| 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. |
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