Device and method for preparing cis-ZEN by converting trans-ZEN through combination of ultraviolet and plasma

Abstract

The invention discloses a device and a method for preparing cis-ZEN by transforming trans-ZEN through combination of ultraviolet and plasma, and the method for preparing the cis-ZEN comprises the following steps: step 1, dissolving a trans-ZEN standard substance in acetonitrile to prepare a trans-ZEN stock solution; step 2, placing the trans-ZEN stock solution on a plate, and naturally volatilizing the trans-ZEN stock solution in a fuming cupboard to obtain a trans-ZEN sample flatly laid at the bottom of the plate; step 3, placing the plate filled with the trans-ZEN sample on a sample plate; 4, starting a plasma generation device and an ultraviolet light source, and enabling plasma wind and ultraviolet light to act on the trans-ZEN sample at the same time until the set treatment time is reached; wherein in the trans-ZEN sample treatment process, the box door is kept closed; and 5, collecting the substrate and the product treated by the trans-ZEN by using a solid-phase extraction column, and separating the product by using ultra-high performance liquid chromatography to obtain the cis-ZEN.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural chemical industry, and particularly relates to a device and method for preparing cis-ZEN by converting trans-ZEN using ultraviolet light and plasma. Background Art

[0002] Zearalenone (ZEN) is a secondary metabolite produced by various Fusarium species, such as Fusarium graminearum, Fusarium trilineum and Fusarium luteum. It is a widely distributed environmental pollutant. ZEN has a similar structure to 17β-estradiol, can competitively bind to estrogen receptors, damage the reproductive system and immune system of animals, and has certain hepatotoxicity and cytotoxicity. It is listed as a Class III carcinogen by the International Agency for Research on Cancer. ZEN can enter the human or animal body through food or skin contact, posing a huge safety hazard to public health. Therefore, controlling ZEN pollution has become a major national need to ensure public health safety. The reduction of ZEN has also attracted the attention of scientists from various countries.

[0003] At present, the methods for eliminating ZEN include physical, chemical and biological methods. Under the action of these methods, ZEN will be converted into other substances. However, although people are more aware of the toxicity and hazards of ZEN, little is known about its conversion products. According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), ZEN actually refers to (3S, 11E)-14,16-dihydroxy-3-methyl-3,4,5,6,9,10-hexahydro-1H-2-benzothiazole cyclotetradecane-1,7(8H)-dione, that is, trans-ZEN. And trans-ZEN also has an isomer, namely cis-ZEN. It is the isomerization product of trans-ZEN under light and still has toxic and estrogenic effects. Studies have shown that trans-ZEN in food oil may be converted into cis-ZEN when exposed to light for a long time. However, people now know very little about the toxicity of cis-ZEN, and it may become a potential environmental or food contaminant. The reason for this phenomenon is that the preparation process of cis-ZEN pure substance is relatively complicated and the preparation process is cumbersome.

[0004] Cis-ZEN is an isomerization product of trans-ZEN, but the efficiency of conversion from trans-ZEN to cis-ZEN under light is low, resulting in a longer action time, increasing the probability of producing impurity molecules, and reducing the stability of substrates and products, which increases the difficulty of later separation and purification. In addition, cis-ZEN and trans-ZEN have similar molecular structures, and the two are difficult to separate in high-performance liquid chromatography, which also increases the difficulty of cis-ZEN preparation. Therefore, the current cis-ZEN preparation technology is not yet mature, the cis-ZEN preparation efficiency is low, and the preparation process is difficult to control. Due to the difficulty in preparing pure cis-ZEN, there is a lack of pure cis-ZEN, which makes it difficult to use pure cis-ZEN substances for cell and other toxicological experiments. This ultimately leads to a lack of understanding of the toxicity and safety of cis-ZEN. The possibility of environmental pollutants causing harm to humans or animals is increased. Therefore, the current cis-ZEN preparation process is in urgent need of improvement, and a product preparation method that can quickly convert trans-ZEN into cis-ZEN and introduce no or very little other impurities needs to be developed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for preparing cis-ZEN by converting trans-ZEN using a combination of ultraviolet and plasma, which can prepare a high-purity cis-ZEN standard, greatly simplify the cis-ZEN preparation process, shorten the cis-ZEN preparation time, and the preparation process is easy to control.

[0006] The present invention also provides a device for preparing cis-ZEN by converting trans-ZEN using a combination of ultraviolet and plasma, which can provide a trans-ZEN processing environment containing both plasma wind and ultraviolet light, has a simple structure, and is easy to operate.

[0007] The technical solution provided by the present invention is:

[0008] A device for converting trans-ZEN to prepare cis-ZEN by combining ultraviolet and plasma, comprising:

[0009] A box body, which is provided with an openable door;

[0010] A plasma generating device is installed in the box and located on the top of the box;

[0011] An ultraviolet light source is installed in the box and is disposed below the plasma generating device and is spaced apart from the plasma generating device;

[0012] Two sets of fans, which are respectively installed on both sides of the box and arranged close to the top of the box, for dissipating heat from the ultraviolet light source;

[0013] A multi-layer support frame is installed in the box and arranged below the ultraviolet light source with a gap between the multi-layer support frame and the ultraviolet light source;

[0014] Wherein, the multi-layer support frames are arranged at intervals along the height direction of the box body;

[0015] The sample plate can be selectively arranged on different support frames for placing the trans-ZEN sample.

[0016] Preferably, the plasma generating device adopts a high-voltage pulse orifice plate.

[0017] Preferably, the ultraviolet light source is one or more of UVA and UVC.

[0018] Preferably, the ultraviolet light source is composed of a plurality of evenly spaced ultraviolet light strips;

[0019] Each of the ultraviolet light strips comprises: a first light source module and two second light source modules; the first light source module and the second light source module are both arranged along the length direction of the ultraviolet light strip, and the first light source module is located between the two second light source modules;

[0020] The ultraviolet light wavelength of the first light source module is 365 nm, and the ultraviolet light wavelength of the second light source module is 275 nm.

[0021] A method for preparing cis-ZEN by converting trans-ZEN using ultraviolet light and plasma, and a device for preparing cis-ZEN by converting trans-ZEN using ultraviolet light and plasma, comprising the following steps:

[0022] Step 1: Dissolve the trans-ZEN standard in acetonitrile to prepare a trans-ZEN stock solution;

[0023] Step 2: Place the trans-ZEN stock solution on a plate and evaporate it naturally in a fume hood to obtain a trans-ZEN sample spread on the bottom of the plate;

[0024] Step 3: Place the plate containing the trans-ZEN sample on the sample plate;

[0025] Step 4: Turn on the plasma generator and the ultraviolet light source, so that the plasma wind and ultraviolet light act on the trans-ZEN sample simultaneously until the set processing time is reached;

[0026] Among them, during the trans-ZEN sample processing, the chamber door remained closed;

[0027] Step 5: After collecting the substrate and product after trans-ZEN treatment using a solid phase extraction column, the product is separated by ultra-high performance liquid chromatography to obtain cis-ZEN.

[0028] Preferably, the concentration of the trans-ZEN stock solution is 100 μg / mL.

[0029] Preferably, the level of the trans-ZEN stock solution in the dish does not exceed 0.5 mm.

[0030] Preferably, the distance between the UV light source and the trans-ZEN sample is 5 cm-10 cm.

[0031] Preferably, in step 4, the processing time is set to 5 min-10 min.

[0032] The beneficial effects of the present invention are:

[0033] (1) The method provided by the present invention for converting trans-ZEN to prepare cis-ZEN by combining ultraviolet and plasma can prepare high-purity cis-ZEN standard products. The preparation method is time-saving, low-cost, and the reaction process is easy to control, and can realize the large-scale production and preparation of cis-ZEN.

[0034] (2) The device provided by the present invention for converting trans-ZEN to prepare cis-ZEN by combining ultraviolet light and plasma can provide a trans-ZEN processing environment containing both plasma wind and ultraviolet light, has a simple structure, and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the overall structure of the device for converting trans-ZEN to prepare cis-ZEN by combining ultraviolet and plasma as described in the present invention.

[0036] Figure 2 A is the total ion current diagram of trans-ZEN treated with the control group in the present invention

[0037] Figure 2 B is the total ion current diagram of trans-ZEN processed in Example 1 of the present invention.

[0038] Figure 3 This is a chromatogram of separation of trans-ZEN and cis-ZEN by ultra-high performance liquid chromatography in the present invention.

[0039] Figure 4 A in the figure is the total ion current diagram of the prepared cis-ZEN in the mass spectrum of the present invention.

[0040] Figure 4B is the primary mass spectrum of the prepared cis-ZEN in the mass spectrum of the present invention.

[0041] Figure 5 A is the prepared product 1 H– 1 H COSY and HMBC correlation diagram.

[0042] Figure 5 B in the figure is the change from substrate trans-ZEN to product cis-ZEN. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0044] like Figure 1 As shown, the present invention provides a device for converting trans-ZEN to prepare cis-ZEN by combining ultraviolet and plasma, including: a box body 110, a box door 120, a plasma generating device 130, an ultraviolet light source 140, a fan 150, a bracket 160 and a sample plate 170.

[0045] The box body 110 is provided with an openable door 120. The plasma generator 130 is installed in the box body 110 and is located at the top of the box body 110. The ultraviolet light source 140 is installed in the box body 110 and is arranged below the plasma generator 120, with a gap between the ultraviolet light source 140 and the plasma generator 120. Two sets of fans 150 are respectively installed on both sides of the box body 110 and are arranged near the top of the box body 110 to dissipate heat for the ultraviolet light source.

[0046] The multi-layer support frame 160 is installed in the box body and is arranged below the ultraviolet light source 140, with a gap between the ultraviolet light source 140. The multi-layer support frame 160 is arranged at intervals along the height direction of the box body 110. In this embodiment, the multi-layer support frame 160 is composed of two long strip baffles, and the two strip baffles are respectively fixedly mounted on two opposite inner walls in the box body 110. The sample plate 170 is used to place the trans-ZEN sample. The sample plate 170 can be selectively arranged on different support frames 160 to meet the processing distance requirements between the ultraviolet light source 140 and the trans-ZEN sample. The box door 110 needs to be opened when placing the sample plate 170. The box door 110 remains closed during the processing so that the box body 110 is in a closed state.

[0047] In one embodiment, a high-voltage pulse orifice plate capable of emitting low-temperature plasma wind is fixed on the top of the box 110 as a plasma generator. The orifice plate is square with a side length of 28 cm. The plasma generator uses a high-voltage pulse power generation method to generate negative ions. The input voltage of the high-voltage module is 220V, the current is 170mA, and the output high voltage is between -8KV and -12.8KV. The concentration of negative ions generated is 1.9×10 7 to 5.2×10 7 pcs / cm 3 In this embodiment, three ultraviolet light strips are used as ultraviolet light sources. The three ultraviolet light strips are evenly spaced and placed below the plasma wind generating device without direct contact with the plasma generating device. The light strip is 260cm long and 30cm wide. 25 lamp beads (second light source module) that can emit 275nm (UVC) wavelengths are evenly distributed on both sides of each light strip along the length direction of the light strip, and 13 lamp beads (first light source module) that can emit 365nm (UVA) wavelengths are evenly distributed in the middle position of the light strip along the length direction of the light strip. Therefore, each ultraviolet light strip is loaded with 50 lamp beads emitting 275nm wavelengths and 13 lamp beads emitting 365nm wavelengths, and the UVC and UVA power consumption of each light strip is 100W and 20W respectively. Fans 150 are installed on the left and right sides of the box 110 to timely dissipate the heat generated in the ultraviolet lamp working diagram and protect the working system. A removable sample plate 160 is installed below the ultraviolet light strip for placing the processed trans-ZEN samples. By placing the sample plate 170 on different support brackets 160, the distance between the trans-ZEN sample and the UV lamp can be adjusted. The plasma orifice plate and the UV light bar are equipped with switches, which can be turned on or off separately. When the plasma orifice plate or the UV light bar is working, the box door 110 is closed to form a closed space, so that the reaction is carried out in the closed space, so that the generated active substances do not overflow, and the sample conversion efficiency is improved.

[0048] The present invention also provides a method for preparing cis-ZEN by converting trans-ZEN by combining ultraviolet and plasma, and the preparation is carried out using the device for converting trans-ZEN by combining ultraviolet and plasma. The preparation method is described below in conjunction with specific examples.

[0049] 1. Combined UV and plasma treatment of trans-ZEN samples

[0050] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The content of trans-ZEN in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally. Then place the disposable plate on the sample plate. The sample plate is 5 cm-10 cm away from the UV light bar. Turn on the UV light source and plasma generator at the same time to treat the trans-ZEN standard for 5 min-10 min. In the control group, trans-ZEN is not treated with UV and plasma equipment. The rest of the steps are the same. Each treatment is repeated three times.

[0051] 2. Testing of the effect of UV and plasma on trans-ZEN transformation

[0052] Use 2mL of methanol to re-dissolve the treated sample on the plate and transfer it to a centrifuge tube, and remove impurities by high-speed centrifugation. Take the supernatant in the above centrifuge tube, dilute it appropriately, and use UPLC-MS to detect the residual amount of trans-ZEN in the system. Calculate the degradation rate of trans-ZEN in the treatment group according to formula 1. Use the external standard method to calculate the changes in the concentrations of trans-ZEN and the generated cis-ZEN, and comprehensively evaluate the effect of ultraviolet and plasma combination on trans-ZEN conversion under the current reaction conditions.

[0053]

[0054] The transformation of trans-ZEN to cis-ZEN is due to a photosensitive reaction, which is essentially a cis-trans isomerization reaction of a molecule. When a molecule absorbs electromagnetic radiation of different energies, it can reach the first excited state (S1) or the high excited state (S n ). The excited state is a high-energy and unstable state of a molecule. The higher the excited state, the shorter its life span and the more unstable it is, so it will quickly deactivate and return to the ground state or other low-energy state through various photochemical and physical processes. Under ultraviolet light, the electrons in the substrate absorb energy and transition to the excited state. There are two main ways for electrons to deactivate from the excited state to the ground state: one is to convert it into heat energy or radiation energy (fluorescence, phosphorescence, thermally delayed fluorescence) through photophysical processes; the other is that the excited state undergoes isomerization, cracking, chemical reactions with the solvent or another solute, and other secondary chemical reactions. This is determined by the structural characteristics of the molecule itself. Trans-ZEN has a C=C double bond and can isomerize under ultraviolet irradiation.

[0055] Part of the reason why plasma can promote the transformation of trans-ZEN to cis-ZEN is that photons are also generated during the plasma generation process, and photons have energy. The substrate molecule, i.e., trans-ZEN, can absorb photons, accelerating the transition of electrons from the ground state to the excited state, and also accelerating the transition of excited state electrons to the ground state, thereby accelerating the isomerization process. In addition, due to the absorption of more energy, ground state electrons may jump to a higher excited state, resulting in a shortened lifetime, accelerating their transition to the ground state, thereby accelerating the photoisomerization reaction process and promoting the transformation of trans-ZEN to cis-ZEN.

[0056] 3. Search and analysis of trans-ZEN transformation products by UV and plasma

[0057] The trans-ZEN standard was treated according to the above conditions, and different reaction times were set. After the treatment, the trans-ZEN on the plate was re-dissolved, centrifuged to remove impurities, and then transferred to a liquid phase vial. The UPLC-MS method was used to detect the product formation in the sample. The Agilent 1290 series UPLC system equipped with an autosampler and a four-stage UPLC pump was used to analyze the product formation in the sample. The mobile phase was a methanol-water solution with formic acid and ammonium formate added, and the gradient elution method was used to effectively separate the chemicals in the reaction system. The mass spectrometry detection used an Agilent 6545ESIQ-TOF device. The Agilent MassProfiler Professional (MPP) multivariate statistical analysis package was used to perform cluster analysis on the obtained data to determine the possible main effect transformation products. The main effect transformation products mined were subjected to ionization strikes with an ionization energy of 20ev. By analyzing the total ion flow, product primary mass spectrum, and secondary fragment distribution obtained by UPLC-MS / MS detection, it was determined that the main effect degradation product of trans-ZEN was cis-ZEN after UV and plasma treatment.

[0058] 4. Isolation and purification of the transformation product cis-ZEN

[0059] The transformation products were separated using an ultra-high performance liquid chromatography (UPLC) system. The column size was 2.1 mm × 100 mm, with a pore size of 1.9 μm (Thermo, Hypersil GOLD). The detector was an UltiMate 3000DAD UV detector with a scanning wavelength of 265 nm. The mobile phase was acetonitrile-water (50:50, v / v) with a flow rate of 0.2 mL / min. The sample volume was 5 μL each time. According to the peak position and peak time, the transformation product cis-ZEN was collected into a 50 mL centrifuge tube, and the purity of the prepared transformation product was detected using UHPLC-MS. The collected product solution was evaporated to dryness at 45 °C using a centrifugal concentrator, freeze-dried using a vacuum freeze dryer, and stored at –20 °C. The solvent was evaporated to dryness at a relatively mild temperature, and then freeze-dried to remove the remaining water for later product structure identification.

[0060] 5. Structural Identification of Transformation Products

[0061] Part of the prepared product was dissolved in 1 mL of methanol and centrifuged at 10,000 r / min for 5 min to remove impurities. The supernatant was aspirated and the molecular structure of the conversion product was identified using the UPLC-MS / MS method. The conversion product was analyzed using an Agilent 1290 series UPLC system equipped with an autosampler and a four-stage UPLC pump. Chromatographic analysis was performed on an Agilent Poroshell 120EC-C18 column with a diameter of 2.1 mm × 100 mm and a pore size of 2.7 μm. The injection volume was 2 μL, the flow rate was 0.2 mL / min, and the temperature was set to 30°C. The mobile phase was divided into A and B, phase A was an aqueous solution with 0.1% formic acid and 5 mM ammonium formate, and phase B was a methanol solution with 0.1% formic acid. The mobile phase gradient is as follows: In order to better separate the substrate and the conversion product, the LC-MS detection method is improved, and the detection time of the liquid phase is extended to 30 minutes, that is, the B phase is maintained at 10% from 0 minutes to 1 minute, and the B phase gradually increases to 100% from 1 minute to 27 minutes. After the B phase is maintained at 100% for 3 minutes, it is reduced back to 10% within 0.5 minutes. The A phase is changed accordingly, and the total ratio of the two is maintained at 100%.

[0062] The mass spectrometry detection used an Agilent 6545ESIQ-TOF device. The optimized conditions were as follows: the ionization source was an electrospray ionization source negative source (ESI–), the nebulizer pressure was 40psig, and the capillary voltage was 4.0kv. The fragmentor voltage was 175v, and the cone voltage was 65v. The gas temperature was 300°C, and the auxiliary gas (N2) was 5L / min. The sheath gas temperature was 325°C, and the flow rate was 11L / min. The mass spectrometer was operated in full scan mode, collecting data with a mass-to-charge ratio (m / z) between 100-1200, and the scanning frequency was 2 spectra / s. After mass spectrometry detection, it can be judged from the total ion flow that the target degradation product cis-ZEN is the only target peak, indicating that the product has a high purity. The primary mass spectrum showed that the mass-to-charge ratio of the prepared degradation product was 317.1394, which was consistent with the molecular mass of cis-ZEN.

[0063] The prepared degradation products were dissolved in DMSO and analyzed using a Bruker 600 spectrometer ( 1 H, 600MHz; 13 C, 125MHz) to measure the 1 H NMR, 13 C NMR, 1 H- 1 H COSY, HSQC, HMBC and ROESY spectra. By analyzing the one-dimensional NMR spectrum, that is, 1 H NMR, 13 C NMR can determine the chemical shifts of C and H in the prepared degradation product molecules and determine the carbon skeleton. And according to the response signals of C and H in NMR, it can be judged that the prepared sample has a high purity. Further analysis of the two-dimensional NMR spectrum 1 H- 1 H COSY, HSQC, HMBC and ROESY spectra can clarify the connection between C and H and determine the molecular structure of the prepared product. Finally, it was determined that the prepared degradation product was cis-ZEN.

[0064] Example 1

[0065] Trans-ZEN standard was treated with UV and plasma for 5 minutes at 5 cm.

[0066] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 5 cm. Turn on the UV light source and plasma generator at the same time, and set the processing time to 5 min. Set up three parallels, and take the average of the three parallel results as the final result.

[0067] Example 2

[0068] Trans-ZEN standard was treated with UV and plasma at 5 cm for 10 min

[0069] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 5 cm. Turn on the UV irradiation and plasma generator at the same time, and set the treatment time to 10 min. Set up three parallels, and take the average of the three parallel results as the final result.

[0070] Example 3

[0071] Trans-ZEN standard was treated with UV and plasma for 7 minutes at 8cm

[0072] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 8 cm. Turn on the UV irradiation and plasma generator at the same time, and set the treatment time to 7 minutes. Set up three parallels, and take the average of the three parallel results as the final result.

[0073] Example 4

[0074] Trans-ZEN standard was treated with UV and plasma at 10cm for 10min

[0075] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 10 cm. Turn on the UV irradiation and plasma generator at the same time, and set the treatment time to 10 min. Set up three parallels, and take the average of the three parallel results as the final result.

[0076] Comparative Example 1

[0077] Trans-ZEN standard was treated with UV and plasma at 3cm for 12min

[0078] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 3 cm. Turn on the UV irradiation and plasma generator at the same time, and set the treatment time to 12 minutes. Set up three parallels, and take the average of the three parallel results as the final result.

[0079] Comparative Example 2

[0080] Trans-ZEN standard was treated with UV and plasma at 12cm for 15min

[0081] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 12 cm. Turn on the UV irradiation and plasma generator at the same time, and set the treatment time to 15 min. Set up three parallels, and take the average of the three parallel results as the final result.

[0082] Comparative Example 3

[0083] Trans-ZEN standard was treated with UV and plasma for 20 minutes at 5 cm

[0084] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 5 cm. Turn on the UV irradiation and plasma generator at the same time, and set the treatment time to 20 min. Set up three parallels, and take the average of the three parallel results as the final result.

[0085] Comparative Example 4

[0086] Plasma treatment of trans-ZEN standard at 5cm for 5min

[0087] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 5 cm. Turn on the plasma generator and set the treatment time to 5 min. Set up three parallels, and take the average of the three parallel results as the final result.

[0088] Comparative Example 5

[0089] UV treatment of trans-ZEN standard at 5cm for 5min

[0090] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 5 cm. Turn on the UV irradiation device and set the treatment time to 5 min. Set up three parallels, and take the average of the three parallel results as the final result.

[0091] Comparative Example 6

[0092] UV treatment first, plasma treatment later

[0093] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 5 cm. First, turn on the UV irradiation of the sample for 5 minutes, then turn off the UV irradiation and turn on the plasma generator to treat the sample for 5 minutes. Set up three parallels, and take the average of the three parallel results as the final result.

[0094] Comparative Example 7

[0095] Plasma treatment first, UV treatment later

[0096] Dissolve 5 mg of trans-ZEN standard in 50 mL of acetonitrile to prepare a stock solution with a concentration of 100 μg / mL. Pipette 3.18 mL of trans-ZEN stock solution onto a disposable plate with a diameter of 9 cm. The height of the liquid in the plate is 0.5 mm. The trans-ZEN content in the plate is 318 μg. Place the plate in a fume hood to allow the organic reagent to evaporate naturally, then place the disposable plate on the sample plate. The vertical distance between the sample plate and the UV light bar in the middle is 5 cm. First, turn on the plasma generator alone to treat the sample for 5 minutes, and then turn on the UV irradiation device alone to treat the sample for 5 minutes. Set up three parallels, and take the average of the three parallel results as the final result.

[0097] Test example

[0098] The samples treated with Examples 1-4 and Comparative Examples 1-7 were dissolved in 2 mL of methanol, transferred to a centrifuge tube, and centrifuged at 10,000 r / min for 5 min to remove impurities. The supernatant was aspirated, and the trans-ZEN residue and cis-ZEN generation in the detection system were detected using UPLC-MS. The changes in the concentrations of the substrate trans-ZEN and the product cis-ZEN were calculated using the external standard method. The degradation rate of trans-ZEN was calculated using reference formula (1). The effect of converting trans-ZEN to cis-ZEN under this method was comprehensively evaluated. The test results of each embodiment and comparative example are shown in Table 1.

[0099] Table 1 The conversion of tran-ZEN to cis-ZEN in various examples and comparative examples

[0100]

[0101]

[0102] As can be seen from Table 1, in Examples 1 to 4, ultraviolet and plasma acted on trans-ZEN simultaneously, the action distance was 5cm-10cm, the action time was 5min-10min, and the final trans-ZEN degradation rates obtained were all greater than 70%, namely 71.73%, 81.56%, 77.66% and 75.38%, respectively. The amounts of cis-ZEN obtained on each plate were 227.65μg, 220.02μg, 221.82μg and 215.31μg, respectively, all greater than 210μg, achieving a better cis-ZEN preparation effect. Among them, the best effect was achieved in Example 1, where the distance between the sample and the ultraviolet light bar was 5cm, and the action time was 5min. Figure 2 As shown, trans-ZEN exists in the mass spectrum in the form of [M–H] – , mass-to-charge ratio was 317.1426, and retention time was 7.71min. After 5min treatment, its peak area decreased significantly. The retention time of the generated cis-ZEN was 7.86min, and its ion peak in the mass spectrum existed in the form of [M–H] – The mass-to-charge ratio is 317.1404. After 5 minutes of UV and plasma treatment, the obvious generation of cis-ZEN can be observed.

[0103] It is worth noting that in Example 1 and Example 2, the action distance of ultraviolet and plasma is 5 cm, and the action time is 5 min and 10 min, respectively. For the degradation rate of trans-ZEN, Example 2 is greater than Example 1, but the amount of cis-ZEN obtained after the reaction is completed is greater in Example 1 than in Example 2. This is because, within a certain range, after the reaction time is extended, although the amount of trans-ZEN is still decreasing, the degradation rate of trans-ZEN is still increasing. However, cis-ZEN will also be converted into other substances under the action of ultraviolet or plasma, resulting in a reduction in the amount of the target product cis-ZEN generated in the end. However, when the action time is within 10 min, the product loss rate is small, which is conducive to achieving the purpose of preparing the final product.

[0104] In Comparative Example 3, when the action distance is 5 cm and the action time is 20 min, the degradation rate of trans-ZEN is 79.81%, which is lower than the degradation rate when the action time is 10 min (Example 2), indicating that the reaction will reach an equilibrium point at 10 min of reaction. Even if the reaction time is extended in the later stage, the degradation rate of trans-ZEN will not increase. And because the action time is too long, the generated cis-ZEN is converted into other substances with smaller molecular weight, resulting in unsatisfactory cis-ZEN production. Therefore, it is not the case that the longer the reaction time, the better the preparation effect of cis-ZEN. It can be obtained that the optimal time for the combined action of ultraviolet and plasma to convert trans-ZEN to cis-EN is 5min-10min.

[0105] In Comparative Example 1, the action distance of ultraviolet and plasma was 3 cm and the action time was 12 min. The degradation rate of Trans-ZEN was 57.69%, and the mass of cis-ZEN generated was 183.09 μg. In this scenario, the substrate degradation rate and the amount of product generated were lower than those in Examples 1 to 4. This is because the distance between the substrate and the ultraviolet light bar and the plasma plate in Comparative Example 1 is too close, which reduces the irradiation area of ​​the ultraviolet light bar, and the close distance easily leads to the accumulation of negative ions generated by the plasma, which is not conducive to its synergistic and abatement effect.

[0106] In Comparative Example 2, the action distance between ultraviolet and plasma is 12 cm, and the action time is 15 min. The degradation rate of trans-ZEN is 55.68%, and the mass of cis-ZEN produced is 176.71 μg. The degradation rate of substrate and the amount of product generated are lower than those in Examples 1 to 4. The reason for the analysis is that when the distance between the sample and the ultraviolet light bar is 12 cm, the intensity of ultraviolet light received is greatly reduced, resulting in insufficient energy source for isomerization of trans-ZEN, resulting in its degradation and conversion rate being slowed down. Therefore, an action distance of 5 cm-10 cm is more appropriate, which can give full play to the accelerating synergistic effect of plasma and enable the sample to obtain a larger irradiation area and irradiation intensity.

[0107] Comparative Example 4 is a case where plasma acts on trans-ZEN alone. When the treatment distance is 5 cm, the degradation rate of trans-ZEN after 5 minutes of action is 18.44%, which is much lower than the effect achieved by the combined action of ultraviolet and plasma for 5 minutes (Example 1). In addition, the generation of cis-ZEN was not detected in the reaction system. This indicates that cis-ZEN cannot be generated when plasma acts alone.

[0108] In Comparative Example 5, ultraviolet light was used alone to act on trans-ZEN. After 5 minutes of action at a distance of 5 cm, the degradation rate of the substrate trans-ZEN was 26.12%, which was significantly lower than the effect produced by the simultaneous action of ultraviolet light and plasma at the same action distance and time. The cis-ZEN content obtained by the reaction was 74.76 μg, which was also significantly lower than the product obtained by the joint action of ultraviolet light and plasma (Example 1). Moreover, the sum of the trans-ZEN degradation rates obtained in Comparative Examples 4 and 5 was lower than the degradation effect obtained by the joint action of ultraviolet light and plasma in Example 1. Similarly, the cis-ZEN yield obtained by adding the two was also lower than the product yield obtained in Example 1. This shows that the efficiency of the simultaneous action of ultraviolet light and plasma is higher than the sum of the effects of ultraviolet light or plasma alone. When ultraviolet light and plasma act simultaneously, the two are not simply added together, but there is a synergistic effect.

[0109] Comparative Examples 6 and 7 are interactive modes of ultraviolet and plasma. That is, ultraviolet and plasma do not act at the same time, but in an alternating manner. The results show that even with the optimal action distance of 5 cm and the optimal action time of 5 min, the degradation rate of trans-ZEN obtained is still not ideal, at 47.20% and 43.16%, respectively, which is much lower than the effect when ultraviolet and plasma act simultaneously in the embodiment. The cis-ZEN contents obtained are 99.86 μg and 91.32 μg, respectively. The product preparation effect is relatively unsatisfactory. It is explained that the key to the mechanism that can promote the conversion of trans-ZEN to cis-ZEN lies in the accelerated transition and energy release of electrons under high-voltage electric fields, which intensifies the isomerization effect on the substrate, thereby promoting the reaction to proceed in the forward direction. The active substances produced in the plasma or ultraviolet system are not the key to determining the degree and speed of the conversion of trans-ZEN to cis-ZEN. If you want to achieve a higher conversion effect of trans-ZEN to cis-ZEN, you need to increase the ultraviolet action time so that the ultraviolet continues to act. However, long-term UV exposure consumes a lot of power, and due to the long time, cis-ZEN may be converted into other substances, reducing the yield of the product. Therefore, it is necessary to have UV exposure throughout the process and improve the efficiency of UV exposure on trans-ZEN. Therefore, simultaneous UV and plasma exposure is the best way to combine UV and plasma.

[0110] The treated samples in the plate in Example 1 are separated and purified to obtain cis-ZEN standards. Use 2 mL of methanol to reconstitute and transfer to a centrifuge tube. After filtering through microfiber filter paper, transfer to a solid phase extraction column (SPE column). Trans-ZEN and its transformation products will be adsorbed to the column filler. Wash twice with ultrapure water to remove impurities that are not hanging on the column. Add 2 mL of methanol in batches to wash the SPE column multiple times to collect trans-ZEN and its transformation products. During this period, the UPLC-MS method is used to detect the content of the target transformation product in the collected eluate, and the elution is stopped when the content is very low. The collected eluate is mixed evenly and placed in a centrifugal concentrator to evaporate to dryness at 45°C. After evaporation, the residue is reconstituted with 3 mL of methanol.

[0111] The conversion products were separated using the UPLC system. The specifications of the chromatographic column were 2.1 mm × 100 mm, with a pore size of 1.9 μm (Thermo, Hypersil GOLD). The detector was an UltiMate 3000DAD UV detector with a scanning wavelength of 265 nm. In order to widen the retention time difference between trans-ZEN and cis-ZEN and reduce the difficulty of product separation and purification, the mobile phase ratio was optimized. The mobile phase was set to acetonitrile-water (50:50, v / v) with a flow rate of 0.2 mL / min. The sample volume was 5 μL each time. Figure 3 As shown, the retention time of trans-ZEN, i.e., the elution time, is 4.23 min, and the elution time of cis-ZEN is 4.59 min. According to the different elution times, cis-ZEN and trans-ZEN can be well separated. Collect cis-ZEN at 4.59 min in a 50 mL centrifuge tube. Use a centrifugal concentrator to evaporate the collected product solution at 45 ° C to remove most of the solvent. Then use a vacuum freeze dryer to freeze-dry the product to completely remove the water in the sample to obtain a cis-ZEN standard.

[0112] To identify the purity and molecular structure accuracy of the prepared degradation products, UPLC-MS and nuclear magnetic resonance spectroscopy were used. The prepared cis-ZEN standard was dissolved in DMSO, and the purity and mass number of the prepared product were detected using the above UPLC-MS method. Figure 4 Where A is the total ion current of the prepared degradation product in the mass spectrum. It can be seen that the prepared degradation product only presents an obvious single peak at the retention time of 7.89 min in the mass spectrum, and the peak area at this location accounts for more than 99.99% of the total peak area of ​​all substances in the ion current diagram. Therefore, it can be judged that the prepared degradation product has excellent purity. Figure 4 B is the primary mass spectrum of the prepared degradation product. It can be seen that the substance is expressed as [M–H] –The ion peak of 2-hydroxy-1-nitropropene was present, and the mass-to-charge ratio was 317.1422, so its molecular mass was 318.1422, which was consistent with cis-ZEN. It can be preliminarily determined that the substance is cis-ZEN.

[0113] The chromatograms were then obtained using a Bruker 600 spectrometer ( 1 H, 600MHz; 13 C, 125MHz) to measure the 13 C NMR, 1 HNMR, 1 H- 1 The molecular structure of the prepared degradation product was further determined by H COSY, HSQC, HMBC and ROESY spectra. 13 C NMR and 1 HNMR, compare the similarities and differences between trans-ZEN and the prepared degradation products in the primary spectrum. Table 2 shows the carbon spectrum and hydrogen spectrum data of trans-ZEN and the prepared degradation products. The carbon spectrum and hydrogen spectrum data of both are clear and have no impurity peaks, indicating that both are of high purity. In addition, the chemical shifts of carbon atoms and hydrogen atoms and the coupling constants of hydrogen atoms in the carbon spectrum and hydrogen spectrum data can preliminarily indicate the distribution of carbon and hydrogen in the molecular structure of the substance.

[0114] The results in Table 2 show that the chemical shifts of carbon at positions 10 and 11 in the prepared degradation products are shifted to high fields compared with trans-ZEN, from 5.80ppm and 6.60ppm in trans-ZEN to 5.31ppm and 6.50ppm. In addition, the coupling constant generated by the coupling effect of the hydrogen connected to the carbon at position 11 is reduced from 15.5Hz to 11.4Hz by the hydrogen at the carbon at position 10, which is consistent with the coupling constant law of trans- and cis-related hydrogen in olefins.

[0115] Figure 5 A in the figure is the two-dimensional NMR spectrum of the prepared degradation product 1 H- 1 H COSY, HSQC, HMBC and ROESY spectral analysis results. The two-dimensional NMR spectrum results can further clarify the direct and indirect connection relationship between carbon and hydrogen in the molecule and depict a clear product molecular structure. The molecular structure of the prepared degradation product is the same as that of cis-ZEN, and the molecular formula is C 18 H 22O5. Compared with trans-ZEN, its molecular structure is different in the distribution of hydrogen atoms on both sides of the double bond at positions 10 and 11. This result is consistent with the change in the coupling constant of hydrogen atoms at this position in Table 2. Comprehensive analysis of the results can determine that the prepared degradation product is cis-ZEN, which is generated by the cis-trans isomerization reaction of trans-ZEN under the combined action of ultraviolet and plasma. The process of trans-ZEN changing to cis-ZEN is as follows Figure 5 As shown in B.

[0116] Table 2 Trans-ZEN and the prepared products 1 H-NMR and 13 C-NMR spectral data

[0117]

[0118]

[0119] In summary: when ultraviolet and plasma act on trans-ZEN simultaneously, the degradation rate of trans-ZEN and the yield of cis-ZEN generated are significantly higher than the effects achieved by ultraviolet or plasma acting alone. The present invention uses ultraviolet and plasma combined technology to quickly convert trans-ZEN into cis-ZEN, greatly shortening the time for product preparation, reducing the probability of impurity generation in the system, reducing the difficulty of purification, and finally obtaining a standard product with a purity of more than 99.99%. This method is easy to use, has no secondary pollution, and has the potential for application in actual production.

[0120] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the embodiments shown and described herein.

Claims

1. A device for converting trans-ZEN to cis-ZEN by combining ultraviolet and plasma, characterized in that: include: A box body, which is provided with an openable door; A plasma generating device is installed in the box and located on the top of the box; An ultraviolet light source is installed in the box and is disposed below the plasma generating device and is spaced apart from the plasma generating device; Two sets of fans, which are respectively installed on both sides of the box and arranged close to the top of the box, for dissipating heat from the ultraviolet light source; A multi-layer support frame is installed in the box and arranged below the ultraviolet light source with a gap between the multi-layer support frame and the ultraviolet light source; Wherein, the multi-layer support frames are arranged at intervals along the height direction of the box body; The sample plate can be selectively arranged on different support frames for placing the trans-ZEN sample.

2. The device for converting trans-ZEN to cis-ZEN by combining ultraviolet and plasma according to claim 1, characterized in that: The plasma generating device adopts a high-voltage pulse orifice plate.

3. The device for converting trans-ZEN to cis-ZEN by combining ultraviolet and plasma according to claim 2, characterized in that: The ultraviolet light source is one or more of UVA and UVC.

4. The device for converting trans-ZEN to cis-ZEN by combining ultraviolet and plasma according to claim 3, characterized in that: The ultraviolet light source is composed of a plurality of ultraviolet light strips evenly spaced apart; Each of the ultraviolet light strips comprises: a first light source module and two second light source modules; the first light source module and the second light source module are both arranged along the length direction of the ultraviolet light strip, and the first light source module is located between the two second light source modules; The ultraviolet light wavelength of the first light source module is 365 nm, and the ultraviolet light wavelength of the second light source module is 275 nm.

5. A method for preparing cis-ZEN by converting trans-ZEN by combining ultraviolet and plasma, using the device for converting trans-ZEN by combining ultraviolet and plasma to prepare cis-ZEN as claimed in any one of claims 1 to 4, characterized in that: The steps include: Step 1: Dissolve the trans-ZEN standard in acetonitrile to prepare a trans-ZEN stock solution; Step 2: Place the trans-ZEN stock solution on a plate and evaporate it naturally in a fume hood to obtain a trans-ZEN sample spread on the bottom of the plate; Step 3: Place the plate containing the trans-ZEN sample on the sample plate; Step 4: Turn on the plasma generator and the ultraviolet light source, so that the plasma wind and ultraviolet light act on the trans-ZEN sample simultaneously until the set processing time is reached; Among them, during the trans-ZEN sample processing, the chamber door remained closed; Step 5: After collecting the substrate and product after trans-ZEN treatment using a solid phase extraction column, the product is separated by ultra-high performance liquid chromatography to obtain cis-ZEN.

6. The method for preparing cis-ZEN by converting trans-ZEN by combining ultraviolet and plasma according to claim 5, characterized in that: The concentration of trans-ZEN stock solution was 100 μg / mL.

7. The method for preparing cis-ZEN by converting trans-ZEN by combining ultraviolet and plasma according to claim 6, characterized in that: The liquid level of trans-ZEN stock solution in the dish should not exceed 0.5 mm.

8. The method for preparing cis-ZEN by converting trans-ZEN by combining ultraviolet and plasma according to claim 7, characterized in that: The distance between the UV light source and the trans-ZEN sample was 5cm-10cm.

9. The method for preparing cis-ZEN by converting trans-ZEN by combining ultraviolet and plasma according to claim 8, characterized in that: In step 4, the processing time is set to 5 min-10 min.

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