| ObjectiveCurrently, all the farmers who planted Aquilaria senensis (Lour.) Gilg face some serious problems or even burden, such as the natural method needed a long period and the. yields were generally low to induce the Aquilaria senensis (Lour.) Gilg to secrete aromatic compounds, and the traditional artificial method was also low-tech. Moreover, it was lack of utilization measures in the whole processing of cultivation. In order to improve the yields of agilawood and significantly promote the comprehensive utilization levels. Our, research team plan to design a new system method to induce the Aquilaria senensis (Lour.) Gilg to secrete much more aromatic compounds, and design a method to promote the comprehensive utilization levels, in the hope of providing a thoretical basis and research paradigms for the yield of agilawood.Methods1 Secrete aromatic compounds induced researching1.1 Administration and reagents of the induced method screening1.1.1 Induced administration screeningDesigned the different groups of induced administration way by rossing, punching injection, infusion,dialeave, girdling, ect, treated the 8-year-old healthy Aquilaria sinensis trees by different administration way. Then observed and recorded the changes of the trunks, branches and foliage growth at the time point of one week, one month, three months, six months after different treatment. Simultaneously, observed and recorded the changes of the color, degree and burned smell of the xylem surrounding the site of the administration at the time point of three months, six months, nine months and one year. Screen the best induced way of administration.1.1.2 Induced reagents screeningDesign different induced reagent groups of No.1, Thidiazuron,2,4-D, A-NAA, 2,3,5-triiodobenzoate, maleic hydrazide,3-indole Ding potassium, hydrogen peroxide, acetic acid, jasmine methyl and other reagents, treated the 8-year-old healthy Aquilaria sinensis trees by different reagents. Then observed and recorded the changes of the trunks, branches and foliage growth at the time point of one week, one month, three months, six months after different treatment. Simultaneously, observed and recorded the changes of the color, degree and burned smell of the xylem surrounding the site of the administration at the time point of three months, six months, nine months and one year. Screen the best induced reagent. 1.2 Systematic induced methods researching1.2.1 Security evaluation of the induced reagentDesign 4%,6%,8%,10% concentration of the NO.1 induced reagent, treated the 8-year-old healthy Aquilaria sinensis trees. Then observed and recorded the changes of the trunks, branches and foliage growth at the time point of one week, one month, three months, six months after different treatment. Simultaneously, observed and recorded the changes of the color, degree and burned smell of the xylem surrounding the site of the administration at the time point of three months, six months, nine months and one year. Evaluate the security of the induced reagent.1.2.2 Induced concentration of the reagent screeningDesign 3%,4%,5%,6% concentration of No.1 reagent, treated the 8-year-old healthy Aquilaria sinensis trees. Then observed and recorded the changes of the trunks, branches and foliage growth at the time point of one week, one month, three months, six months after different treatment. Simultaneously, observed and recorded the changes of the color, degree and burned smell of the xylem surrounding the site of the administration at the time point of three months, six months, nine months and one year. Screen the best concentration of the induced reagent.1.2.3 Treated number for one tree screeningDesign 2,4,8 of treated holes, treated the 8-year-old healthy Aquilaria sinensis trees. Then observed and recorded the changes of the trunks, branches and foliage growth at the time point of one week, one month, three months, six months after different treatment. Simultaneously, observed and recorded the changes of the color, degree and burned smell of the xylem surrounding the site of the administration at the time point of three months, six months, nine months and one year. Screen the best concentration of the induced reagent.1.2.4 Harvest time and yield evaluationTreated the trees using the best induced way, observed and recorded the changes of the color, degree and burned smell of the xylem surrounding the site of the administration at the time point of three months, six months, nine months and one year. And estimate the yield at the end of each period to screen the harvest time.1.2.5 Quality of the best way induced agilawood evaluationCollect different induced samples of the agilawood, such as the scalding, off dried, chopped, ants borers, insects drain, snags and the fungal infected, together with the reference herb. According to the required schedule of agilawood, detect the characteristic traits, moisture, content of the ethanol extraction and the content of diethyl ether extraction recorded in Chinese Pharmacopoeia (2010). Simultaneously, determine the contents of Aquilarone B, Aquilarone C, Aquilarone D, Aquilarone E and (5S,6R,7R, 8S)-2-[2-(4-methoxy-phenyl) ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydro using HPLC-PDA, and calculate the total content of chromones.2 utilization researching and development of the leaves of Aquilaria sinensis2.1 Identification and separation of the componentsUsing the separation techniques of reflux extraction, extraction, semi-preparative column, combined with HPLC testing techniques to isolate monomer components from the leaves of Aquilaria sinensis, and identified their structures.2.2 Content, temporal and spatial variation of the separated components studySelect a characteristic component as mangiferin, established the assay method using HPLC-PDA and UPLC-PDA. And then to detect the different samples as distribution parts, age, etc., to explore the dynamic changes of the characteristic component to screen the harvest time, storage allotted time.2.3 Extraction Technology studyUsing the orthogonal experimental design, based on the inspection, ethanol concentration, liquid-solid ratio, extraction time, extraction temperature and other single factors, and used the extraction rate as the index, to explore the optimum extraction of the characteristic component.2.4 Industrial process of the characteristic component researchingConsidered the purity and yield of the characteristic component and based on the extraction Technology, and the simplicity and economic requirements of the processing, to develop a industrial process for extracting the high-purity of characteristic component. Results1 Results of the induced experiment1.1 Administration and reagents of the induced method screening1.1.1 Induced administration screeningThe growth of the trees and leaves, and inside of the trunk of the scraped blank, scraped negative control, scraped with formic acid, scraped with No.1 reagent, punch blank group, punch negative blank control, infusion of blank, infusion the negative control, defoliation, ring peeling treated groups were no obvious changes during the observation period at one week, one month, three months or six months. While the growth of the trees and leaves, and inside of the trunk of the other treated groups (punched formic acid, punched No.1, infusion formic acid, infusion No.l, etc.) changed obviously, and the impacted area increased gradually, color gradually deepen, burned smell thicken with the induced time prolong. And the No.l treated groups changed more worsen, even parts of the branchless were death.1.1.2 Results of induced reagentsThe growth of the trees and leaves of the blank control, negative control, hydrogen peroxide and methyl jasmine acid treated groups were no obvious changes during observation period after induced one week, a month, three months or six months. The growth of the trees of the No.1, Thidiazuron,2,4-D,A-NAA,2,3,5-triiodobenzoate, maleic hydrazide,3-indole Ding potassium, hydrogen peroxide, acetic acid treated groups were normally, but the leaves turned yellow or fall off, the No.1 treated group was worse almost fall off completely and parts of branchless were death.There were no obvious color changes and light flavor after the hydrogen peroxide, methyl jasmine acid treated groups and the blank control, negative control groups at three months, six months, nine months or one year. The changes the No.1, Thidiazuron, 2,4-D,A-NAA,2,3,5-triiodobenzoate, maleic hydrazide,3-indole Ding potassium, hydrogen peroxide, acetic acid treated groups were obvious at three months, and the impacted area increased gradually, color gradually deepen, burned smell thicken with the induced time prolong. And the No.1 reagent was the best one.1.2 Systematic induced methods researching1.2.1 Security evaluation of the induced reagentThe 4%,6% concentration of No.l reagent treated group appeared obvious color changes at three months. And the impacted area increased gradually, color gradually deepen, burned smell thicken with the induced time prolong. Parts of the 6% concentration treated group were died, which lead to the yield was less than that of the 4% concentration treated group. And the trees of the 8%,10% concentration treated group were died, bad color and burned smells.1.2.2 Induced concentration of the reagent screening3%,4%,5%,6% concentration of No.1 reagent treated group appeared obvious color changes at three months. And the impacted area increased gradually, color gradually deepen, burned smell thicken with the induced time prolong, while the color, area size, burned smell of 3%,4% concentration treated group were no significant difference, and parts of the 5%,6% concentration of treated group die in local site, lead to the yield was less than the other groups. The induced fragrant area of 3%,4%,5%,6% treated group was 4792,4920,4460,4184 cubic centimeters, respectively.1.2.3 Treated holes number for one tree screeningThe two holes, four holes and eight holes treated groups appeared obvious color changes at three months. And the impacted area increased gradually, color gradually deepen, burned smell thicken with the induced time prolong. And the two holes treated group was the better.1.2.4 Harvest time and yield evaluationThe No.1 induced trunk can be observed grey color, a low sweet area, and badly burned smell in the third month, half a year, nine months or a year. And the area was 3022, 4358,3022 cubic centimeters, respectively. According to the burned smell, it could be divided into low-, under medium- and medium-grade, so the highest yield and level was induced one year. 1.2.5 Quality of the best way induced agilawood evaluationThe scalding samples were shuttle, brown yellow, hard, burn fragrant. The off dried samples were thick slices off dry, tan, light, fragile, burn fragrant. The chopped samples were fragments, tan, light, fragile, burned fragrant. Ants borers samples were obtained strip or pieces, yellowish-white, strong but pliable, burn fragrant. Insects drain samples were chunk or fragments, brownish black, hard and fragile, burned smell. Snags infected samples were fragments, light brown, light texture, medium burned smell. Fungal infected samples were thin, light brown, light texture, light burned smell. And the No.1 induced samples were the elongated, yellow brown, wooden pole form soft, medium burned smell.The moisture content of all samples was between 6.51%-9.07%, the reference material was 8.68%, and the No.1 induced samples was between 7.67%-8.54%.The alcohol soluble extract content of all the samples was between 10.03%-24.88%, the reference material content was 11.19%, and the No.1 induced samples was between 10.61%-10.61%.The ether soluble extract content of all samples was between 0.61%-8.06%, the reference material was 1.07%, and the No.1 induced samples was between 1.43%-1.92%.The results of the HPLC determination showed that the content of Aquilarone B, Aquilarone C, Aquilarone D, Aquilarone E, (5S,6R and 7R,8S)-2-[2-(4-metonym-phenyl) ethyl]-5,6,7,8-4 hydroxyl-5,6,7,8-tetrahydro of all the samples were significantly different. The content of total chromate was between 1.9257-12.1821 mg/g, the reference material materials was 11.7852 mg/g, and the NO.1 induced samples was between 4.4592-5.1076 mg/g.2 Results of comprehensive utilization and development researchingA compound was isolated from 70% ethanol extraction of the leaves, and identified as mangiferin by the physical and chemical properties, spectrum.And the content of mangiferin detected method was by HPLC-DAD, in this method the separation degree of the chromatographic peak was greater than 1.5, the linear range of 68.8-688 ng, regression equation Y= 4972.6X-22524 (R2= 0.9999), and the recovery rate was 98.25%, RSD was 0.55%. And the UPLC-PDA method, separation degree of chromatographic peak was also greater than 1.5, the linear range of 3.44-172.00 ng, regression equation Y=15819.3263+16717.5036X, (R2=0.9999), recovery rate was 98.43%, RSD was 0.58%.Each part of the mangiferin content as follows, primary leaf was 5.12%, old leaf was 2.96%, primary branches was 4.81%, primary branch wood was 2.13%, flowers was 4.54% and green peel was 1.23%, while the seeds, old branch skin, bark, old branches and trunk were all not detected. The content of different samples from Enping, Zhanjiang, Dianbai, Yao Wang Shan of Guangzhou university of Chinese medicine was 3.73%,3.86%,3.43% and 4.34%, respectively. There was no significant different in the origin restrictions.The content of mangiferin was between 3.64%-4.06% between the two years to ten years old tree, no obvious restriction. The high content of mangiferin in March was 4.37% but total output was relative small, and in September was 3.91% and the output was high. Under the condition of suitable storage was 1 year.With the ethanol concentration increasing, the extracted content of mangiferin increases, and the highest content of mangiferin was at the ethanol concentration of 70%. With liquid-solid ratio increasing, the content of mangiferin increases, and the extract content of mangiferin was trend to balance at 20 times of liquid-solid ratio. And the same as the hot reflux time and extraction temperature.Orthogonal experiment results showed that the extraction of ethanol concentration had the greatest influence on the extraction rate, extraction temperature the minimum, the influence degree of various factors on the extraction yield was ethanol concentration> solid-liquid ratio> extraction time> extraction temperature, each influence factor has no significant difference, the best extraction technology conditions was 20 times of 70% ethanol, extracted time for 1 h, extraction temperature 90 ℃, extracted 2 times, the extracted rate of mangiferin was 3.25%.The industrialized preparation of high puritied mangiferin from the leaves of Aquilaria sinensis as follows:1000 g powder, add 7000 ml 70% ethanol, hot reflux 1 h, repeat one time, merge the filtrate, concentrated at 70 ℃ in a reduced pressure equipment to no alcohol, add distilled water to 2000 ml, place at room temperature for 12 h; the supernatant was concentrated to 500 mL at 80 ℃ in a reduced pressure equipment, slowly add in 2500 ml of ethanol, place at room temperature for 12 h; the supernatant was concentrated to 1000 mL at 75 ℃ in a reduced pressure equipment, place at room temperature for 24 h, wash the crystallize with a small amount of ethanol and distilled water, the filtrate was concentrated to 75 ml at 75 ℃, place at room temperature for 24 h, wash the crystallize with a small amount of ethanol and distilled water, merge the crystallize and to be the crude products; the crude products dissolve in 50% ethanol solution to be saturated state, adjust pH value to 3.0 with 5% hydrochloric acid solution, place at room temperature for 24 h, wash the crystallize with a small amount of ethanol and distilled water, dried the crystallize under 70 ℃ and the product was prepared.Finally each 1000 g of leaves of Aquilaria sinensis can extract mangiferin 26.4648 g, and the purity was 97.71%, the transfer rate was 75.39%. Conclusion1 Conclusion of the induced wayThe best induced way was 3% of No.1 reagent by whole infusion administration and two holes at one time, can make the whole tree to secrete aromatic compounds, and could earn a significantly improved yield on average of 4792 cm3. Judged the alcohol-soluble extract content, ether-soluble extract content, content of Aquilarone B, Aquilarone C, Aquilarone D, AquilaroneE,(5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl) ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydro and the content of total chromones, most of the NO.l induced sample can be classified the medium quality.2 Utilization researching and development of the leaves of Aquilaria sinensisMangiferin was isolated from the leaves of Aquilaria sinensis, and the quantitative identify methods were established by HPLC-PDA and UPLC-PDA. The harvest time was September and storage period for one year.The optimum extraction conditions for mangiferin was 20 times of 70% ethanol, extraction time 1 h, and temperature 90 ℃, extracted for two times. And each 1000 g leaves of Aquilaria sinensis can obtain a purity of 97.71% of mangiferin 26.4648 g, the transfer rate was 75.39%. This process was a fixed low investment, short production cycle, little pollution and harmful substances, can be provided a scientific method and means for the utilization researching and development of the leaves of Aquilaria sinensis... |
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