A rapid determination method of belladonna herb content

Abstract

The present application relates to the technical field of traditional Chinese medicine analysis and detection, and in particular to a rapid determination method of belladonna herb content, which uses a processing device including a kettle body. The kettle body is used in cooperation with a liquid guiding channel and a heating mechanism. A liquid pump can deliver the heated liquid medium into the liquid guiding channel through a first liquid guide pipe, a second liquid guide pipe and a rotary joint. At this time, the liquid medium can heat the stirrer, and the stirrer can be quickly heated. This can make the liquid medium heat the mixture through the kettle body inner wall and the stirrer, so that all parts in contact with the mixture can be heated, thereby improving the heating effect on the mixture. Belladonna herb is hydrolyzed by plant extraction enzyme to obtain a test solution in the kettle body. The kettle body can make the effective components of belladonna herb be stably and quickly dissolved, avoid incomplete extraction and degradation of the effective components, and use sulfuric acid hyacinthamine as a control sample. The content can be quickly determined by using high performance liquid chromatography.

Description

Technical Field

[0001] This invention relates to the field of Chinese medicinal herb analysis and testing technology, and more specifically, to a rapid method for determining the content of belladonna. Background Technology

[0002] The current method for determining the content of belladonna oleracea is as follows: Accurately weigh approximately 10g of belladonna oleracea powder and place it in a Soxhlet extractor. Add an appropriate amount of a mixed solution of 10ml ethanol, 8ml concentrated ammonia solution, and 20ml ether. Let stand for 12 hours, then add 70ml ether and heat under reflux for 3 hours until the alkaloids are completely extracted. Evaporate most of the ether from the extract on a water bath. Transfer the extract to a separatory funnel and extract with 10ml of 0.5mol / L sulfuric acid solution in several portions, shaking each time. Extract the alkaloids completely, combine the acid solutions, and extract with chloroform in several portions, 10 ml each time, until the chloroform layer is colorless. Combine the chloroform solutions and extract with 10 ml of 0.5 mol / L sulfuric acid solution, discarding the chloroform solution. Combine the two acid solutions, filter, wash the filter with 0.5 mol / L sulfuric acid solution, combine the washings and the filtrate, add excess concentrated ammonia solution to make it alkaline, and quickly extract with chloroform in several portions, until the alkaloids are completely extracted. Each chloroform solution was washed with 10 ml of the same water, the washings were discarded, the chloroform solutions were combined, evaporated to dryness, 3 ml of ethanol was added, evaporated to dryness again, and dried at 80°C for 2 hours. The residue was dissolved in 2 ml of chloroform by gentle heating if necessary. 20 ml of 0.01 mol / L sulfuric acid titrant was accurately added, and the mixture was heated in a water bath to remove the chloroform. After cooling, 1-2 drops of methyl red indicator were added, and titration was performed with 0.02 mol / L sodium hydroxide titrant. Current methods involve complex sample processing steps, multiple extraction and desorption steps, are time-consuming, and have low specificity. Therefore, there is an urgent need to develop a rapid sample processing device and analytical detection method.

[0003] Belladonna grass is processed into a sample solution by hydrolysis with plant extract enzymes in a reactor. The reactor allows for the stable and rapid dissolution of the active ingredients of belladonna grass, avoiding incomplete extraction and degradation of the active ingredients, and forming a test solution. Using hyoscyamine sulfate as a control, the content can be rapidly determined by high performance liquid chromatography. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid method for determining the content of belladonna grass in order to solve the above-mentioned problems, thereby improving the existing methods for determining the content of belladonna grass by addressing issues such as cumbersome processing steps, long processing time, and low extraction efficiency.

[0005] In a first aspect, the present invention achieves the above-mentioned objective through the following technical solution: a rapid determination method for belladonna content, wherein the sample processing device used in the method includes a vessel body, a sealing cover is installed on the top of the vessel body, a processing device is installed on the top of the sealing cover, one end of the processing device penetrates the sealing cover and extends into the interior of the vessel body; wherein the processing device includes a stirring mechanism installed on the top of the sealing cover, one end of the stirring mechanism penetrates the sealing cover and extends into the interior of the vessel body, a heating mechanism disposed inside the sealing cover is installed on the surface of the stirring mechanism, one end of the heating mechanism penetrates the sealing cover and extends into the interior of the vessel body, and a limiting mechanism disposed inside the interior of the vessel body is installed on the surface of the stirring mechanism, one end of the limiting mechanism penetrates into the exterior of the sealing cover.

[0006] Preferably, the stirring mechanism includes a stirrer and a drive motor, both of which are rotatably connected to the inside of the sealing cover. The other end of the stirrer penetrates the sealing cover and extends to the inner side of the vessel body. One end of the drive motor penetrates to the outside of the sealing cover and is fixedly connected to it. A first gear is fixedly connected to both the output shaft of the drive motor and the surface of the stirrer, with two first gears meshing together. A liquid guiding channel is provided at the other end of the stirrer. The heating mechanism includes a liquid storage box and a ring frame. The liquid storage box is embedded inside the vessel body. A smart temperature-controlled heater is fixedly connected inside the liquid storage box. One end of the smart temperature-controlled heater extends to the outside of the liquid storage box. One end of the liquid storage box is fixedly connected to and connected to a liquid pump. The other end of the liquid pump is fixedly connected to and connected to a first liquid guiding pipe embedded inside the vessel body. The other end of the first liquid guiding pipe is connected to a second liquid guiding pipe embedded inside the sealing cover. The other end of the second liquid guiding pipe is fixedly connected to and connected to a rotating mechanism fixedly connected to the stirrer. The joint has one end connected to one opening of the liquid guiding channel. The ring frame is rotatably connected to the surface of the stirrer. The other opening of the liquid guiding channel is located inside the ring frame. A third liquid guiding pipe is fixedly connected and connected to the surface of the ring frame. The other end of the third liquid guiding pipe is connected to a fourth liquid guiding pipe embedded in the interior of the vessel. The other end of the fourth liquid guiding pipe is fixedly connected and connected to the storage box. Through the cooperation of the liquid guiding channel and the heating mechanism, the pump can deliver the heated liquid medium into the liquid guiding channel through the first liquid guiding pipe, the second liquid guiding pipe and the rotary joint. At this time, the liquid medium heats the stirrer through the liquid guiding channel, causing the stirrer to heat up rapidly. This allows the liquid medium to heat the mixture through both the inner wall of the vessel and the stirrer, so that all parts in contact with the mixture can heat the mixture, thereby improving the heating efficiency of the mixture. At the same time, this also avoids the stirrer absorbing the temperature of the mixture inside the vessel due to the low temperature at the beginning of operation, which delays the probability of the mixture heating up to the specified temperature.

[0007] Preferably, a first sealed bearing is fixedly connected to the surface of the rotary joint and embedded in the inner wall of the liquid guiding channel. A second sealed bearing is embedded in both the inner top wall and the inner bottom wall of the ring frame. The inner edge of the second sealed bearing is sleeved and fixedly connected to the surface of the stirrer. This can reduce the probability of liquid medium leaking through the gap between the rotary joint and the liquid guiding channel or the gap between the stirrer and the ring frame. This not only reduces the waste of liquid medium, but also reduces the probability of liquid medium contaminating or corroding the surrounding structure.

[0008] Preferably, the first liquid guide tube and the second liquid guide tube, and the third liquid guide tube and the fourth liquid guide tube, are all fixedly connected and communicated by connecting components. These connecting components are embedded between the vessel body and the sealing cap. One of the connecting components includes two interconnected mounting cylinders. The other end of one mounting cylinder is fixedly connected and communicates with the other end of the first liquid guide tube, and the other end of the other mounting cylinder is fixedly connected and communicates with the other end of the second liquid guide tube. Each of the two mounting cylinders has an intersecting first mounting cavity at its opposite ends. An opening and closing module is installed on the inner wall of the first mounting cavity. One end of the opening and closing module passes through the first mounting cavity and is rotatably connected to the inner wall of the mounting cylinder. The opening and closing module includes a first connecting shaft rotatably connected to the inner wall of the first mounting cavity. The other end of the first connecting shaft passes through the first mounting cavity and is rotatably connected to the inner wall of the adjacent mounting cylinder. A first sealing plate is fixedly connected to the surface of the first connecting shaft. The vertical cross-sectional shape of the first sealing plate is a circle matching the inner wall of the mounting cylinder. The end face of the first sealing plate is at this time connected to the stirrer. The axes are perpendicular to each other. A second gear is fixedly connected to the surface of the first connecting shaft and disposed inside the first mounting cavity. A rack slidably connected to the inner wall of the first mounting cavity is meshed with the surface of the second gear. One end of the rack passes through the first mounting cavity and contacts the adjacent mounting cylinder. The rack drives the second gear to rotate within an effective range of ninety degrees. The other end of the rack is fixedly connected to a spring fixedly connected to the first mounting cavity. The spring is in a compressed state. By setting the connecting components, when the operator separates the vessel body from the sealing cap, the opening and closing module can automatically close the mounting cylinder. This can prevent the liquid medium inside the mounting cylinder from leaking, which can not only reduce the waste of liquid medium, but also reduce the probability of liquid medium contaminating the mixture or the external environment, so as to ensure the effect of subsequent test data. When the operator connects the vessel body and the sealing cap respectively, the mounting cylinder can open the corresponding mounting cylinder in cooperation with the corresponding opening and closing module, so that the two adjacent mounting cylinders always remain unobstructed and interconnected, thereby ensuring the normal flow of liquid medium.

[0009] Preferably, the other end of the rack has a placement cavity, and one end of the spring passes through the interior of the placement cavity and is fixedly connected to the inner wall of the placement cavity. This can prevent the spring from being over-compressed and reduce the probability of spring damage.

[0010] Preferably, a limiting cavity is formed on the inner wall of the mounting cylinder away from the vessel body. The vertical cross-sectional shape of the limiting cavity is fan-shaped, with the arc-shaped inner wall of the limiting cavity near the first liquid guide tube facing upward and the arc-shaped inner wall of the limiting cavity near the third liquid guide tube facing downward. The limiting cavity is located between two adjacent first mounting cavities. Two second mounting cavities, both communicating with the limiting cavity, are formed inside the mounting cylinder away from the vessel body. A sealing module is installed on the inner wall of the limiting cavity, with both ends of the sealing module penetrating between the two second mounting cavities. The sealing module includes a second sealing element rotatably connected to the inner wall of the limiting cavity. The second sealing plate has a second connecting shaft fixedly connected to both ends. The other end of the second connecting shaft passes through the interior of the second mounting cavity and is rotatably connected to the second mounting cavity. A spring fixedly connected to the second mounting cavity is fixedly connected to the surface of the second connecting shaft. The spring is always in a compressed state. By setting the sealing module, when the liquid medium is not flowing, the spring can drive the second sealing plate through the second connecting shaft to seal the corresponding mounting cylinder, thus preventing the flow of liquid medium inside the second or third liquid guide tube in advance, further reducing the probability of liquid medium leakage when the vessel body and the sealing cap are separated.

[0011] Preferably, the second connecting shaft intersects with the inner side of the mounting cylinder, and the second sealing plate can rotate at a 90-degree angle inside the limiting cavity, which ensures that the sealing module can play a normal sealing role.

[0012] Preferably, the limiting mechanism includes an electric hydraulic cylinder fixedly connected to the other end of the sealing cover. One end of the electric hydraulic cylinder passes through the sealing cover and extends to the inner side of the vessel body. A rotating ring is fixedly connected to one end of the electric hydraulic cylinder. A heat insulation frame sleeved on the surface of the stirrer is rotatably connected to the surface of the rotating ring. By setting the limiting mechanism, the operator can use the electric hydraulic cylinder to move the heat insulation frame as needed. During the normal operation of the subsequent hydrolysis reaction, the stirrer drives the heat insulation frame to rotate inside the vessel body. At this time, the heat insulation frame can not only prevent the heat of the mixture from dissipating upwards, but also prevent the mixture lifted by the stirrer, so as to avoid some of the mixture adhering to the high inner wall of the vessel body, thereby affecting the subsequent hydrolysis reaction rate.

[0013] Preferably, a silicone sleeve is fixedly connected to the surface of the heat insulation frame, and the surface of the silicone sleeve is in contact with the stirrer. This can fill the gap between the heat insulation frame and the stirrer to improve the heat insulation effect of the heat insulation frame.

[0014] Secondly, this invention provides a rapid method for determining the content of belladonna. In addition to the apparatus described above, it also includes a method for determining the content of the active ingredient (hyoscyamine) in belladonna hydrolyzed by plant enzymes.

[0015] The content of hyoscyamine in belladonna samples was determined by high performance liquid chromatography (HPLC). The chromatographic column was a C18 reversed-phase column, and the mobile phase was a mixed solution of acetonitrile and 0.25% sodium dodecyl sulfate in 0.004% phosphoric acid.

[0016] Preferably, the volume ratio of the mobile phase, consisting of acetonitrile and 0.25% sodium dodecyl sulfonate in 0.004% phosphoric acid solution, is 40:60.

[0017] In an optional implementation, the reference standard used in the high-performance liquid chromatography (HPLC) method is hyoscyamine sulfate; the concentration of the reference standard used in the HPLC method is 0.24-0.26 mg / ml.

[0018] In an optional embodiment, before detection using the high performance liquid chromatography method, the belladonna sample is hydrolyzed with plant enzymes to form a test solution.

[0019] Preferably, the plant extract enzyme hydrolysate is prepared by weighing 0.2g of plant extract enzyme, stirring thoroughly, adding 600ml of purified water, and adjusting the pH of the solution to 4.0-5.0 with 10% hydrochloric acid, preferably 4.5.

[0020] Preferably, the preparation steps of the test solution include: accurately weighing 3g of belladonna powder, accurately measuring 50ml of plant enzyme hydrolysate and adding it to the belladonna powder, hydrolyzing for 1 hour, and filtering to obtain the test solution.

[0021] The main active ingredient in belladonna is hyoscyamine. Hyoscyamine sulfate was used as a reference standard. After the cell walls of belladonna were hydrolyzed by plant extract enzymes, the active ingredient was fully dissolved and prepared into a solution, which was then compared with the reference standard to ensure the accuracy of the test results.

[0022] After the test was completed, the chromatogram was analyzed. The test sample showed a chromatographic peak with the same retention time as the reference standard of hyoscyamine sulfate. The results were compared with those of the original detection method. The deviation of the detection result of hyoscyamine sulfate content was within 0.02%.

[0023] The beneficial effects of this invention are:

[0024] 1. By using the liquid guiding channel in conjunction with the heating mechanism, the infusion pump can deliver the heated liquid medium into the liquid guiding channel through the first liquid guiding pipe, the second liquid guiding pipe and the rotary joint. At this time, the liquid medium heats the stirrer through the liquid guiding channel, causing the stirrer to heat up rapidly. This allows the liquid medium to heat the mixture through both the inner wall of the vessel and the stirrer, so that all parts in contact with the mixture can be heated, thereby improving the heating efficiency of the mixture. At the same time, this also avoids the stirrer absorbing the temperature of the mixture inside the vessel due to the low temperature at the beginning of operation, which delays the probability of the mixture heating up to the specified temperature.

[0025] 2. By setting up a connecting component, when the operator separates the vessel body from the sealing cap, the opening and closing module can automatically close the installation cylinder. This can prevent the leakage of liquid medium inside the installation cylinder, which can not only reduce the waste of liquid medium, but also reduce the probability of liquid medium contaminating the mixture or the external environment, so as to ensure the effect of subsequent test data. When the operator connects the vessel body to the sealing cap, the installation cylinder can open the corresponding installation cylinder in conjunction with the corresponding opening and closing module, so that the two adjacent installation cylinders always remain unobstructed and interconnected, thereby ensuring the normal flow of liquid medium.

[0026] 3. By setting a limiting mechanism, the staff can use an electric hydraulic cylinder to move the heat insulation frame as needed. During the normal operation of the subsequent hydrolysis reaction, the stirrer drives the heat insulation frame to rotate inside the vessel. At this time, the heat insulation frame can not only prevent the heat of the mixture from dissipating upwards, but also prevent the mixture from being lifted by the stirrer, so as to avoid some of the mixture adhering to the high part of the inner wall of the vessel, thereby affecting the subsequent hydrolysis reaction rate.

[0027] 4. This invention enables the stable and rapid dissolution of active ingredients in belladonna. Current methods use titration to detect the content of hyoscyamine in belladonna, which is not specific and time-consuming. The main active ingredient of belladonna fluid extract is hyoscyamine. As belladonna is the raw material for belladonna fluid extract, using hyoscyamine as an indicator component for control is more in line with quality control requirements. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of the present invention;

[0029] Figure 2 This is a cross-sectional schematic diagram of the present invention;

[0030] Figure 3 This is a schematic diagram of the processing device in this invention;

[0031] Figure 4 This is a schematic diagram showing the connection between the stirring mechanism and the limiting mechanism in this invention;

[0032] Figure 5 This is a partial cutaway schematic diagram showing the connection between the stirring mechanism and the heating mechanism in this invention;

[0033] Figure 6 This is an exploded view of a partial section of the heating mechanism in this invention;

[0034] Figure 7 This is a horizontal cross-sectional view of a partial section of the connecting component in this invention;

[0035] Figure 8 This is an exploded view of the opening and closing module in this invention;

[0036] Figure 9 This is a vertical cross-sectional view of a partial section of the connecting component structure in this invention;

[0037] Figure 10 The chromatogram for the detection of hyoscyamine sulfate reference standard;

[0038] Figure 11 The chromatogram of the test sample provided in Example 1;

[0039] Figure 12 This is the chromatogram of the negative sample detection provided in Example 1.

[0040] In the diagram: 1. Kettle body; 2. Sealing cap; 3. Processing device; 4. Stirring mechanism; 401. Stirrer; 402. Drive motor; 403. First gear; 404. Liquid guiding channel; 5. Heating mechanism; 501. Liquid storage box; 502. Intelligent temperature-controlled heater; 503. Infusion pump; 504. First liquid guiding pipe; 505. Second liquid guiding pipe; 506. Rotary joint; 507. Ring frame; 508. Third liquid guiding pipe; 509. Fourth liquid guiding pipe; 510. First sealed bearing; 511. Second sealed bearing; 512. Connecting assembly; 5121. Anchor. 5122, First mounting cavity; 5123, Opening and closing module; 51231, First connecting shaft; 51232, First sealing plate; 51233, Second gear; 51234, Rack; 51235, Spring; 51236, Placement cavity; 5124, Limiting cavity; 5125, Second mounting cavity; 5126, Sealing module; 51261, Second sealing plate; 51262, Second connecting shaft; 51263, Spring; 6, Limiting mechanism; 601, Electric hydraulic cylinder; 602, Rotary ring; 603, Heat insulation frame; 604, Silicone sleeve. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] In practical implementation: such as Figure 1-9As shown, a rapid method for determining belladonna content includes a vessel body 1, a sealing cover 2 installed on the top of the vessel body 1, and a processing device 3 installed on the top of the sealing cover 2. One end of the processing device 3 penetrates the sealing cover 2 and extends into the interior of the vessel body 1. The processing device 3 includes a stirring mechanism 4 installed on the top of the sealing cover 2, one end of the stirring mechanism 4 penetrating the sealing cover 2 and extending into the interior of the vessel body 1. A heating mechanism 5 disposed inside the sealing cover 2 is installed on the surface of the stirring mechanism 4, one end of the heating mechanism 5 penetrating the sealing cover 2 and extending into the interior of the vessel body 1. A limiting mechanism 6 disposed inside the vessel body 1 is installed on the surface of the stirring mechanism 4, one end of the limiting mechanism 6 penetrating to the outside of the sealing cover 2.

[0043] like Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 9As shown, the stirring mechanism 4 includes a stirrer 401 and a drive motor 402. Both the stirrer 401 and the drive motor 402 are rotatably connected to the inside of the sealing cover 2. The other end of the stirrer 401 penetrates the sealing cover 2 and extends to the inner side of the vessel body 1. One end of the drive motor 402 penetrates to the outside of the sealing cover 2 and is fixedly connected to it. First gears 403 are fixedly connected to both the output shaft of the drive motor 402 and the surface of the stirrer 401. Two first gears 403 mesh together. A liquid guiding channel 404 is provided at the other end of the stirrer 401. The heating machine... Structure 5 includes a liquid storage box 501 and a ring frame 507. The liquid storage box 501 is embedded inside the vessel body 1. An intelligent temperature-controlled heater 502 is fixedly connected inside the liquid storage box 501. One end of the intelligent temperature-controlled heater 502 extends to the outside of the liquid storage box 501. One end of the liquid storage box 501 is fixedly connected to and communicates with an infusion pump 503. The other end of the infusion pump 503 is fixedly connected to and communicates with a first liquid guide pipe 504 embedded inside the vessel body 1. The other end of the first liquid guide pipe 504 communicates with a second liquid guide pipe 505 embedded inside the sealing cap 2. The other end of 05 is fixedly connected to and communicates with a rotary joint 506 fixedly connected to the stirrer 401. The other end of the rotary joint 506 communicates with one opening of the liquid guiding channel 404. The ring frame 507 is rotatably connected to the surface of the stirrer 401. The other opening of the liquid guiding channel 404 is located inside the ring frame 507. The surface of the ring frame 507 is fixedly connected to and communicates with a third liquid guiding pipe 508. The other end of the third liquid guiding pipe 508 communicates with a fourth liquid guiding pipe 509 embedded in the inside of the vessel body 1. The other end of the fourth liquid guiding pipe 509 is fixedly connected to the liquid storage box 501. The internal structure of the liquid storage box 501 is filled with enough liquid medium to completely fill the first liquid guide tube 504, the second liquid guide tube 505, the liquid guide channel 404, the third liquid guide tube 508, and the fourth liquid guide tube 509. The specific material of the liquid medium needs to be selected according to the actual situation and will not be elaborated here. The surface of the rotary joint 506 is fixedly connected to a first sealing bearing 510 embedded in the inner wall of the liquid guide channel 404. The inner top wall and inner bottom wall of the ring frame 507 are both embedded with second sealing bearings 511. The inner edge of the second sealing bearing 511 is sleeved and fixedly connected to the surface of the stirrer 401.

[0044] like Figure 6 , Figure 7 , Figure 8 and Figure 9As shown, the first liquid guide tube 504 and the second liquid guide tube 505, and the third liquid guide tube 508 and the fourth liquid guide tube 509 are all fixedly connected and communicated by a connecting assembly 512. The connecting assembly 512 is embedded between the vessel body 1 and the sealing cover 2. One of the connecting assemblies 512 includes two interconnected mounting cylinders 5121. The other end of one mounting cylinder 5121 is fixedly connected and communicates with the other end of the first liquid guide tube 504, and the other end of the other mounting cylinder 5121 is fixedly connected to one end of the second liquid guide tube 505. The two mounting cylinders 5121 are fixedly connected and interconnected. Each of the two mounting cylinders 5121 has an intersecting first mounting cavity 5122 at its opposite ends. An opening / closing module 5123 is installed on the inner wall of each first mounting cavity 5122. One end of the opening / closing module 5123 passes through the first mounting cavity 5122 and is rotatably connected to the inner wall of the mounting cylinder 5121. The opening / closing module 5123 includes a first connecting shaft 51231 rotatably connected to the inner wall of the first mounting cavity 5122. The other end of the first connecting shaft 51231 passes through the first mounting cavity 5122 and is connected to the adjacent mounting cylinder 5121. The inner wall of the cylinder 5121 is rotatably connected. A first sealing plate 51232 is fixedly connected to the surface of the first connecting shaft 51231. The vertical cross-sectional shape of the first sealing plate 51232 is a circle that matches the inner wall of the mounting cylinder 5121. The end face of the first sealing plate 51232 is perpendicular to the axis of the mounting cylinder 5121. A second gear 51233, which is disposed inside the first mounting cavity 5122, is fixedly connected to the surface of the first connecting shaft 51231. The surface of the second gear 51233 is engaged with a sliding connection to the first mounting cavity 5122. A rack 51234 is attached to the inner wall of cavity 5122. One end of the rack 51234 passes through the first mounting cavity 5122 and contacts the adjacent mounting cylinder 5121. The effective rotation range of the rack 51234 driving the second gear 51233 is 90 degrees. (Both opposite ends of the two adjacent mounting cylinders 5121 are fitted with sealing rings, which are made of fluororubber material.) The other end of the rack 51234 is fixedly connected to a spring 51235, which is fixedly connected to the first mounting cavity 5122. The spring 51235 is in a compressed state at this time.

[0045] like Figure 6 and Figure 7As shown, the other end of the rack 51234 has a placement cavity 51236. One end of the spring 51235 passes through the interior of the placement cavity 51236 and is fixedly connected to the inner wall of the placement cavity 51236. When the operator connects the vessel body 1 and the sealing cap 2, the two adjacent mounting cylinders 5121 move closer to each other. The mounting cylinders 5121 respectively press the corresponding rack 51234. The rack 51234 drives the second gear 51233 to rotate 90 degrees. The second gear 51233 drives the first connecting shaft 51231 to rotate 90 degrees. The first connecting shaft 51231 drives the first sealing plate 51232 to rotate 90 degrees. At this point, the first sealing plate 51232 rotates from its initial state parallel to the stirrer 401 to a state perpendicular to the stirrer 401. At this time, the inner wall of the mounting cylinder 5121 is no longer obstructed, and the two mounting cylinders 5121 are connected to each other. When the operator separates the vessel body 1 from the sealing cover 2, the vessel body 1 and the sealing cover 2 respectively drive the connected mounting cylinders 5121 to move. At this time, the distance between two adjacent mounting cylinders 5121 gradually increases, the rack 51234 also loses resistance, the spring 51235 pushes the rack 51234 back, and the rack 51234 drives the second gear 51233 to rotate 90 degrees. The second gear 51233 then... The first connecting shaft 51231 rotates 90 degrees, causing the first sealing plate 51232 to rotate 90 degrees. At this time, the first sealing plate 51232 is perpendicular to the axis of the stirrer 401, and it also seals the mounting cylinder 5121. This prevents the liquid medium inside the mounting cylinder 5121 from seeping out, reducing waste of the liquid medium and the probability of contaminating the mixture or the external environment, thus ensuring the accuracy of subsequent test data. A limiting cavity 51 is formed on the inner wall of the mounting cylinder 5121 away from the vessel body 1. 24. The vertical cross-sectional shape of the limiting cavity 5124 is fan-shaped. The inner arc wall of the limiting cavity 5124 near the first liquid guide pipe 504 faces upward, and the inner arc wall of the limiting cavity 5124 near the third liquid guide pipe 508 faces downward. The limiting cavity 5124 is located between two adjacent first mounting cavities 5122. Two second mounting cavities 5125 are opened inside the mounting cylinder 5121 away from the vessel body 1, and both are connected to the limiting cavity 5124. A sealing module 5126 is installed on the inner wall of the limiting cavity 5124. The two ends of the sealing module 5126 pass through the two second mounting cavities 5125 respectively.The sealing module 5126 includes a second sealing plate 51261 rotatably connected to the inner wall of the limiting cavity 5124. Both ends of the second sealing plate 51261 are fixedly connected to a second connecting shaft 51262. The other end of the second connecting shaft 51262 extends into the interior of the second mounting cavity 5125 and is rotatably connected to the second mounting cavity 5125. A spring 51263 is fixedly connected to the surface of the second connecting shaft 51262 and is fixedly connected to the second mounting cavity 5125. The spring 51263 is always in a compressed state. During normal flow of the liquid medium, the liquid medium impacts the second sealing plate 51261, causing the second sealing plate to... When plate 51261 loses its obstruction to the inner wall of mounting cylinder 5121, the liquid medium can pass through mounting cylinder 5121 normally. When the liquid medium stops flowing, the second sealing plate 51261 loses its resistance, the spring 51263 loses its resistance, and the spring 51263 drives the second connecting shaft 51262 to rotate. The second connecting shaft 51262 drives the second sealing plate 51261 to rotate and contact the inner wall of the limiting cavity 5124, resealing the mounting cylinder 5121. The second connecting shaft 51262 intersects with the inner side of the mounting cylinder 5121, and the second sealing plate 51261 can rotate 90 degrees inside the limiting cavity 5124.

[0046] like Figure 3 and Figure 4 As shown, the limiting mechanism 6 includes an electric hydraulic cylinder 601 fixedly connected to the other end of the sealing cover 2. One end of the electric hydraulic cylinder 601 penetrates the sealing cover 2 and extends to the inner side of the vessel body 1. A rotating ring 602 is fixedly connected to one end of the electric hydraulic cylinder 601. A heat insulation frame 603, sleeved on the surface of the stirrer 401, is rotatably connected to the surface of the rotating ring 602. A silicone sleeve 604 is fixedly sleeved on the surface of the heat insulation frame 603. The surface of the silicone sleeve 604 contacts the stirrer 401. During normal operation of the device, the stirrer 401 drives the heat insulation frame 603 to... The reactor body 1 rotates inside. At this time, the heat insulation frame 603 can not only prevent the heat of the mixture from dissipating upwards, but also block the mixture that is lifted by the stirrer 401, so as to avoid some of the mixture adhering to the inner wall of the reactor body 1, thereby affecting the subsequent hydrolysis reaction rate. At the same time, the operator can adjust the length of the electric hydraulic cylinder 601 according to the volume of the mixture. The electric hydraulic cylinder 601 drives the rotating ring 602 to rise and fall, and the rotating ring 602 drives the heat insulation frame 603 to rise and fall along the stirrer 401, adjusting the distance between the heat insulation frame 603 and the mixture to a suitable position.

[0047] In use, after adding the mixture of crushed belladonna, buffer solution, and cellulase into the vessel body 1, the operator adjusts the intelligent temperature-controlled heater 502 to the power required for the experiment. Then, the intelligent temperature-controlled heater 502, the infusion pump 503, and the drive motor 402 are simultaneously turned on. The drive motor 402 drives the stirrer 401 to rotate via the first gear 403. The stirrer 401 rotates and stirs the mixture inside the vessel body 1 to accelerate the hydrolysis reaction. Simultaneously, the intelligent temperature-controlled heater 502 heats the liquid medium inside the storage box 501 to the appropriate temperature. The infusion pump 503 delivers the heated liquid matrix into the first liquid guide pipe 504. During the flow of the liquid medium through the first liquid guide pipe 504, the inner wall of the vessel body 1 is heated, and the inner wall of the vessel body 1 heats the mixture inside the vessel body 1 to accelerate the hydrolysis reaction. The liquid medium flows along the first liquid guide pipe 504 into the second liquid guide pipe 505, which then... The liquid medium is fed into the rotary joint 506, which then feeds the liquid medium into the liquid guiding channel 404. At this time, the liquid medium heats the stirrer 401 through the liquid guiding channel 404, causing the stirrer 401 to heat up rapidly. This allows the liquid medium to heat the mixture through both the inner wall of the vessel 1 and the stirrer 401, ensuring that all parts in contact with the mixture can be heated, thereby improving the heating efficiency of the mixture. At the same time, this also avoids the stirrer 401 absorbing the temperature of the mixture inside the vessel 1 due to its low initial temperature, which delays the probability of the mixture heating up to the specified temperature. After the liquid medium flows out along the liquid guiding channel 404, the ring frame 507 guides the liquid medium into the third liquid guiding pipe 508, the third liquid guiding pipe 508 guides the liquid medium into the fourth liquid guiding pipe 509, and the fourth liquid guiding pipe 509 guides the liquid medium into the storage box 501, enabling the intelligent temperature-controlled heater 502 to circulate and heat the liquid medium.

[0048] It should be noted that the drive motor 402, intelligent temperature control heater 502, infusion pump 503, and electric hydraulic cylinder 601 mentioned above are all devices with relatively mature existing technology. The specific models can be selected according to actual needs. At the same time, the drive motor 402, intelligent temperature control heater 502, infusion pump 503, and electric hydraulic cylinder 601 can be powered by the built-in power supply or by the mains power supply. The specific power supply method is selected according to the situation and will not be elaborated here.

[0049] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0050] The features and performance of this invention patent will be further described in detail below with reference to the above-described implementation technical solutions:

[0051] Given that existing methods for determining belladonna content are cumbersome, have low specificity, and are time-consuming, this invention provides a rapid method for determining belladonna content, comprising: using high-performance liquid chromatography (HPLC) to rapidly detect the content of hyoscyamine sulfate in belladonna. This invention's detection method has high sensitivity, strong specificity, and is easy to operate. It also has good reproducibility and durability, avoiding chemical harm to analysts.

[0052] Example 1

[0053] Preparation of reference solution: Accurately weigh hyoscyamine sulfate reference standard (provided by China National Institutes for Food and Drug Control, batch number: 111982-202103), add 50% methanol (analytical grade) to prepare solutions containing 0.25 mg of hyoscyamine sulfate per 1 ml, thus obtaining hyoscyamine sulfate reference solutions.

[0054] Preparation of the test solution: Accurately weigh more than 3g of pulverized belladonna (from Jiangxi province), hydrolyze with 50ml of cellulase for 1 hour, filter, and collect the filtrate.

[0055] Preparation of negative control solution: The negative control solution without belladonna grass was prepared according to the above preparation method of the test sample.

[0056] Chromatographic conditions for the determination of hyoscine sulfate content: Octadecylsilane-bonded silica gel was used as the stationary phase, and acetonitrile-0.25% sodium dodecyl sulfate in 0.004% phosphoric acid solution (40:60) was used as the mobile phase; the detection wavelength was 210 nm. The theoretical plate number, calculated based on the hyoscine sulfate peak, should not be less than 2000. The injection volume was 10 μL. Acetonitrile was of chromatographic grade, water was purified water, and sodium dodecyl sulfate and phosphoric acid were of analytical grade.

[0057] Assay method: Take the reference solution, test solution, and negative control solution separately and determine according to the above method.

[0058] See test results Figures 10-12 , Figure 10 This is the chromatogram for the detection of hyoscyamine sulfate reference standard. Figure 11 To detect the chromatogram of the test sample, Figure 12 This is the chromatogram of a negative sample solution.

[0059] according to Figures 10-12 The test sample solution showed a chromatographic peak with the same retention time as the reference standard hyoscyamine sulfate, and the resolution met the requirements. The content was then calculated using an external method based on the peak area of ​​the test chromatogram and the peak area of ​​the reference standard. No chromatographic peak was detected in the negative sample solution at the same relative retention time as the reference standard.

[0060] Investigation of content determination methods

[0061] 1. Linearity Study: Accurately weigh hyoscyamine sulfate reference standard and prepare solutions in 50% methanol at concentrations of 240, 480, 960, 1920, 3840, and 7680 μg / ml. -1 Standard solutions; accurately weigh scopolamine lactone and prepare solutions in 50% methanol at concentrations of 15.3, 30.6, 61.2, 122.4, 244.8, and 489.6 μg / ml. -1 Standard solutions. Inject the above standard solutions under the chromatographic conditions of Example 1. Perform regression analysis with peak area as the ordinate (Y) and standard solution concentration as the abscissa (X), obtaining the regression equations, as shown in Table 1.

[0062] Table 1 Linear range of hyoscyamine sulfate

[0063]

[0064] 2. Precision test: 10 μl of hyoscyamine sulfate reference solution was accurately pipetted and injected 6 times consecutively under the chromatographic conditions of Example 1. The peak area RSD of hyoscyamine sulfate was measured to be 0.38%, indicating that the instrument precision was good.

[0065] 3. Repeatability test: The same sample of belladonna was hydrolyzed with plant extract enzyme to prepare 6 test solutions. The same chromatographic conditions were used, and 10 μl of sample was injected. The relative standard deviation of the solution was calculated based on the sample chromatogram. The RSD value of hyoscyamine sulfate and its content was 0.85%, indicating that the method has good repeatability.

[0066]

[0067] 4. Stability Study: Samples of the same test solution were taken at 0, 0.5, 1, 2, 4, 8, 16, and 24 hours, and analyzed under the chromatographic conditions of Example 1. The RSD value of the peak area of ​​hyoscyamine sulfate was 0.42%, indicating that the test solution was stable within 24 hours.

[0068]

[0069] 5. Accuracy Test: Accurately weigh approximately 12 mg, 15 mg, and 18 mg of hyoscyamine sulfate reference standard into three portions and place them into nine 50 ml volumetric flasks. Prepare nine test solutions of the same concentration from belladonna powder according to the test solution preparation method. Add hyoscyamine sulfate reference standard to every three portions at 80%, 100%, and 120% of the original amount, respectively. Determine the content of hyoscyamine sulfate under the chromatographic conditions of Example 1 and calculate the recovery rate.

[0070] The accuracy results are as follows: Results of the recovery rate determination of hyoscyamine sulfate.

[0071]

[0072] Example 2

[0073] Applicability study of plant-extracted enzymes

[0074] Three batches of belladonna samples from each of five production areas were treated with the aforementioned processing device using plant-extracting enzymes for hydrolysis. The content of hyoscyamine sulfate was determined according to the detection method provided in Example 1. The chromatographic peak areas of 15 batches of belladonna samples from the five production areas were examined, and the peak areas with retention times consistent with hyoscyamine sulfate were calculated. The content was then calculated using the external standard method.

[0075] The test results are as follows:

[0076] belladonna origin batch number Hyoscine sulfate content (%) Jiangxi 20220605 0.25％ Jiangxi 20220607 0.21％ Jiangxi 20220608 0.22％ Xinjiang 20220612 0.19％ Xinjiang 20220615 0.18％ Xinjiang 20220618 0.20％ Hunan 20220619 0.15％ Hunan 20220620 0.17％ Hunan 20220623 0.17％ Shandong 20220710 0.21％ Shandong 20220715 0.23％ Shandong 20220717 0.21％ Hebei 20220801 0.31％ Hebei 20220809 0.28％ Hebei 20220810 0.29％

Claims

1. A rapid method for determining the content of belladonna grass, wherein the method uses a vessel (1) to treat belladonna grass with plant extract enzymes, so that the effective active ingredients in belladonna grass can be stably and rapidly dissolved, characterized in that: A sealing cover (2) is installed on the top of the vessel body (1), and a processing device (3) is installed on the top of the sealing cover (2). One end of the processing device (3) passes through the sealing cover (2) and extends into the interior of the vessel body (1). The processing device (3) includes a stirring mechanism (4) installed on the top of the sealing cover (2). One end of the stirring mechanism (4) passes through the sealing cover (2) and extends to the inside of the vessel body (1). A heating mechanism (5) is installed on the surface of the stirring mechanism (4) and is located inside the sealing cover (2). One end of the heating mechanism (5) passes through the sealing cover (2) and extends to the inside of the vessel body (1). The stirring mechanism (4) includes a stirrer (401) and a drive motor (402). Both the stirrer (401) and the drive motor (402) are rotatably connected to the inside of the sealing cover (2). The other end of the stirrer (401) passes through the sealing cover (2) and extends to the inside of the vessel body (1). One end of the drive motor (402) passes through the outside of the sealing cover (2) and is fixedly connected to the sealing cover (2). The output shaft of the drive motor (402) and the surface of the stirrer (401) are both fixedly connected with a first gear (403). The two first gears (403) are meshed together. The other end of the stirrer (401) is provided with a liquid guiding channel (404). The heating mechanism (5) includes a liquid storage box (501) and a ring frame (507). The liquid storage box (501) is embedded inside the vessel body (1). An intelligent temperature-controlled heater (502) is fixedly connected inside the liquid storage box (501). One end of the intelligent temperature-controlled heater (502) extends to the outside of the liquid storage box (501). One end of the liquid storage box (501) is fixedly connected to and connected to an infusion pump (503). The other end of the infusion pump (503) is fixedly connected to and connected to a first liquid guide pipe (504) embedded inside the vessel body (1). The other end of the first liquid guide pipe (504) is connected to a second liquid guide pipe (505) embedded inside the sealing cap (2). The other end of the liquid guide tube (505) is fixedly connected to and communicates with a rotary joint (506) fixedly connected to the stirrer (401). The other end of the rotary joint (506) is communicated with one of the openings of the liquid guide channel (404). The ring frame (507) is rotatably connected to the surface of the stirrer (401). The other opening of the liquid guide channel (404) is located on the inner side of the ring frame (507). The surface of the ring frame (507) is fixedly connected to and communicates with a third liquid guide tube (508). The other end of the third liquid guide tube (508) is communicated with a fourth liquid guide tube (509) embedded in the inside of the vessel body (1). The other end of the fourth liquid guide tube (509) is fixedly connected to and communicates with the liquid storage box (501). After the mixture of crushed belladonna grass, buffer solution and cellulase is added into the interior of the vessel body (1), the liquid medium heats the inner wall of the vessel body (1) as it flows through the first liquid guide tube (504). The inner wall of the vessel body (1) heats the mixture inside the vessel body (1) to accelerate the hydrolysis reaction. The liquid medium flows into the second liquid guide tube (505) along the first liquid guide tube (504). The second liquid guide tube (505) delivers the liquid medium into the rotary joint (506). The rotary joint (506) delivers the liquid medium into the liquid guide channel (404). At this time, the liquid medium heats the stirrer (401) through the liquid guide channel (404), causing the stirrer (401) to heat up rapidly.

2. The rapid determination method for belladonna content according to claim 1, characterized in that: The first liquid guide tube (504) and the second liquid guide tube (505) and the third liquid guide tube (508) and the fourth liquid guide tube (509) are fixedly connected and connected by a connecting component (512). The connecting component (512) is embedded between the vessel body (1) and the sealing cover (2). One of the connecting components (512) includes two connected mounting cylinders (5121). The other end of one mounting cylinder (5121) is fixedly connected to and communicates with the other end of the first liquid guide tube (504). The other end of the other mounting cylinder (5121) is fixedly connected to and communicates with one end of the second liquid guide tube (505). The opposite ends of the two mounting cylinders (5121) are provided with staggered first mounting cavities (5122). An opening and closing module (5123) is installed on the inner wall of the first mounting cavity (5122). One end of the opening and closing module (5123) passes through the first mounting cavity (5122) and is rotatably connected to the inner wall of the mounting cylinder (5121). The opening and closing module (5123) includes a first connecting shaft (51231) rotatably connected to the inner wall of the first mounting cavity (5122). The other end of the first connecting shaft (51231) passes through the first mounting cavity (5122) and is rotatably connected to the inner wall of the adjacent mounting cylinder (5121). A first sealing plate (51232) is fixedly connected to the surface of the first connecting shaft (51231). The vertical cross-sectional shape of the first sealing plate (51232) is a circle that matches the inner wall of the mounting cylinder (5121). The end face of the first sealing plate (51232) is flush with the inner wall of the mounting cylinder (5121). The axis of the mounting cylinder (5121) is perpendicular to each other. The surface of the first connecting shaft (51231) is fixedly connected to a second gear (51233) disposed inside the first mounting cavity (5122). The surface of the second gear (51233) is meshed with a rack (51234) that is slidably connected to the inner wall of the first mounting cavity (5122). One end of the rack (51234) passes through the first mounting cavity (5122) and contacts the adjacent mounting cylinder (5121). The effective rotation range of the rack (51234) driving the second gear (51233) is ninety degrees.

3. The rapid determination method for belladonna content according to claim 2, characterized in that: A limiting cavity (5124) is provided on the inner wall of the mounting cylinder (5121) away from the vessel body (1). The vertical cross-sectional shape of the limiting cavity (5124) is fan-shaped. The inner arc of the limiting cavity (5124) near the first liquid guide pipe (504) faces upward, and the inner arc of the limiting cavity (5124) near the third liquid guide pipe (508) faces downward. The limiting cavity (5124) is located between two adjacent first mounting cavities (5122). Two second mounting cavities (5125) are provided inside the mounting cylinder (5121) away from the vessel body (1) and both of them are connected to the limiting cavity (5124). A sealing module (5126) is installed on the inner wall of the limiting cavity (5124). The two ends of the sealing module (5126) respectively penetrate between the two second mounting cavities (5125). The sealing module (5126) includes a second sealing plate (51261) rotatably connected to the inner wall of the limiting cavity (5124). Both ends of the second sealing plate (51261) are fixedly connected to a second connecting shaft (51262). The other end of the second connecting shaft (51262) passes through the interior of the second mounting cavity (5125) and is rotatably connected to the second mounting cavity (5125). A spring (51263) is fixedly connected to the surface of the second connecting shaft (51262) and is fixedly connected to the second mounting cavity (5125). The spring (51263) is always in a compressed state.

4. A rapid method for determining the content of belladonna according to claim 1, comprising a method for detecting hyoscyamine sulfate, characterized in that... The content of hyoscyamine sulfate was calculated based on the chromatogram obtained from high performance liquid chromatography (HPLC). The chromatographic column was a C18 reversed-phase column, and the mobile phase was a mixed solution of acetonitrile and 0.25% sodium dodecyl sulfate in 0.004% phosphoric acid, with a volume ratio of 40:

60.

5. The rapid determination method for belladonna content according to claim 4, characterized in that... The reference standard used in the high performance liquid chromatography (HPLC) method is hyoscyamine sulfate, with a concentration of 0.12-0.15 mg / ml.

6. The rapid determination method for belladonna content according to claim 5, characterized in that, The belladonna grass was treated with plant-extracting enzymes using a processing device, and the belladonna grass sample had to be a dry coarse powder.

7. The rapid method for determining the content of belladonna according to claim 4, characterized in that... Preparation of plant extract enzyme hydrolysate: Weigh 0.2g of plant extract enzyme, stir thoroughly, add 600ml of purified water, and adjust the pH of the solution to 4.5 with 10% hydrochloric acid.

8. The rapid determination method for belladonna content according to claim 4, characterized in that... Based on the chromatogram detected by high performance liquid chromatography, the test sample showed a chromatographic peak with the same retention time as the reference standard hyoscyamine sulfate.

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