Extraction method of active substance of Ulidipine and application of active substance in anti-tumor drugs

Abstract

The invention discloses an extraction method of an active substance of Uligang and application of the active substance in anti-tumor drugs, and relates to the technical field of tumor treatment, dried Uligang is put into a smashing device to be smashed, and Uligang particles can pass through a 18-mesh sieve; putting the crushed Wuligang in a beaker, adding distilled water according to a material-liquid ratio of 1: 8, and then adding n-hexane to form a mixed solution; distilling the mixed solution, and layering the mixed solution after distillation to obtain a water layer and an n-hexane layer; adding analytically pure solid sodium chloride into a water layer of the mixed solution, extracting the saturated water layer by using diethyl ether, and drying after the extraction is completed; according to the present invention, the active substance phenylacetaldehyde is extracted from Urindigesting, and the phenylacetaldehyde can inhibit the energy metabolism pathway of tumor cells, such that the active substance phenylacetaldehyde is extracted from Urindigesting, such that the activity of the Urindigesting is improved, and the activity of the Urindigesting is improved.

Description

Technical Field

[0001] The present application relates to the technical field of tumor treatment, and in particular to a method for extracting the active substance of Ulidige and its application in anti-tumor drugs. Background Art

[0002] Malignant tumors, like a dangerous ticking time bomb, remain a core challenge posing a serious threat to human health and life. With the rapid advancement of modern medicine, the field of anti-cancer drug research has undergone tremendous changes, moving from the previous, extensive approach relying on cytotoxic chemotherapy to a new era of precision, personalized treatment. However, the road to anti-cancer treatment is not always smooth. Tumor heterogeneity, a major challenge, results in significant variations in gene and protein expression in tumor cells from patient to patient, significantly increasing the complexity of treatment. Against this backdrop, Traditional Chinese Medicine (TCM) has reemerged at the center of global anti-cancer research with its unique appeal. Derived from nature, TCM boasts the significant advantage of being sourced from natural sources, boasting a rich and diverse composition that retains its natural biological activity. Compared to chemical drugs, TCM is more readily absorbed and metabolized by the body, preventing long-term residual activity and thus avoiding toxic reactions caused by drug accumulation. Furthermore, its multi-component, multi-target synergistic mechanism reduces the risk of tumor cell resistance.

[0003] However, although Chinese herbal medicines have enormous therapeutic potential, how to identify and efficiently extract effective active substances from them has become a key technical problem that needs to be overcome urgently by technical personnel in this field; not only that, in the process of utilizing the active substances of Ulitia ulmoides, the raw materials usually contain impurities (such as solid debris, crude salt solution), and there is a certain amount of rust and suspended matter in the equipment pipelines, resulting in poor subsequent extraction accuracy. How to overcome the technical bottlenecks of extraction efficiency and accuracy in the preparation process is also an important issue that needs to be solved urgently. Summary of the Invention

[0004] This device provides a method for extracting the active substance of Ulidige and its application in anti-tumor drugs. The specific implementation method is as follows:

[0005] This device provides a method for extracting the active substance of Ulidige, wherein the extraction method uses an extraction device, which includes:

[0006] A tank body for containing the mixed liquid, which is divided into an upper part and a lower part;

[0007] An ultrasonic generator is located at the bottom of the tank;

[0008] A sieve drum is built into the upper part of the tank, and the mixed liquid and solid sodium chloride are introduced from the inside of the sieve drum. A stirring component is also provided inside the sieve drum to accelerate the extraction.

[0009] An infrared spectrometer and a reflective plate are horizontally arranged at the lower part of the tank body. The reflective plate is coaxially arranged with the stirring assembly through a pull rod, and the reflective plate axially passes through the screen cylinder.

[0010] Preferably, the extraction device also includes a cover plate arranged on the top of the tank body, an isolation valve and a drive assembly are provided on the outside of the cover plate, the isolation valve and the stirring assembly are arranged coaxially, and the drive assembly is transmission-connected to the stirring assembly; the stirring assembly includes a fixed ring sleeve and a rotating ring sleeve that are integrally mounted, the rotating ring sleeve is provided with an impeller, and the rotating ring sleeve is connected to the output end of the drive assembly.

[0011] Based on the above technical solution, the extraction efficiency can be effectively improved by setting a sieve drum to block impurities before extraction, and with the help of the stirring assembly in the sieve drum and the ultrasonic generator at the bottom of the tank; the infrared spectrometer and the reflector at the bottom of the tank can be used to control the timing of the completion of the extraction; when the extraction is not completed, there are incompletely dissolved solutes, impurities or stratification in the material, resulting in uneven material composition at this height. At this time, when infrared light is irradiated on the material, different components have different absorption and reflection characteristics of light, which will produce unstable spectral signals; when the extraction is completed, the target component has been fully transferred from the raw material to the solvent, and the material at this height reaches a uniformly mixed state. After the infrared light is irradiated on the material, the spectral signal received by the reflector will tend to be stable and single, indicating that the material composition is uniform and the extraction reaction has ended.

[0012] Preferably, the top end of the isolation valve is connected to a receiving kit for a pull-out rod, and the pull-out rod is axially slidably connected to the fixed ring sleeve, the isolation valve and the receiving kit in sequence, and its end is passed through the receiving kit. The pull-out rod is divided into a hollow section and a solid section along its axial direction, and an unlocking button is built into the hollow section; a plurality of annular limit grooves are provided on the inner side of the fixed ring sleeve along its axial direction, and a conducting port is opened laterally at the bottom of the hollow section. The unlocking button is released so that its end passes through the conducting port through the unlocking structure, and is clamped to act on any annular limit groove to lock the installation height of the reflector plate.

[0013] Preferably, a two-section lifting structure is provided between the pulling rod and the fixed ring sleeve, and the second section of the lifting displacement is unlocked after the pulling rod is rotated; the solid section is provided with an angle guide groove along its axial direction, and the inner side of the fixed ring sleeve is provided with a protrusion block that is slidably connected to the angle guide groove; a brush is provided on the inner side of the bottom end of the rotating ring sleeve along its circumference, and the brush self-cleans the reflective plate after the pulling rod completes the first section of the lifting displacement.

[0014] Based on the above technical solution, by setting up a accommodating kit, an isolation valve and an axially movable pull-out rod, if the infrared spectrometer detects a weak reflection signal during a single extraction process, the pulling action of the pull-out rod can be used to self-clean or replace the reflection plate in sequence, effectively reducing the problem of decreased extraction accuracy caused by contamination of the reflection plate.

[0015] Preferably, the accommodating kit is sequentially provided with a hollow sleeve body and a threaded seat body along its axial direction, and the threaded seat body and the isolation valve are connected via a vertical plate with a lateral gap, the lateral gap being used for replacement of the reflector plate after removal, and an isolation sleeve for closing the lateral gap being threadedly connected to the outside of the threaded seat body, and an interlocking structure is provided between the isolation sleeve and the isolation valve;

[0016] Preferably, the isolation valve includes a valve body and a radially arranged valve, a flap is provided on the side of the valve body for rotation, and a reset torsion spring is provided at the center of rotation, the valve is integrally provided with a synchronization ring with a notch, the top of one end of the flap is in abutment with the abutment ring body on the outside of the isolation sleeve, and when the isolation sleeve is screwed open, one end of the flap is vertically engaged with the notch.

[0017] Preferably, the interlocking structure also includes a cylindrical interlocking part that is covered on the valve. The interlocking part and the valve are integrated. The abutting ring body is provided with a limiting protrusion along its tangent direction. The open end of the interlocking part is provided with an arc-shaped locking block. When the isolation valve is in the open state, the limiting protrusion abuts against the limiting protrusion laterally, and the isolation sleeve is locked.

[0018] Based on the above technical solution, by linking and interlocking the isolation sleeve, interlocking parts and isolation valve, it is ensured that the valve body is in a closed state when the isolation sleeve moves upward, and it is also avoided that the valve body is accidentally opened after the isolation sleeve moves upward, thereby reducing the overall contact time between the active extract in the tank and the external environment.

[0019] In summary, this application has the following beneficial technical effects:

[0020] 1. The present invention extracts the active substance phenylacetaldehyde from Ulidige. Phenylacetaldehyde can inhibit the energy metabolism pathway of tumor cells, reduce the energy supply of tumor cells, and thus regulate the metabolic state of tumor cells to a certain extent;

[0021] 2. This invention uses a sieve drum to block impurities before extraction. The stirring assembly inside the sieve drum and the ultrasonic generator at the bottom of the tank can improve the extraction efficiency. Furthermore, the infrared spectrometer and reflective plate at the bottom of the tank can accurately control the timing of extraction completion.

[0022] 3. By providing a housing kit, an isolation valve, and an axially movable pull rod, the present invention allows for self-cleaning or replacement of the reflector plate by pulling the pull rod when a single extraction mid-infrared spectrometer fails, thereby reducing the problem of reduced extraction accuracy due to reflector plate contamination.

[0023] 4. The present invention has a simple structure. By interlocking the isolation sleeve, the interlocking member and the isolation valve, it is ensured that the valve body is in a closed state when the isolation sleeve moves upward, and it is also ensured that the valve body is not accidentally opened after the isolation sleeve moves upward, thereby reducing the contact time between the active extract in the tank body and the external environment as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the LC-MS identification spectrum of the methanol phase and the ethanol phase of the flower part of Ulitiga uler;

[0025] Figure 2 This is the enriched substance diagram of the methanol phase and ethanol phase of the flower part of Ulitiga ulerata identified by mass spectrometry;

[0026] Figure 3 This is a diagram showing the results of plate cloning of the human laryngeal cancer cell line in the present invention;

[0027] Figure 4 This is a diagram showing the invasion and metastasis results of the human nasopharyngeal carcinoma cell line of the present invention;

[0028] Figure 5 This is a control experiment diagram of different tumors in the present invention;

[0029] Figure 6 This is a comparison chart of the results of the seahorse experiment in the present invention;

[0030] Figure 7 This is a graph showing the effect of phenylacetaldehyde treatment on U87MG in the present invention;

[0031] Figure 8 This is a graph showing the effect of U251 under phenylacetaldehyde treatment in the present invention;

[0032] Figure 9 This is the analysis result diagram of differentially expressed genes mediated by phenylacetaldehyde in VENN and KEGG;

[0033] Figure 10 This is a diagram showing the effect of phenylacetaldehyde on brain glioma cells in the present invention;

[0034] Figure 11 This is a diagram showing the effect of phenylacetaldehyde mediating mitochondrial metabolism in the present invention in inhibiting brain glioma;

[0035] Figure 12 This is a diagram showing the effect of phenylacetaldehyde on ATP formation in brain glioma cells in the present invention;

[0036] Figure 13 It is a schematic structural diagram of the extraction device of the present invention;

[0037] Figure 14 It is an enlarged view of a part of the structure of the extraction device of the present invention;

[0038] Figure 15 is a cross-sectional view of the extraction device structure of the present invention;

[0039] Figure 16 This invention Figure 15 A magnified view of the middle part of the structure;

[0040] Figure 17 This is a schematic diagram of the structure of the screen drum in the present invention Figure 1 ;

[0041] Figure 18 This is a schematic diagram of the structure of the screen drum in the present invention Figure 2 ;

[0042] Figure 19 This invention Figure 17 Schematic diagram of explosion structure in ;

[0043] Figure 20 is a cross-sectional view of the structure of the unlocking button and stirring component in the present invention;

[0044] Figure 21 This is a schematic diagram of the exploded structure of the unlocking button and stirring component in the present invention. Figure 1 ;

[0045] Figure 22 This is a schematic diagram of the exploded structure of the unlocking button and stirring component in the present invention. Figure 2 ;

[0046] Figure 23 is a cross-sectional view of the unlocking button and stirring assembly structure of the present invention;

[0047] Figure 24 This is a side view of the structure of the housing kit and the reflector in the present invention. Figure 1 ;

[0048] Figure 25 It is a right side structural schematic diagram of the accommodation kit and the reflective plate in the present invention;

[0049] Figure 26 This is a side view of the structure of the housing kit and the reflector in the present invention. Figure 2 ;

[0050] Figure 27 It is a schematic diagram of the explosion structure of the accommodation kit and the isolation sleeve in the present invention;

[0051] Figure 28 It is a schematic diagram of the exploded structure of the housing assembly and the unlocking button cross section of the present invention;

[0052] Figure 29 It is a structural diagram of the unlock button in the present invention;

[0053] Figure 30 It is a cross-sectional view of the pull-out rod and unlock button structure of the present invention;

[0054] Figure 31 It is a structural schematic diagram of the interlocking parts in the closed state of the isolation valve of the present invention;

[0055] Figure 32It is a structural schematic diagram of the interlocking parts in the open state of the isolation valve in the present invention.

[0056] Description of reference numerals:

[0057] 1. Tank, 2. Drive assembly, 3. Isolation valve, 4. Accommodation kit, 5. Pull rod, 6. Isolation sleeve, 7. Interlocking piece, 8. Unlock button, 9. Stirring assembly, 10. Infrared spectrometer, 11. Sieve drum, 12. Reflector, 13. Ultrasonic generator, 14. Neck retaining ring, 15. Mounting plate, 16. Cover plate,

[0058] 101, discharge hole, 201, motor, 202, gear, 301, valve body, 302, valve, 303, synchronization ring, 304, flip plate, 305, positioning hoop, 306, reset torsion spring, 401, sleeve, 402, threaded seat, 403, vertical plate, 501, hollow section, 502, solid section, 503, angle guide groove, 504, guide port, 601, abutting ring body, 602, limiting protrusion, 701, arc locking block, 801, movable rod, 802, wedge-shaped mounting block, 803, wedge-shaped slider, 804, spring, 805, pressing block, 901, fixed ring sleeve, 902, rotating ring sleeve, 903, impeller, 904, brush, 905, annular limiting groove, 906, protrusion block,

[0059] 3031, notch, 8021, latch. DETAILED DESCRIPTION

[0060] The specific implementation of the present invention is described below with reference to the accompanying drawings and embodiments:

[0061] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the conditions under which the present invention can be implemented. Any structural modification, change in proportional relationship or adjustment of size should still fall within the scope of the technical content disclosed in the present invention without affecting the efficacy and purpose that can be achieved by the present invention.

[0062] At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description and are not used to limit the scope of implementation of the present invention. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of the present invention without substantially changing the technical content.

[0063] The following is combined with Figure 1-32 This application is described in further detail.

[0064] The examples of the present application disclose a method for extracting the active substance of Ulidige and its application in anti-tumor drugs.

[0065] Example 1

[0066] Reference Figures 1 to 16 This embodiment discloses a method for extracting the active substance of Ulidige, which specifically comprises the following steps:

[0067] Step 100: Place the dried Ulidige in a grinding device for grinding, so that the Ulidige particles can pass through an 18-mesh sieve;

[0068] Step 200: Put the crushed ulitige into a beaker, add distilled water at a material-liquid ratio of 1:8, and then add n-hexane to form a mixed solution;

[0069] Step 300: Distill the mixed solution and keep it in a slightly boiling state for 6 hours;

[0070] Step 400: After the distillation is completed, the mixed solution is allowed to stand, and the mixed solution is separated into a water layer and a n-hexane layer;

[0071] Step 500: adding analytically pure solid sodium chloride to the aqueous layer of the mixed solution until the sodium chloride reaches saturation;

[0072] Step 600: extracting the saturated aqueous layer with ether, and repeating the extraction operation three times;

[0073] Step 700: After the extraction is completed, the ether extract is dried using anhydrous sodium sulfate;

[0074] Step 800: After drying, filter to obtain a filtrate, and combine the water layer and the n-butanol layer.

[0075] Step 900: Perform chromatographic analysis on the filtrate to extract the target component phenylacetaldehyde.

[0076] When performing chromatographic analysis in step 900 in this structure, a DB-1 type chromatographic column is selected and three heating temperatures are set. The first heating temperature is a starting temperature of 40°C, which is maintained for 1 minute; the second heating temperature is heated to 100°C at a rate of 5°C / min and maintained for 5 minutes; and the third heating temperature is heated to 210°C at a rate of 5°C / min and maintained for 10 minutes.

[0077] The carrier gas in the DB-1 type chromatographic column is high-purity He, the carrier gas flow rate is set to 1 ml / min, the injection port temperature is controlled at 200°C, the injection volume is 1 μL, the solvent delay time is set to 4 min, and in terms of mass spectrometry detection, an EI source is used, the electron energy is 70 eV, the ion source temperature is 230°C, and the mass scanning range is set to 40-800u.

[0078] Example 2

[0079] Reference Figures 13 to 19 Based on the above embodiment, this embodiment also discloses an extraction device used in the extraction method of the active substance of Ulidige, which includes a tank body 1 for containing a mixed liquid, an ultrasonic generator 13 provided at the bottom of the tank body 1, and a cover plate 16 provided at the top of the tank body 1. The tank body 1 is divided into an upper part and a lower part. The upper part of the tank body 1 is equipped with a sieve drum 11, and the mixed liquid and solid sodium chloride are both introduced from the inside of the sieve drum 11. The sieve drum 11 is provided with a stirring component 9 for accelerating the extraction. The lower part of the tank body 1 is provided with an infrared light source. The spectrometer 10 and the reflector 12 are coaxially arranged with the stirring assembly 9 through the pulling rod 5, and the reflector 12 axially passes through the sieve cylinder 11. The outside of the sieve cylinder 11 and the top of the tank body 1 are the upper layer of the extracted liquid, and the outside of the sieve cylinder 11 and the bottom of the tank body 1 are the lower layer of liquid, which are discharged to the outside through the discharge hole 101; the infrared spectrometer 10 adopts existing conventional devices. The infrared spectrometer can indirectly detect the extraction effect. Its core principle is to use the infrared characteristic absorption peak of the substance to judge the concentration change or separation degree of the target component.

[0080] An isolation valve 3 and a drive assembly 2 are provided on the outside of the cover plate 16. The isolation valve 3 is coaxially arranged with the stirring assembly 9, and the drive assembly 2 is transmission-connected to the stirring assembly 9. The stirring assembly 9 includes a fixed ring sleeve 901 and a rotating ring sleeve 902 which are integrally mounted. The rotating ring sleeve 902 is provided with an impeller 903, and the rotating ring sleeve 902 is connected to the output end of the drive assembly 2. In this structure, a mounting plate 15 is provided above the cover plate 16, and the drive assembly 2 is sandwiched between the two. The isolation valve 3 is provided above the mounting plate 15. The drive assembly 2 includes a motor 201, and its output end is engaged with the rotating ring sleeve 902 of the stirring assembly 9 through a gear 202.

[0081] The specific extraction process is as follows: the mixed liquid and solid sodium chloride are both introduced from the inside of the sieve cylinder 11, and the ultrasonic generator 13 and the stirring assembly 9 are started to accelerate the extraction; the sieve cylinder 11 is used to block impurities, and the extracted liquid is vertically layered outside the sieve cylinder 11; during this period, the infrared spectrometer 10 uses the light reflection of the reflective plate 12 to detect the extracted single layer of material, and then judge the extraction effect, which greatly improves the success rate of single-side closed extraction.

[0082] Example 3

[0083] Reference Figures 13 to 24Based on the above embodiment, this embodiment also discloses an extraction device used in the extraction method of the active substance of Ulitiga. The top of the isolation valve 3 is connected to the accommodating sleeve 4 of the pull-out rod 5. The pull-out rod 5 is axially slidably connected to the fixed ring sleeve 901, the isolation valve 3 and the accommodating sleeve 4 in sequence, and its end passes through the accommodating sleeve 4. The pull-out rod 5 is divided into a hollow section 501 and a solid section 502 along its axial direction, and the hollow section 501 is equipped with an unlocking button 8. The inner side of the fixed ring sleeve 901 is provided with a plurality of annular limiting rings along its axial direction. The bottom of the hollow section 501 is provided with a conducting port 504. The unlocking button 8 is released to allow its end to pass through the conducting port 504 through the unlocking structure, and is engaged with any annular limiting groove 905 to lock the installation height of the reflector 12. In this structure, a membrane-type sealing structure can be provided at the end of the fixed ring sleeve 901. After the reflector 12 is pulled out, the membrane-type sealing component can prevent liquid from entering the fixed ring sleeve 901 as much as possible, and a sealing ring is also provided between the pulling rod 5 and the fixed ring sleeve 901.

[0084] A two-stage lifting structure is provided between the pulling rod 5 and the fixed ring sleeve 901. After rotating the pulling rod 5, the second stage of the lifting displacement is unlocked. The solid section 502 is provided with an angled guide groove 503 along its axial direction, and the inner side of the fixed ring sleeve 901 is provided with a protruding block 906 that is slidably connected to the angled guide groove 503. A brush 904 is provided on the inner side of the bottom end of the rotating ring sleeve 902 along its circumference. The brush 904 self-cleans the reflective plate 12 after the pulling rod 5 completes the first stage of the lifting displacement. In this structure, through the two-stage lifting structure, the first lifting after pressing the unlocking button 8 is the self-cleaning position of the reflective plate 12. After rotating the pulling rod 5, the second lifting after pressing the unlocking button 8 is the reflective plate 12 reaching the replacement position.

[0085] During a single extraction process, if the reflection signal of the infrared spectrometer 10 is not good, it may be that there is a lot of dirt attached to the reflective plate 12 and it needs to be cleaned; preliminary cleaning can be done with the help of the rotating brush 904 in the stirring assembly 9; press the unlock button 8, pull the pull rod 5 for the first time, so that the stepped layer of the angle guide groove 503 hits the raised block 906, and the collision proves that the reflective plate 12 has moved into place; after the self-cleaning is completed, push down the pull rod 5 to make the reflective plate 12 return to its position to detect the extraction effect of light reflection.

[0086] Example 4

[0087] Reference Figures 28 to 30 Based on the above embodiments, this embodiment also discloses an extraction device used in the extraction method of the active substance of Ulitiga. The top of the accommodating sleeve 4 is threaded with a neck retaining ring 14, and the pulling rod 5 is axially isodiametric, which can maintain the stability of the movement of the pulling rod 5 during the axial pulling process.

[0088] The unlocking button 8 includes a pair of movable rods 801 arranged in the hollow section 501, a spring 804 is provided between the two movable rods 801, and a wedge-shaped mounting block 802 and a pressing block 805 are provided at both ends of the movable rod 801. The three form a C-shaped structure, and a wedge-shaped slider 803 is radially slidably provided at the conducting opening 504. The wedge slider 803 and the wedge-shaped mounting block 802 are abutted by an inclined surface. In this structure, the bottom of the wedge-shaped mounting block 802 slides against the bottom of the conducting opening 504, and the side of the wedge-shaped mounting block 802 is provided with a latch that is locked in the annular limiting groove 905. 8021, when the wedge-shaped slider 803 is in the pushed-out state, its overall diameter is larger than the caliber of the necking retaining ring 14. After the pressing block 805 is pressed, the wedge-shaped mounting block 802 is retracted and the wedge-shaped slider 803 is pushed out, so that the wedge-shaped mounting block 802 is only used for position locking. The top end of the wedge-shaped slider 803 is used for the impact of being pulled to the extreme position, which can effectively improve the durability of the wedge-shaped mounting block 802. The collision feeling between the wedge-shaped slider 803 and the necking retaining ring 14 can remind the reflector 12 that it has moved to the position to be replaced, that is, at the same height as the isolation sleeve 6.

[0089] If the reflection signal of the infrared spectrometer 10 is still poor, the reflection plate 12 needs to be replaced; press the unlock button 8, pull the pull rod 5 for the first time, so that the step layer of the angle guide groove 503 hits the protruding block 906; then rotate the pull rod 5, so that the pull rod 5 is pulled a second time after pressing the unlock button 8; during the pulling process, the wedge-shaped mounting block 802 is retracted, the pin 8021 is released from the annular limit groove 905, and the wedge-shaped slider 803 is pushed out radially; after the end of the wedge slider 803 hits the neck retaining ring 14, it proves that the reflection plate 12 has reached the replacement position; after closing the isolation valve 3, replace the reflection plate 12; after the replacement is completed, it is reinserted after dust removal to detect the signal of the infrared spectrometer 10.

[0090] Example 5

[0091] Reference Figures 24 to 32 Based on the above embodiments, this embodiment also discloses an extraction device used in the extraction method of the active substance of Ulitig, wherein the accommodating kit 4 is sequentially provided with a hollow sleeve body 401 and a threaded seat body 402 along its axial direction, and the threaded seat body 402 is connected to the isolation valve 3 through a vertical plate 403 with a lateral gap, and the lateral gap is used for replacing the reflective plate 12 after disassembly. The external threaded seat body 402 is threaded with an isolation sleeve 6 for sealing the lateral gap, and an interlocking structure is provided between the isolation sleeve 6 and the isolation valve 3. This structure can ensure that the isolation valve 3 is opened during the process of moving the isolation sleeve 6 upward and replacing the reflective plate 12, and can also ensure that before the isolation sleeve 6 is moved upward and opened, the pull rod 5 needs to be pulled out of the isolation valve 3 and the isolation valve 3 is in a closed state, thereby improving the independence of the mixed liquid inside the tank body 1.

[0092] The interlocking structure includes a cylindrical interlocking member 7 which is covered on the valve 302. The isolation valve 3 includes a valve body 301 and a radially arranged valve 302. A flip plate 304 is provided on the side of the valve body 301 for rotation, and a return torsion spring 306 is provided at the center of rotation. The valve 302 is integrally provided with a synchronization ring 303 with a notch 3031. The top of one end of the flip plate 304 abuts against the abutting ring body 601 on the outside of the isolation sleeve 6. When the isolation sleeve 6 is screwed open, one end of the flip plate 304 is vertically engaged with the notch 3031. The interlocking member 7 is integrally provided with the valve 302. The abutting ring body 601 is provided with a limiting protrusion 602 along its tangent direction. The open end of the interlocking member 7 is provided with an arc locking block 701. When the isolation valve 3 is open, the limiting protrusion 602 abuts against the limiting protrusion 602 laterally, and the isolation sleeve 6 is locked.

[0093] During the process of pulling up and replacing the reflective plate 12, the specific implementation process is: the reflective plate 12 reaches the replacement position and the valve 302 is closed; the arc locking block 701 rotates synchronously with the valve 302, so that the limiting protrusion 602 is released; the isolation sleeve 6 is rotated, and the isolation sleeve 6 moves up relative to the vertical plate 403, and the reflective plate 12 can be replaced laterally; after the isolation sleeve 6 moves up, the flip plate 304 and the abutment ring body 601 lose the abutment effect, and the flip plate 304 is stuck in the notch 3031 of the synchronous ring 303, so that the valve 302 is locked when the isolation sleeve 6 does not fall.

[0094] Example 6

[0095] Reference Figures 1 to 12 Based on the above embodiments, this embodiment also discloses the application of the active substance of Ulidiger in anti-tumor drugs, which applies the method for extracting the active substance of Ulidiger. The active substance of Ulidiger is phenylacetaldehyde, and phenylacetaldehyde is used in anti-glioma drugs.

[0096] Example 7

[0097] Reference Figures 1 to 12 Based on the above embodiment, this embodiment also discloses a method for verifying the effect of the active substance Ulidige in anti-tumor drugs. Figure 1 By performing mass spectrometry and chromatography on the methanol and ethanol phases of the effective anticancer fractions of Prunus mume, we identified the highly abundant compound phenylacetaldehyde, which we will further investigate. Initial cell experiments have led us to conclude that the natural compounds phenylacetaldehyde and tetradecanol have potential anti-tumor effects.

[0098] based on Figure 2Existing technologies consider the principle of non-toxicity to humans and the exploration of natural antibacterial and anticancer drugs. Phenylacetaldehyde is a colorless liquid extracted from various species, including roses, bagpipes, Aleppo pines, and orange blossoms. It has a pleasant odor and is also an autologous antibiotic produced by Candida albicans. It can be used as a preservative in artificial fragrances, soaps, and as a cigarette additive. It has potential applications as an antibacterial, antiviral, and natural antibiotic. However, the compound's role in tumor prevention and treatment is supported by only two publications, and its mechanism of action remains unclear. Therefore, the following method was used to verify its mechanism.

[0099] To further investigate the tumor-suppressing effects of phenylacetaldehyde, we applied it to two glioma cell lines (U87MG and U251). We first verified its effect on glioma cell viability using the MTT cell viability assay (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The experimental principle is that succinate dehydrogenase, present in the mitochondria of living cells, can be reduced by MTT molecules to form water-insoluble, blue-purple crystalline formazan, which then deposits within the cells. Dead cells lack this enzyme activity and are therefore unable to undergo this reduction reaction. By adding dimethyl sulfoxide (DMSO) to dissolve the intracellular formazan crystals, the absorbance (OD) at a wavelength of 490 nm was measured, providing an indirect indicator of viable cell count. Within a certain cell population range, the amount of formazan crystals formed is proportional to the number of viable cells. The experimental method involves plating 5,000 U87MG / U251 cells in the logarithmic growth phase in a 96-well plate (100 μl volume) per well. After overnight adherence, the cells are treated with the drug at varying concentrations and durations. After the target treatment time, the prepared MTT solution (6 μl per well) is added and the cells are incubated in a cell culture incubator for another 4 hours. The cell culture medium is then aspirated, and 100 μl of DMSO is added to each well. The absorbance is measured at 490 nm using a microplate reader.

[0100] To investigate the effect of phenylacetaldehyde on the ability of tumor single-cell clone formation, a plate cloning assay was performed. The experimental method was as follows: logarithmically growing cells were collected and counted, and 1000 cells (2 ml of culture medium) were plated per well of a 6-well plate. After the cells adhered (approximately 1 day later), the drug was added (by changing the medium) for 10-14 days, and the clone formation was observed. The cell culture medium was discarded, and paraformaldehyde was added for fixation for 15 minutes, followed by crystal violet staining for 15 minutes. The excess crystal violet stain was washed off, and the cells were allowed to dry. The size and number of clones formed were observed.

[0101] Combine Figure 3A represents the colony formation graph of the blank control group, B represents the colony formation graph of the phenylacetaldehyde-treated group, C represents the microscopic image of the single clones of the blank control group, D represents the microscopic image of the single clones of the phenylacetaldehyde-treated group, and E represents the histogram of the colony number of the blank control on the phenylacetaldehyde plate. According to the results of the plate cloning experiment, it can be found that phenylacetaldehyde has a significant inhibitory effect on the formation of tumor cell colonies and has a significant impact on the proliferation of tumor cells. It can effectively reduce the number of colonies and inhibit the increase of colony size. This shows that phenylacetaldehyde has potential application value in inhibiting tumor cell growth and provides a strong experimental basis for further research on its anti-tumor mechanism and the development of new anti-cancer drugs.

[0102] To investigate the effects of phenylacetaldehyde on the invasion and migration of tumor cells, the researchers collected logarithmically growing cells, counted them, and plated 10-50w cells per well in the upper chamber of a transwell plate. Different treatment factors were then added to the lower chamber. The cells were divided into a control group (cultured in DMEM high-glucose medium) and a phenylacetaldehyde-treated group (treated with 2μM for 48 hours).

[0103] Combine Figure 4 The left side of the figure shows the results of the transwell experiment under the microscope; the right side of the figure shows the bar chart of the transwell experiment counting results. The experimental results show that by comparing the invasion and metastasis capabilities of different cells after treatment with phenylacetaldehyde, the research results show that the invasion and metastasis capabilities of cells treated with phenylacetaldehyde were significantly reduced, which suggests that phenylacetaldehyde has a potential inhibitory effect on the invasion and metastasis process of tumor cells, which may be achieved by interfering with the migration-related mechanisms of cells or affecting the biological characteristics of cells. This provides important clues for the subsequent in-depth exploration of its mechanism of action and its application prospects in tumor treatment.

[0104] To investigate the ability to inhibit cell proliferation. Since tumor cell proliferation requires a lot of energy to maintain, and ATP is the main source of cellular energy, the ability to inhibit cell proliferation was indirectly investigated by detecting changes in total ATP content after drug treatment. The experimental method was as follows: cells treated with drugs (5 μM concentration, 2 hours) and blank control cells (normal growth in DMEM high-glucose medium) were collected, washed with PBS, and operated according to the instructions of the Biyuntian ATP Enhanced Detection Kit.

[0105] Combine Figure 5After treating different tumor cells with phenylacetaldehyde, the test results showed that the total ATP content in the drug-treated tumor cells changed significantly, that is, the ATP content decreased significantly. This suggests that the drug may inhibit the energy metabolism pathway of tumor cells and reduce the energy supply of tumor cells, thereby changing the metabolic state of tumor cells to a certain extent. This is of great significance for understanding the mechanism of drug effects on tumor cell growth and survival, and also provides experimental basis for subsequent research on the potential application value of the drug in tumor treatment.

[0106] Further explore the effect of phenylacetaldehyde on cell metabolism. The seahorse experiment was used to explore and compare the energy metabolism of each stage to further determine its effect on brain glioma cell lines; combined with Figure 6 The experimental method is as follows: according to different treatment groups, the blank control group (DMEM high-glucose complete medium culture) and the phenylacetaldehyde drug treatment group (concentration of 5μM, 30min) were divided into groups according to the instructions of the Seahorse exclusive kit;

[0107] Combine Figure 7 Upper half and Figure 8 The upper half shows the effects of phenylacetaldehyde treatment on U87MG and U251, respectively. A represents basal respiration, B represents ATP production, C represents maximum respiration, and D represents residual respiration. n = 3, results are expressed as mean ± standard deviation, * represents P < 0.05 compared with the control group, ns represents P > 0.05 compared with the control group. Note: U87C is the blank control group (Control group); U87B is the phenylacetaldehyde-treated group;

[0108] Combine Figure 7 Lower half and Figure 8 The lower panel shows the effects of phenylacetaldehyde treatment on U87MG and U251, respectively. A represents the glycolytic rate; B represents the glycolytic capacity; and C represents the glycolytic reserve. n = 3. Results are expressed as mean ± standard deviation. * indicates P < 0.05 compared with the control group; ns indicates P > 0.05 compared with the control group. Note: U87C is the blank control group (Control group); U87B is the phenylacetaldehyde-treated group.

[0109] The effects of phenylacetaldehyde on the transcription of U87MG and U251 gliomas were analyzed by transcriptome and VENN. Figure 9The results of the analysis of differentially expressed genes mediated by phenylacetaldehyde in VENN and KEGG were obtained. The transcriptome data were analyzed to find the common differentially expressed genes in glioma cells after treatment with phenylacetaldehyde, and Wayne enrichment and KEGG analysis were performed. The results showed that 35 common differentially expressed genes were found in the two cell lines after phenylacetaldehyde treatment. KEGG bioinformatics analysis revealed that the common differentially expressed genes were closely related to cell aging, apoptosis, p53, MAPK, and NF-KB signaling pathways. The specific results are as follows:

[0110] Combine Figure 10 The first row, transcriptome analysis of phenylacetaldehyde mediated the oxidative phosphorylation process of brain glioma cells (consistent with the results of OCAR). Transcriptome sequencing analysis showed that phenylacetaldehyde treatment of brain glioma cells can effectively reduce the transcription level of oxidative phosphorylation-related genes; combined with Figure 10 The second row, transcriptome analysis of phenylacetaldehyde mediated the glycolysis process of brain glioma cells (consistent with the ECAR results). Phenylacetaldehyde treatment of brain glioma cells can effectively reduce the transcription levels of glycolysis-related genes; combined with Figure 10 The third row shows the effect of phenylacetaldehyde on the MAPK signaling pathway in brain glioma cells.

[0111] Phenylacetaldehyde mediates mitochondrial metabolism and participates in the inhibition of brain glioma. Figure 11 The effect of phenylacetaldehyde on cell ROS was detected by flow cytometry, and the changes in mitochondrial morphology after drug addition were observed by transmission electron microscopy. The results showed that after drug addition, intracellular ROS increased, mitochondrial morphology shrank, and the inner cristae structure disappeared, resulting in a decrease in ATP content. Figure 12 , showing the effect of phenylacetaldehyde on ATP formation in brain glioma cells. Phenylacetaldehyde significantly reduced ATP production in brain glioma cells.

[0112] Many other changes and modifications can be made without departing from the spirit and scope of the present invention. It should be understood that the present invention is not limited to the specific embodiments, and the scope of the present invention is defined by the appended claims.

Claims

1. A method for extracting active substances from Ulidige, characterized in that: The specific steps include: Step 100: Place the dried Ulidige in a grinding device for grinding, so that the Ulidige particles can pass through an 18-mesh sieve; Step 200: Put the crushed ulitige into a beaker, add distilled water at a material-liquid ratio of 1:8, and then add n-hexane to form a mixed solution; Step 300: Distill the mixed solution and keep it in a slightly boiling state for 6 hours; Step 400: After the distillation is completed, the mixed solution is allowed to stand, and the mixed solution is separated into a water layer and a n-hexane layer; Step 500: adding analytically pure solid sodium chloride to the aqueous layer of the mixed solution until the sodium chloride reaches saturation; Step 600: extracting the saturated aqueous layer with ether, and repeating the extraction operation three times; Step 700: After the extraction is completed, the ether extract is dried using anhydrous sodium sulfate; Step 800: After drying, filter to obtain a filtrate, and combine the water layer and the n-butanol layer. Step 900: Perform chromatographic analysis on the filtrate to extract the target component phenylacetaldehyde.

2. The extraction method according to claim 1, wherein When performing chromatographic analysis in step 900, a DB-1 type chromatographic column is selected and three heating temperatures are set.

3. The extraction method according to claim 2, characterized in that The first heating temperature is 40℃, which is maintained for 1 min. The second heating temperature was raised to 100°C at a rate of 5°C / min and maintained for 5 min; The third heating temperature was raised to 210° C. at a rate of 5° C. / min and maintained for 10 min.

4. The extraction method according to claim 2, characterized in that The carrier gas in the DB-1 column was high-purity He, the carrier gas flow rate was set at 1 ml / min, the inlet temperature was controlled at 200 °C, the injection volume was 1 μL, and the solvent delay time was set at 4 min; In terms of mass spectrometry detection, an EI source was used with an electron energy of 70 eV, an ion source temperature of 230 °C, and a mass scanning range set at 40-800 u.

5. The extraction method according to claim 1, wherein The extraction of the aqueous layer in step 600 utilizes an extraction apparatus comprising: A tank body (1) for containing a mixed liquid, wherein the tank body (1) is divided into an upper part and a lower part; an ultrasonic generator (13) disposed at the bottom of the tank (1); A sieve drum (11) is built into the upper portion of the tank body (1), and the mixed liquid and solid sodium chloride are introduced from the inside of the sieve drum (11). A stirring assembly (9) for accelerating extraction is provided in the sieve drum (11); An infrared spectrometer (10) and a reflective plate (12) are horizontally provided at the lower portion of the tank body (1); the reflective plate (12) is coaxially arranged with the stirring assembly (9) via a pull rod (5), and the reflective plate (12) axially passes through the sieve drum (11); The extraction device further comprises a cover plate (16) arranged on the top of the tank body (1), an isolation valve (3) and a drive assembly (2) are provided on the outside of the cover plate (16), the isolation valve (3) and the stirring assembly (9) are coaxially arranged, and the drive assembly (2) is transmission-connected to the stirring assembly (9); The stirring assembly (9) comprises a fixed ring sleeve (901) and a rotating ring sleeve (902) which are sleeved together. The rotating ring sleeve (902) is provided with an impeller (903), and the rotating ring sleeve (902) is connected to the output end of the driving assembly (2).

6. The extraction method according to claim 5, characterized in that The top end of the isolation valve (3) is connected to the accommodating sleeve (4) of the pull-out rod (5), and the pull-out rod (5) is axially slidably connected to the fixed ring sleeve (901), the isolation valve (3) and the accommodating sleeve (4) in sequence, and its end passes through the accommodating sleeve (4). The pull-out rod (5) is divided into a hollow section (501) and a solid section (502) along its axial direction, and the hollow section (501) is equipped with an unlocking button (8); The inner side of the fixing ring sleeve (901) is provided with a plurality of annular limiting grooves (905) along its axial direction, and a conducting opening (504) is provided on the side of the bottom of the hollow section (501). When the unlocking button (8) is released, the end thereof passes through the conducting opening (504) through the unlocking structure, and is engaged with any of the annular limiting grooves (905) to lock the installation height of the reflector (12).

7. The extraction method according to claim 6, characterized in that A two-stage lifting structure is provided between the pulling rod (5) and the fixed ring sleeve (901), and the lifting displacement of the second stage is unlocked by rotating the pulling rod (5); The solid section (502) is provided with an angled guide groove (503) along its axial direction, and the inner side of the fixed ring sleeve (901) is provided with a protruding block (906) that is slidably connected to the angled guide groove (503); A brush (904) is provided on the inner side of the bottom end of the rotating ring sleeve (902) along its circumference, and the brush (904) performs a self-cleaning action on the reflective plate (12) after the pulling rod (5) completes the first stage of pulling displacement.

8. The extraction method according to claim 7, characterized in that The accommodating kit (4) is sequentially provided with a hollow sleeve body (401) and a threaded seat body (402) along its axial direction, and the threaded seat body (402) and the isolation valve (3) are connected via a vertical plate (403) with a lateral gap, the lateral gap being used for replacement of the reflecting plate (12) after disassembly, and an isolation sleeve (6) for closing the lateral gap is threadedly connected to the outside of the threaded seat body (402), and an interlocking structure is provided between the isolation sleeve (6) and the isolation valve (3); The isolation valve (3) comprises a valve body (301) and a radially arranged valve (302); a flap (304) is rotatably provided on the side of the valve body (301), and a return torsion spring (306) is provided at the rotation center; a synchronization ring (303) with a notch (3031) is integrally provided on the valve (302); the top of one end of the flap (304) abuts against an abutting ring (601) on the outside of the isolation sleeve (6); and when the isolation sleeve (6) is screwed open, one end of the flap (304) is vertically engaged with the notch (3031); The interlocking structure further comprises a cylindrical interlocking member (7) which is covered on the valve (302). The interlocking member (7) and the valve (302) are integrally formed. The abutting ring (601) is provided with a limiting protrusion (602) along its tangent direction. The open end of the interlocking member (7) is provided with an arc-shaped locking block (701). When the isolation valve (3) is in the open state, the limiting protrusion (602) abuts against the limiting protrusion (602) laterally, and the isolation sleeve (6) is locked.

9. Use of the active substance of Ulidige in anti-tumor medicine, which uses the method for extracting the active substance of Ulidige according to claim 1, characterized in that: The active ingredient of Ulidige is phenylacetaldehyde.

10. Use of the active substance of Ulidige in antitumor medicine according to claim 9, characterized in that Application of phenylacetaldehyde in anti-glioma drugs.

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