【作者】 姜贵全；

【导师】 方桂珍；

【作者基本信息】 东北林业大学 ， 林产化学加工工程， 2013， 博士

【摘要】 合理利用落叶松树皮及提高其综合利用率,对于我国木材和造纸工业来说都有着极其现实和深远的意义。落叶松树皮中含有丰富的原花青素类化合物,在抗氧化、清除自由基、抗肿瘤、保护心血管及改善微循环等方面具有广泛的生物活性。但是,利用价值高的低聚体部分所占比重少,主要是以多聚体的形式存在,从而降低了其生物利用度,影响其生物活性。因此,对低聚原花青素的分离纯化及对多聚原花青素进行催化降解等精细化利用研究是当前亟待解决的问题。本论文以落叶松树皮为研究对象,进行了化学成分对比分析评价、原花青素的提取条件优化、分级纯化、催化降解、抗氧化性能分析研究。论文主要分为五部分。第一部分对长白落叶松(长白山自然保护区)、兴安落叶松(内蒙古阿尔山林业局)及长白落叶松(吉林市二道林场)树皮的化学成分进行分析。长白落叶松(长白山自然保护区)、兴安落叶松及长白落叶松(吉林市二道林场)树皮的鞣质含量分别为11.91%、11.5%、11.43%；总黄酮含量分别为16.59%、9.7%、7.57%；原花青素含量分别为：10.84%、9.66%、9.91%；二氢槲皮素含量分别为1.87%、0.92%、1.45%。结果表明：长白落叶松树皮(长白山自然保护区)中总黄酮和二氢槲皮素含量明显高于兴安落叶松和长白落叶松(吉林市二道林场)；鞣质和原花青素含量相差不大,含量都较高,都具有很好的提取价值。此部分的研究为落叶松树皮综合开发利用奠定了基础。第二部分为采用不同的提取方法对落叶松树皮中的生物活性物质原花青素进行提取分析。首先,采用超临界CO2萃取技术用于落叶松树皮脱脂,树脂平均萃取收率为2.16%。其次,针对脱脂落叶松树皮,采用超声波辅助法提取原花青素,经单因素和正交实验优化条件为：原料粒度80～100目、功率320W、料液比1：12、时间20min、超声2次,此条件下原花青素提取率为10.44%；采用纤维素酶法提取原花青素,较优条件为：原料粒度80～100目、纤维素酶添加量0.6%、料液比为1：14、pH为5.0、酶解温度40℃、酶解时间2h,此条件下原花青素提取率为7.34%；在纤维素酶法较优条件下并结合超声波辅助法,在超声功率240W和超声时间20min下原花青素提取率为11.05%。对比几种提取工艺,结果表明：超声波提取工艺方法简单,时间短,提取率高,其原花青素粗提物(LPC)的产率、原花青素纯度和平均聚合度分别为22.85%、44.29%和4.52。采用SEM对三种方法提取前后的树皮表面微观形貌分析,表明提取后树皮的纤维结构皆有不同程度的破坏；采用FTIR对三种方法所提取的原花青素进行结构分析其均具有原花青定型结构特征。第三部分对原花青素进行分级纯化分析。采用溶剂逆流萃取法对脱脂落叶松树皮原花青素分级,获得乙酸乙酯萃取级分(LOPC)、乙酸乙酯萃余水级分(LPPC)和醇溶级分(LPR)产率分别为7.36%、5.86%和9.63%；分级级分各占比率为32.21%、25.65%和42.14%；原花青素纯度分别为51.72%、26.37%和39.21%；平均聚合度分别为2.76、7.47和13.25。采用HP2MGL树脂对LPC、LOPC、LPPC级分纯化后产物的累积回收率分别为96.19%、98.54%、99.09%；三种级分纯化后产物中LPC-1、LOPC-1、 LPPC-1纯度分别为84.70%、92.20%、99.79%,平均聚合度分别为6.78、2.66、7.99。采用FTIR确证了LPC-1、LOPC-1、LPPC-1皆具有原花青定型结构。采用HPLC分析了LOPC-1、LPC-1、市售葡萄籽和松树皮提取物中儿茶素的含量分别为5.05%、2.85%、2.77%、0.71%；表儿茶素含量分别为2.02%、0.56%、2.34%、0.10%；原花青素B1分别为0.71%、0.30%、0.61%、0.89%。采用线性模式MALDI-TOF质谱法分析表明-LOPC-1是以二聚体为主到十聚体分布的低聚物；LPPC-1是以六聚体为主的四聚体到十四聚体分布的多聚物。第四部分为多聚原花青素的催化降解研究。采用钯碳为催化剂对LPPC-1进行催化氢解,降解的较优条件为：催化剂用量0.25g/100mL、压力3.5Mpa、温度100℃、时间3h,此条件下降解产物的平均聚合度为2.66,降解率为67.46%,残留率为72.1%；CR57树脂为催化剂对LPPC-1的催化降解较优条件为：树脂用量20mL/100mL、温度70℃、时间1h,此时降解产物的平均聚合度为3.98,降解率为50.19%,残留率为78.4%。采用HPLC分析了降解产物LHOPC-1、LHOPC-2中儿茶素的含量分别为5.27%、4.29%；表儿茶素含量分别为0.31%、1.40%；原花青素B1含量分别为3.45%、0.67%,结果表明降解产物LHOPC-1、LHOPC-2中儿茶素、原花青素B1含量远高于葡萄籽和松树皮提取物。采用FTIR对降解后的产物结构分析表明,两种催化降解得到的产物结构均具有原花青定型结构特征。采用线性模式MALDI-TOF质谱法分析了降解后的LHOPC-1和LHOPC-2产物分子量分布,结果表明LHOPC-1是以三聚体为主到十一聚体分布的低聚物；LHOPC-2是以四聚体为主从三聚体到十二聚体分布的低聚物。第五部分为落叶松树皮原花青素分级纯化后各级分、多聚级分降解产物以及各对照品的还原能力、DPPH自由基、OH自由基和ABTS自由基的清除能力。分析结果为：还原能力大小顺序为LHOPC-1>GS>LOPC-1>LHOPC-2>Vc>PB>LPPC-1;各产物对DPPH自由基清除能力分析表明,Vc能力最低,其它各产物能力差异不显著；各产物对OH自由基清除能力的差异比较显著,钯碳催化氢解产物LHOPC-1清除.OH自由基能力最强,分别是LOPC-1、LPPC-1、GS、PB、VC和TBHQ的1.58倍、13.33倍、1.38倍、7.64倍、8.72倍和8.24倍；GS、LHOPC-1、LHOPC-2、LOPC-1、LPPC-1和PB对ABTS+自由基清除能力的IC5o值分别为2.05ug/mL、2.26ug/mL、2.52ug/mL、2.45ug/mL、2.57ug/mL和2.81ug/mL,皆高于Vc对ABTS+自由基的清除能力。以上结果分析表明,落叶松树皮原花青素分级纯化后级分及降解产物的抗氧化能力较强。更多还原

【Abstract】 The rational and effective utilization of larch bark have a very realistic and far-reaching significance for wood and paper industry in China. There are abundant proanthocyanidins compounds in larch bark, which has a wide range of biological activity in terms of antioxidation, scavenging free radicals, anti-tumor, cardiovascular protection and improving microcirculation. However, the content of oligomers with high utilization value is low, and the main content is polymer, which results the decrease of its bioavailability and biological activity. Therefore, the separation and purification for oligomeric proanthocyanidins and catalytic degradation for polymer proanthocyanidins become the urgent problem. In this paper, the larch bark aced as the research object and we analysized the chemical composition, optimized the extraction conditions of proanthocyanidins, classification and purification,catalytic degradation, and antioxidant performance analysis. The Thesis is mainly including five parts.In the first part, we analysized the chemical composition of the bark of Larix olgensis var. koreana (Changbai Mountain Nature Reserve), Larix gmelinii (Aershan forest bureau in mongolica) and Larix olgensis var. koreana(Erdao forest farms in Jilin City). The content of tannin in bark is11.91%,11.5%,11.43%, the content of flavonoid is16.59%,9.7%,16.59%,, the content of proanthocyanidins is3.84%,2.66%,2.91%and the content of dihydroquercetin is1.87%,0.92%,1.45%, respectively. The results indicated that the content of total flavonoid and dihydroquercetin in the bark of Larix olgensis var. koreana (natural protected areas of Changbai Mountain) is higher than that in Larix gmelinii and Larix olgensis var. koreana(Erdao forest, jilin city); the content of tannin and proanthocyanidins is high and similar, which had good extract value. These research laid the foundation for the development and utilization of larch bark.In the second part, we extraction of proanthocyanidins from larch bark with different extraction methods. Firstly, the larch bark was degreased by the extraction technique of supercritical CO2, and the average extraction yield of resin was2.16%. Secondly, the proanthocyanidins was extracted from the degreased larch bark through the ultrasonic-assisted method. The result of single factor and orthogonal experiments revealed that the optimum extracted conditions were as follows:the granularity of raw materials is80-100mesh, Power is360W, the ratio of material to liquid is1:14, extracted time is20min and twice Ultrasounded.Under these conditions,the extraction rate of proanthocyanidins was10.94%. The optimum conditions for extracting proanthocyanidins by the method of cellulase as follows:the granularity of raw materials is80-100mesh, the loading of cellulase is0.6%, the ratio of materials to liquid is1:14, pH value is5, the temperature and time of enzymatic hydrolysis is 40℃and2h, respectively. The extraction rate of proanthocyanidins was7.34%.Under these conditions,The extraction rate of proanthocyanidins was11.05%by the the method of cellulase combined with ultrasonic assisted when the ultrasonic power is240W and ultrasonic time is20min. Comparing the three kinds of extraction method, the ultrasonic extraction method was simplest, effective and the extraction rate was highest. The yield, purity and the average degree of polymerization of the crude extract proanthocyanidins (LPC) were22.85%,44.29%and4.52, respectively. The surface morphology of the bark had been damaged with different degrees after the extraction with the different three methods by SEM analysis. The structural features of proanthocyanidins is confirmed by FTIR analysised.The graded purification and analysis of proanthocyanidins present in the third part. The procyanidins was fractional separated from degreased larch bark through the extraction method of solvent countercurrent and ethyl acetate fraction (LOPC), ethyl acetate fraction from extraction raffinate wate (LPPC) and alcoholic solution (LPR) were obtaied, the yield of LOPC, LPPC and LPR were7.36%,5.86%and9.63%,respectively. The content ratios of each product were32.21%,25.65%and42.14%, respectively. The purities of proanthocyanidins of each product were51.72%,26.37%and39.21%and The average degree of polymerization was2.76,7.47and13.25, respectively. The cumulative recovery rates of purified product of LPC, LOPC, LPPC fractions were96.19%,98.54%and99.09%, respectively by the adsorption method of HP2MGL resin,. The purities of LPC-1, LOPC-1, LPPC-1were84.74%,92.20%and99.79%and The average degree of polymerization was6.78,2.66,7.99, respectively. The structures of LPC-1, LOPC-1, LPPC-1were procyanidins forming structure, confirmed by FTIR analysis. Through the HPLC analysis method, the content of catechin in LOPC-1, LPC-1commercially available grape seed and the extraction of pine bark were5.05%,2.85%,2.77%,0.71%, respectively. The contents of epicatechin were2.02%,0.56%,2.34%,0.10%, the contents of proanthocyanidins B1were0.71%,0.30%,0.61%,0.89%, respectively. The results revealed that LOPC-1was an oligomer with dimers mainly to Decamers distribution and LPPC-1was a polymer with hexamer mainly from tetramer to fourteen oligomer distribution through the analysis of the linear model of MALDI-TOF mass spectrometry.We researched the catalytic degradation of polymeric proanthocyanidins in the fourth part. LPPC-1was degradated by palladium carbon catalyst and the optimum conditions of degradation were obtained, as following:catalyst amount is0.25g/100mL, pressure is3.5Mpa, temperature is100℃and reaction time is3h. Under these conditions, the degradation rate was67.46%, the residual rate was72.1%and the average degree of degradation products was2.66. When the CR57resin acted as the catalyst, the optimum conditions for degradation of LPPC-1were as follows:the resin dosage is20mL/100mL, temperature is70℃and reaction time is1h. Under these conditions, the average polymerization degree of the degradation products is3.98, the degradation rate is50.19%and the residual rate is78.4%. By the analysis method of HPLC, the contents of catechin were5.27%and4.29%, epicatechin were0.31%and1.40%, procyanidin B1were45%,0.67%in the degradation products of LHOPC-1and LHOPC-2, respectively. The results demonstrated that the content of catechin and proanthocyanidins B1in LHOPC-1and LHOPC-2were higher than those in grape seed and pine bark extract. The products of catalytic degradation were characted by FTIR and confirmed to procyanidins features. The molecular weight distribution of the product of LHOPC-1and LHOPC-2after degradation was analysized by the linear model of MALDI-TOF mass spectrometry. The results revealed that LHOPC-1was an oligomer with trimer mainly to eleven oligomer distribution and LHOPC-2was also an oligomer with tetramer mainly from trimer to twelve oligomer distribution.In the fifth part, We researched the reducing capacity and scavenging ability of grading components and degradation products of proanthocyanidins extracted from Larch bark to DPPH·,·OH and ABTS+·. The tested results showed that the reducing capacity as following: VC> LHOPC-1> GS> LOPC-1> LHOPC-2> TBHQ> PB> LPPC-1; the scavenging ability to DPPH·of Vc was low and the other was almost similar; The scavenging ability of all products to-OH was significant difference, LHOPC-1had the strongest scavenging ability on OH and the scavenging ability of LHOPC-1was1.58times、13.33times、1.38times、7.64times、8.72times and8.24times that of the LOPC-1,LPPC-1,GS,PB,VC and TBHQ,respectively; the scavenging ability to ABTS+.of GS,LHOPC-1,LHOPC-2,LOPC-1,LPPC-1and PB is2.05ug/mL,2.26ug/mL,2.52ug/mL,2.45ug/mL,2.57ug/mLand2.81ug/mL, respectively. They were all higher than the scavenging ability of Vc to ABTS+·.These results indicated that the grading components and degradation products of proanthocyanidins from Larch bark has more excellent antioxidant ability.更多还原