Application of mesaconitine in medicine for treating chronic inflammatory pain and extraction method of mesaconitine

Abstract

The invention provides application of mesaconitine in a medicine for treating chronic inflammatory pain and an extraction method of the mesaconitine, and belongs to the technical field of mesaconitine extraction. An acid alcohol-alkali alcohol gradient elution method is adopted for elution and impurity removal, the obtained extract is a mesaconitine monomer component, then column chromatography refining and purification are carried out, mesaconitine with the purity being 95% or above is obtained, the extracted component is single, the preparation quality can be accurately controlled, and pharmacokinetic research and reasonable medication are facilitated. And the new aconitine has a strong effect and provides a new target spot for analgesia.

Description

Technical Field

[0001] This invention relates to the field of neoaconitine extraction technology, and in particular to the application of neoaconitine in drugs for chronic inflammatory pain and its extraction method. Background Technology

[0002] Mesaconine is a C19 diterpenoid alkaloid, and existing research has focused on its cardiovascular activity: in various cardiomyopathy models, mesaconine can improve heart failure and reduce cardiotoxicity.

[0003] The aconitine derivatives obtained from the hydrolysis of aconitine are complex. After component separation and structural identification, the main components are: aconitine, neoaconitine, hypoaconitine, aconitine, sine, 15-hydroxyaconicine, aconicine, and tararaconine. Their structures are as follows:

[0004]

[0005]

[0006]

[0007]

[0008] Patent 202110384047.2 discloses a method for extracting and refining aconitine: Aconite slices are heated under reflux in alkaline water, refined through a macroporous resin, eluted with 10-20% ethanol to remove impurities, the column is acidified, and then desorbed and eluted with an acidic 10-20% ethanol solution to obtain an extract with a combined content of aconitine and 15-hydroxyaconitine greater than 50%. Research has found that this method removes neoaconitine and aconitine during elution with 10-20% ethanol, resulting in the near-total loss of neoaconitine.

[0009] Neoaconitine is present in high amounts in aconitine hydrolysate and has the strongest analgesic activity for chronic inflammatory pain. How to extract it and broaden its application range is a problem that needs to be solved. Summary of the Invention

[0010] In view of this, the purpose of the present invention is to provide an application of neoaconitine in a drug for chronic inflammatory pain.

[0011] To achieve the above objectives, the present invention discloses the following technical solutions:

[0012] The application of a neoaconitine in a chronic inflammatory pain medication, wherein the neoaconitine is used to prepare a chronic inflammatory pain medication.

[0013] Preferably, the chronic inflammatory pain medication contains neoaconitine or its pharmaceutically acceptable salt.

[0014] Preferably, the chronic inflammatory pain includes joint inflammation pain, lumbago, neuropathic pain, sprain or traumatic chronic inflammatory pain, tumor pain or postoperative chronic inflammatory pain; the chronic inflammatory pain medication is any one of tablets, capsules, granules, oral liquids, targeted liposomes, long-acting sustained-release agents, topical gels, sprays or patches.

[0015] Another object of the present invention is to provide a method for extracting aconitine, comprising the following steps:

[0016] (1) Crush raw Aconitum carmichaelii root, add calcium hydroxide and water and decoct for 1-2 hours, then filter; add water to the residue and decoct for 0.5-1 hours, then filter, and combine the two filtrates; the mass ratio of calcium hydroxide to Aconitum carmichaelii root is 1-2:25-50, and the amount of water added at one time is 6-12 times the mass of Aconitum carmichaelii root.

[0017] (2) Pack the AB-8 macroporous resin column into the chromatography column, wash with water, and then load the filtrate from step (1) onto the chromatography column, controlling the flow rate to 2-6 BV / hr;

[0018] (3) Use a combination of acid, alkali and alcohol to remove impurities;

[0019] (4) Desorption was performed by eluting with an ethanol solution containing ammonia, the eluent was collected, concentrated and dried to obtain crude aconitine;

[0020] (5) The crude aconitine is mixed with silica gel or alumina and packed into a column for column chromatography purification to obtain aconitine.

[0021] Preferably, the specific method for elution and impurity removal using the acid, alkali, and alcohol combination method in step (3) is as follows: water, 0.5-1% acetic acid aqueous solution, water, 0.1-1% sodium hydroxide solution, and 5-7.5% ethanol aqueous solution are used as eluents in sequence for elution and impurity removal.

[0022] More preferably, in the elution and impurity removal process described in step (3), the amount of the eluent used is 2-8 times the column volume.

[0023] Preferably, in the ethanol solution containing ammonia in step (4), the mass concentration of ethanol is 10-15% and the mass concentration of ammonia is 0.1-0.3%.

[0024] Preferably, the amount of ethanol solution containing ammonia in step (4) is 3-8 times the column volume.

[0025] Preferably, the mass ratio of the crude aconitine in step (5) to silica gel or alumina is 1:5-20.

[0026] Preferably, the purification method in step (5) is as follows: using ethyl acetate and anhydrous ethanol as the mobile phase, gradient elution is performed to combine the fractions of the target component, recover and dry them to obtain neoacinol;

[0027] Alternatively, first remove impurities with ethyl acetate, then elute with a 1:1 mixture of ethyl acetate and anhydrous ethanol. The eluent is recovered and dried to obtain neoaconitine.

[0028] Alternatively, an alumina column can be used for elution with anhydrous ethanol, and the eluent can be recovered and dried to obtain neoaconitine.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention provides an application of neoaconitine in chronic inflammatory pain medications and its extraction method. An acid-alcohol-alkaline gradient elution method is used to remove impurities, yielding an extract containing the monomeric neoaconitine. This extract is then purified by column chromatography to obtain neoaconitine with a purity of over 95%. Furthermore, this invention extracts a single component, allowing for precise control of formulation quality, facilitating pharmacokinetic studies, and enabling rational drug use. Neoaconitine also exhibits potent activity, providing a new target for analgesia. Attached Figure Description

[0031] Figure 1 The results are the HPLC analysis results of the crude aconitine from Example 1;

[0032] Figure 2 The results are the HPLC analysis results of high-purity aconitine in Example 1;

[0033] Figure 3 The results of HPLC analysis of neoaconitine in Comparative Example 1 are shown.

[0034] Figure 4 The results of HPLC analysis of neoaconitine in Comparative Example 2 are shown.

[0035] Figure 5 The results of HPLC analysis of neoaconitine in Comparative Example 3 are shown.

[0036] Figure 6 The HPLC analysis results of neoaconitine in Comparative Example 4 are shown.

[0037] Figure 7 The results of the heat-induced paw retraction latency in mice in Experiment 5;

[0038] Figure 8 The results represent the 50% mechanical pain threshold of mice in Experiment 5;

[0039] Figure 9 The concentration of TNF-α in the mouse spinal cord was determined by ELISA in Experiment 6.

[0040] Figure 10 The concentration of IL-6 in the mouse spinal cord was determined by ELISA in Experiment 6;

[0041] Figure 11 The concentration of IL-1β in the mouse spinal cord was determined by ELISA in Experiment 6.

[0042] Figure 12 The results of ELISA determination of SP expression in mouse spinal cord in Experiment 6;

[0043] Figure 13 The results of ELISA determination of CGRP expression in mouse spinal cord in Experiment 6;

[0044] Figure 14 In Experiment 6, Nissl staining was used to observe morphological changes in mouse DRG neurons. Detailed Implementation

[0045] The present invention will be further described below with reference to the embodiments.

[0046] Example 1

[0047] A method for extracting a new aconitine, comprising the following steps:

[0048] (1) Take 6 kg of raw Aconitum carmichaelii root and crush it (crushing is a conventional method in this field, crush Aconitum carmichaelii root to less than 2 mm, the same below), add 240 g of calcium hydroxide and 12 times the amount of water of raw Aconitum carmichaelii root, decoct at 100°C for 1 hour, and filter; add 10 times the amount of water of raw Aconitum carmichaelii root to the dregs and decoct for 0.5 hours, filter, and combine the two filtrates;

[0049] (2) Take 2LAB-8 macroporous resin (purchased from Xi’an Lanxiao Technology New Material Co., Ltd.) and pack it into the chromatography column. Wash with water until the effluent does not contain alcohol. Load the filtrate from step (1) onto the chromatography column and control the flow rate to 3 BV / hr.

[0050] (3) Elute with water, 0.5% acetic acid aqueous solution, water, 0.2% sodium hydroxide solution and 6% ethanol solution in sequence to remove impurities (the volume of each eluent used is 6 times the column volume);

[0051] (4) Elute with 8 column volumes of 12% ethanol solution containing 0.2% ammonia, collect the eluent, recover, concentrate and dry to obtain crude aconitine;

[0052] (5) Take 10g of crude aconitine prepared in step (4), grind it into fine powder, add 10g of 200-mesh silica gel, mix well, grind evenly, and pass through a 100-mesh sieve to obtain a mixture of aconitine and silica gel; take 160g of 200-mesh silica gel, and pack the silica gel and mixture into a glass chromatography column with an inner diameter of 5cm in sequence, and perform gradient elution with ethyl acetate: anhydrous ethanol (10:0, 8:2, 7:3, 6.5:3.5, 6:4, 5.5:4.5, 5:5, 1000ml each), collect the eluent in 250ml fractions, detect by HPLC, combine the fractions of one peak of aconitine, recover the solvent, dissolve in water and dispense into 10mL vials, place in a -80℃ low temperature environment for pre-freezing for 24 hours, immediately transfer to a vacuum freeze dryer for freeze drying, collect the sample after 48 hours, grind it into powder, and obtain high-purity aconitine.

[0053] Experiment 1: Component Detection

[0054] The experiment was repeated twice, and the crude aconitine obtained in step (4) of the two experiments was combined. 39.7 g of crude aconitine was obtained, and the yield was calculated to be 0.31%. HPLC analysis showed the following results: Figure 1 The content of neoacinogenase was 37.8%, from... Figure 1 It can be seen that the neoacin peak is the base peak, while the others are small peaks, and the peak areas of the other components are less than 5% of the total area.

[0055] The high-purity neoaconitine obtained in step (5) of Example 1 was weighed to be 1.9 g, and the yield was calculated to be 19%.

[0056] Neoaconitumine was detected by HPLC. The instrument used was a Waterse2695 high-performance liquid chromatograph with an ELSD6000 evaporative light scattering detector. The chromatographic conditions were as follows: column: Hedera ODS-2C18 (4.6 mm × 250 mm, 5 μm); gradient elution was performed using acetonitrile (A) - 0.2% trifluoroacetic acid (B) as the mobile phase. The flow rate for gradient elution was 1 mL / min; the gas flow rate for the evaporative light scattering detector was 3 L / min; and the drift tube temperature was 110 °C. The gradient changes during gradient elution are shown in Table 1.

[0057] Table 1

[0058] Time (min) Mobile phase A (%) Mobile phase B (%) 0 8 92 40 18 82

[0059] Liquid chromatography results as follows Figure 2 As shown, only one peak was detected in the liquid chromatography, and the purity of neoacin was calculated to be 96.1% by the normalization method.

[0060] Experiment 2, Structural Identification:

[0061] The new aconitine prepared in step (5) of Example 1 was vacuumed for 8 hours. 5 mg of the sample was weighed and added to deuterated pyridine to prepare a sample of 10 mg / ml. The sample was then subjected to proton, carbon and two-dimensional spectra.

[0062] Target compound: A white powder after lyophilization. ESI-MS m / z: 486.6 [M+H]+, molecular formula: C 24 H 39 NO.9 1 H-NMR (C5D5N, 500MHz) δ: 2.70 (s,3H,N-CH3), 3.19 (s,3H,1-OCH3), 3.55 (s,3H,6-OCH3), 3.70 (s,3H,16-OCH3), 3.34 (s,3H,18-OCH3); 1 C-NMR (C5D5N, 125MHz) δ: 82.65 (C-1), 34.83 (C-2), 68.86 (C-3), 44.31 (C-4), 45.96 (C-5), 8 3.73 (C-6), 47.56 (C-7), 78.97 (C-8), 50.01 (C-9), 42.11 (C-10), 50.26 (C-11), 38.45 (C-12) The values ​​are: 77.13 (C-13), 79.34 (C-14), 81.94 (C-15), 92.90 (C-16), 63.98 (C-17), 74.70 (C-18), 50.59 (C-19), 42.70 (C-20), 55.30 (1-OCH3), 57.74 (6-OCH3), 60.73 (16-OCH3), and 58.63 (18-OCH3). These data are consistent with literature reports, and the compound is identified as neoaconitine.

[0063] Example 2

[0064] A method for extracting a new aconitine, comprising the following steps:

[0065] Take 10g of crude aconitine from step (4) of Example 1, grind it into a fine powder, mix it with 20g of 200-mesh silica gel, grind it evenly, and pass it through a 100-mesh sieve to obtain aconitine silica gel mixture; take 150g of 200-mesh silica gel, and successively pack the silica gel and the mixture into a glass chromatography column with an inner diameter of 5cm, and elute successively with ethyl acetate and ethyl acetate: anhydrous ethanol (volume ratio of 1:1) 1000ml each, collect the mixed solvent eluent, recover the solvent, dissolve it in water, dispense it into 10mL vials, place it in a -80℃ low temperature environment for pre-freezing for 24 hours, and immediately transfer it to a vacuum freeze dryer for freeze drying. After 48 hours, collect the sample, grind it into powder, and obtain aconitine. Weigh out 3.4g of aconitine, with a yield of 34%, and store it in a sealed container. HPLC analysis showed that the purity of aconitine was 83.5%.

[0066] Example 3

[0067] A method for extracting a new aconitine, comprising the following steps:

[0068] Weigh 10g of the crude aconitine from step (4) of Example 1, grind it into a fine powder, mix it with 20g of 100-200 mesh alumina, grind it evenly, and pass it through a 100-mesh sieve to obtain a mixture of aconitine and alumina. Take 60g of 100-200 mesh alumina, and successively pack the alumina and the mixture into a glass chromatography column with an inner diameter of 5cm. Elute with 1000ml of anhydrous ethanol, collect the eluent, recover and concentrate it, dry it under reduced pressure at 60℃, scrape off the dried product and grind it into a fine powder to obtain 3.8g of aconitine, with a yield of 38%, and store it in a sealed container. HPLC analysis showed that the purity of aconitine was 69.4%.

[0069] Comparative Example 1

[0070] A method for extracting a new aconitine, comprising the following steps:

[0071] Take 6 kg of raw Aconitum carmichaelii root, crush it, add 240 g of calcium hydroxide and 12 times the amount of water to the raw Aconitum carmichaelii root, decoct at 100℃ for 1 hour, and filter. Add 10 times the amount of water to the residue and reflux for 0.5 hours, then combine the filtrates. Load the filtrate onto an AB-8 macroporous resin column (2 L of resin), and elute successively with 8 column volumes of water, 2 column volumes of 0.5% NaOH, 4 column volumes of water, 4 column volumes of 15% ethanol, and 6 column volumes of 0.1% acetic acid aqueous solution to remove impurities. Then elute with 6 column volumes of a mixed acetic acid-ethanol solution (acetic acid concentration of 0.5% and ethanol concentration of 15%) for desorption. Collect the eluent, concentrate under reduced pressure, and vacuum dry to obtain 27.1 g of brown extract, with a yield of 0.45%. HPLC results are as follows. Figure 3 ,Depend on Figure 3 The chromatogram showed that aconitine was the highest peak, while aconitine and 15-hydroxyaconitine were the second highest peaks.

[0072] Comparing the HPLC chromatograms, it can be seen that the extract of Example 1 mainly shows a single peak of neoaconitine, while the chromatographic peak of neoaconitine in the extract of Comparison 1 is extremely low.

[0073] Comparative Example 2

[0074] A method for extracting a new aconitine, comprising the following steps:

[0075] Take 300g of crushed raw Aconitum carmichaelii root, add 18g of calcium hydroxide, place in a 5000ml round-bottom flask, add 12 times the amount of water, heat with a heating mantle for 1 hour, and filter the extract. Then add 10 times the amount of water and extract for half an hour, filter, and combine the two extracts. Take 100ml of AB-8 macroporous resin, pack it into a chromatography column, wash with water until the eluent is free of alcohol, load the filtrate at a flow rate of 4 BV / hr. After loading, elute sequentially with 6 column volumes of water, 0.2% sodium hydroxide solution, water, and 6% ethanol solution to remove impurities, then elute with 8 column volumes of 15% ethanol solution. Collect the eluent, dilute with methanol, and analyze by HPLC as follows: Figure 4 ,from Figure 4 It can be seen that neoaconitine is the highest peak, but there are also the Econicamine peak and other smaller peaks.

[0076] As can be seen from Example 1, desorption using an alkaline 10-15% ethanol solution is required. This avoids the desorption of components of the chromatographic peak after neoaconitine (such as hypoaconitine, aconitine, songguoning, 15-hydroxyacinol, aconitine, etc.) during desorption using neutral or acidic ethanol solutions, which would seriously affect the purity of neoaconitine in the extract.

[0077] Comparative Example 3

[0078] A method for extracting a new aconitine, comprising the following steps:

[0079] Take 300g of crushed raw Aconitum carmichaelii root, add 18g of calcium hydroxide, place in a 5000ml round-bottom flask, add 12 times the amount of water, heat with a heating mantle for 1 hour, and filter the extract. Then add 10 times the amount of water and extract for half an hour, filter, and combine the two extracts. Take 100ml of AB-8 macroporous resin, pack it into a chromatography column, wash with water until the eluent is free of alcohol, load the filtrate at a flow rate of 6 BV / hr. After loading, elute sequentially with 6 column volumes of water, 0.2% sodium hydroxide solution, water, and 6% ethanol solution to remove impurities, then elute with 8 column volumes of ethanol solution containing ammonia (ethanol concentration: 0.1%, ammonia concentration: 0.1%), collect the eluent, and concentrate. Dilute with methanol, and perform HPLC analysis as follows: Figure 5 ,from Figure 5 It can be seen that neoaconitine is the highest peak, but it also contains iconica peak.

[0080] As can be seen from Example 1, if the elution process lacks 0.5% acetic acid aqueous solution, the aconitine component cannot be removed, thus affecting the purification of the single component of neoaconitine.

[0081] Comparative Example 4

[0082] A method for extracting a new aconitine, comprising the following steps:

[0083] Take 300g of crushed raw Aconitum carmichaelii root, add 18g of calcium hydroxide, place in a 5000ml round-bottom flask, add 12 times the amount of water of raw Aconitum carmichaelii root, heat with an electric heating mantle for 1 hour, and filter out the extract; then add 10 times the amount of water of raw Aconitum carmichaelii root, extract for half an hour, filter, and combine the two extracts. Take 100 ml of pretreated AB-8 macroporous resin, pack it into a chromatography column, wash with water until the eluent has no alcohol odor, load the filtrate, control the flow rate, and after loading, elute sequentially with 6 column volumes of water, a mixed acetic acid-ethanol solution (acetic acid concentration of 0.5%, ethanol concentration of 2.5%), water, 0.2% sodium hydroxide solution, water, and 6% ethanol solution to remove impurities. Then elute with 8 column volumes of ammonia-containing ethanol solution (ethanol mass concentrations of 5%, 7.5%, 10%, 12.5%, 15%, and ammonia mass concentration of 0.1%), and a mixed acetic acid-ethanol solution (acetic acid concentration of 0.5%, ethanol concentration of 15%). Collect the eluents for HPLC analysis. The results are as follows: Figure 6 ,from Figure 6 It can be seen that neoaconitine was completely eluted when eluted with 10-12.5% ​​alkaline ethanol, and no neoaconitine component could be detected when eluted with 15% alkaline ethanol solution. However, other components after the peak were not eluted either; but they could be eluted by acidic 15% ethanol solution.

[0084] This indicates that during the elution process, the ethanol concentration should not exceed 7.5%; an alkaline solution of 10-15% ethanol can desorb the alkaloid of Aconitum carmichaelii, but other components after the peak cannot be desorbed; other components can be desorbed by an acidic ethanol solution of 10-15% ethanol.

[0085] Experiment 3: Acetic acid writhing test for analgesia

[0086] Experimental animals: Male ICR mice, weighing 18-22g. Animal housing environment: room temperature 22℃, relative humidity about 50%, free access to water and food, and regular replacement of bedding and cages. The experiment complied with GLP standards, animal ethics number: 202305A011; neoaconitine was the sample prepared in step (5) of Example 1.

[0087] ICR mice were randomly divided into 6 groups (5 experimental groups and 1 control group) according to body weight, with 10 mice in each group. The experimental groups were administered doses of 0.05, 0.1, 0.2, 0.4, and 0.8 mg / kg, respectively, according to the design of the previous acute toxicity experiment. Except for the control group, which was given an equal volume of physiological saline by gavage daily, the other groups were given different doses of neoacinuron by gavage once a day for 7 consecutive days. On the seventh day, an appropriate amount of unopened glacial acetic acid was taken and dissolved in physiological saline to prepare a 0.6% glacial acetic acid solution, which was prepared and used immediately.

[0088] One hour after the end of gavage on the seventh day, mice in each group were injected intraperitoneally with 0.6% glacial acetic acid solution at a dose of 0.1 ml / 10 g. The number of writhing movements (hip lifting, hind limb extension, and abdominal concavity) of each mouse within 15 minutes was observed and recorded. The pain inhibition rate of different doses of neoacinol on acetic acid-induced pain was calculated using the following formula (1):

[0089] (1)

[0090] Statistical analysis was performed using GraphPadPrism 8.0 software, with quantitative data presented in [the format of the software]. This indicates that one-way ANOVA was used for comparisons of multiple groups, and t-tests were used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant. The results are shown in Table 2.

[0091] Table 2 Effects of neoaconitine on the acetic acid writhing test in mice (n=10)

[0092]

[0093] Compared with the blank group, ;

[0094] As shown in Table 2, different concentrations of neoaconitine reduced the number of writhing movements in mice, indicating that neoaconitine can alleviate the writhing response induced by 0.6% acetic acid in mice. Compared with the blank control group, neoaconitine at concentrations of 0.1, 0.2, and 0.4 mg / kg showed good analgesic effects. Therefore, it was finally decided to set up neoaconitine at concentrations of 0.1, 0.2, and 0.4 mg / kg as low, medium, and high concentration groups to study its therapeutic effect on mice with CFA-induced chronic inflammatory pain.

[0095] Experiment 4: Comparison of Neoaconitine and Aconitine:

[0096] The experimental animals were the same as in Experiment 3. ICR mice were randomly divided into 9 groups (1 experimental group, 7 control groups, and 1 blank group, Mod) according to body weight, with 10 mice in each group. The experimental group received aconitine at a dose of 0.2 mg / kg, while the control groups received aconitine at doses of 0.2, 0.4, 0.8, 1.6, and 3.2 mg / kg, or aconitine at 3.2 mg / kg, or aconitine at 3.2 mg / kg. Except for the blank group, which received an equal volume of physiological saline by gavage daily, the other groups received aconitine or different doses of aconitine by gavage once daily for 7 consecutive days. On the seventh day, an appropriate amount of unopened glacial acetic acid was taken and dissolved in physiological saline to prepare a 0.6% glacial acetic acid solution, which was prepared and used immediately.

[0097] One hour after gavage, mice in each group were intraperitoneally injected with 0.6% acetic acid at a dose of 0.1 ml / 10 g. The number of writhing movements (hip lifting, hind limb extension, and abdominal retraction) per mouse within 15 minutes was observed and recorded. The pain inhibition rate of different doses of aconitine compounds on acetic acid-induced pain was calculated (calculation method same as in Experiment 6). The results are shown in Table 3.

[0098] Table 3 Effects of neoaconitine on the acetic acid writhing test in mice ( )

[0099]

[0100] Compared with the control group, Compared with the aconitine group at the same dosage, ### P < 0.001.

[0101] As shown in Table 3, neoaconitine, along with aconitine, acetaminophen, and acetaminophen, reduced the number of writhing movements in mice. However, the neoaconitine group showed a significantly greater alleviating effect than the same dose of aconitine, with statistical significance. The inhibitory rate of neoaconitine was significantly better than that of aconitine at the same dose (0.2 mg / kg), and also better than the highest doses of aconitine, acetaminophen, and acetaminophen at 16 times the dose. Therefore, neoaconitine is more effective in treating inflammatory pain.

[0102] Experiment 5: Effects of neoaconitine on CFA-induced chronic inflammatory pain in mice

[0103] The experimental animals were the same as in Experiment 3. Mice were randomly divided into Con group (negative group), CFA group (model group), Nar group (positive drug group), and low, medium, and high dose neoaconitumine groups according to their body weight. CFA (10 μl) was slowly injected subcutaneously into the left posterior foot of mice in the CFA group, Nar group, and low, medium, and high dose neoaconitumine groups to induce a chronic inflammatory pain model. An equal volume of physiological saline was injected into the left posterior foot of the control group. Two days after modeling, naproxen (naproxen tablets: Nanjing Baijingyu Pharmaceutical Co., Ltd., 221104; dosage: 75 mg / kg) and neoaconitine (0.1, 0.2 and 0.4 mg / kg) were administered to mice in each experimental group. The Con group and CFA group were given the same dose of physiological saline. From day 3 to day 14, the thermal pain threshold and mechanical pain threshold of the mice were measured on days 0, 3, 6, 9 and 13. After the behavioral test was completed on the last day, all mice were sacrificed and samples were collected for further testing.

[0104] Before the mechanical pain test, mice were acclimatized to the test environment for 30 minutes. Mice were placed on a metal grid (100cm × 50cm), and their paw withdrawal threshold was measured using a set of Von Frey filaments (0.04–2g) to assess mechanical aberration pain. The filaments were applied vertically to the plantar surface from the bottom for up to 3 seconds on day 0 before CFA injection and on days 3, 7, 10, and 14 post-injection. The mechanical withdrawal reflex threshold of 50% was determined using a previously reported up-and-down method.

[0105] Before the hot plate test, mice were allowed to acclimatize to the test environment for 30 minutes. Mice were placed on a hot / cold plate analgesia device (IITC Inc. Life Sciences, USA) and an organic plastic box with a transparent lid. Thermal hyperalgesia was assessed on days 0, 3, 7, 10, and 14. The temperature of the metal plate was set to 50°C. Response latency, i.e., the time required to observe noxious behavior (retreat of the hind paw or licking / stomping), was recorded. Each test was repeated three times, and the average value was taken.

[0106] The effects of neoaconitine on the mechanical and thermal pain thresholds in mice with chronic inflammatory pain, such as... Figure 7-8 As shown, Figure 7 This refers to the heat-induced paw retraction latency period in mice. Figure 8 The mechanical pain threshold for mice was 50%.

[0107] Compared with the Con group, ####P<0.001; compared with the CFA group, .

[0108] from Figure 7-8It can be seen that the sensitivity of animals treated with CFA showed a significant upward trend in mechanical stimulation response and hot plate test (####P<0.001). After administration of different doses of aconitine (Mac) and naproxen (Nar), the mechanical pain sensitivity and thermal stimulation tolerance time of mice were significantly improved (P<0.001), and the analgesic effect increased with prolonged administration time. The efficacy of 0.1, 0.2, and 0.4 mg / kg aconitine and Nar was comparable. During the experiment, the mechanical pain and thermal pain thresholds of mice in the control group did not change significantly (P>0.05). The results indicate that aconitine plays a positive role in CFA-induced chronic inflammatory pain in mice and can effectively alleviate CFA-induced mechanical pain and thermal hyperalgesia. The mechanical pain thresholds in the 0.1, 0.2, and 0.4 mg / kg groups all recovered to near baseline on day 14, with no statistically significant difference compared to the naproxen group.

[0109] Experiment 6: Anti-inflammatory effects and mechanisms of neoaconitine

[0110] After euthanizing the mice in Experiment 5, the L4-L6 segment of the spinal cord was immediately removed, homogenized in phosphate-buffered saline (PBS), centrifuged at 15000g for 15 minutes at 4°C, and the supernatant was collected. The concentrations of TNF-α, IL-1β, IL-6, CGRP, and SP in the mouse spinal cord were measured according to the kit instructions.

[0111] The specific method is as follows: After anesthetizing the mice with 10% chloral hydrate, the thoracic cavity is cut open, and PBS is injected into the left ventricle. After the outflowing liquid is colorless, the spinal cord and DRG of the mice at L4-L6 are removed and fixed and preserved in centrifuge tubes containing 4% paraformaldehyde.

[0112] The fixed tissue was then embedded in paraffin and sectioned. The sections were then dehydrated using xylene and ethanol of different concentrations. After dehydration, the tissue was stained with toluidine blue, mounted with neutral resin, and then examined under a microscope.

[0113] (1) Effects of neoaconitine on TNF-α, IL-1β and IL-6 in the spinal cord of mice with chronic inflammatory pain

[0114] After successfully inducing a chronic inflammatory pain model in mice by injecting CFA into the body, from Figure 4It can be seen that, compared with the Con group, the levels of TNF-α, IL-1β, and IL-6 in the spinal cord of mice in the CFA group were significantly increased (P < 0.001), while the levels of TNF-α, IL-1β, and IL-6 in the spinal cord of mice in the Nar group and different concentrations of neoacin showed a significant decreasing trend compared with the CFA group (P < 0.01); the levels of TNF-α, IL-1β, and IL-6 in the spinal cord of the 0.2 mg / kg neoacin group decreased by 62%, 58%, and 55% respectively compared with the CFA group (all P < 0.001). The results indicate that neoacin can inhibit the secretion of pro-inflammatory factors in the spinal cord of mice with CFA-induced chronic inflammatory pain, and has an inhibitory effect on neuroinflammation.

[0115] The effects of neoaconitine on pro-inflammatory factors in the spinal cord of mice with CFA-induced inflammatory pain, such as... Figure 9-11 . Figure 9 To determine the concentration of TNF-α in the spinal cord of mice using ELISA; Figure 10 To determine the concentration of IL-6 in the spinal cord of mice using ELISA; Figure 11 To determine the concentration of IL-1β in the mouse spinal cord using ELISA; (n=6), compared with the Con group, ###P<0.001, compared with the CFA group, .

[0116] (2) Effects of neoaconitine on CGRP and substance P in the spinal cord of mice with chronic inflammatory pain

[0117] Studies have shown that CGRP and substance P often co-occur in the spinal cord, but the mechanism of their interaction remains unclear. Some research indicates that they play an important role in the regulation and transmission of pain signals. Therefore, ELISA kits were used to detect the levels of CGRP and substance P in the mouse spinal cord, and the results are as follows: Figure 12-13 As shown, compared with the Con group, the levels of CGRP and substance P in the spinal cord of CFA group mice were significantly increased (P < 0.001). However, after intervention with Nar and different concentrations of neoaconitine, the levels of CGRP and substance P in the spinal cord were significantly decreased (P < 0.05); the CGRP and SP contents in the spinal cord of the 0.2 mg / kg group decreased by 49% and 53% respectively compared with the CFA group (both P < 0.001). The results indicate that neoaconitine may exert its analgesic effect by inhibiting the secretion of CGRP and substance P in the spinal cord of CFA mice.

[0118] The effects of neoaconitine on CGRP and substance P in the spinal cord of mice with CFA-induced inflammatory pain are shown in Figures 12-13.

[0119] Figure 12 The expression of SP in the mouse spinal cord was determined by ELISA; Figure 13To determine the expression of CGRP in the mouse spinal cord using ELISA; (n=6). Compared with the Con group, ###P<0.001, compared with the CFA group: .

[0120] (3) Effects of neoaconitine on DRG in mice with chronic inflammatory pain

[0121] The dorsal root ganglion (DRG) is formed by the aggregation of cell bodies of primary sensory neurons. It primarily undertakes the integration and transmission of sensory information, playing a crucial role, especially in the reception of nociceptive signals and the transmission of pain, and is closely related to the occurrence and development of acute and chronic pain. Therefore, Nissl staining was performed on the DRGs of mice in each group to investigate the changes in DRG neurons under the intervention of drugs such as CFA injection and neoacin. The results are as follows: Figure 14 As shown, compared with the Con group, the number of Nissl bodies in the neuronal cells of the DRG of mice in the CFA group was significantly reduced. However, after intervention with Nar and different concentrations of neoaconitine, the downward trend of Nissl bodies was reversed. The above results indicate that neoaconitine has a certain neuroprotective effect and can reverse the downward trend of Nissl bodies in mouse DRG caused by CFA.

[0122] The effects of neoacin on the DRG in mice with chronic inflammatory pain were investigated, and morphological changes in mouse DRG neurons were observed using Nissl staining. Figure 14 (Scale bar: 20μm).

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of neosarcracine in a medicament for chronic inflammatory pain, characterized in that, The new protoaconitine is used for preparing a chronic inflammatory pain medicine.

2. The use of neosarpermidine according to claim 1 in the pharmaceutical treatment of chronic inflammatory pain, characterized in that, The chronic inflammatory pain medicine contains new protoaconitine or a pharmaceutically acceptable salt thereof.

3. The use of neosarpermidine according to claim 1 in the pharmaceutical treatment of chronic inflammatory pain, characterized in that, The chronic inflammatory pain includes arthritic pain, lumbar and leg inflammatory pain, neuropathic pain, chronic inflammatory pain caused by sprain or trauma, tumor pain or postoperative chronic inflammatory pain; and the chronic inflammatory pain medicine is any one of a tablet, a capsule, a granule, an oral solution, a targeted liposome, a long-acting sustained-release preparation, an external gel, a spray or a plaster.

4. A method for extracting neosinasapalidine, characterized by, The method comprises the following steps: (1) grinding raw Radix Aconiti Kusnezoffii, adding calcium hydroxide and water to decoct for 1-2 hours, filtering; adding water to the residue to decoct for 0.5-1 hour, filtering, and combining the two filtrates; the mass ratio of calcium hydroxide to Radix Aconiti Kusnezoffii is 1-2:25-50, and the amount of water added at a time is 6-12 times the mass of Radix Aconiti Kusnezoffii; (2) loading an AB-8 macroporous resin column into a chromatographic column, washing with water, and then loading the filtrate in step (1) into the chromatographic column, and controlling the flow rate to be 2-6 BV / hr; (3) performing elution and impurity removal by using an acid, a base and an alcohol in combination; (4) performing desorption by using an ethanol solution containing ammonia water, collecting the eluate, and concentrating and drying to obtain crude new protoaconitine; (5) mixing the crude new protoaconitine with silica gel or alumina, loading into a column, and performing column chromatography to purify and obtain new protoaconitine.

5. The method of claim 4, wherein the new neoline is extracted by the following steps of: The specific method for elution and impurity removal by using an acid, a base and an alcohol in combination in step (3) is as follows: water, 0.5-1% acetic acid aqueous solution, water, 0.1-1% sodium hydroxide solution and 5-7.5% ethanol aqueous solution are sequentially used as eluents for elution and impurity removal. ​ 6. The method of claim 5, wherein the new neoline is extracted by the process of, In the process of elution and impurity removal in step (3), the amount of eluent used at a time is 2-8 times the column volume.

7. The method for extracting neoacin according to claim 4, characterized in that, In the ethanol solution containing ammonia water in step (4), the mass concentration of ethanol is 10-15%, and the mass concentration of ammonia is 0.1-0.3%.

8. The method of claim 4, wherein the new neoline is extracted by the process of, The amount of the ethanol solution containing ammonia water used in step (4) is 3-8 times the column volume.

9. The method of claim 4, wherein the new neoline is extracted by the process of, In step (5), the mass ratio of the crude new protoaconitine to silica gel or alumina is 1:5-20.

10. The method of claim 4, wherein the new neoline is extracted by the process of, The method for purification in step (5) is as follows: ethyl acetate and anhydrous ethanol are used as mobile phases to perform gradient elution and obtain new protoaconitine; or, ethyl acetate is used for impurity removal first, and then a solution of ethyl acetate-anhydrous ethanol with a volume ratio of 1:1 is used for elution to obtain new protoaconitine; or, an alumina column is used for anhydrous ethanol elution to obtain new protoaconitine.

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