Efficient preparation method of methyl jasmonate

Abstract

The invention discloses a synthesis method of methyl jasmonate, which comprises the following steps: preparing a catalyst from 1-butyl-3-methylimidazolium tetrafluoroborate, nano mesoporous silica and cerous sulfate according to the weight ratio of (10-15): (3-5): 1, and adding 2-(1-cyclopentenyl) methyl acetate, 1, 3-pentadiene and the catalyst according to the weight ratio of (6-10): (1.5-2.5): 1 into a reactor; removing oxygen in the reactor; reacting for 4 to 6 hours under the conditions that the temperature is 30 to 40 DEG C and the speed is 200 to 300r / min, so as to obtain a methyl jasmonate crude product; centrifuging the methyl jasmonate crude product to obtain supernate; extracting the supernate with an inorganic salt aqueous solution, and standing to obtain an organic phase; distilling the organic phase, and collecting fractions at 120-130 DEG C to obtain a methyl jasmonate primary product; and crystallizing and drying the jasmonic acid methyl ester primary product by using absolute ethyl alcohol to obtain a jasmonic acid methyl ester finished product.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology and relates to a method for preparing methyl jasmonate. Background Technology

[0002] Methyl jasmonate (MeJA) is an important plant hormone and fragrance component. MeJA plays an irreplaceable role in agricultural stress resistance induction and fragrance blending. It is also widely used as a key inducer in the synthesis of plant secondary metabolites, exhibiting significant activity, particularly in the induced synthesis of ginsenosides. MeJA can activate key enzymes in the ginsenoside synthesis pathway in ginseng cells (such as farnesyl pyrophosphate synthase and squalene synthase), increasing the total yield of ginsenosides by more than 30%.

[0003] Currently, the preparation methods are mainly divided into three categories: chemical synthesis, plant extraction, and microbial fermentation. However, all existing technologies have significant drawbacks: the plant extraction method cannot meet industrial requirements because the content of methyl jasmonic acid in the raw materials is extremely low (e.g., less than 10 μg in 1 kg of fresh tomato leaves). Traditional chemical synthesis methods use cis-4-heptenic acid, ethyl acetate, etc., as raw materials, with reaction yields generally below 30%, and require harsh conditions such as high temperature and high pressure. At the same time, the products are racemic mixtures, in which only specific configurations have ginsenoside-inducing activity, which limits their application in the field of secondary metabolism regulation of medicinal plants. Even the ionic liquid catalytic method developed in recent years (such as using (E)-4-oxodec-7-enal as raw material) achieves room temperature reaction, but the raw material cost is high, and the removal effect on specific impurities is poor, making it difficult to break through 92% purity of the product. Low-purity products can cause fluctuations in ginsenoside-inducing efficiency exceeding 15%. While microbial fermentation can generate bioactive isomers, traditional processes only achieve yields of 1-1.5 g / L under static culture conditions. During stirred fermentation, abnormal mycelial morphology leads to a sharp drop in yield, and the methyl jasmonic acid content in the fermentation broth is only 20%-60%. Subsequent purification requires silica gel chromatography, molecular distillation, and other methods, which suffer from high organic solvent consumption, low separation efficiency, and high costs. Furthermore, when the prepared product is used for ginsenoside induction, residual organic solvents easily inhibit ginseng cell activity, leading to a high induction failure rate. Therefore, developing a method for preparing methyl jasmonic acid with readily available raw materials, mild reaction conditions, balanced yield and purity, and stable maintenance of ginsenoside induction activity, especially developing a highly efficient and low-cost catalyst, has become a pressing technical challenge in this field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, such as high raw material costs, low reaction yields, complex purification processes, and unstable ginsenoside-inducing activity in the products, this invention provides a high-yield method for preparing methyl jasmonate. Through the integrated innovation of a composite catalytic system and a targeted purification process, it achieves a synergistic improvement in raw material utilization, reaction selectivity, and product purity, while ensuring that the product possesses stable and efficient ginsenoside-inducing activity.

[0005] The core innovation of this invention lies in the combination of the construction of a composite catalytic system and a "distillation-recrystallization" directional purification process. By enhancing catalytic activity through a nanocarrier and directionally promoting the formation of methyl cis-jasmonic acid, the salt washing-distillation-recrystallization process synergistically improves purity and the proportion of active configurations, overcoming the technical bottlenecks of existing technologies where yield and purity are difficult to balance, and the low proportion of active configurations leads to poor ginsenoside induction efficiency. Example data shows that this method achieves high-yield and high-purity preparation under mild conditions, with a methyl cis-jasmonic acid content ≥95% in the finished product. When used for ginsenoside induction, the yield is increased by 40%-55%, and the catalyst is recyclable, demonstrating significant technical advantages and industrial value.

[0006] The first aspect of the present invention provides a catalyst for the synthesis of methyl jasmonate, wherein the catalyst is made of 1-butyl-3-methylimidazolium tetrafluoroborate, nanoporous silica and cerium sulfate, wherein the weight ratio of the 1-butyl-3-methylimidazolium tetrafluoroborate, the nanoporous silica and the cerium sulfate is 10-15:3-5:1.

[0007] In some embodiments, the specific surface area of ​​the nanoporous silica is 1950-1150 m². 2 / g, with a pore size of 2.4-2.5nm and a particle size of 90-110nm.

[0008] The second aspect of the present invention provides a method for preparing the catalyst described in the first aspect of the present invention, wherein the preparation method comprises: mixing the 1-butyl-3-methylimidazolium tetrafluoroborate, the nanoporous silica and the cerium sulfate to obtain the catalyst.

[0009] In some embodiments, the preparation method includes the following steps:

[0010] S1: Mix the 1-butyl-3-methylimidazolium tetrafluoroborate and the nanoporous silica to obtain a first mixture;

[0011] S2: The first mixture is subjected to ultrasonic dispersion treatment to obtain a second mixture;

[0012] S3: Mix the second mixture with the cerium sulfate to obtain a third mixture;

[0013] S4: Stir the third mixture until homogeneous to obtain the catalyst.

[0014] In some embodiments, in S2, the ultrasonic power is 200-400W, and the ultrasonic time is 20-30min; and / or

[0015] In S4, the stirring rate is 130-170 r / min, the stirring temperature is 60-80℃, and the stirring time is 1-2 h.

[0016] The third aspect of this invention discloses a method for synthesizing methyl jasmonate, wherein the method comprises: using methyl 2-(1-cyclopentenyl)acetate and 1,3-pentadiene as substrates, and using the catalyst described in the first aspect of this invention or the catalyst prepared by the preparation method described in the second aspect of this invention as a catalyst, to synthesize the methyl jasmonate.

[0017] In some embodiments, the methyl 2-(1-cyclopentenyl)acetate, the 1,3-pentadiene, and the catalyst are used in a weight ratio of 6-10:1.5-2.5:1.

[0018] The method includes the following first three, first four, or five steps:

[0019] T1: The methyl 2-(1-cyclopentenyl)acetate, the 1,3-pentadiene, and the catalyst are added to the reactor simultaneously or sequentially.

[0020] T2: Remove oxygen from the reactor to obtain an anoxic reaction system;

[0021] T3: In the hypoxic reaction system, the reaction is carried out at 30-40℃ and 200-300r / min for 4-6h to obtain crude methyl jasmonate.

[0022] T4: Centrifuge the crude methyl jasmonate to obtain a supernatant; extract the supernatant with an aqueous inorganic salt solution, and allow it to stand to obtain an organic phase; distill the organic phase and collect the fraction at 120-130℃ to obtain the crude methyl jasmonate.

[0023] T5: Dissolve the methyl jasmonate crude product in anhydrous ethanol, crystallize and dry to obtain the methyl jasmonate finished product.

[0024] In some implementations, one or more of the following are selected: A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10;

[0025] A1: In T1, the purity of the methyl 2-(1-cyclopentenyl)acetate is ≥98%, and the purity of the 1,3-pentadiene is ≥99%;

[0026] A2: In T2, the method for removing oxygen from the reactor is to replace the air in the reactor with nitrogen;

[0027] In A3:T4, the centrifugation conditions are 4000-6000 r / min for 5-20 min;

[0028] In A4:T4, the inorganic salt aqueous solution is a sodium chloride aqueous solution with a mass fraction of 5-10%;

[0029] In A5:T4, the step of distilling the organic phase is as follows: the organic phase is injected into a three-stage distillation column, loaded with glass spring packing with a diameter of 8-12 mm, and the temperature inside the column is controlled at 140-160℃, the vacuum degree at 4.5-5.5 Pa, the condensation temperature at 4-8℃, and the reflux ratio at 1:25-30.

[0030] A6: In T5, the primary methyl jasmonate is added to anhydrous ethanol for recrystallization, wherein the weight ratio of the primary methyl jasmonate to the anhydrous ethanol is 100:3-5; the mixture is allowed to stand at 0-5℃ for 8-12 hours; the mixture is filtered to crystallize; the crystals are then vacuum dried at a vacuum degree of 8-12 Pa and a temperature of 40-50℃ for 2-3 hours to obtain the finished methyl jasmonate product.

[0031] The fourth aspect of this invention provides a method for preparing total ginsenosides, the method comprising the following first four or five steps:

[0032] X1: Clean and disinfect the ginseng adventitious roots to obtain disinfected ginseng adventitious roots;

[0033] X2: The disinfected ginseng adventitious roots were inoculated into ginseng adventitious root liquid culture medium for suspension culture to obtain a fixed ginseng adventitious root line;

[0034] The solvent of the ginseng adventitious root liquid culture medium is water; based on the final concentration of the materials used, the ginseng adventitious root liquid culture medium contains 900 mg / L ammonium nitrate, 1125 mg / L potassium nitrate, 90 mg / L potassium dihydrogen phosphate, 170 mg / L calcium chloride dihydrate, 370 mg / L magnesium sulfate heptahydrate, and 30 mg / L... Disodium EDTA, 22 mg / L ferrous sulfate, 0.93 mg / L potassium iodide, 0.35 mg / L sodium molybdate dihydrate, 9.6 mg / L zinc sulfate heptahydrate, 0.125 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate tetrahydrate, 2 mg / L glycine, 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L vitamin B6, 0.1 mg / L vitamin B1, 50 mg / L glutamine, 300 mg / L hydrolyzed casein, 9.0 mg / L IBA, and 20 g / L sucrose; pH 5.5-6.0;

[0035] X3: Add jasmonic acid, as described in the third aspect of this invention, to the reaction system of ginseng adventitious root fixed line at a final concentration of 80-120 μM to obtain proliferated ginseng adventitious roots.

[0036] X4: The adventitious roots of ginseng were treated by ethanol reflux extraction to obtain crude total ginsenosides.

[0037] X5: The crude total ginsenosides were purified by a combination of column chromatography and high performance liquid chromatography to obtain the pure total ginsenosides.

[0038] In some implementations, one or more of the following are selected: B1, B2, B3, B4, B5, B6, B7, B8, B9, and B10.

[0039] B1: In X1, the ginseng adventitious roots are rinsed clean in water, washed with 70-80% alcohol aqueous solution for 50-70 seconds, and rinsed with sterile distilled water 3-5 times to obtain the sterilized ginseng adventitious roots.

[0040] B2: In X2, 10-20g of ginseng adventitious roots are inoculated into each L of the ginseng adventitious root liquid culture medium.

[0041] B3: In X2, the light condition is dark culture;

[0042] B4: In X2, the culture temperature is 25-28℃; filtered air is introduced into the culture system at a flow rate of 100-200 mL / min;

[0043] B5: In X2, the incubation time is 5-10 days;

[0044] B6: In X3, the light condition is dark culture;

[0045] B7: In X3, the incubation period is 30-40 days;

[0046] B8: In X3, the culture temperature is 22-24℃; filtered air is introduced into the culture system at a flow rate of 100-200 mL / min;

[0047] B9: In X3, the ginseng variety is Da Ma Ya ginseng;

[0048] B10: In X4, take the dried ginseng adventitious roots, add 0.8-1.2% of the weight of the dried ginseng adventitious roots of Tween, and perform reflux extraction using 75% ethanol aqueous solution. The extract is the crude total ginsenosides of the ginseng.

[0049] Beneficial effects of the present invention

[0050] 1. Innovation in materials and catalytic system: 2-(1-cyclopentenyl)methyl acetate, which is inexpensive and readily available, is selected as the starting material, reducing the cost by more than 40% compared to the existing (E)-4-oxodec-7-enal raw material; in the composite catalyst, nanoporous silica provides a dispersion carrier for ionic liquid and cerium sulfate, and the synergistic effect of the three improves the stereoisomer selectivity of the reaction to more than 90%, and can directionally promote the formation of the active configuration (cis-jasmonic acid methyl ester). The proportion of the active configuration is increased by 25%-30% compared with the traditional method, which solves the problems of poor selectivity and low proportion of active configuration in the traditional catalytic system, and lays the foundation for efficient induction of ginsenosides.

[0051] 2. Significantly improved reaction efficiency: Under mild conditions of 30-40℃, the reaction yield can reach 85%-90%, which is significantly higher than that of traditional chemical synthesis methods (yield <30%) and existing ionic liquid methods (yield 70%-75%). Moreover, the composite catalyst can be recycled more than 5 times, with a catalytic activity retention rate of ≥88% and an active configuration selectivity retention rate of ≥85%. Long-term use can still stably ensure the ginsenoside induction potential of the product.

[0052] 3. Optimized purification process: Polar impurities are removed through salt washing pretreatment, followed by three-stage distillation for directional separation, and then low-temperature recrystallization to further enrich the active configuration and remove trace impurities. The product purity can reach 96%-98%, with an active configuration content of ≥95%, which is significantly improved compared to the purity (≤92%) and active configuration ratio (≤70%) of existing ionic liquid methods. At the same time, it avoids the consumption of organic solvents in processes such as silica gel chromatography, and the residual organic solvent content of the finished product is ≤0.005%. It has no cytotoxicity when used for ginseng cell culture, and is more environmentally friendly and biosafety-free.

[0053] 4. Excellent ginsenoside induction activity: When the finished product is used for ginsenoside induction, the total yield of ginsenosides in ginseng suspension cells can reach 12.8-14.5 g / L, which is 40%-55% higher than that of methyl jasmonate prepared by traditional methods (induction yield 8.2-9.5 g / L). Moreover, the fluctuation range of the induction effect is ≤5%, which solves the problems of low induction efficiency and poor stability of existing products.

[0054] 5. High industrial feasibility: The entire reaction process is mild, requiring no high-temperature or high-pressure equipment. Catalyst recovery is simple, and the distillation-recrystallization process is mature and easy to scale up. The overall production cost is reduced by 35%-45% compared to existing technologies. It has application value in both the fragrance industry and the field of secondary metabolism regulation of medicinal plants, and has extremely strong prospects for industrial application. Attached Figure Description

[0055] Figure 1 This is the HPLC chromatogram of the methyl jasmonate product.

[0056] Figure 2 The NMR spectrum of the finished product, methyl jasmonate.

[0057] Figure 3 The image shows the HPLC chromatogram of ginsenosides detected after co-culturing the finished product of Example 1 with ginseng suspension cells. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0059] Example 1: Preparation of composite catalytic system

[0060] Take 30g of 1-butyl-3-methylimidazolium tetrafluoroborate (code name [BMIM]BF4 or BMIMBF4, CAS number 174501-65-6) and 10g of nano-mesoporous silica (prepared by sol-gel method, specific surface area 1103.65m²). 2 The mixture of 2.45 nm average pore size and 100 nm average particle size was ultrasonically dispersed at 300 W for 25 min, and then 2.4 g cerium sulfate was added. The mixture was stirred and activated at 70 °C and 150 r / min for 1.5 h. After cooling to room temperature, 42.4 g of composite catalyst was obtained.

[0061] Example 2: Preparation of methyl jasmonate

[0062] I. Chemical Reaction Equations

[0063] The chemical reaction is shown in Formula 1. Using methyl 2-(1-cyclopentenyl)acetate and cis-1,3-pentadiene as substrates, and the composite catalyst prepared in Example 1 of this invention as catalyst, the enzyme-catalyzed reaction produces methyl cis-jasmonic acid (commonly referred to as methyl jasmonic acid).

[0064]

[0065] (Equation 1)

[0066] II. Catalytic Synthesis Reactions

[0067] In a 500 mL reflux reactor, 20 g of methyl 2-(1-cyclopentenyl)acetate (purity 98.5%) and 80 g of 1,3-pentadiene (purity 99.2%) were added, followed by 10 g of composite catalyst (prepared in Example 1 of this invention). After the internal space of the reactor was purged with nitrogen to remove oxygen three times, the reaction was stirred at 35 °C and 250 r / min for 5 h to obtain 108.2 g of crude reaction solution.

[0068] III. Targeted Purification Process

[0069] The crude reaction solution prepared in Section 2 was centrifuged at 5000 r / min for 10 min, and the precipitate (i.e., catalyst) was collected. 9.6 g of catalyst was recovered (recovery rate 96%).

[0070] Take 100 mL of the supernatant obtained from the centrifugation, add 50 mL of 8% sodium chloride aqueous solution and extract three times. After standing and separating the layers, take 82 mL of the organic phase. Inject the organic phase into a three-stage distillation column, load it with 10 mm diameter glass spring packing, control the temperature inside the column at 150 °C, the vacuum degree at 5.0 Pa, the condensation temperature at 6 °C, and the reflux ratio at 1:28. Collect the fraction at 125-128 °C to obtain 29.8 g of methyl jasmonate as the initial product. Add 1.19 g of anhydrous ethanol to the initial product, stir to dissolve, and let it stand at 2 °C for 10 h. Filter to obtain 28.2 g of crystals. Dry the crystals under vacuum of 10 Pa and 45 °C for 2.5 h to obtain 28.0 g of methyl jasmonate as the final product.

[0071] HPLC analysis was performed on the methyl jasmonic acid product. Specifically, a C18 reversed-phase column (4.6×150mm, 2.7μm) was used, with a mobile phase of 0.1% formic acid solution-0.1% formic acid acetonitrile solution (volume ratio 40:60), a flow rate of 0.75mL / min, an injection volume of 5μL, a detection wavelength of 214nm, a column temperature of 35℃, and a product purity of 97.5%, of which methyl cis-jasmonic acid content was 95.3%. Figure 1The HPLC chromatogram of the methyl jasmonate product in Example 2 is shown below (the target peak of methyl cis-jasmonate is at a retention time of 10.0 min, accounting for 95.3% of the peak area; the impurity peaks at 8.21 min, 8.96 min, and 9.34 min account for ≤0.5% of the peak area).

[0072] NMR analysis of methyl jasmonate is shown in the following spectrum. Figure 2 . Figure 2 To replace chloroform with deuterium (CDCl) 3 ) as solvent 1 In the H-NMR spectrum, the horizontal axis "f1 (ppm)" represents the chemical shift (range -1.0 to 9.0 ppm, representing the position of hydrogen nuclei in different chemical environments), and the vertical axis represents the signal intensity (reflecting the relative number of hydrogen nuclei). The red curve in the figure is the proton signal peak, and the blue / green curve is the integral curve (the step height corresponds to the peak area, i.e., the proportion of hydrogen atoms). The values ​​marked next to the peaks, such as 3.00 and 2.97, are the chemical shifts of the corresponding peaks. The distribution of the peaks corresponds to different hydrogen structures: around 5.0 to 6.0 ppm corresponds to double-bonded hydrogen, around 3.0 ppm corresponds to hydrogen bonded to electronegative atoms (such as O), and 0.5 to 2.0 ppm corresponds to alkyl hydrogen. Overall, it is used to characterize the molecular structure of the compound.

[0073] Figure 2 It is a product of the synthesis of methyl jasmonate. 1 The H-NMR spectrum has the following core parameters: the solvent used is deuterated chloroform (CDCl3), the chemical shift (x-axis) range covers -1.0~9.0 ppm (unit is ppm), and the y-axis is the relative signal intensity; the spectrum includes an integral curve (blue / green curve, the step height corresponds to the proportion of hydrogen atoms), and the peak chemical shift distribution corresponds to the structural characteristics of methyl jasmonate: the peak in the 5.0~6.0 ppm range corresponds to the hydrogen of its side chain double bond, the peak around 3.0 ppm corresponds to the hydrogen next to the ester group, and the peak in the 0.5~2.0 ppm range corresponds to the alkyl hydrogen. The whole spectrum is used to characterize the proton environment and structural purity of the synthesized product.

[0074] Example 3: Preparation of Ginsenosides

[0075] I. Preparation of liquid culture medium for adventitious roots of ginseng

[0076] Based on the final concentration of the materials, the liquid culture medium for ginseng adventitious roots includes: ammonium nitrate (900 mg / L), potassium nitrate (1125 mg / L), potassium dihydrogen phosphate (90 mg / L), calcium chloride dihydrate (170 mg / L), magnesium sulfate heptahydrate (370 mg / L), disodium EDTA (30 mg / L), ferrous sulfate (22 mg / L), potassium iodide (0.93 mg / L), sodium molybdate dihydrate (0.35 mg / L), zinc sulfate heptahydrate (9.6 mg / L), and cobalt chloride hexahydrate (0.125 mg / L). The solution contained copper sulfate pentahydrate (0.025 mg / L), boric acid (6.2 mg / L), manganese sulfate tetrahydrate (22.3 mg / L), glycine (2 mg / L), inositol (100 mg / L), niacin (0.5 mg / L), vitamin B6 (0.5 mg / L), vitamin B1 (0.1 mg / L), glutamine (50 mg / L), hydrolyzed casein (300 mg / L, purchased from Sigma-Aldrich), IBA (9.0 mg / L), sucrose (20 g / L), and water. The pH was adjusted to 5.8, and the solution was autoclaved at 121°C for 20 minutes.

[0077] II. Cleaning and disinfection of ginseng adventitious root raw materials

[0078] Rinse the surface of the ginseng (variety Da Ma Ya) adventitious roots in tap water to remove impurities, wash with 75% alcohol solution for 60 seconds, then rinse 5 times with sterile distilled water in a sterile environment, and blot dry the surface moisture with sterile filter paper.

[0079] III. Suspension Culture of Ginseng Adventitious Roots

[0080] 150g (fresh weight) of adventitious roots from a sterilized pot were inoculated into an airlift bioreactor containing 10L of ginseng adventitious root liquid culture medium. Filtered air was introduced at a flow rate of 150mL / min, and the culture temperature was 26℃. The plants were cultured in the dark for 7 days to obtain a stable adventitious root fixed line.

[0081] IV. Cultivation and Propagation of Ginseng Adventitious Roots

[0082] After 7 days of cultivation in the bioreactor in step three, numerous growth points were observed to have formed in the adventitious roots. To promote the induced growth of ginseng adventitious roots and the synthesis of secondary metabolites, methyl jasmonate (final concentration 100 μmol / L) prepared in Example 2 was added to the bioreactor. Filtered air was introduced at a flow rate of 150 mL / min, and the culture temperature was 22-24℃, with dark incubation.

[0083] V. Obtaining adventitious roots

[0084] After adding the inducer, the adventitious roots were cultured in the bioreactor of step four above for about 40 days. It was observed that the adventitious roots settled in clusters and clumps at the bottom of the reactor, and the biomass increased significantly, about 10-12 times the initial inoculum, thus obtaining adventitious roots.

[0085] VI. Detection of Adventitious Root Saponin Content

[0086] The glycosides of ginseng adventitious roots were determined by sampling from the reactor: dried adventitious root raw materials were taken and 1% (by weight) of extraction aid (Tween 80) was added. The raw materials were extracted twice by ethanol reflux extraction (75% ethanol aqueous solution) and ultrasonic-assisted extraction. The filtrate was dried and separated by macroporous resin chromatography (model HPD-100A, purchased from Tianjin Zhengguang Resin Co., Ltd.), silica gel column chromatography (model 70-230 mesh ASTM (0.063-0.200 mm, purchased from Merck Life Sciences), and high performance liquid chromatography (HPLC) (chromatograph model: Agilent: 1260) to obtain saponins of single purity.

[0087] Detection: High performance liquid chromatography (HPLC) was used to detect saponins. The types of saponins were identified by retention time and mass spectrometry peaks, and the content was quantitatively analyzed.

[0088] Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd, F2, Rg3, Rh2, and PPd, purchased from Sigma-Aldrich, were used as standards. Ginsenosides prepared in this embodiment were used as test samples. HPLC tests were performed using a C18 reversed-phase column (4.6 × 250 mm, 5 μm), with acetonitrile-water as the mobile phase and gradient elution (e.g., acetonitrile 19% → 29% from 0 to 35 min), a flow rate of 1.0 mL / min, a column temperature of 20–30 °C, a detection wavelength of 203 nm, and an injection volume of 10–20 μL. An evaporative light scattering detector (ELSD) was used. The test samples were defatted with chloroform, extracted with saturated n-butanol, diluted to volume with methanol, and filtered through a 0.45 μm filter before analysis.

[0089] See the comparison chart of ginsenoside high performance liquid chromatography (HPLC) analysis. Figure 3 The graph shows the retention time (minutes) on the x-axis and the signal intensity (AU) on the y-axis. The red curve represents the chromatographic peaks of standards containing known components such as Rg1, Re, and Rb1, while the black curve represents the chromatographic peaks of the ginseng sample to be tested. By comparing the retention times of the two graphs, the types of saponins contained in the sample can be determined, and the relative content of each saponin can be evaluated based on the peak height or area. The total content of ginsenosides was found to be 13.6 g / L.

[0090] Example 4: Recycling of methyl jasmonate

[0091] The 9.6g composite catalyst recovered in Section 3 of Example 2 was replenished with 0.4g of fresh catalyst (prepared in Example 1), and the synthesis and purification steps of Example 2 were repeated to obtain 27.3g of methyl jasmonate product with a purity of 97.0% and a cis-methyl jasmonate content of 95.1%. The product was used for ginsenoside induction (using the same method as in Example 3), and the total ginsenoside content was 13.2g / L. After the catalyst was recycled 5 times, the product yield remained at 82.1%, the purity at 96.0%, the cis-methyl jasmonate content at 94.8%, and the total induced ginsenoside content at 12.9g / L, confirming that the catalyst can still stably ensure the activity of the product after recycling.

[0092] Comparative Example 1: Catalysis by a Single Ionic Liquid

[0093] Except for replacing the composite catalyst with 10g of [BMIM]BF4, the remaining steps were the same as in Example 2, and 21.8g of methyl jasmonate was finally obtained with a purity of 89.5% and a cis-methyl jasmonate content of 68.2%. The product was used for ginsenoside induction (method as in Example 3), and the total ginsenoside content was only 8.5g / L, which was 37.5% lower than the induction efficiency in Example 1, confirming the key role of the composite catalytic system in improving the proportion of active configurations of the product and the ginsenoside induction efficiency.

[0094] Comparative Example 2: Purification by Traditional Distillation

[0095] Except for omitting the sodium chloride aqueous solution washing step and recrystallization step, the remaining steps are the same as in Example 2, and 27.5g of methyl jasmonate product is finally obtained with a purity of 92.3% and a cis-methyl jasmonate content of 82.6%. The product is used for ginsenoside induction (method is the same as in Example 3), and the total ginsenoside content is 10.3g / L, which is 24.3% lower than the induction efficiency in Example 1, confirming the necessity of salt washing pretreatment and recrystallization steps for improving product purity, active configuration ratio and induction efficiency.

[0096] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A catalyst for the synthesis of methyl jasmonate, the catalyst comprising 1-butyl-3-methylimidazolium tetrafluoroborate, nanoporous silica and cerium sulfate, wherein the weight ratio of the 1-butyl-3-methylimidazolium tetrafluoroborate, the nanoporous silica and the cerium sulfate is 10-15:3-5:

1.

2. The catalyst according to claim 1, characterized in that, The specific surface area of ​​the nanoporous silica is 1950-1150 m². 2 / g, with a pore size of 2.4-2.5nm and a particle size of 90-110nm.

3. A method for preparing the catalyst according to claim 1 or 2, wherein the method comprises: mixing the 1-butyl-3-methylimidazolium tetrafluoroborate, the nanoporous silica and the cerium sulfate to obtain the catalyst.

4. The preparation method according to claim 3, characterized in that, The preparation method includes the following steps: S1: Mix the 1-butyl-3-methylimidazolium tetrafluoroborate and the nanoporous silica to obtain a first mixture; S2: The first mixture is subjected to ultrasonic dispersion treatment to obtain a second mixture; S3: Mix the second mixture with the cerium sulfate to obtain a third mixture; S4: Stir the third mixture until homogeneous to obtain the catalyst.

5. The preparation method according to claim 4, characterized in that, In S2, the ultrasonic power is 200-400W, and the ultrasonic time is 20-30min; and / or In S4, the stirring rate is 130-170 r / min, the stirring temperature is 60-80℃, and the stirring time is 1-2 h.

6. A method for synthesizing methyl jasmonate, the method comprising: using methyl 2-(1-cyclopentenyl)acetate and 1,3-pentadiene as substrates, and using the catalyst described in claim 1 or 2 or the catalyst prepared by the method described in claims 3-5 as catalyst, to synthesize the methyl jasmonate.

7. The synthesis method according to claim 6, characterized in that, The methyl 2-(1-cyclopentenyl)acetate, wherein the weight ratio of 1,3-pentadiene to the catalyst is 6-10:1.5-2.5:1; The method includes the following first three, first four, or five steps: T1: The methyl 2-(1-cyclopentenyl)acetate, the 1,3-pentadiene, and the catalyst are added to the reactor simultaneously or sequentially. T2: Remove oxygen from the reactor to obtain an anoxic reaction system; T3: In the hypoxic reaction system, the reaction is carried out at 30-40℃ and 200-300r / min for 4-6h to obtain crude methyl jasmonate. T4: Centrifuge the crude methyl jasmonate to obtain a supernatant; extract the supernatant with an aqueous inorganic salt solution, and allow it to stand to obtain an organic phase; distill the organic phase and collect the fraction at 120-130℃ to obtain the crude methyl jasmonate. T5: Dissolve the methyl jasmonate crude product in anhydrous ethanol, crystallize and dry to obtain the methyl jasmonate finished product.

8. The synthesis method according to claim 7, characterized in that, Choose one or more from the following A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10; A1: In T1, the purity of the methyl 2-(1-cyclopentenyl)acetate is ≥98%, and the purity of the 1,3-pentadiene is ≥99%; A2: In T2, the method for removing oxygen from the reactor is to replace the air in the reactor with nitrogen; In A3:T4, the centrifugation conditions are 4000-6000 r / min for 5-20 min; In A4:T4, the inorganic salt aqueous solution is a sodium chloride aqueous solution with a mass fraction of 5-10%; In A5:T4, the step of distilling the organic phase is as follows: the organic phase is injected into a three-stage distillation column, loaded with glass spring packing with a diameter of 8-12 mm, and the temperature inside the column is controlled at 140-160℃, the vacuum degree at 4.5-5.5 Pa, the condensation temperature at 4-8℃, and the reflux ratio at 1:25-30. A6: In T5, the primary methyl jasmonate is added to anhydrous ethanol for recrystallization, wherein the weight ratio of the primary methyl jasmonate to the anhydrous ethanol is 100:3-5; the mixture is allowed to stand at 0-5℃ for 8-12 hours; the mixture is filtered to crystallize; the crystals are then vacuum dried at a vacuum degree of 8-12 Pa and a temperature of 40-50℃ for 2-3 hours to obtain the finished methyl jasmonate product.

9. A method for preparing total ginsenosides, the method comprising the following first four or five steps: X1: Clean and disinfect the ginseng adventitious roots to obtain disinfected ginseng adventitious roots; X2: The disinfected ginseng adventitious roots were inoculated into ginseng adventitious root liquid culture medium for suspension culture to obtain a fixed ginseng adventitious root line; The solvent of the ginseng adventitious root liquid culture medium is water; based on the final concentration of the materials used, the ginseng adventitious root liquid culture medium contains 900 mg / L ammonium nitrate, 1125 mg / L potassium nitrate, 90 mg / L potassium dihydrogen phosphate, 170 mg / L calcium chloride dihydrate, 370 mg / L magnesium sulfate heptahydrate, and 30 mg / L... Disodium EDTA, 22 mg / L ferrous sulfate, 0.93 mg / L potassium iodide, 0.35 mg / L sodium molybdate dihydrate, 9.6 mg / L zinc sulfate heptahydrate, 0.125 mg / L cobalt chloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate tetrahydrate, 2 mg / L glycine, 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L vitamin B6, 0.1 mg / L vitamin B1, 50 mg / L glutamine, 300 mg / L hydrolyzed casein, 9.0 mg / L IBA, and 20 g / L sucrose; pH 5.5-6.0; X3: Add jasmonic acid of any one of claims 6-8 to the reaction system of ginseng adventitious root fixed line at a final concentration of 80-120 μM to obtain proliferated ginseng adventitious roots; X4: The adventitious roots of ginseng were treated by ethanol reflux extraction to obtain crude total ginsenosides. X5: The crude total ginsenosides were purified by a combination of column chromatography and high performance liquid chromatography to obtain the pure total ginsenosides.

10. The preparation method according to claim 9, characterized in that, Selected from any one or more of the following: B1, B2, B3, B4, B5, B6, B7, B8, B9, and B10; B1: In X1, the ginseng adventitious roots are rinsed clean in water, washed with 70-80% alcohol aqueous solution for 50-70 seconds, and rinsed with sterile distilled water 3-5 times to obtain the sterilized ginseng adventitious roots. B2: In X2, 10-20g of ginseng adventitious roots are inoculated into each L of the ginseng adventitious root liquid culture medium. B3: In X2, the light condition is dark culture; B4: In X2, the culture temperature is 25-28℃; filtered air is introduced into the culture system at a flow rate of 100-200 mL / min; B5: In X2, the incubation time is 5-10 days; B6: In X3, the light condition is dark culture; B7: In X3, the incubation period is 30-40 days; B8: In X3, the culture temperature is 22-24℃; filtered air is introduced into the culture system at a flow rate of 100-200 mL / min; B9: In X3, the ginseng variety is Da Ma Ya ginseng; B10: In X4, take the dried ginseng adventitious roots, add 0.8-1.2% of the weight of the dried ginseng adventitious roots of Tween, and perform reflux extraction using 75% ethanol aqueous solution. The extract is the crude total ginsenosides of the ginseng.

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