| Juglans mandshurica Maxim.is an important timber species in the Northeast,and it also contains a medicinal ingredient-juglone.In order to make the walnut tree grow better,it is necessary to prun the walnut tree branches,but the pruned branches and leaves are generally thrown away or used as firewood.The branches of walnut tree contain a large amount of juglone.Many studies have shown that jugquinone has Anti-tumor,antibacterial and other activity,discarding or incinerating branches not only wastes resources but also pollutes the environment.In this study,the branches and leaves of Juglans mandshurica are used as experimental materials to determine the dynamic changes in the contents of juglone,endogenous hormones and non-structural carbohydrates(NSC)in various parts of different growth months.In this study,branches and leaves of Juglans mandshurica were used as raw materials,the dynamic changes of juglone,endogenous hormones and non-structural carbohydrates(NSC)contents in different parts of Juglans mandshurica in different growth months were measured,the correlation between juglone,endogenous hormones and NSC contents was analyzed to determine the optimal harvest time.Taking the pruned branches as the research object,a jugquinone microemulsion system was prepared as the extraction solvent instead of traditional organic solvents,and the microwave-assisted microemulsion extraction process was optimized by response surface methodology.The GO/ND/Fe3O4 magnetic composite material was prepared,which replaced the traditional purification materials,and used reverse micellar microemulsion extraction combined with magnetic solid phase extraction technology to efficiently separate and purify the juglone.A sample of juglone with a purity of 90.68%was obtained.The main findings are as follows:1.Content of juglone were determinated by HPLC method.HIQ sil C18W Column,injection volume 20 μL,column temperature 30℃ flow rate 0.8 mL/min,detection wavelength 250 nm,mobile phase methanol(50%):0.2%phosphoric acid water(50%)isocratic Elute for 30 min.In the detection range,juglone has a good linear relationship.4 endogenous hormones’(3-indole acetic acid,3-indole butyric acid,naphthalene acetic acid,abscisic acid)was determinated by HPLC method:Injection volume 20 μL,column temperature:25℃,flow rate 1.0 mL/min,detection wavelength:280 nm,0.06%formic acid water(A):methanol(B),gradient elution conditions:0-15 min,51.3%A;15～22 min,51.3-49.3%A;22～26 min,49.3-49.2%A;26～30 min,49.2-44.2%A;30～34 min,44.2-43.4%A;34～36 min,43.4-45.0%A;36～45 min,45.0-70%A.Methodological verification shows that all four hormones have a good linear relationship within the measurement range(R2>0.999),and the reproducibility,precision and stability of the analytical method are good.2.Using Juglans mandshurica as the experimental material,we tested the contents of juglone,hormones and NSC in different months(from May to October)and different parts(branches and leaves).The results showed that the juglone,hormone and NSC content measured differed greatly due to the different tissues.The content of juglone in the branches showed an increasing trend from May to October,and the slight difference between September and October was 2.88±0.10 mg/g and 2.93±0.11 mg/g,respectively;the content of juglone in the leaves first increased and then decreased,the highest content is 0.53±0.03 mg/g in July;The contents of IAA and NAA in branches showed a bimodal pattern,and IB A showed a single peak pattern.The contents of IAA,IB A and NAA were the highest,which were 70.23 ± 0.19 ng/g,58.99± 0.10 ng/g and 28.46±0.14 ng/g,respectively.The variation trends of IAA and IBA contents in leaves were similar,and the highest contents of the three hormones in May were 115.26 ± 0.46 ng/g,105.11±0.23 ng/g and 84.07 ± 0.14 ng/g,respectively.The variation trend of ABA content in branches and leaves of Juglans mandshurica was similar and gradually increased.The maximum ABA content in branches in October was 88.84 ± 2.58 ng/g,and the maximum ABA content in leaves in September was 96.63±2.02 ng/g.The variation trend of NSC content in branches and leaves was similar,which decreased first and then increased.The NSC content was the lowest in June,which was 73.17±1.06 mg/g and 51.05 ±1.33 mg/g,respectively.The study found that four endogenous hormones in branches or leaves of Juglans mandshurica had no correlation with juglone,and NSC was positively correlated with Juglone(P<0.05)3.A special microemulsion system for extracting juglone was established,various excipients were preliminarily processed according to the dissolution ability of excipients to juglone and the mutual dissolution into emulsion.Then the excipients were further screened by ternary phase diagram.Finally,the best microemulsion system ratio we got was Tween 80(27%):n-propyl alcohol(13.5%):n-hexane(4.5%):water(55%).This microemulsion preparation method is simple,and the prepared microemulsion is a clear and transparent W/O microemulsion with an average particle size of 46.8 nm.It does not break emulsion under low-speed or high-speed centrifugation and long-term storage.Flocculation occurs when placed at high temperature for a long time.It is recommended to be stored at room temperature.4.The optimal extraction conditions were determined with the optimization of process parameters of the microemulsion-assisted extraction of juglone from Juglans mandshurica branches:microemulsion pH:5.60,extraction time:63 s,microwave temperature:40℃,microwave power:400 W,liquid-solid ratio:20:1 mL/g.Under these conditions,the average yield of juglone was 4.58 mg/g.After comparing with different extraction methods,we found that the yields of juglone after microwave-assisted microemulsion extraction were 1.86 and 6.65 times that of ethanol microwave extraction and ethanol heat reflux extraction respectively.The microwave-assisted microemulsion extraction technology is a fast and efficient extraction technology.After extraction,the jugquinone microemulsion was subjected to a stripping experiment,and the optimal stripping conditions were determined as the phase ratio of 4(Vw/Vo,mL/mL),the KCl concentration:1.6 mol/L,and the shaking time:25 min.shaker speed:140 r/min.Under these conditions,the stripping experiment was carried out,and the stripping yield of juglone was 65.32%.Experiments have proved that the reconstituted microemulsion after stripping can be recycled 3 times.5.The magnetic composite material GO/ND/Fe3O4 was prepared,and the process parameters of magnetic solid phase extraction combined with reverse micelle microemulsion extraction and purification of juglone were optimized.The optimal adsorption conditions were determined to be 14 h;material-to-liquid ratio 30 mg/mL;pH 5.5;shaker rotation speed 200 r/min.The best desorption conditions are:desorption time 16 h;desorption liquid type is methanol;desorption liquid integral number 100%;pH 8;material-to-liquid ratio 40 mg/mL.Under these conditions,magnetic solid phase extraction combined with reverse micellar microemulsion extraction was repeated three times,and the purity of the juglone could reach 90.68%,the yield based on the raw material was 3.82 mg/g,and the recovery yield was 83.32%.Experiments show that the magnetic composite material can be used 9 recycles.Based on the correlation analysis of jugquinone and endogenous hormones and non-structural carbohydrate content,this study determines the best harvest time to obtain juglone;The idea of extracting juglone from pruning branches was proposed to make full use of waste resources.Jugquinone is extracted to make full use of the idea of waste resources.At the same time,the extraction and purification process proposed in this study solves the problems of traditional extraction and purification methods such as large solvent consumption,low efficiency,cumbersome,high toxicity,and difficult recovery;and developed a novel and efficient,low solvent consumption,green and recyclable extraction and purification system.This study provides theoretical guidance for the efficient extraction and separation of juglone from the branches and leaves of Juglans mandshurica and the rational utilization of juglone resources. |
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