Studies On The Dalbergia Odor Ifera T.Chen Stumulated By "Biological Stimulation Method" And Quality Assessment By Biological Stimulation

ObjectiveThe fragrant resin of D.Odorifera T.Chen can formated by external stimulated,currently,the artificial stimulating method that has been knowed can not achieve a good result in quality and yield.To develop a new stimulating method for stimulating fragrant resin in D.Odorifera T.Chen-"Biological stimulation method"which can reduce fomating time and improve heartwood production,Further more,the research of fragrant resin heartwood made by "Bio-physical stimulation method"evaluating can provide more scientific and advanced quality evaluation system.Methods1 Experimental study of stimulate Method 1.1 The effect of stimulation way in dalbergia heartwood formation The 4-years-old healthy dalbergia has been select for subjects,Designed three groups:one hole injection fluid,two holes injection fluid and transfusion groups.The growth about leaves,branches and trunk have been recorded after 1 week,2 months,4 months,6 months,12 months.Moreover,the best situmulating way was been choosed by different indexes of fragrant hearwood,such as color,flavor and hardness anfer 2 months,4 months,6 months,12 months.1.2 The effect of stimulation reagents in dalbergia heartwood formationThe 4-years-old healthy dalbergia has been select for subjects,Designed six groups:a-Reagent,Maleic hydrazide,Hydrogen peroxide,Acetic acid,Formic acid,Thidiazuron.The growth about leaves,branches and trunk have been recorded after 1 week,2 months,4 months,6 months,12 months.Moreover,the optimal situmulating regent was been selected by different indexes of fragrant hearwood,such as color,flavor and hardness anfer 2 months,4 months,6 months,12 months.1.3 Induced optimal concentration and dose of a-reagent experimentThe 4-years-old healthy dalbergia has been select for subjects,Designed six optimal concentration of a-reagent groups:4%、6%、8%、10%、12%、14%concentration of a-reagent.Designed six optimal dose of a-reagent groups:80、100、120、140、160mL dose of a-reagent.The growth about leaves,branches and trunk have been recorded after 1 week,2 months,4 months,6 months,12 months.Moreover,the optimal concentration and dose were choosed by different indexes of fragrant hearwood,such as color,flavor and hardness anfer 2 months,4 months,6 months,12 months.1.4 Verify and optimization the dalbergia optimal stimulation techniqueThe 4-years-old healthy dalbergia has been select for subjects,Designed three groups:conventional group;infusion 10%concentration of a-reagent for 60mL what position were 1.2 and 2.4m by dalbergia trunk;infusion 10%concentration of a-reagent for 120mL what are perforated trunk infusion liquid.The growth about leaves,branches and trunk have been recorded after 1 week,2 months,4 months,6 months,12 months.Moreover,the different indexes of fragrant hearwood were recored,such as color,flavor and hardness after 2 months,4 months,6 months,12 months.While production in 12 months were evaluated of stimulation technology.2 Quality analysis of dalbergia heartwood sample by "Bio-physical stimulation method"2.1 Preliminary evaluation of dalbergia heartwood sample by "Bio-physical stimulationmethod"Quality analysis sample colected from different city Commercial materials;Bio-physical stimulation method" for 6 or 12 months samples;dalbergia control samples.Traits characteristic,microscopic characteristics,TLC,were evaluated followed Chinese pharmacopoeia 2010(a)edition.The water content was determinated According to Chinese pharmacopoeia 2010 edition(a)under appendix IX H "Water Mensuration"-The third method(hypobaric drying method).Alcohol extract content was determinated by Chinese pharmacopoeia 2010 edition(A)appendix X-"extract mensuration-ethanol soluble extraction" that item "hot dipping method".detect the content of diethyl ether extraction and volatile oil content in different samples recorded in Chinese Pharmacopoeia(2010).2.2 Dalbergia Liquiritigenin,Isoliquiritigenin Content determination and establish HPLCfingerprint.Determined by HPLC dalbergia liquiritigenin,different content liquiritigenin,column Symmetry C18(4.6mm × 250mm,5.0μm);mobile phase:acetonitrile(B),0.3%phosphoric acid(C,v/v)gradient elution;flow rate:1 mL/min;glycyrrhizin,isoliquiritigenin detection wavelength were 275nm,363nm;column temperature:35℃;injection volume:10μL.HPLC-UV method was used for establishing different dalbergias samples fingerprint,the use of "traditional medicine fingerprint similarity evaluation software version 2004A"in 23 samples were analyzed patterns and determine common peaks,similarity,SPSS20.0 software was used to conduct cluster analysis.2.3 Dalbergia oil composition analysis and Establishing GC-MS fingerprintingAnalysis of 15 dalbergia samples chemical composition by GC-MS method.HP-5 capillary column;inlet temperature of 250 ℃;interface temperature 280 ℃,carrier gas:helium flow rate:1mL/min;split ratio of 50:1;injection volume:1μL.Programmed temperature was used to establish GC-MS fingerprint,Meanwhil,the similarity determined by "South University similarity evaluation software",SPSS20.0 software was used to conduct cluster analysis.Results1 Experimental study of stimulate Method1.1 The effect of stimulation way in dalbergia heartwood formationIt was found that three experimental groups occurs showed deciduous phenomenon after a week,recovering slowly after about a month,two months later,the site of stimulation hole Exudated purple-brown and sticky substance,which has a similar burning smell with the Dalbergia heartwood.Four months later,we could see Bark burst around stimulation hole,while it could not see in the control and negative control groups.Chiseling and cutting off the tree after 6 months and 12 months.Red-brown hearwood could be seen in one hole injection fluid and two holes injection fluid.The heartwood substances yield of three kinds of stimulation methods’ significant differences in region.Affected by the infusion method was significantly higher than one hole and two-hole liquid injection method,and the method of liquid infusion delivery fastest.1.2 The effect of stimulation reagents in dalbergia heartwood formationThe results shows that six groups such as Maleic hydrazide,Formic acid,Acetic acid,Thidiazuron,a-Reagent groups could form fragrant resin,whereas,Hydrogen peroxide group could not produce results incense.Stimulants production betreen different reagents ranging from high to low were a-Reagent>Thidiazuron>Formic acid>Acetic acid>Hydrogen peroxide,and the 1 year Stimulants heartwood had a good quality,such as crimson color,smell strong,hard.1.3 Induced optimal concentration and dose of a-reagent experimentThe heartwood under 10%concentrations’ lengths can reach 258cm,diameter:8.9cm,red color,hard,strong aroma.The quality is better than 4%,6%and 8%concentration experimental groups.However,the trees under 12%and 14%concentrations groups can be seen fade and death.in addition,the formation under 10%concentration,120mL group had a good quality and high production which heartwood length of 157cm,width 8.7cm.1.4 Verify and optimization the dalbergia optimal stimulation techniqueConventional group production reached 15,767.1/year,respectively,significantly higher than the group infusioned 10%concentration of a-reagent for 60mL what position were 1.2 and 2.4m by dalbergia trunk(14842.2/year);infusion 10%concentration of a-reagent for 120mL what are perforated trunk infusion liquid(14937.8/year).Furether more,Heartwood by "Bio-phisical sitimulation method" had red coloe,stronger odor and good hardness,which has reached nine-year-old growth naturally,and incense breakthrough low point for at least four years in advance,and the whole heartwood fragrant roots to the trunk at 3 meters.2 Quality analysis of dalbergia heartwood sample by "Biological stimulation method"2.1 Preliminary evaluation of dalbergia heartwood sample by "Biological stimulation method"Character identification showed six months heartwood produced better quality,average yield about 2100cm3,and the color was light brown red,firmer,burning whit white smoke,strong smellr.Microscopic identification was found that stimulation heartwood has all the substances Pharmacopoeia specified,such as Bordered Pit,a prismatic crystal of calcium oxalate,Crystal fiber sheath.but there are some differences between size and morphology,relatively,small sample stimulate pigment blocks in stimulation samples,the colour of crystal sheath fiber,wood fiber and wood ray is light yellow,and there are many square crystals;TLC found that stimulating samples for six months reference Rf at different positions in the same spot.water content,alcohol extract,ether extract results showed that the stimulation for 2,4,6 months sample water content 7.71%～8.74%,alcohol extract content were 9.08%～21.67%,ether extract at 4.82%-8.96%;volatile oil containing test results shows that the content of six months stimulate sample was 1.92%,while 12 months sample stimulating content of 3.85%.2.2 Dalbergia Liquiritigenin,Isoliquiritigenin Content determination and establish HPLCfingerprint.HPLC was used to determine the content of dalbergia liquiritigenin Isoliquiritigenin for its convenience and highly repetitive.The content of 6 months stimulation heartwood were respectively 2.353,0.848mg/g,while the averages content of commercial dalbergia samples liquiritigenin and Isoliquiritigenin were 2.04,0.818mg/g.HPLC fingerprint display that the quality of dalbergia stimulating samples have to meet quality standards,and establishment the quality standards by 23 batches dalbergia which similarity between 0.306-0.974.Cluster analysis showed the samples can classified into five types,which stimulate for 12 months samples was divided into a class with most commercially available samples,stimulate for 2,4 months samples were divide into a sample class with 9 years,12 years growth naturally.2.3 Dalbergia oil composition analysis and Establishing GC-MS fingerprintingComponent analysis showed that the control dalbergia volatile oil mainly contained β-bisabolene,trans-Nerolidol and epoxidation humulene up to 85%on average,the content of six months stimulating samples maily contained trans-nerolidol,β-bisabolene,while epoxidation lower humulene content,and 12 months stimulating samples containing the three main components up to 82%.Volatile oil Component’s similarity between 0.3984-0.9127,making a big difference,of which only No.9(Guangdong Maoming commercially available)sample’s similarity above 0.9,the "Bio-phsical stimulating method" sample heartwood’s similarity between 0.4385-0.6649,compared similarity low.cluster analysis results will be divided into three class between 13 batches samples,stimulating 6-months and 12-month reference samples with volatile oil as a class in the cluster analysis.Conclusion1 Experimental study of stimulate MethodThe best technology Stimulate method:infusioning 10%concentration of a-reagent for 120mL.In this method,dalbergia heartwood was hard,occasional texture,red color,strong scent;heartwood length of 3m,heartwood diameter 9cm.the production increase significantly which estimated at 15767.1cm3 for one annual production.knot pilgrimage at least four years in advance,which has a great production and utilization.However,the the optimal Stimulating season,heartwood harvest time,deodorant fragrant tree transporting need further research in the future.2 Quality analysis of dalbergia heartwood sample by "Biological stimulation method""Bio-physical stimulation method" samples have been met the pharmacopoeia standard,and better than the quality of some commercial samples.A simple and convenient method was made for determining content of glycyrrhizin,liquiritigenin.6 and 12 months stimulating samples’ liquiritigenin and Isoliquiritigenin were higher than average levels of commercial samples to verify the feasibility and reliabilityof"Biological stimuly method".Dalbergia HPLC fingerprint can reflect the overall characteristics dalbergia flavonoids,and HPLC fingerprint has been established successfully to stimulating samples,commercial samples and control samples,which provided more scientific and effective quality evaluation methods for the further reaseach.Volatile oil was the main chemical ingredient in dalbergia,GC-MS fingerprinting method is simple and convenient,high sensitivity,repeatability,which provided more scientific evaluation quality system for controlling varying quality dalbergia samples stimulating dalbergia samples collected were limited,so,in future studies,different stimuli can increase the time period,different origin stimulation,etc.starting from the spectral effect relationship point of view,comprehensive chemical analysis would be used to quality,in order to establish a bio-physical stimulus method in dalbergia which can be a more effective,comprehensive,scientific quality control methods.