A process for extracting an active ingredient of smilax glabra that inhibits xanthine oxidase activity and an extraction device thereof

Abstract

The application discloses a kind of smilax glabra inhibiting xanthine oxidase active ingredient extraction process and its extraction device. By once reflux extraction, the fine powder of smilax glabra is placed in the material supporting filter frame, ethanol solution is placed in the extraction tank, so that the fine powder of smilax glabra and ethanol solution are separated by the material supporting filter frame, then the obtained extraction liquid after extraction is extracted into the solvent turnover box, then the solvent in the solvent turnover box is extracted into the extraction tank to repeat the step S3 to circulate reflux extraction 3-4 times, so that the extraction liquid can be separated from the fine powder of smilax glabra without pouring out for filtration. Compared with the traditional pouring-out filtration method, the extraction efficiency is improved, the operation is simple, the heat release and heat absorption of the heating liquid are fully utilized during the whole process, thereby realizing resource recycling, achieving the purpose of energy saving, and improving economic benefits.

Description

Technical Field

[0001] This invention relates to the field of extraction technology, specifically to an extraction process and apparatus for the active ingredient in Smilax glabra that inhibits xanthine oxidase. Background Technology

[0002] Hyperuricemia has become the second leading metabolic disease worldwide, second only to diabetes. Long-term hyperuricemia is a key factor in the development of gout and can also cause various complications. XOD (xanthine oxidase) is a key rate-limiting enzyme catalyzing the metabolism of xanthine and hypoxanthine into uric acid. Inhibiting its activity can reduce uric acid production in the body, thus treating hyperuricemia. Therefore, XOD has become an important drug target for the treatment of hyperuricemia. However, currently used Western medicine-based XOD inhibitors have many toxic side effects, seriously threatening human health. With in-depth research on natural product XOD inhibitors, finding highly effective and low-toxicity XOD inhibitors from plants to treat hyperuricemia and gout has gradually become a research hotspot.

[0003] Smilax glabra Roxb., a plant in the Liliaceae family, is a dried rhizome that has the effects of removing dampness and promoting joint mobility. Flavonoids in Smilax glabra have significant anti-inflammatory and analgesic effects. Pharmacological studies have shown that Smilax glabra extract has a significant inhibitory effect on XOD activity in vitro. However, as a traditional Chinese medicine with great potential for XOD inhibitor development, the current extraction of Smilax glabra extract is generally carried out by reflux extraction to avoid solvent evaporation. The existing reflux extraction process involves placing the material in a flask, adding solvent to submerge the surface of the material, soaking for a certain period of time, installing a condenser at the mouth of the flask and connecting it to cooling water, then heating the flask in a water bath and refluxing for a specified time. After filtering out the reflux liquid, new solvent is added and refluxed again. After a second and third heating and reflux, the effective components are extracted. This process requires repeated pouring and filtering, which is labor-intensive, time-consuming, cumbersome, and has low extraction efficiency. Moreover, the extraction of the effective components of the drug is not sufficient. At the same time, the existing extraction devices use extraction tanks during reflux, but the existing extraction tanks do not have a cleaning function, resulting in residues in the extraction tank, which affects the next extraction. Therefore, it is necessary to propose an extraction process and extraction device for the inhibitory xanthine oxidase activity components of Smilax glabra. Summary of the Invention

[0004] The purpose of this invention is to solve the problems in the background art and provide an extraction process and extraction device for the active ingredient of Smilax glabra that inhibits xanthine oxidase.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0006] An extraction process for the active ingredient of Smilax glabra that inhibits xanthine oxidase includes the following steps:

[0007] S1, Grind, pulverize the Smilax glabra medicinal material into fine powder;

[0008] S2, Cleaning, cleaning the extraction tank;

[0009] S3, single reflux extraction: The fine powder of Smilax glabra is put into the material receiving filter frame of the extraction tank, and then ethanol solution is added to the extraction tank for heating and reflux extraction. During the reflux process, the motor is turned on every 12 to 15 minutes, and the material receiving filter frame rotates for 20 to 50 seconds at a speed of 0.5 to 2 revolutions per second to stir the fine powder of Smilax glabra and the extraction solvent in the material receiving filter frame. This is also carried out in the heating coil of the extraction tank.

[0010] S4, reflux extraction: The solvent formed after one reflux extraction in step S3 is extracted into a solvent transfer tank. Then, the solvent in the solvent transfer tank is extracted into the extraction tank and the steps of step S3 are repeated for reflux extraction 3 to 4 times.

[0011] S5, preliminary filtration: The extract obtained after the cyclic reflux extraction in step S4 is drawn into the storage tank, and the fine powder of Smilax glabra and the extract are preliminarily filtered and separated through the material-supporting filter frame.

[0012] S6, Dehydration: Start the motor to quickly rotate the material-bearing filter frame, discard the extract from the Smilax glabra powder, and extract the obtained extract into the storage tank.

[0013] S7, Secondary filtration: The extract obtained in steps S5 and S6 is separated a second time through the filter screen in the storage tank.

[0014] S8, Reduced pressure concentration: The obtained Smilax glabra extract is concentrated under reduced pressure to obtain a dry extract for later use.

[0015] The reflux extraction step involves turning on the motor every 12-15 minutes, rotating the filter frame for 20-50 seconds at a speed of 0.5-2 rpm, stirring the fine powder of Smilax glabra and the extraction solvent, which is beneficial for the full extraction of the effective components of the Chinese medicinal material.

[0016] Preferably, the concentration of the ethanol solution is 66-72%, and the extraction temperature is 80-85℃, which is beneficial for the extraction of active ingredients.

[0017] A reflux extraction device for inhibiting xanthine oxidase activity from Smilax glabra includes an extraction tank, a solvent transfer tank, a storage tank, a condenser, a first water pump, a material-receiving filter frame, and an internally hollow connecting rod. The material-receiving filter frame has a mesh barrel structure. The top of the extraction tank has a condenser installation port, a liquid inlet, and a cleaning port. A rotating nozzle is installed near the inside of the extraction tank at the cleaning port, which can spray and clean the inside of the extraction tank. The rotating nozzle is existing technology and will not be described in detail here. The bottom of the extraction tank has a drain port and a water outlet. The condenser is located at the condenser installation port. The solvent transfer tank... The inlet of the container is connected to the outlet of the extraction tank via a pipe. The inlet of the first water pump is connected to the outlet of the solvent turnover box via a pipe. The outlet of the first water pump is connected to the inlet via a pipe. The inlet of the storage tank is connected to the outlet of the extraction tank via a pipe. The connecting rod is fixed to the feed inlet and located inside the extraction tank. The material-supporting filter frame is fixed to the bottom of the connecting rod. The outlet of the extraction tank is equipped with a control valve. The outlet is connected to the inlet of the solvent turnover box and the inlet of the storage tank via a three-way valve. This is existing technology and will not be described in detail in this case.

[0018] Place the Smilax glabra (Tufuling) in a filter frame, and add solvent into the extraction tank through the inlet. The solvent will submerge the herb, and the extraction will be carried out by heating and reflux. The solvent will then be discharged into a solvent transfer tank through a drain pipe. The solvent in the solvent transfer tank will then be transferred into the extraction tank for a second heating and extraction. This process will be repeated two to three times to complete the reflux extraction of the xanthine oxidase-inhibiting component of Smilax glabra. During extraction, the filter frame keeps the material and solvent separated, eliminating the need for filtration. This not only results in fast and efficient extraction with simple operation, but also allows for more complete extraction of the active ingredients of the herb due to the rotating filter frame.

[0019] Preferably, the extraction tank is surrounded by a heating coil, with the water inlet of the heating coil located at the bottom and the water outlet located at the top. The water inlet of the heating coil is equipped with a heating box, and the water inlet of the heating box is connected to the water outlet of the heating coil through a pipe. The water outlet of the heating box is connected to the water inlet of the heating coil through a pipe. The water in the heating coil is circulated and heated through the heating box to achieve a constant temperature.

[0020] Preferably, the heating chamber includes a chamber body, the top of which is covered by a cover plate, and two partitions are vertically fixed to the bottom of the chamber body. A water pipe is provided inside the chamber body, and the partitions have mounting holes for the water pipe to pass through. Two water pipe through holes are provided on the side wall of the chamber body. Both ends of the water pipe pass through the water pipe through holes, and one end of the water pipe is connected to the water inlet of the heating coil, while the other end is connected to the water outlet of the heating coil. Several annular heating elements are fitted on the water pipe. A temperature sensor is provided in the water pipe through hole near the water outlet of the heating coil. Water flows through the water pipe and is heated by the heating elements, thereby further heating the water to ensure that the water temperature meets the requirements for heating the extraction tank.

[0021] By incorporating several ring-shaped heating elements, the number of heating elements can be controlled based on the temperature detected by the temperature sensor, thereby achieving energy saving.

[0022] Preferably, the extraction tank is equipped with a cleaning mechanism, which includes a first cleaning rod, a first driven gear, a second cleaning rod, a second driven gear, a support base, a rotary bearing, and a first driving gear. The first cleaning rod and the second cleaning rod are located on opposite sides of the material receiving filter frame. The first driven gear is located at the top of the first cleaning rod, and the second driven gear is located at the top of the second cleaning rod. The support base is located at the bottom of the extraction tank, and the rotary bearing is fixed to the support base. The bottom of the second cleaning rod is fixed to the rotary bearing, and the bottom of the first cleaning rod is fixed to the rotary bearing. The first driving gear is sleeved and fixed to the outer wall of the connecting rod, and the first driving gear meshes with the first driven gear and the second driven gear, respectively.

[0023] Preferably, the connecting rod is provided with a vertical moving and rotating mechanism, which includes an electric push rod, a connecting rod, a hollow fixed box, a rotary motor, a second driving gear, a third driven gear, and a sealed bearing. The electric push rod is located at the top of the extraction tank, and the fixed box is fixedly connected to the push rod end of the electric push rod. The rotary motor is located inside the fixed box, the second driving gear is located on the output shaft of the rotary motor, and the third driven gear meshes with the second driving gear. The connecting rod passes through the third driven gear and the fixed box and is fixedly connected to the third driven gear. The fixed box has mounting holes from its upper surface to its lower surface, and the sealed bearing is fixed in the mounting holes. The inner ring of the sealed bearing is fixedly connected to the outer wall of the connecting rod. The material-receiving filter frame is located at the bottom of the connecting rod. The connecting rod has a hollow internal structure, and the outer ring of the sealed bearing is fixedly connected to a feed telescopic tube, which can be a flexible hose. The other end of the feed telescopic tube extends to the outside of the extraction tank.

[0024] When the extraction tank needs cleaning, the electric push rod drives the connecting rod upward, causing the first driving gear, the first driven gear, and the second driven gear to mesh. Then, the rotary motor rotates, driving the second driving gear, which in turn drives the third driven gear, causing the connecting rod to rotate. The rotation of the connecting rod causes the material-bearing filter frame, the first cleaning rod, and the second cleaning rod to rotate. This rotation of the material-bearing filter frame thoroughly cleans it, while the cleaning rods agitate and clean the inside of the extraction tank. This comprehensive cleaning of the extraction tank and the material-bearing filter frame prevents residue from affecting subsequent extractions and improves extraction purity. When cleaning is not needed, simply moving the electric push rod downward disengages the first driving gear, the second driven gear, and the second driven gear, allowing the connecting rod to control only the material-bearing filter frame. This facilitates subsequent rotation of the material-bearing filter frame, ensuring the cleaning mechanism is activated only when cleaning is required, making it more convenient.

[0025] The feed telescopic tube and the connecting rod are connected by a sealed bearing, which not only makes it easy for the material to enter the material receiving filter frame from the feed telescopic tube, but also does not affect the rotation of the material receiving filter frame.

[0026] Preferably, the liquid storage tank includes a liquid storage tank body, and a baffle ring is provided on the inner side wall of the liquid storage tank body. The baffle ring is provided with a filter screen mechanism to filter the solvent.

[0027] Preferably, the filter mechanism includes a frame, a honeycomb-shaped filter is provided inside the frame, and a handle is fixedly connected to the frame. The honeycomb-shaped filter makes the filter less prone to deformation, has a large contact area, and has high purification efficiency. The handle allows for easy removal from the main body of the turnover box.

[0028] In summary, the beneficial effects of this invention are as follows:

[0029] 1. This invention involves placing fine powder of Smilax glabra in a receiving filter frame and an ethanol solution in an extraction tank through a single reflux extraction process. This allows the fine powder and ethanol solution to be separated by the receiving filter frame. The extracted liquid is then transferred to a solvent transfer tank, and the solvent in the solvent transfer tank is transferred back to the extraction tank. This process of repeating step S3 is repeated 3-4 times. This allows the extract to be separated from the fine powder of Smilax glabra without having to be poured out for filtration. Compared to the traditional method that requires pouring out for filtration, this method improves extraction efficiency and simplifies operation.

[0030] 2. When the extraction tank needs cleaning, the electric push rod drives the connecting rod upward, causing the first driving gear, the first driven gear, and the second driven gear to mesh. Then, the rotary motor rotates, driving the second driving gear to rotate, which in turn drives the third driven gear to rotate, causing the connecting rod to rotate. The rotation of the connecting rod causes the material-bearing filter frame, the first cleaning rod, and the second cleaning rod to rotate. The rotation of the material-bearing filter frame thoroughly cleans it, and the cleaning rods agitate and clean the inside of the extraction tank. This comprehensive cleaning of the extraction tank and the material-bearing filter frame prevents residue from affecting the next extraction. When cleaning is not needed, simply move the electric push rod downward, disengaging the first driving gear, the second driven gear, and the second driven gear. This allows the connecting rod to control only the material-bearing filter frame, facilitating its rotation later. This ensures that the cleaning mechanism is activated only when cleaning is required, making it more convenient.

[0031] 3. This invention uses a water pipe to flow water through, and a heating element to heat the water pipe, thereby further heating the water to ensure that the water temperature meets the heating requirements of the extraction tank. By using several ring-shaped heating elements, the number of heating elements can be controlled according to the temperature detected by the temperature sensor, thus achieving energy saving. Attached Figure Description

[0032] Figure 1 This is an overall schematic diagram of the reflux extraction device of the present invention;

[0033] Figure 2 This is a cross-sectional schematic diagram of the extraction tank of the present invention;

[0034] Figure 3 This is a schematic diagram of the connection between the connecting rod and the feed telescopic tube of the present invention;

[0035] Figure 4 This is a cross-sectional schematic diagram of the heating box of the present invention;

[0036] Figure 5 This is a schematic diagram of the heating chamber of the present invention;

[0037] Figure 6 This is a cross-sectional schematic diagram of the liquid storage tank of the present invention;

[0038] Figure 7 This is a top view schematic diagram of the filter screen mechanism of the present invention;

[0039] Figure 8 Schematic diagram of the effect of ethanol concentration on the inhibition of XOD activity;

[0040] Figure 9 Schematic diagram of the effect of extraction time on XOD activity inhibition;

[0041] Figure 10 Schematic diagram of the effect of extraction temperature on the inhibition of XOD activity;

[0042] Figure 11 Schematic diagram of the effect of liquid-to-solid ratio on XOD activity inhibition;

[0043] Figure 12 Response surface methodology for the interaction of various factors;

[0044] Figure 13 Schematic diagram of the effect of different mass concentrations of Smilax glabra extract on XOD activity

[0045] Figure 14 Schematic diagram showing the effect of different enzyme concentrations on reaction rate;

[0046] Figure 15 Line-weaver-Burk plot of the inhibitory kinetics of the extract on XOD;

[0047] Figure 16 A schematic diagram illustrating the determination of the inhibitory effect of the extract on XOD in Changshu. Detailed Implementation

[0048] The following specific embodiments are merely illustrative of the present invention and are not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of the present invention.

[0049] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0050] Example 1

[0051] like Figure 1-7 As shown, an extraction process for the active ingredient of Smilax glabra that inhibits xanthine oxidase is characterized by the following steps:

[0052] S1, Grind, pulverize the Smilax glabra medicinal material into fine powder;

[0053] S2, Cleaning, cleaning the extraction tank 11;

[0054] S3, single reflux extraction: The fine powder of Smilax glabra is put into the receiving filter frame 12 of the extraction tank 11. Then, an ethanol solution with a concentration of 69% is added to the extraction tank 11. The extraction temperature is 82℃ and the extraction time is 89min. Heating reflux extraction is carried out. During the reflux process, the motor is turned on every 12 to 15 minutes. The receiving filter frame 12 rotates for 20 to 50 seconds. The rotation speed of the receiving filter frame 12 is 0.5 to 2 rpm. The fine powder of Smilax glabra and the extraction solvent in the receiving filter frame 12 are stirred. At the same time, the extraction tank 11 is heated by the heating coil.

[0055] S4, reflux extraction: The solvent formed after one reflux extraction in step S3 is extracted into the solvent turnover box 13. Then, the solvent in the solvent turnover box 13 is extracted into the extraction tank 11 and the steps of step S3 are repeated for reflux extraction 3 to 4 times.

[0056] S5, preliminary filtration: the extract obtained after the cyclic reflux extraction in step S4 is drawn into the storage tank 14, and the fine powder of Smilax glabra and the extract are preliminarily filtered and separated through the material-supporting filter frame 12.

[0057] S6, Dehydration: Start the motor to quickly rotate the material-bearing filter frame 12, remove the extract from the Smilax glabra powder, and extract the obtained extract into the storage tank 14.

[0058] S7, Secondary filtration: The extract obtained in steps S5 and S6 is separated a second time through the filter screen in the storage tank 14.

[0059] S8, Reduced pressure concentration: The obtained Smilax glabra extract is concentrated under reduced pressure to obtain a dry extract for later use.

[0060] Studies have reported that Smilax glabra extract has inhibitory effects on XOD activity, with flavonoids being the most effective inhibitors, mainly including astilbin, scutellarin, isoflavone, epicatechin, quercetin, and naringenin. To ensure the complete extraction of these active components, this experiment employed ethanol reflux extraction and selected response surface methodology to optimize the extraction process, ultimately obtaining extracts with high XOD inhibitory activity. Enzyme kinetic analysis showed that the inhibitory effect of Smilax glabra extract on XOD is similar to that of allopurinol. Therefore, for the treatment of hyperuricemia, the appropriate use of natural XOD inhibitors from Smilax glabra could replace some Western medicines to reduce toxic side effects.

[0061] like Figure 1-7As shown, a reflux extraction device for inhibiting xanthine oxidase activity from Smilax glabra includes an extraction tank 11, a solvent transfer box 13, a storage tank 14, a condenser 15, a first water pump 16, a material-supporting filter frame 12, and an internally hollow connecting rod 62. The top of the extraction tank 11 is provided with a condenser installation port 111, a liquid inlet 113, a cleaning port 112, and a feed inlet 118. The bottom of the extraction tank 11 is provided with a drain port 110 and a water outlet 114. The condenser 15 is installed on the condenser installation port 111. The inlet of the solvent turnover tank 13 is connected to the outlet 110 of the extraction tank 11 via a pipe. The inlet of the first water pump 16 is connected to the outlet of the solvent turnover tank 13 via a pipe. The outlet of the first water pump 16 is connected to the inlet 113 via a pipe. The inlet of the storage tank 14 is connected to the outlet 110 of the extraction tank 11 via a pipe. The connecting rod 62 is fixed on the feed inlet 118 and located inside the extraction tank 11. The material-receiving filter frame 12 is fixed to the connecting rod 62. At the bottom of the extraction tank 11, a heating coil 19 surrounds the extraction tank 11. The inlet of the heating coil 19 is located at the bottom, and the outlet is located at the top. A heating box 2 is installed at the inlet of the heating coil 19. The inlet of the heating box 2 is connected to the outlet of the heating coil 19 through a pipe, and the outlet of the heating box 2 is connected to the inlet of the heating coil 19 through a pipe. The heating box 2 includes a box body 21. A cover plate is fitted on the top of the box body 21, and two partitions 22 are vertically fixed to the bottom of the box body 21. The housing 21 is equipped with a water pipe 23. The partition 22 has an installation hole 24 for the water pipe 23 to pass through. The side wall of the housing 21 has two water pipe through holes 25. The two ends of the water pipe 23 pass through the water pipe through holes 25 respectively. One end of the water pipe 23 is connected to the water inlet of the heating coil 19, and the other end is connected to the water outlet of the heating coil 19. Several annular heating elements 26 are fitted on the water pipe 23. The liquid storage tank 14 includes a liquid storage tank body 141. The inner side wall of the liquid storage tank body 141 is provided with a baffle ring 142. The baffle ring 142 is provided with a filter screen mechanism 133. The filter screen mechanism 133 includes a frame 134. The frame 134 is provided with a honeycomb filter screen 135. A handle 136 is fixedly connected to the frame 134.

[0062] like Figure 1-7As shown, the extraction tank 11 is equipped with a cleaning mechanism 3. The cleaning mechanism 3 includes a first cleaning rod 31, a first driven gear 32, a second cleaning rod 33, a second driven gear 34, a support base 35, a rotary bearing 36, and a first driving gear 38. The first cleaning rod 31 and the second cleaning rod 33 are located on opposite sides of the material receiving filter frame 12. The first driven gear 32 is located at the top of the first cleaning rod 31, and the second driven gear 34 is located at the top of the second cleaning rod 33. The support base 35 is located at the bottom of the extraction tank 11. The rotary bearing 36 is fixed to the support base 35, and the bottom of the second cleaning rod 33 is fixed to the rotary bearing 36. The bottom of the first cleaning rod 31 is fixed to the rotary bearing 36. The first driving gear 38 is sleeved and fixed to the outer wall of the connecting rod 62. The first driving gear 38 meshes with the first driven gear 32 and the second driven gear 34 respectively. The connecting rod 62 is equipped with a vertical moving and rotating mechanism 6, which includes an electric... The extraction tank 11 consists of a moving push rod 61, a hollow fixed box 63, a rotary motor 64, a second driving gear 65, a third driven gear 66, and a sealed bearing 67. The electric push rod 61 is located on top of the extraction tank 11. The fixed box 63 is fixedly connected to the push rod end of the electric push rod 61. The rotary motor 64 is located inside the fixed box 63. The second driving gear 65 is located on the output shaft of the rotary motor 64. The third driven gear 66 meshes with the second driving gear 65. The connecting rod 62 passes through the third driven gear. The wheel 66 and the fixed box 63 are fixedly connected to the third driven gear 66. The fixed box 63 has a mounting hole 631 on its upper and lower surfaces. The sealed bearing 67 is fixed on the mounting hole 631. The inner ring of the sealed bearing 67 is fixedly connected to the outer wall of the connecting rod 62. The material receiving filter frame 12 is located at the bottom of the connecting rod 62. The connecting rod 62 has a hollow structure inside. The outer ring of the sealed bearing 67 is fixedly connected to the feed telescopic tube 68. The other end of the feed telescopic tube 68 extends to the outside of the extraction tank 1.

[0063] Working principle: such as Figure 1-8As shown, the Smilax glabra herb is pulverized into a fine powder and set aside. Then, the electric push rod 61 drives the connecting rod 62 upwards, causing the first driving gear 38, the first driven gear 32, and the second driven gear 34 to mesh. Cleaning liquid is then added to the cleaning port 112. Next, the rotary motor 64 rotates, driving the second driving gear 65 to rotate, which in turn drives the third driven gear 66 to rotate, causing the connecting rod 62 to rotate. The rotation of the connecting rod 62 drives the material-receiving filter frame 12, the first cleaning rod 31, and the second cleaning rod 33 to rotate, thereby cleaning the extraction tank and the material-receiving filter. The filter frame is thoroughly cleaned to prevent residue from remaining in the extraction tank and affecting the next extraction. After cleaning, the electric push rod 61 moves downward, disengaging the first driving gear 38, the second driven gear 32, and the second driven gear 34. This allows the connecting rod 62 to control only the material-bearing filter frame. Ethanol is then added through the inlet 113, and the fine powder of *Smilax glabra* is fed into the material-bearing filter frame of the extraction tank through the feed extension tube. The heating liquid is then delivered to the heating coil 19 of the extraction tank 11 to heat the extraction tank to 82°C. The temperature was set at ℃ for reflux extraction. During the reflux extraction, the motor was turned on every 12-15 minutes, and the filter frame was rotated for 20-50 seconds at a speed of 0.5-2 rpm. The fine powder of Smilax glabra and the extraction solvent were stirred in the filter frame to ensure thorough extraction. After the first reflux extraction, the solvent formed was transferred to the solvent transfer tank 13. Then, the solvent in the solvent transfer tank 13 was transferred to the extraction tank 11 to repeat the reflux extraction. After the reflux extraction was completed, the extracted liquid was transferred to the storage tank 14. The material-bearing filter frame 12 allows for initial filtration and separation of the Smilax glabra powder and the extract. Then, the motor is started to rapidly rotate the material-bearing filter frame 12, discarding the extract from the Smilax glabra powder. The obtained extract is then drawn into a storage tank. Finally, the obtained extract is passed through a filter screen in the storage tank for secondary separation. The obtained Smilax glabra extract is concentrated under reduced pressure to obtain a dry extract for later use. This method allows for separation of the extract and Smilax glabra powder without the need for filtration, improving extraction efficiency and simplifying operation compared to traditional filtration methods that require filtration.

[0064] Establishment of an in vitro model for XOD activity inhibition rate determination

[0065] Xanthine can be catalyzed by XOD to produce uric acid, which has a characteristic absorption peak at 295 nm. The amount of uric acid produced per unit time is directly proportional to the activity of XOD. Four treatment groups were selected, and samples were added according to Table 1. Phosphate-buffered saline (PBS, pH=7.5), enzyme solution (0.05 U / mL), and different concentrations of Smilax glabra extract solutions were added sequentially to the reaction system. After sample addition, the mixture was incubated at 37℃ for 10 min. Then, a pre-warmed xanthine solution (1 mmol / L) was added, and the mixture was thoroughly mixed. Timing was started from the addition of the xanthine solution, and the reaction was continued for 1 min. The absorbance was measured at 295 nm. A All samples were tested in triplicate, and the data are expressed as mean ± standard deviation. Inhibition rate % = [1 - ( A 1 - A 2 ) / ( A 3 - A 4 )]×100%.

[0066] Table 1. Reaction system for XOD activity test

[0067]

[0068] Single-factor experiments: The effects of four factors—ethanol concentration, liquid-to-solid ratio, extraction time, and extraction temperature—on XOD activity were investigated. Reflux extraction was performed at different levels of the four factors: extraction time (30 min, 60 min, 90 min, 120 min, and 150 min), ethanol concentration (40%, 50%, 60%, 70%, and 80%), extraction temperature (50℃, 60℃, 70℃, 80℃, and 90℃), and liquid-to-solid ratio (10:1, 20:1, 30:1, 40:1, and 50:1). The resulting dry extract was prepared into a 6 mg / mL solution, and its inhibition rate was measured.

[0069] ①The effect of ethanol concentration on the inhibition of XOD activity is determined by Figure 8 It can be seen that when the ethanol concentration is between 40% and 80%, the inhibition rate first increases and then decreases with increasing ethanol concentration. The inhibition rate reaches its maximum at an ethanol concentration of 70%. This may be because low-concentration ethanol solutions easily dissolve polar components in cells, such as proteins, tannins, and starch, which have virtually no inhibitory effect on XOD activity. Appropriately increasing the ethanol concentration is beneficial for the dissolution of XOD-inhibiting active ingredients, but excessively high ethanol concentrations do not effectively dissolve these ingredients. Therefore, ethanol concentration is a crucial factor affecting the extraction of XOD-inhibiting active substances from Smilax glabra.

[0070] ②The effect of extraction time on the inhibition of XOD activity is determined by Figure 9 It can be seen that the inhibition rate first increases and then decreases with extraction time, reaching its maximum at 90 minutes. It is possible that if the extraction time is too short, the XOD inhibitory active ingredients in Smilax glabra cannot be fully extracted, thus causing the inhibition rate to gradually increase with prolonged extraction time. At 90 minutes, the active ingredients in Smilax glabra are essentially completely extracted, hence the maximum inhibition rate. Excessive extraction time leads to the destruction or oxidative decomposition of the enzyme-inhibiting active ingredients, causing the inhibition rate to gradually decrease.

[0071] ③ The effect of extraction temperature on the inhibition of XOD activity is determined by Figure 10 It can be seen that within the extraction temperature range of 50-80℃, the higher the temperature, the higher the inhibition rate; the inhibition rate reaches its maximum at 80℃. Within the range of 80-90℃, the inhibition rate decreases with increasing extraction temperature. This may be because within the 50-80℃ range, higher temperatures accelerate the condensation and reflux of ethanol, which is beneficial for the extraction of active ingredients, thus gradually increasing the inhibition rate; when the extraction temperature exceeds 80℃, the active ingredients in the extract are decomposed by the high temperature, leading to a decrease in the inhibition rate.

[0072] ④ The effect of liquid-to-solid ratio on the inhibition of XOD activity is determined by Figure 11 It is observed that when the liquid-to-solid ratio is between 10:1 and 40:1, the inhibition rate increases with increasing liquid-to-solid ratio; the inhibition rate reaches its maximum at a liquid-to-solid ratio of 40:1; and when the liquid-to-solid ratio exceeds 40:1, the inhibition rate no longer increases with increasing liquid-to-solid ratio. This is likely because when the liquid-to-solid ratio is less than 40:1, increasing the liquid-to-solid ratio results in more dissolution of the XOD active inhibitory component; when the liquid-to-solid ratio is 40:1, the active component may be completely extracted, the inhibition rate reaches its maximum, and an equilibrium is reached. Further increasing the liquid-to-solid ratio would only increase solvent waste and energy consumption in subsequent concentration. Therefore, the liquid-to-solid ratio is fixed at 40:1.

[0073] Box-Behnken Design - Response Surface Optimization: Based on single-factor experiments, the Box-Behnken experimental design method in Design-Expert 8.05b software was used for experimental design, selecting the ethanol concentration (… A Extraction time () B ) and extraction temperature ( C Three factors were considered as influencing factors, with XOD activity inhibition rate ( Y A response surface methodology (RSM) experiment with 3 factors and 3 levels, totaling 17 experimental points, was designed to further optimize the process. The factors and levels are shown in Table 2, and the RSM experimental design and results are shown in Table 3.

[0074] The experimental design and results are shown in Table 3.

[0075] Table 2 Response Surface Experiment Factors and Levels

[0076]

[0077] Table 3 Response Surface Analysis Scheme and Experimental Results

[0078]

[0079] The data in Table 3 were subjected to regression fitting using Design-Expert 8.05b software, and the regression equation was obtained. Y =61.40-0.86 A -0.40 B +1.44 C +0.80 AB -4.38 AC +1.50 BC -6.64 A 2 -5.81 B 2 -6.24 C 2 The analysis of variance is shown in Table 4. As can be seen from Table 4, the model... P <0.0001 indicates that the model has reached a highly significant level. Furthermore, the model lacks a certain level of significance. P >0.05, not significant. R 2 =0.9908, indicating that the model fits well and the relative error of the experiment is small. This model can be used to analyze and predict the XOD activity inhibition rate. F The larger the value, the greater the influence of the variable on the response value. The influence of each factor on the XOD activity inhibition rate is in the order of extraction temperature > ethanol concentration > extraction time.

[0080] Table 4 Analysis of Variance

[0081]

[0082] See response surface analysis Figure 12 .Depend on Figure 12 It can be seen that the interaction between extraction temperature and ethanol concentration is the strongest, with elliptical contour lines and steep curves, which has a significant impact on the XOD activity inhibition rate; the interaction between extraction time and extraction temperature is the second strongest, with elliptical contour lines and relatively steep curves, which has a certain impact on the XOD activity inhibition rate; the interaction between ethanol concentration and extraction time is the weakest, with approximately circular contour lines and relatively steep curves, which has a certain impact on the XOD activity inhibition rate.

[0083] The established mathematical model was analyzed using Design-Expert 8.05b software. The optimal extraction conditions were found to be an ethanol concentration of 68.83%, an extraction time of 89.33 min, and an extraction temperature of 81.53℃. Under these conditions, the predicted enzyme inhibition rate was 61.57%. Based on the model analysis and practical results, an ethanol concentration of 69%, an extraction time of 89 min, and an extraction temperature of 82℃ were selected. The actual average value of three verification experiments was 62.25%. The goodness of fit between the actual and predicted values ​​reached 98.91%, indicating that the response surface methodology for optimizing the extraction process is reasonable and feasible.

[0084] Application examples

[0085] Inhibitory activity of extract against XOD and IC50 50 Determination of values: A graph was plotted with the concentration of the active component extracted from Smilax glabra under optimal extraction conditions as the independent variable and the inhibition rate as the dependent variable. The results are shown below. Figure 13 The inhibition rate of Smilax glabra extract increased continuously with increasing concentration, indicating a dose-dependent inhibition of XOD. From 0 to 2 mg / mL, the inhibition rate increased linearly with increasing extract concentration. From 2 to 6 mg / mL, the increasing trend of inhibition rate decreased with increasing extract concentration. Above 6 mg / mL, the inhibition rate tended to plateau, and there was no significant difference between two adjacent concentrations. The fitting equation was obtained through linear fitting: Y =8.54 X +11.56 ( R 2 =0.9965), IC 50 The concentration was 4.50 mg / mL.

[0086] Inhibition kinetics of the extract against XOD activity: In an enzymatic reaction system, with the xanthine substrate concentration fixed at 1 mmol / L, the reaction rates of 1, 3, and 5 mg / mL *Smilax glabra* extract in enzyme concentration systems of 0.025, 0.05, 0.10, and 0.15 U / mL were measured. A graph was plotted with XOD concentration on the x-axis and reaction rate on the y-axis. A set of straight lines that basically intersect at the origin were obtained, and the slope of the lines decreased with increasing *Smilax glabra* extract concentration. (See figure). Figure 14 This indicates that the inhibitory effect of Smilax glabra extract on XOD is reversible, and the enzyme activity can be restored using physical methods.

[0087] In the reaction system, keeping the XOD concentration constant at 0.05 U / mL, the reaction rates of 1, 3, and 5 mg / mL Smilax glabra extract in systems with different xanthine substrate concentrations of 0.25, 0.5, 1.0, and 2.0 mmol / L were measured. A double reciprocal plot was used, with the reciprocal of the substrate concentration [1 / S[1 / ] is the x-axis, and the reciprocal of the reaction rate [1 / ] V Plotting the vertical axis yields a set of straight lines, see... Figure 15 All the straight lines intersect at a single point on the vertical axis, and the location of the intersection point hardly changes with the concentration of the extract, meaning Vmax remains constant, which conforms to the competitive inhibition characteristics of enzymes. Therefore, Smilax glabra extract is a competitive and reversible inhibitor of XOD.

[0088] The Dixon plot method was used, selecting two substrate concentrations of 0.5 mmol / L and 1.0 mmol / L, with the extract concentration as the independent variable and the reciprocal of the reaction rate as the dependent variable. Two intersecting straight lines were obtained, as shown in the figure. Figure 16 The inhibition constant K of the extract on XOD is calculated based on the intersection of the two lines. i It is 1.92 mg / mL.

[0089] Based on single-factor experiments, ethanol concentration, extraction time, and extraction temperature were used as influencing factors, and the xanthine oxidase activity inhibition rate was used as the evaluation index. The Box-Behnken design-response surface methodology was employed to optimize the extraction process of the XOD-inhibiting active ingredient in Smilax glabra. The inhibition type and inhibition kinetic constant K were determined using Lineweaver-Burk plots and Dixon plots. i Results: The optimal extraction conditions were 69% ethanol concentration, 89 min extraction time, and 82℃ extraction temperature, with an XOD activity inhibition rate of 62.25% for the extracted component. The inhibition type of XOD activity by the Smilax glabra extract was competitive and reversible, and the inhibition kinetic constant K... i The concentration was 1.92 mg / mL. Conclusion: This method is stable and reliable, and can be used to extract components from Smilax glabra that inhibit XOD activity, providing a theoretical basis for the development of XOD activity inhibitors.

[0090] The biggest advantage of the natural XOD inhibitor in Smilax glabra is that it has no side effects and is widely available. In addition, the chemical components in Smilax glabra have a comprehensive therapeutic effect on multiple targets, such as inhibiting XOD activity, promoting uric acid excretion and relieving inflammatory response, in the treatment of gout and hyperuricemia. Therefore, it has a natural advantage in the treatment of such diseases, and also has broad prospects in the development of related health products.

Claims

1. An extraction process for the active ingredient of Smilax glabra that inhibits xanthine oxidase, characterized in that, Includes the following steps: S1, Grind, grind the Smilax glabra medicinal material into fine powder; S2, Cleaning, cleaning the extraction tank (11); S3, one-time reflux extraction, put the fine powder of Smilax glabra into the material receiving filter frame (12) of the extraction tank (11), then add ethanol solution into the extraction tank (11) and perform heating reflux extraction. Turn on the motor once every 12~15 minutes, rotate the material receiving filter frame (12) for 20~50 seconds, the rotation speed of the material receiving filter frame (12) is 0.5~2 revolutions / second, stir the fine powder of Smilax glabra and the extraction solvent in the material receiving filter frame (12); S4, reflux extraction: The solvent formed after one reflux extraction in step S3 is extracted into the solvent turnover box (13), and then the solvent in the solvent turnover box (13) is extracted into the extraction tank (11) and the steps of step S3 are repeated for reflux extraction 3 to 4 times. S5, preliminary filtration: the extract obtained after the cyclic reflux extraction in step S4 is drawn into the storage tank (14), and the fine powder of Smilax glabra and the extract are preliminarily filtered and separated through the material-bearing filter frame (12). S6, dehydration, start the motor to quickly rotate the material-bearing filter frame (12), shake off the extract in the fine powder of Smilax glabra, and extract the obtained extract into the storage tank (14); S7, Secondary filtration: The extract obtained in steps S5 and S6 is separated a second time through the filter screen in the storage tank (14); S8, Reduced pressure concentration: The obtained Smilax glabra extract is concentrated under reduced pressure to obtain a dry extract for later use. The extraction process for the xanthine oxidase-inhibiting active ingredient from Smilax glabra employs a reflux extraction device, which includes an extraction tank (11), a solvent transfer box (13), a storage tank (14), a condenser (15), a first water pump (16), a material-supporting filter frame (12), and a hollow connecting rod (62). The top of the extraction tank (11) is provided with a condenser installation port (111), a liquid inlet (113), a cleaning port (112), and a feed inlet (118). The bottom of the extraction tank (11) is provided with a drain port (110) and a water outlet (114). The condenser (15) is installed on the condenser installation port (111). The inlet of the solvent turnover box (13) is connected to the outlet (110) of the extraction tank (11) through a pipe. The inlet of the first water pump (16) is connected to the outlet of the solvent turnover box (13) through a pipe. The outlet of the first water pump (16) is connected to the inlet (113) through a pipe. The inlet of the storage tank (14) is connected to the outlet (110) of the extraction tank (11) through a pipe. The connecting rod (62) is fixed on the feed inlet (118) and located inside the extraction tank (11). The material-bearing filter frame (12) is fixed at the bottom of the connecting rod (62). The extraction tank (11) is equipped with a cleaning mechanism (3), which includes a first cleaning rod (31), a first driven gear (32), a second cleaning rod (33), a second driven gear (34), a support base (35), a rotary bearing (36), and a first driving gear (38). The first cleaning rod (31) and the second cleaning rod (33) are located on opposite sides of the material receiving filter frame (12). The first driven gear (32) is located at the top of the first cleaning rod (31), and the second driven gear (34) is located at the bottom of the second cleaning rod (12). The top of the cleaning rod (33) is provided with the support base (35) at the bottom of the extraction tank (11), the rotary bearing (36) is fixed on the support base (35), the bottom of the second cleaning rod (33) is fixed on the rotary bearing (36), the bottom of the first cleaning rod (31) is fixed on the rotary bearing (36), the first driving gear (38) is sleeved and fixed on the outer side wall of the connecting rod (62), and the first driving gear (38) meshes with the first driven gear (32) and the second driven gear (34) respectively; The connecting rod (62) is provided with a vertical moving and rotating mechanism (6), which includes an electric push rod (61), a hollow fixed box (63), a rotary motor (64), a second driving gear (65), a third driven gear (66), and a sealed bearing (67). The electric push rod (61) is located on the top of the extraction tank (11), and the fixed box (63) is fixedly connected to the push rod end of the electric push rod (61). The rotary motor (64) is located inside the fixed box (63), and the second driving gear (65) is located inside the rotary motor (67). 4) The output shaft, the third driven gear (66) meshes with the second driving gear (65), the connecting rod (62) passes through the third driven gear (66) and the fixed box (63) and is fixedly connected to the third driven gear (66), the fixed box (63) has a first mounting hole (631) from the upper surface to the lower surface, the sealing bearing (67) is fixed in the first mounting hole (631), the inner ring of the sealing bearing (67) is fixedly connected to the outer side wall of the connecting rod (62), and the material-bearing filter frame (12) is located at the bottom of the connecting rod (62); The connecting rod (62) has a hollow structure inside. The outer ring of the sealed bearing (67) is fixedly connected to the feed telescopic tube (68), and the other end of the feed telescopic tube (68) extends to the outside of the extraction tank (11).

2. The extraction process of the active ingredient of Smilax glabra that inhibits xanthine oxidase according to claim 1, characterized in that, The concentration of the ethanol solution is 66-72%, and the extraction temperature is 80-85℃.

3. The extraction process of the active ingredient of Smilax glabra that inhibits xanthine oxidase according to claim 1, characterized in that, The extraction tank (11) is surrounded by a heating coil (19). The inlet of the heating coil (19) is located at the bottom and the outlet is located at the top. The inlet of the heating coil (19) is equipped with a heating box (2). The inlet of the heating box (2) is connected to the outlet of the heating coil (19) through a pipe. The outlet of the heating box (2) is connected to the inlet of the heating coil (19) through a pipe.

4. The extraction process of the active ingredient of Smilax glabra that inhibits xanthine oxidase according to claim 3, characterized in that, The heating box (2) includes a box body (21), the top of the box body (21) is covered with a cover plate, and two partitions (22) are vertically fixed to the bottom of the box body (21). A water pipe (23) is provided inside the box body (21). A second mounting hole (24) is provided on the partition (22) to facilitate the passage of the water pipe (23). Two water pipe through holes (25) are provided on the side wall of the box body (21). Both ends of the water pipe (23) pass through the water pipe through holes (25) respectively, and one end of the water pipe (23) is connected to the water inlet of the heating coil (19), and the other end is connected to the water outlet of the heating coil (19). Several ring-shaped heating elements (26) are sleeved on the water pipe (23).

5. The extraction process of the active ingredient of Smilax glabra that inhibits xanthine oxidase according to claim 1, characterized in that, The liquid storage tank (14) includes a liquid storage tank body (141), and a retaining ring (142) is provided on the inner side wall of the liquid storage tank body (141). A filter screen mechanism (133) is provided on the retaining ring (142).

6. The extraction process of the xanthine oxidase-inhibiting active ingredient from Smilax glabra according to claim 5, characterized in that, The filter mechanism (133) includes a frame (134), a honeycomb filter (135) is provided inside the frame (134), and a handle (136) is fixedly connected to the frame (134).

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