辣椒素酯类物质在高浓度下对PPARγ受体良好的激活作用；天然辣椒素类物质(capsaicinoids)的MTS实验毒性范围小于50μg／mL，因而在高浓度下由于细胞毒性大，与低浓度相比，反而失去激活作用。
(3)样品在10μg／mL时，对小鼠前脂肪细胞3T3-L1的脂肪分化进程没有明显的抑制作用。
(4)天然辣椒素类物质(capsaicinoids)及酶法制备的天然辣椒素酯类物质(capsinoids)在高浓度条件下(capsaicinoids在50μg／mL以上，capsinoids在100μg／mL以上)对实验的乳腺癌细胞(MCF-7)和肝癌细胞(HepG2)有明显的抑制作用，实验还表明天然辣椒素类物质的抑瘤能力要强于天然辣椒素酯类物质。
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 resinⅠhas 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 C_(18)column (150mm×4.6mm, 5μm). The column temperature was 35℃and the moble phase wasV(CH_3OH):V(H_2O)=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(Ⅰ) 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 BF_3·Et_2O 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 NaHCO_3 capturing the acid by-product instead of pyridine with theyield of 35.8% and characterized by the UV, FT-IR, ~1HNMR, ~(13)CNMR 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,~(13)CNMR 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) method
High 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.
辣椒素酯类(capsinoids)物质是近年发现的结构类似辣椒素的物质，没有辣味且细胞毒性比辣椒素更低，但具有与辣椒素相似的药理作用及生理活性，认为是辣椒素在医药及保健食品行业的替代品。由于辣椒素酯类物质资源非常有限，因而严重限制它的应用研究。
本研究在辣椒素的制备方法，辣椒素糖苷化的分子修饰，辣椒素转化为辣椒素酯类化合物的生物催化方法，以及辣椒素与辣椒素酯类物质的糖脂代谢等生物活性的高通量筛选四个方面进行了研究，取得了以下实验研究结果。
1．辣椒素的提取方法研究
(1)建立了同时提取辣椒素与红色素的工艺：乙醇浓度95％，温度为50℃，提取时间为1h，固液比为1：11分三次提取(提取两次时辣椒素提取率为95.9％，色素提取率为79.3％；提取三次时色素提取率可达到96.4％)。
(2)纤维素酶预处理法提取辣椒素的研究：考察了用纤维素酶预处理辣椒粉后以30％和95％乙醇分步提取辣椒素的方法。首先，以单因素实验和4因素3水平的正交实验为基础，确定了辣椒粉的酶解条件为：酶解液初始pH值=5.3，酶解温度为40℃，酶用量为10mg／g，酶解时间为3h。辣椒粉酶解后用乙醇提取时，辣椒素的提取率与超声提取法接近，比乙醇直接提取时高出7％左右。更重要的是酶解液及30％乙醇提取液(占提取辣椒素总量的45.7％)可直接用吸附树脂进行分离纯化。
2．辣椒素的分离方法研究
(1)考察了11种大孔吸附树脂对辣椒素类物质的吸附分离性能，以乙醇溶液为洗脱剂，筛选了一种适合富集辣椒素的大孔吸附树脂Ⅰ。该树脂的静态饱和吸附容量及动态饱和吸附容量分别为26.1mg／g及16.0 mg／g。以40％乙醇溶解辣椒浸膏后上Ⅰ型大孔吸附树脂柱，以70％乙醇解吸，HPLC分析显示纯化样品的辣椒素从辣椒浸膏中的1.5％富集到81.7％，回收率为78.3％～87.6％。
(2)探索了吸附树脂拌样法分离辣椒素和色素的实验方法，选用的2号树脂对辣椒素、辣椒色素的分离效果较好，过柱后产品中辣椒素含量从提取物中1.2％提高到28.1％，辣椒素富集23倍；辣椒色素的色价为93.1，富集17.7倍。
(3)利用硅胶柱色谱法以石油醚-丙酮(8：2)洗脱，结合重结晶方法，实现了辣椒素纯化的目的，所得结晶辣素总含量达到99.9％(其中辣椒素含量为48.9％，二氢辣椒素含量为42.4％，降二氢辣椒素含量为8.7％)，相对于辣椒干果的得率为0.13％。
(4)用制备性HPLC色谱分离得到降二氢辣椒素、辣椒素及二氢辣椒素，纯度分别为98.1％、99.9％及100.7％(HPLC方法)。
(5)建立了制备性HSCCC色谱分离辣椒素与二氢辣椒素的方法，得到纯度为99.8％的capsaicin及97.5％的dihydrocapsaicin(HPLC方法)。
3．辣椒素的分析方法比较研究
(1)建立了以甲醇-水为流动相的同时测定样品中的两种主要辣椒素类物质含量的RP-HPLC分析方法。辣椒素进样量在0.0115～2.30μg范围内，峰面积与进样量之间呈线性关系，回归方程为y=813237x+21490，相关系数r=0.9997(n=8)，以3倍信噪比计算最低检出限为0.61ng；二氢辣椒素进样量在0.006～1.20μg范围内，峰面积与进样量之间呈线性关系，回归方程为y=696861x+2908，相关系数r=0.9997(n=8)，以3倍信噪比计算最低检出限为0.72ng。
(2)建立了辣椒素的荧光定量分析方法。在Ex为278nm，Em为312nm荧光条件下，辣椒素在0.58～5.8μg／mL浓度范围内其浓度C(μg／L)与荧光强度I具有良好线性关系，回归方程c=1.0377×10~(-3)I-0.3667，r=0.9994，精密度RSD=0.08％(n=5)。平均回收率95.39％。
该方法具有快速、操作简便等特点，特别适合大量样品的快速分析。
4．辣椒素分子结构的化学修饰方法研究
(1)辣椒素强烈的辛辣味制约它在医药及保健品方面的广泛应用。本研究利用分子修饰方法，采用Heferich法以BF_3乙醚液为催化剂将辣椒素糖苷化，合成了四乙酰辣椒素葡萄糖苷中间体，经IR、HNMR、MS及HPLC-MS表征结构；中间体经碱性水解+得到辣椒素葡萄糖苷，以IR表征结构。
(2)以丙酮为溶剂、NaHCO_3为缚酸剂替代有毒、恶臭的吡啶的方法，合成了壬酸(3-甲氧基-4-羟基)苄基酯(即壬酸香草醇酯)，以UV、IR、~1HNMR、~(13)CNMR、MS表征了结构。
5．辣椒素的生物催化转化为辣椒素酯的方法研究
(1)以香草醇和壬酸甲酯为底物，通过对脂肪酶、有机溶剂、反应温度、酶用量、底物摩尔比、反应时间、溶剂中微量水及酰基供体中的酯基结构等单因素考察，选择Novozyme 435作为催化用酶，丙酮为反应介质；并对反应温度、酶用量、底物摩尔比、反应时间进行了四因素三水平正交实验，优化其反应条件：在1mL反应体系中，底物VA∶MN=50∶75(mmol／L)、酶用量20mg、温度30℃、反应时间24h。在该反应条件下合成壬酸香草醇酯时，反应10 h产率达65.9％(二氧六环为反应介质反应21 h时产率可达81.4％)。
将体系扩大至100mL，合成了碳链长为C2-C18的一系列辣椒素酯类化合物，以硅胶柱色谱方法纯化得到目标产物；产物经UV、IR、~1HNMR、~(13)CNMR及MS表征结构。
(2)用化学-酶法实现了辣椒素向辣椒素酯的转化。辣椒素经甲醇分解转化为脂肪酸甲酯，然后在脂肪酶催化下与香草醇反应得到产物，经硅胶柱层析(石油醚-乙酸乙酯洗脱)纯化，HPLC分析表明不含原料辣椒素，两种主要辣椒素酯的含量之和达到97.4％；用制备性HPLC分离得到两个主要成分——capsiate及dihydrocapsiate，以~1HNMR及MS表征结构。
(3)以食用菜油及花生油为酰基供体，在有机介质中以脂肪酶催化合成了混合长链脂肪酸的辣椒素酯类物质，产品经硅胶柱层析纯化后，HPLC分析表明主要脂肪酸香草醇酯的含量分别达到96.6％及99.7％(面积归一法)；菜油酰基香草醇酯类物质以制备性HPLC分离得到5个主要成分，其中包括亚油酸香草醇酯、亚麻酸香草醇酯、芥酸香草醇酯，以~1HNMR表征结构。
(4)对脂肪酶Novozyme 435催化壬酸甲酯与香草醇转化为壬酸香草醇酯的反应机制进行了探讨，建立了转酯反应的动力学模型，根据反应机制推导并求解得到初速度方程。
(5)研究了发酵产脂肪酶的方法。自制的固定化酶与商品酶具有相近的催化性质，能进行有机相中的转酯反应。
6．辣椒素及辣椒素酯的高通量筛选研究
(1)MTS结果表明，辣椒素及辣椒素酯类物质的细胞毒性范围大于10μg／mL，但辣椒素的毒性范围小于50μg／mL，其中样品N1-N12为辣椒素酯类化合物(N10为天然辣椒素酯)，这些样品的细胞毒性较小；N13为天然辣椒素类物质(capsaicinoids)及N14为天然辣椒素(capsaicin，C)，细胞毒性较大。
(2)样品浓度为10μg／mL，天然辣椒素类物质(capsaicinoids)及天然辣椒素(capsaicin，C)两个样品激活PPARγ受体在2倍以上；辣椒素酯类物质则对PPARγ受体基本没有作用；在部分样品初筛浓度为50μg／mL的对比实验中，壬酸香草醇酯、癸酸香草醇酯、天然辣椒素酯类物质、菜油基香草醇酯类物质四个样品的MTS实验毒性范围大于50μg／mL，激活PPARγ受体分别达到10.7、9.2、9.9、1.5倍，显示辣椒素酯类物质在高浓度下对PPARγ受体良好的激活作用；天然辣椒素类物质(capsaicinoids)的MTS实验毒性范围小于50μg／mL，因而在高浓度下由于细胞毒性大，与低浓度相比，反而失去激活作用。
(3)样品在10μg／mL时，对小鼠前脂肪细胞3T3-L1的脂肪分化进程没有明显的抑制作用。
(4)天然辣椒素类物质(capsaicinoids)及酶法制备的天然辣椒素酯类物质(capsinoids)在高浓度条件下(capsaicinoids在50μg／mL以上，capsinoids在100μg／mL以上)对实验的乳腺癌细胞(MCF-7)和肝癌细胞(HepG2)有明显的抑制作用，实验还表明天然辣椒素类物质的抑瘤能力要强于天然辣椒素酯类物质。
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 resinⅠhas 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 C_(18)column (150mm×4.6mm, 5μm). The column temperature was 35℃and the moble phase wasV(CH_3OH):V(H_2O)=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(Ⅰ) 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 BF_3·Et_2O 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 NaHCO_3 capturing the acid by-product instead of pyridine with theyield of 35.8% and characterized by the UV, FT-IR, ~1HNMR, ~(13)CNMR 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,~(13)CNMR 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) method
High 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.
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