| Objective:Agarwood is famous for its precious value and function of relieving pain.It is the resin part of the heartwood in Aquilaria sinensis formed after external stimulations(known as Jie Xiang).For a long period of time,agarwood has been in short supply because of the long growing process,low yield,as well as poor quality of the product.The latest artifically stimulating methods cannot solve the key problem,together with the high cost on both money and time.Biologically inducing method is a new method for agarwood formation created by our team.Since the preliminary experiments had shown the method can induce the whole tree to form Agarwood within a short period.To perfect the new method to produce agarwood through the whole tree,the author developed this study.Methods:Eighteen four-year-old trees of Aquilaria sinensis were selected randomly in an agarwood planting base.They were devided into seven groups,including biologically inducing group,fire treating group,ant biting group,partly-trunk-prunning method group,drilling method group and mould inducing method group.6 mouths after stimulation,samples of objects were collected and dried for quality evaluation.Methods for quality evaluation in China Pharmacopeia for agarwood were applied.Methods of GC-MS,Phosphotungstic acid lithium molybdenum-sulfuric acid colorimetry and HPLC were added to compare the chemical composition of essencial oil,total chromones and fingerprint of chromones respectively.In addition those methods were also applied to evaluate the different parts of agarwood by biologically inducing method.Quantitative Real-Time PCR(qRT-PCR)was used to detect the seven genes'expression after biologically inducing stimulation and fire treating stimulation on Aquilaria sinensis trees in 6 months.The expressions were compared with the chemical constituents in essencial oil every month.Differential proteomics before and after stimulations were studied.The total protein of heartwood from biologically inducing and fire treating groups were extracted,separated,and dyed by CBB(Coomassie brilliant blue).Different protein points were spotted by Progenesis Same SPots software,identified by MALDI-TOF-MS.Result:1 Quality evaluation study on agarwood by different stimulation methods1.1 Common identical study on agarwood by different stimulation methodsObvious differences were shown among agarwood samples induced by differentstimulation methods.In yield aspect,objects of biologically inducing method group were able to produce 233.8g agarwood on average,others 10.74-58.41g.Agarwood of highest quality from biologically inducing method group was in almost a whole trunk which is 7.5cm high and 3cm in diameter.Quality study showed that agarwood of biologically inducing method group had reached the standards of China Pharmacopeia.Their percentage of ethanol-soluble extractives reached to 11.62%,above the standard(10%),while the fire treating group contained only 4.44%.Samples from both biologically inducing group and fire treating group showed the same TLC behavior of the criterion.In addition behavior of biologically inducing method group were visually closer to the criterion than fire treating group.1.2 Determination of total chromones' contents on agarwood by different stimulation methodsThe criterion contained the highest content of total chromones,which was 19.35± 1.06mg·g-1,when white wood heartwood contained the lowest,which was 2.33±1.25 mg · g-1.Samples from biologically inducing method group got the second-highest content which was 16.67 ± 1.35 mg · g-1.Besides,samples of fire treating group ranked the third(8.34±0.64mg · g-1).Other groups got the contents between 3.12±0.83mg·g-1 and 6.45± 1.47mg·g-1.1.3 GC-MS study on the agarwood by different stimulation methodsCompared with other groups,samples from biologically inducing method and fire treating groups got over 500%characterstic chemicals in agarwood.Samples from the mould inducing method group contained the lowest chemical compounds,almost the same as the white wood heartwood.Among the traditional methods,fire treating method group gained agarwood of more characterstic compounds,while biologically inducing method group gained agarwood with more sesquiterpenes,aromatic series and chromones,and thus could be regarded as the closest to the criterion.1.4 HPLC fingerprint study of chromones on the agarwood by different stimulation methodsThe Shared Mode of HPLC fingerprint was established,and one among the ten common peaks was identified by reference substance.Evaluation of similarity degree showed that different methods can cause significant differences in chemical compositon.Clustering analysis showed that samples from biologically inducing method and reference substance were in the same cluster.2 Study on different parts of agarwood by biologically inducing method2.1 Identical study of different parts of agarwood by biologically inducing methodThe results of quality evaluation in H1 to H4 parts showed almost the same,so did the Z1 to Z4 parts.However the H part was quite different from the Z part.The result of contents in ethanol extracts revealed that H1 to H4 parts reached to the standard(10%)in Chinese Pharmacopeia,but Z1 to Z4 parts failed to.The TLC behaviors of H1 to H4 parts were the same as the criterion.While the Z1 to Z4 parts shared the same TLC behaviors,but only 3 points showed the same Rf to the criterion.2.2 Determination of total chromone content in different parts of agarwood by biologically inducing methodThe contents of H1 to H4 parts were 13.78±0.38-16.69±0.35mg · g1,and the lowest was from H4(13.78 ± 0.38mg · g-1).However the contents of Z1 to Z4 parts were 1.42 ± 0.05-4.19±0.12mg · g-1,with Z4 containing the lowest content.2.3 Analysis of chemical constituents by GC-MS on different parts of agarwood by biologically inducing methodThe contents of sesquiterpenes and chromones in H part were quite high,while the chemical characteristics of H1 to H3 were nearly the same.For H4 part,beside the unique compounds of agarwood there were still some other aromatic series.More fatty acid and alkanes were detected in Z part with no other unique compounds of agarwood,which was significantly different from H part.2.4 Study on the GC-MS fingerprint of essencial oil from agarwood on different parts of agarwood by biologically inducing methodThe similarity degree from the H and Z parts were up to 0.8638-0.9605%,0.8596-0.9163%respectively.2.5 Determination of 3 chromones in agarwood on different parts of agarwood by biologically inducing methodThe contents of 3 chromones varied in different parts.H1 part contained the highest contents of Aquilarone B and Aquilarone C,3.2282 and 0.9889mg ·g-1 respectively.They were followed by H2 and H4 with contents of Aquilarone B 2.0369 and 1.8024 mg· g-1 respectively,contents of Aquilarone C 0.3439 and 0.6527 mg · g-1 respectively.The lowest contents of Aquilarone B and Aquilarone C were 1.4847and 0.3152mg ·g-1 fromH3.For the contents of CX16,with the highest H4 of 7285 mg · g-1,followed H3 and H1 of 0.4519 and 0.3633 mg · g-1 respectively.The lowest content was 0.24mg · g-1 from H2.3 Study on the mechanism of agarwood formation by biologically induced method3.1 Gene expressions in agarwood by biologically inducing methodThe expressions of DXS1?HMGR and FPS decreased slowly in biologically inducing method group.While they sharply increased in fire treating groupin 5 months.Especially the FPS one increased almost 100 times higher than control and HMGR 30 times respectively.Expressions of ASS1 and TPS increased slowly,and reached to the highest in 3 months in biologically inducing method group.TPS up-expressed in 5 months,reached to the highest in 6 months.The change ranges were not obvious,which were the same as DXS1?HMGR and FPS.However the expression of ASS1 and TPS sharply increased in 5 months with 1000 times higher than control.Expressions of CHS1 decreased slowly in biologically inducing method group while increased sharply in 5 months with almost 600 times bigger than conrol.After biologically inducing stimulation,CHI1 up-expressed slowly,and reached the highest in 3 months,then down-expressed.For fire treating samples CHI1 down-expressed later and sharply up-expressed in 5 months,then down-expressed again.3.2 Compared study on chemical constituents and gene expressions by biologically inducing stimulationThe contents of ethyl ether extract,sesquiterpenes and chromones were 0.Four months later these contents began to increase,and reached to 5.13%6 months later in biologically inducing method group.And the percentage of sesquiterpenes and chromones was 22.04%and 64.41%respectively.Fire treating group behaved almost the same.No chemicals were found in 3 months,and four months later these contents began to increase.In 6 months the contents of ethyl ether extract,sesquiterpenes and chromones were 1.05%,10.55%and 42.02%respectively.Compared with the results of gene expression in biologically inducing method group,except TPS with an obvious up-trend same as the contents,others showed no obvious feature.In fire treating group both the chemical and gene level showed the same increasing trend as time went by.3.3 2-DE study on agarwood by biologically inducing methodThe total protein of agarwood from biologically inducing method and fire treating groups was extracted,seperated and identified.1500-1900 effective protein points were detected on every map.Paired contrast among biologically inducing method,fire treating and white wood groups were analyzed,then 647 points were found,32 points were finally identified.Conclusion:1 Quality of agarwood from different stimulating methods showed significant difference.Agarwood from biologically inducing method satisfied all the quality index from the Chinese Pharmacopia,with its quality obviously better than that of others obtained by traditional methods.The result of appearance,microscopic structure,color reaction and content of ethanolic extract of agarwood from biologically induced method group met the standards of Chinese Pharmacopia.Its TLC behavior,content of total chromone,chemical composition of essencial oil,fingerprint were closer to the criterion.Cluster analysis showed that sample from biologically inducing method group and the criterion were in one cluster.Content of ethanolic extract from other groups were far behind the standards in Chinese Pharmacopia.Samples from other groups showed significant difference from the criterion.As for yield,biologically inducing method was quite superior to other methods.2 Quality of agarwood from different parts by biologically inducing method showed significant difference.Z part may be in the transition state from white heartwood to agarwood.The quality of H part was higher than Z part,almost the same as criterion.But the quality index of different H parts varied,which may be the different effects of the new method to the different parts of the trunk,or influenced by conducting system.Quality of Z parts didn' t reach the standards because of the planned sample collecting timing.But that is enough to provide some information on the optimization of biologically inducing method.The results of total chromone content,essencial oil,GC-MS fingerprint,3 kinds of chromones' contents and HPLC fingerprint revealed that H part were the real agarwood of high-quality.In addition,Z part may be at the transition state from white heartwood to agarwood.3 Significant differences of gene expressions after differnt stimulation in6 months showed some relevance to the chemical contents,together with the distribution of differential expression protein,revealing that the mechanism of biologiclly inducing method may be different from traditionally fire treated method.In biologically inducing method group the changes of different gene expression were unapparent,mainly concentrated in 3 months or 5 months.While in fire treating group a unique feature can be found easily that gene expressions sharply increased in 5 months,some even reached to 1000 times.Study on relevance between gene expression and chemical contents in two groups were quite different.It can be infered that these two methods may induce different biosynthetic pathways for terpinoids and flavonoids,or the sample collecting timing was not accurate enough to overall reveal the inner physiological and biochemical reactions.More target genes and time point should be included for the detection of agarwood fromation mechanism.The results of 2-DE indicated that these two methods may differ in agarwood formating mechanism.Fire treatment can sharply trigger the plant defense reactions,which was accord with the result of gene expressions.For biologically inducing method,althogh differential expression proteins were different from fire treating method,it can also induce the plant efense reactions to form higher quality agarwood on chemical level.That may be the reason that in our research due to the planned sample collection timing no obvious changed were observed,or the new method induced other mechanism different from the traditional one.This issue should be deeply investigated later. |
PDF Full Text Request