香樟木材提取物的成分及其防腐机理的研究
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摘要
香樟木材具有很强的耐腐性能,究其原因主要是由于其内部含有的各种抑菌活性成分,但迄今为止香樟材提取物对木材腐朽菌(彩绒革盖菌和密粘褶菌)的抑制作用机制还不为人知。因此,本论文将各种溶剂提取香樟木质部得到的提取物作用于木材腐朽菌并测试其抑菌及耐腐性能,再采用XRD, FTIR, TG等进行表征,系统分析了香樟木质部提取物抑制木材腐朽菌生长的生理生化机制、分子生物学以及香樟材挥发性成分的释放规律,以揭示香樟材次生代谢产物与木材腐朽菌之间的关系,为香樟木质部提取物及人工合成同类物质在木材防腐方面的应用提供了理论依据。论文研究的主要结论及创新点如下：
1.采用响应面法优化超声波-微波协同提取香樟木质部提取物,得到最佳工艺参数为：液料比60mL/g,提取时间30min,粒径大小40～50目,在此条件下的得率为4.65%。采用生长速率法分析了香樟木质部的5种溶剂提取物对木材腐朽菌的毒力,结果表明：香樟木质部三氯甲烷提取物、甲醇提取物分别对彩绒革盖菌和密粘褶菌的抑制效果最强,在8g/L浓度下的抑菌率分别为57.5%和64.87%,EC50值分别为7.8g/L和0.3g/L。对香樟木质部甲醇提取物的GC/MS分析共鉴定出27种成分,主要包括：芳樟醇(2.9%)、樟脑(14.29%)、a-松油醇(9.88%)、反式-氧化芳樟醇(7.66%)等。在香樟木质部三氯甲烷提取物中共鉴定出20种化学成分,其中含量较高有：芳樟醇(5.9%),樟脑(17.6%),a-松油醇(11.8%),肉豆蔻醛(5.6%),(-)-g-杜松烯(7.4%)等。
2.通过木材防腐剂对腐朽菌毒性实验室试验方法进行耐白腐试验,结果表明：香樟木质部5种溶剂提取物在浓度为10%(w/v)时均达到Ⅰ级强耐腐水平,在浓度为1%时的耐腐效果都达到Ⅱ级。显微镜观察香樟木质部提取物处理白腐菌菌丝,发现其细胞形态皱缩扭曲变形,菌体表面出现破裂。SEM观察发现,三氯甲烷提取物处理试样白腐后在结构方面保持较完整,具有较好的耐白腐效果。XRD对比各试样结晶度大小顺序为：毛白杨素样>乙酸乙酯提取物>ACQ>三氯甲烷提取物>丙酮提取物>甲醇提取物>蒸馏水提取物>樟脑。FTIR结果分析表明：与纤维素和半纤维素相关的谱峰其相对强度变化不明显或略有降低,而表征木质素的一系列特征峰峰高出现不同程度降低。通过耐褐腐试验发现,4%ACQ、4%硼酸、4%樟脑以及10%蒸馏水提取物、10%甲醇提取物、10%乙酸乙酯提取物、10%丙酮提取物处理材的失重率分别为1.78%,5.7%,13.08%,40.85%,9.39%,18.66%和21.45%。香樟木质部甲醇提取物处理褐腐菌菌丝会导致细胞壁变粗糙,细胞扩张变形,说明甲醇提取物影响了菌丝的结构和功能。甲醇提取物处理材其结构相对其他提取物处理材在褐腐后更完整,显示了其较好的防腐效果。XRD对比发现,4种香樟木质部提取物的防腐效果与相对结晶度大小呈正相关。FTIR研究发现表征纤维素和半纤维素的特征峰值越低,则被降解的量也越大,相对应的防腐剂防腐效果越差。热重分析表明,最大失重量越大则对应试样所用的香樟提取物防腐效果越好。各试样的一级热解反应活化能大小比较反映出防腐剂防腐效果越好,则纤维素含量保留得越多,活化能也越高。
3.检测分析了香樟木质部提取物对腐朽菌的生理生化机制,结果表明：香樟木质部甲醇与三氯甲烷提取物均对密粘褶菌和彩绒革盖菌菌丝的氧呼吸作用有抑制效果。乙酸乙酯提取物处理浓度越高,对褐腐菌分泌的纤维素酶的抑制越强。乙酸乙酯提取物会使密粘褶菌的菌丝电导率提高,且浓度越高其电导率越大。乙酸乙酯提取物浓度越高,其处理后的褐腐菌蛋白渗透量就越大。
4.采用双向电泳技术和质谱技术分别对香樟木质部甲醇及丙酮提取物处理白腐菌,甲醇以及乙酸乙酯提取物处理褐腐菌的抑菌机制进行蛋白质组学分析。结果表明：①对香樟木质部甲醇提取物处理白腐菌鉴定出的差异蛋白进行细胞定位,生物学过程以及功能分析发现：9个在细胞质,6个在细胞内,5个在线粒体等；22%为生物合成,15%为小分子代谢,10%为细胞的氨基酸代谢等；25%与离子结合有关,15%与氧化还原酶活性有关,15%与ATP酶活性以及跨膜运输活性有关。②对香樟木质部丙酮提取物处理白腐菌的差异蛋白的细胞定位,生物学过程以及功能分析发现：11个在细胞器,8个在细胞质,8个在蛋白质络合物等；18%参与了小分子代谢过程,14%参与了细胞氮化合物代谢过程,13%参与了生物合成的过程等；30%与离子结合有关,14%与翻译因子活性,核酸结合有关,12%与ATP酶活性有关。③由香樟木质部甲醇提取物处理褐腐菌差异表达蛋白的功能分析可知：酸性核糖体蛋白出现下调表达,说明褐腐菌在蛋白质合成方面受到了抑制,生命稳定性遭到破坏；磷酸丙酮酸水合酶是经过甲醇提取物处理后的褐腐菌中出现的特异性蛋白。④对香樟木质部乙酸乙酯提取物处理褐腐菌差异蛋白进行了鉴定,eIF-5A酶、NAD-苹果酸酶、GMP合成酶、富含甘氨酸RNA结合蛋白等的上调或下调表明褐腐菌在能量代谢、核糖体合成等方面发生了变化。
5.采用固相微萃取结合气质联用(SPME-GC/MS)技术检测了香樟木质部挥发性成分,比较分析了两种色谱柱对分离效果的影响。结果表明：采用弱极性DB-5MS色谱柱共分离出51个组分,解析出43种挥发性成分,其峰面积占总峰面积的97.04%,分离效果和所得的峰形比采用DB-WAXetr色谱柱更好；分析鉴定出含量较高的成分包括：左旋-α-蒎烯(4.57%)、双环[3.1.0]-4-甲基-1-异丙基-2-己烯(4.16%)、D-柠檬烯(7.49%)、桉叶油醇(13.85%)、樟脑(38.71%)等。运用顶空气相色谱(HS-GC)测试分析了香樟木质部樟脑的挥发机制,推导出在40～70℃加热范围,0～1.4h检测时间内,顶空气相色谱检测到的樟脑含量y(μg)与温度T(℃)和时间t(h)的耦合关系为：Y=-75.369+2.3786T+(41.125-1.1972T)t。
Cinnamomum camphora (L.) Presl.(C. camphora) is one of the most important hardwood species indigenous to China that possesses significant antifungal activity. C. camphora contain essential oil which can kill many types of harmful bacteria. Studies have reported on the chemical components of extractives, as well as their antimicrobial and insecticidal effects. The mechanism of the C. camphora xylem extractives inhibit Gloeophyllum trabeum (G trabeum) and Coriolus versicolor (C. versicolor) is unknown up to now. In order to reveal the relation between secondary metabolite of C. camphora and wood-decaying fungi and provide the theory basis to the application of the C. camphora extractives in wood preservatives. In this paper, various C. camphora xylem extractives were used to inhibit G. trabeum and C. versicolor to obtain decay resistance activity. Then all kinds of samples were characterized by XRD, FTIR and TG analysis. Last physiological and biochemical mechanism of the extractives antifungal activity, proteome research and release regulation of the volatile components was conducted by the author. The main conclusions and innovations as follows:
1. The extraction process for microwave-ultrasound assisted extraction of extractives from C. camphora wood by response surface methodology. The results showed that the optimum extraction condition was as follows:liquid-solid ratio60ml/g, extraction time30min, and particle size40～50mesh. Under above optimum condition, the extraction yield was4.65%.The antifungal activity of C. camphora extracts was tested against two wood-rot fungus by the growth rate of poison medium culture method. Chloroform extracts had the best antifungal effect on C. versicolor among these five extracts. This growth inhibition ratio of chloroform extracts was calculated in the ranges of8mg/mL to57.5%for C. versicolor. The growth inhibition ratios for methanol extracts were8mg/mL to64.9%for G. trabeum. Methanol extracts possessed the greatest inhibition of hyphal growth of G. trabeum among these five extracts. Further bioassays of the extracts revealed some inhibitory effect on the two wood-decaying fungi, with the chloroform and the methanol extracts showing the best results. The EC50of chloroform extracts on white-rot fungus and brown-rot fungus were7.8mg/mL and4.53mg/mL, respectively.27different components accounting for73.02%of the total MeOH extracts from the xylem of C. camphora were identified by gas chromatography-mass spectrometry. The major chemical components of MeOH extracts are linalool (2.9%), camphor (14.29%), a-terpineol (9.88%), trans-linalool oxide (furanoid)(7.66%), etc. A total of20compounds were identified which accounting for80.76%of the total chloroform extracts from the xylem of C. camphora, and the major constituents were: cineole (2.0%), linalool (5.9%), camphor (17.6%), a-terpineol (11.8%), tetradecanal (5.6%),(-)-g-cadinene (7.4%), etc.
2. Populus tomentosa was impregnated with different preservatives, and the decay resistance performance was studied. The results showed that the samples impregnated with five preservatives of the concentration10%(w/v) meet the demand of degree Ⅰ. The samples impregnated with five preservatives of the concentration1%meet the demand of degree Ⅱ. G. trabeum was observed microscopically after treatment with methanol extracts at a concentration of8mg/mL. After24h of treatment, the morphological characteristics of aerial hyphae appeared uneven, with swollen and excessive branching; part of the swollen hyphal areas continued to expand, which resulted in the formation of extremely swollen bodies of irregular shape; some of the hyphae had apoptosis and condensed endosomes, which leaked out and formed voids. The SEM micrograph clearly shows the structural integrity of the decay samples which treated with methanol extractive is better than another, treatment with chloroform extracts showed strong decay resistance. The XRD comparison showed that the order of crystal degree was:untreated samples> ethyl acetate extracts> ACQ> chloroform extracts> acetone extracts> methanol extracts> hot water extracts> camphor. The FTIR analysis showed that the spectral peak of cellulose and hemicellulose decreased not obviously, while that of lignin displayed reductions in different degrees compared to the untreated samples. The analysis results proved that the degradation activity of lignin is stronger than that of cellulose and hemicelluloses by white-rot fungi. Ethyl acetate extracts and ACQ inhibited good effect of anti-fungi. Masson pine which was impregnated with these different extracts and the decay resistance performance of the wood treated with the extracts was studied. The results showed that the mass loss of wood treated with4%ACQ,4%boric acid,4%camphor, and10%hot water extracts,10%methanol extracts,10%ethyl acetate extracts, and10%chloroform extracts were1.78%,5.7%,13.08%,40.85%,9.39%,18.66%, and21.45%, respectively. The brown rot fungi mycelium which treated with methanol extracts that hyphae exhibited an obvious morphological change:the cell wall became rough, and the cell expanded, became twisted, and exhibited uneven growth of hyphae, indicating that the extracts affected the structure and function of the hyphae. The samples treated with methanol extract have more complete wood structures than other extracts, shows a good decay resistance effect. Through XRD analysis shows that the preservative effects of four extracts are proportional to the degree of crystallinity. According to FTIR analysis, the amount of degraded cellulose and hemicellulose increased with the decline of characteristic absorption peak of cellulose and hemicellulose, which means that the corresponded preservative is worse. Using the thermal analysis to study and compared the pyrolysis characteristics and thermodynamics of Pinus massoniana and different sapwood of preservative-treated P. massoniana and treatment of brown rot. The more the maximum weight loss, the decay resistance effect of wood preservative is better. The large of activation energy of sample in primary pyrolysis reaction, the decay resistance effect of wood preservative is better.
3. Physiological and biochemical mechanism of the extracts of C. camphor a was detected. The results indicated that the methanol and chloroform extracts had oxygen consumption inhibition effect on C. versicolor and G. trabeum. The inhibition of cellulase secreted by G. trabeum was increased with the augment of the concentration. The ethyl acetate extracts could improve the electric conductivity of the G. trabeum hyphae, and the higher the concentration, the conductivity was higher as well. Protein permeability increased with the promotion of the concentration of the ethyl acetate extracts.
4. Proteomics analysis the antifungal mechanism of C. camphora extracts was conducted by two-dimensional electrophoresis and mass spectrometry. The results as follows:①For the identified protein which was produced by C. versicolor treated with methanol extracts, the cell localization results showed that9protein exited in cytoplasm,6in intracellular and5in mitochondrion; the biological process and functional analysis indicated that22%identified protein involved in the biosynthetic process,15%participated in the process of small molecule metabolic process,10%took part in cellular amino acid metabolic process,25%related to ion binding,15%related to oxidoreductase activity and15%related to ATPase activity and transmembrane transporter activity.②For the identified protein which was produced by C. versicolor treated with acetone extracts, the cell localization results showed that11proteins in organelle,8proteins exited in cytoplasm and8proteins belonged to protein complex; the biological process and function analysis indicated that18%participated in the process of small molecule metabolic process,14%took part in the process of cellular nitrogen compound metabolic process,13%identified protein involved in the biosynthetic process,30%related to ion binding,14%had something to translation factor activity, nucleic acid binding and12%related to ATPase activity.③The function analysis of the identified protein which was produced by G. trabeum treated with methanol extracts results showed that down-regulating the expression of60S acidic ribosomal protein P2was happened, which indicated G. trabeum was inhibited in terms of protein synthesis. Phosphopyruvate hydratase was emerged when G. trabeum was inhibited by methanol extracts.④The appraisal of the identified protein which was produced by G. trabeum treated with ethyl acetate extracts that was conducted by author. The rise or down of eukaryotic translation initiation factor5A, NAD-malate dehydrogenase, GMP synthase, glycine-rich RNA binding protein up-regulation or down-regulation expression which indicated the energy metabolism and ribosomal synthesis of G. trabeum was changed during the treatment.
5. In this paper, solid phase microextraction (SPME) was put to use absorbing the volatile components of C. camphora xylem, separation and identification of the volatile components was conducted by GC/MS, the author compared the effects of different chromatographic column on separation efficiency as well. The results showed that GC/MS spectrometric analysis with DB-5MS isolated51components which contains43kinds of volatile content, the peak area of volatile components accounted for97.04%of the total peak area. The separation efficiency and the peak shape of GC/MS spectrometric analysis with DB-5MS is superior to GC/MS spectrometric analysis with DB-WAXetr. Based on the identification of the volatile components, ls-alpha-Pinene (4.57%), Bicyclo[3.1.0]hex-2-ene,4-methyl-1(1-methylethyl)-(4.16%), D-Limonene (7.49%), Eucalyptol (13.85%), Camphor (38.71%) accounted for most of them. Using the method of headspace gas chromatography (HS-GC) to test and analyze the volatilization mechanism of camphor. The coupling relation among the detection of camphor y(μg), the temperature T (℃) and the time t (h) was deduced:Y=-75.369+2.3786T+(41.125-1.1972T)t.
1.采用响应面法优化超声波-微波协同提取香樟木质部提取物,得到最佳工艺参数为：液料比60mL/g,提取时间30min,粒径大小40～50目,在此条件下的得率为4.65%。采用生长速率法分析了香樟木质部的5种溶剂提取物对木材腐朽菌的毒力,结果表明：香樟木质部三氯甲烷提取物、甲醇提取物分别对彩绒革盖菌和密粘褶菌的抑制效果最强,在8g/L浓度下的抑菌率分别为57.5%和64.87%,EC50值分别为7.8g/L和0.3g/L。对香樟木质部甲醇提取物的GC/MS分析共鉴定出27种成分,主要包括：芳樟醇(2.9%)、樟脑(14.29%)、a-松油醇(9.88%)、反式-氧化芳樟醇(7.66%)等。在香樟木质部三氯甲烷提取物中共鉴定出20种化学成分,其中含量较高有：芳樟醇(5.9%),樟脑(17.6%),a-松油醇(11.8%),肉豆蔻醛(5.6%),(-)-g-杜松烯(7.4%)等。
2.通过木材防腐剂对腐朽菌毒性实验室试验方法进行耐白腐试验,结果表明：香樟木质部5种溶剂提取物在浓度为10%(w/v)时均达到Ⅰ级强耐腐水平,在浓度为1%时的耐腐效果都达到Ⅱ级。显微镜观察香樟木质部提取物处理白腐菌菌丝,发现其细胞形态皱缩扭曲变形,菌体表面出现破裂。SEM观察发现,三氯甲烷提取物处理试样白腐后在结构方面保持较完整,具有较好的耐白腐效果。XRD对比各试样结晶度大小顺序为：毛白杨素样>乙酸乙酯提取物>ACQ>三氯甲烷提取物>丙酮提取物>甲醇提取物>蒸馏水提取物>樟脑。FTIR结果分析表明：与纤维素和半纤维素相关的谱峰其相对强度变化不明显或略有降低,而表征木质素的一系列特征峰峰高出现不同程度降低。通过耐褐腐试验发现,4%ACQ、4%硼酸、4%樟脑以及10%蒸馏水提取物、10%甲醇提取物、10%乙酸乙酯提取物、10%丙酮提取物处理材的失重率分别为1.78%,5.7%,13.08%,40.85%,9.39%,18.66%和21.45%。香樟木质部甲醇提取物处理褐腐菌菌丝会导致细胞壁变粗糙,细胞扩张变形,说明甲醇提取物影响了菌丝的结构和功能。甲醇提取物处理材其结构相对其他提取物处理材在褐腐后更完整,显示了其较好的防腐效果。XRD对比发现,4种香樟木质部提取物的防腐效果与相对结晶度大小呈正相关。FTIR研究发现表征纤维素和半纤维素的特征峰值越低,则被降解的量也越大,相对应的防腐剂防腐效果越差。热重分析表明,最大失重量越大则对应试样所用的香樟提取物防腐效果越好。各试样的一级热解反应活化能大小比较反映出防腐剂防腐效果越好,则纤维素含量保留得越多,活化能也越高。
3.检测分析了香樟木质部提取物对腐朽菌的生理生化机制,结果表明：香樟木质部甲醇与三氯甲烷提取物均对密粘褶菌和彩绒革盖菌菌丝的氧呼吸作用有抑制效果。乙酸乙酯提取物处理浓度越高,对褐腐菌分泌的纤维素酶的抑制越强。乙酸乙酯提取物会使密粘褶菌的菌丝电导率提高,且浓度越高其电导率越大。乙酸乙酯提取物浓度越高,其处理后的褐腐菌蛋白渗透量就越大。
4.采用双向电泳技术和质谱技术分别对香樟木质部甲醇及丙酮提取物处理白腐菌,甲醇以及乙酸乙酯提取物处理褐腐菌的抑菌机制进行蛋白质组学分析。结果表明：①对香樟木质部甲醇提取物处理白腐菌鉴定出的差异蛋白进行细胞定位,生物学过程以及功能分析发现：9个在细胞质,6个在细胞内,5个在线粒体等；22%为生物合成,15%为小分子代谢,10%为细胞的氨基酸代谢等；25%与离子结合有关,15%与氧化还原酶活性有关,15%与ATP酶活性以及跨膜运输活性有关。②对香樟木质部丙酮提取物处理白腐菌的差异蛋白的细胞定位,生物学过程以及功能分析发现：11个在细胞器,8个在细胞质,8个在蛋白质络合物等；18%参与了小分子代谢过程,14%参与了细胞氮化合物代谢过程,13%参与了生物合成的过程等；30%与离子结合有关,14%与翻译因子活性,核酸结合有关,12%与ATP酶活性有关。③由香樟木质部甲醇提取物处理褐腐菌差异表达蛋白的功能分析可知：酸性核糖体蛋白出现下调表达,说明褐腐菌在蛋白质合成方面受到了抑制,生命稳定性遭到破坏；磷酸丙酮酸水合酶是经过甲醇提取物处理后的褐腐菌中出现的特异性蛋白。④对香樟木质部乙酸乙酯提取物处理褐腐菌差异蛋白进行了鉴定,eIF-5A酶、NAD-苹果酸酶、GMP合成酶、富含甘氨酸RNA结合蛋白等的上调或下调表明褐腐菌在能量代谢、核糖体合成等方面发生了变化。
5.采用固相微萃取结合气质联用(SPME-GC/MS)技术检测了香樟木质部挥发性成分,比较分析了两种色谱柱对分离效果的影响。结果表明：采用弱极性DB-5MS色谱柱共分离出51个组分,解析出43种挥发性成分,其峰面积占总峰面积的97.04%,分离效果和所得的峰形比采用DB-WAXetr色谱柱更好；分析鉴定出含量较高的成分包括：左旋-α-蒎烯(4.57%)、双环[3.1.0]-4-甲基-1-异丙基-2-己烯(4.16%)、D-柠檬烯(7.49%)、桉叶油醇(13.85%)、樟脑(38.71%)等。运用顶空气相色谱(HS-GC)测试分析了香樟木质部樟脑的挥发机制,推导出在40～70℃加热范围,0～1.4h检测时间内,顶空气相色谱检测到的樟脑含量y(μg)与温度T(℃)和时间t(h)的耦合关系为：Y=-75.369+2.3786T+(41.125-1.1972T)t。
Cinnamomum camphora (L.) Presl.(C. camphora) is one of the most important hardwood species indigenous to China that possesses significant antifungal activity. C. camphora contain essential oil which can kill many types of harmful bacteria. Studies have reported on the chemical components of extractives, as well as their antimicrobial and insecticidal effects. The mechanism of the C. camphora xylem extractives inhibit Gloeophyllum trabeum (G trabeum) and Coriolus versicolor (C. versicolor) is unknown up to now. In order to reveal the relation between secondary metabolite of C. camphora and wood-decaying fungi and provide the theory basis to the application of the C. camphora extractives in wood preservatives. In this paper, various C. camphora xylem extractives were used to inhibit G. trabeum and C. versicolor to obtain decay resistance activity. Then all kinds of samples were characterized by XRD, FTIR and TG analysis. Last physiological and biochemical mechanism of the extractives antifungal activity, proteome research and release regulation of the volatile components was conducted by the author. The main conclusions and innovations as follows:
1. The extraction process for microwave-ultrasound assisted extraction of extractives from C. camphora wood by response surface methodology. The results showed that the optimum extraction condition was as follows:liquid-solid ratio60ml/g, extraction time30min, and particle size40～50mesh. Under above optimum condition, the extraction yield was4.65%.The antifungal activity of C. camphora extracts was tested against two wood-rot fungus by the growth rate of poison medium culture method. Chloroform extracts had the best antifungal effect on C. versicolor among these five extracts. This growth inhibition ratio of chloroform extracts was calculated in the ranges of8mg/mL to57.5%for C. versicolor. The growth inhibition ratios for methanol extracts were8mg/mL to64.9%for G. trabeum. Methanol extracts possessed the greatest inhibition of hyphal growth of G. trabeum among these five extracts. Further bioassays of the extracts revealed some inhibitory effect on the two wood-decaying fungi, with the chloroform and the methanol extracts showing the best results. The EC50of chloroform extracts on white-rot fungus and brown-rot fungus were7.8mg/mL and4.53mg/mL, respectively.27different components accounting for73.02%of the total MeOH extracts from the xylem of C. camphora were identified by gas chromatography-mass spectrometry. The major chemical components of MeOH extracts are linalool (2.9%), camphor (14.29%), a-terpineol (9.88%), trans-linalool oxide (furanoid)(7.66%), etc. A total of20compounds were identified which accounting for80.76%of the total chloroform extracts from the xylem of C. camphora, and the major constituents were: cineole (2.0%), linalool (5.9%), camphor (17.6%), a-terpineol (11.8%), tetradecanal (5.6%),(-)-g-cadinene (7.4%), etc.
2. Populus tomentosa was impregnated with different preservatives, and the decay resistance performance was studied. The results showed that the samples impregnated with five preservatives of the concentration10%(w/v) meet the demand of degree Ⅰ. The samples impregnated with five preservatives of the concentration1%meet the demand of degree Ⅱ. G. trabeum was observed microscopically after treatment with methanol extracts at a concentration of8mg/mL. After24h of treatment, the morphological characteristics of aerial hyphae appeared uneven, with swollen and excessive branching; part of the swollen hyphal areas continued to expand, which resulted in the formation of extremely swollen bodies of irregular shape; some of the hyphae had apoptosis and condensed endosomes, which leaked out and formed voids. The SEM micrograph clearly shows the structural integrity of the decay samples which treated with methanol extractive is better than another, treatment with chloroform extracts showed strong decay resistance. The XRD comparison showed that the order of crystal degree was:untreated samples> ethyl acetate extracts> ACQ> chloroform extracts> acetone extracts> methanol extracts> hot water extracts> camphor. The FTIR analysis showed that the spectral peak of cellulose and hemicellulose decreased not obviously, while that of lignin displayed reductions in different degrees compared to the untreated samples. The analysis results proved that the degradation activity of lignin is stronger than that of cellulose and hemicelluloses by white-rot fungi. Ethyl acetate extracts and ACQ inhibited good effect of anti-fungi. Masson pine which was impregnated with these different extracts and the decay resistance performance of the wood treated with the extracts was studied. The results showed that the mass loss of wood treated with4%ACQ,4%boric acid,4%camphor, and10%hot water extracts,10%methanol extracts,10%ethyl acetate extracts, and10%chloroform extracts were1.78%,5.7%,13.08%,40.85%,9.39%,18.66%, and21.45%, respectively. The brown rot fungi mycelium which treated with methanol extracts that hyphae exhibited an obvious morphological change:the cell wall became rough, and the cell expanded, became twisted, and exhibited uneven growth of hyphae, indicating that the extracts affected the structure and function of the hyphae. The samples treated with methanol extract have more complete wood structures than other extracts, shows a good decay resistance effect. Through XRD analysis shows that the preservative effects of four extracts are proportional to the degree of crystallinity. According to FTIR analysis, the amount of degraded cellulose and hemicellulose increased with the decline of characteristic absorption peak of cellulose and hemicellulose, which means that the corresponded preservative is worse. Using the thermal analysis to study and compared the pyrolysis characteristics and thermodynamics of Pinus massoniana and different sapwood of preservative-treated P. massoniana and treatment of brown rot. The more the maximum weight loss, the decay resistance effect of wood preservative is better. The large of activation energy of sample in primary pyrolysis reaction, the decay resistance effect of wood preservative is better.
3. Physiological and biochemical mechanism of the extracts of C. camphor a was detected. The results indicated that the methanol and chloroform extracts had oxygen consumption inhibition effect on C. versicolor and G. trabeum. The inhibition of cellulase secreted by G. trabeum was increased with the augment of the concentration. The ethyl acetate extracts could improve the electric conductivity of the G. trabeum hyphae, and the higher the concentration, the conductivity was higher as well. Protein permeability increased with the promotion of the concentration of the ethyl acetate extracts.
4. Proteomics analysis the antifungal mechanism of C. camphora extracts was conducted by two-dimensional electrophoresis and mass spectrometry. The results as follows:①For the identified protein which was produced by C. versicolor treated with methanol extracts, the cell localization results showed that9protein exited in cytoplasm,6in intracellular and5in mitochondrion; the biological process and functional analysis indicated that22%identified protein involved in the biosynthetic process,15%participated in the process of small molecule metabolic process,10%took part in cellular amino acid metabolic process,25%related to ion binding,15%related to oxidoreductase activity and15%related to ATPase activity and transmembrane transporter activity.②For the identified protein which was produced by C. versicolor treated with acetone extracts, the cell localization results showed that11proteins in organelle,8proteins exited in cytoplasm and8proteins belonged to protein complex; the biological process and function analysis indicated that18%participated in the process of small molecule metabolic process,14%took part in the process of cellular nitrogen compound metabolic process,13%identified protein involved in the biosynthetic process,30%related to ion binding,14%had something to translation factor activity, nucleic acid binding and12%related to ATPase activity.③The function analysis of the identified protein which was produced by G. trabeum treated with methanol extracts results showed that down-regulating the expression of60S acidic ribosomal protein P2was happened, which indicated G. trabeum was inhibited in terms of protein synthesis. Phosphopyruvate hydratase was emerged when G. trabeum was inhibited by methanol extracts.④The appraisal of the identified protein which was produced by G. trabeum treated with ethyl acetate extracts that was conducted by author. The rise or down of eukaryotic translation initiation factor5A, NAD-malate dehydrogenase, GMP synthase, glycine-rich RNA binding protein up-regulation or down-regulation expression which indicated the energy metabolism and ribosomal synthesis of G. trabeum was changed during the treatment.
5. In this paper, solid phase microextraction (SPME) was put to use absorbing the volatile components of C. camphora xylem, separation and identification of the volatile components was conducted by GC/MS, the author compared the effects of different chromatographic column on separation efficiency as well. The results showed that GC/MS spectrometric analysis with DB-5MS isolated51components which contains43kinds of volatile content, the peak area of volatile components accounted for97.04%of the total peak area. The separation efficiency and the peak shape of GC/MS spectrometric analysis with DB-5MS is superior to GC/MS spectrometric analysis with DB-WAXetr. Based on the identification of the volatile components, ls-alpha-Pinene (4.57%), Bicyclo[3.1.0]hex-2-ene,4-methyl-1(1-methylethyl)-(4.16%), D-Limonene (7.49%), Eucalyptol (13.85%), Camphor (38.71%) accounted for most of them. Using the method of headspace gas chromatography (HS-GC) to test and analyze the volatilization mechanism of camphor. The coupling relation among the detection of camphor y(μg), the temperature T (℃) and the time t (h) was deduced:Y=-75.369+2.3786T+(41.125-1.1972T)t.
引文
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