Extraction method of total original alkali rich in mesaconitine and application of total original alkali in chronic inflammatory pain medicine

Abstract

The invention provides an extraction method of total original alkali rich in mesaconitine and application of the total original alkali in a chronic inflammatory pain medicine, and belongs to the technical field of extraction of the mesaconitine. The method comprises the following steps: firstly, decocting radix aconiti with alkaline water for extraction, then, removing impurities with resin, sequentially adopting water, a NaOH solution, water and a dilute ethanol solution as eluents for elution and impurity removal in the impurity removal process, and then, carrying out acid-alcoholysis adsorption. According to the method, the concentration and dosage of the eluent in the impurity removal and desorption processes are adjusted, the content of the mesaconitine is increased, the extract can be used for preparing the medicine for treating chronic inflammatory pain, and the analgesic effect is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aconitine extraction, in particular to a total protoaconine extraction method rich in neoprotodaphnine and application thereof in chronic inflammatory pain drugs. BACKGROUND

[0002] There are many plants in the Aconitum genus that are used as traditional Chinese medicine, including 36 species such as Aconitum carmichaelii, Aconitum kusnezoffii, Aconitum carmichaelii, Aconitum kusnezoffii, Aconitum carmichaelii, and Aconitum kusnezoffii. The chemical components mainly include diterpene alkaloids, which are divided into C18, C19 and C20 types of alkaloids, among which C18 type of hypaconitine and C19 type of fuzhuanjiasu have been developed into drugs.

[0003] The aconitine hydrolysis obtained protoaconine components are complex. After component separation and structure identification, the main components are aconicarmine, neoprotodaphnine, hypoprotodaphnine, protoaconine, songguoning, 15-hydroxyaconine, aconine and talatokamine. The structures are as follows:

[0004]

[0005]

[0006]

[0007]

[0008] Neoprotodaphnine improves heart failure and reduces the incidence of arrhythmia in myocardial ischemia reperfusion models, and its single maximum tolerated dose (MTD) reaches 1200mg / kg (rats), indicating a wide safety window.

[0009] Decoction and boiling can break the ester bond and reduce toxicity by 10-100 times. Currently, most of the drugs used in clinical practice are processed products, and raw products are mostly used externally.

[0010] Patent 202110384047.2 discloses a method for extracting protoaconine from aconitine by alkaline water extraction. Aconitum carmichaelii decoction, alkaline water heating reflux, and macroporous resin refining are used to obtain an extract with aconine and 15-hydroxyaconine as the main components, the content of which is more than 50% of the total alkaloids; the eluent is recovered, concentrated and dried to obtain the extract. The characteristics of the extract are that the total content of aconine and 15-hydroxyaconine is 50-70% of the extract.

[0011] The research found that the above method lost a high content of alkaloid component when impurities were removed and eluted by 10-20% ethanol, which was identified as neoprotoaconitine; and another alkaloid component was lost when impurities were further removed by eluting with 0.1-0.5% organic acid (formic acid or acetic acid) aqueous solution, which was identified as C20 type alkaloid aconicarmine. The main components of the extract of aconite boiled with alkaline water were analyzed by HPLC, and the peak order of the main components was: aconicarmine, neoprotoaconitine, protoaconitine, aconine and 15-hydroxyaconine. Through component separation, structure identification and activity test, it was found that the analgesic activity of neoprotoaconitine was the strongest, which showed higher potency and lower effective dose, and the effective dose was reduced by more than 5 times compared with other C19 components.

[0012] The ethanol concentration for removing impurities and elution was screened, and it was found that when the elution ethanol concentration was ≥5%, the alkaloid component was not lost, but the removal rate of solid content (impurities) in the eluent relative to the amount of medicinal material was >0.8%, indicating that the impurities could be effectively removed; when the elution ethanol concentration was >7.5%, neoprotoaconitine began to be lost, and when the elution ethanol concentration was ≥10%, most of the neoprotoaconitine component was eluted and lost, so the elution ethanol concentration for removing impurities should be 5-7.5% to avoid loss of effective components.

[0013] Therefore, the existing patent CN202110384047.2 obtains a C20 / C18 mixture mainly containing aconine and 15-hydroxyaconine by alkaline water extraction and macroporous resin refining, but the most effective neoprotoaconitine is lost during the resin impurity removal, so the analgesic activity is limited and the drug dosage is high.

[0014] Therefore, it is urgent to develop an extract of aconitine alkaloids with stable process, controllable quality, higher activity and higher content of neoprotoaconitine; not only the product yield is increased by about 1 times, but also the drug dosage is significantly reduced, the efficacy and safety of the drug are improved, and the cost of drug raw materials is reduced by more than 10 times. SUMMARY

[0015] Therefore, the present application aims to provide a total alkaloid extraction method rich in neoprotoaconitine.

[0016] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0017] A total alkaloid extraction method rich in neoprotoaconitine, comprising the following steps:

[0018] (1) Alkaline water decoction: grind the radix aconiti kusnezoffii, add calcium hydroxide and water, decoct for 1-2 h, and filter; add water to the residue and decoct for 0.5-1 h, and combine the filtrates;

[0019] (2) resin impurity removal: the filtrate is loaded onto an AB-8 macroporous resin column, and water, 0.1-1% w / w NaOH solution, water, and 5-7.5% v / v ethanol solution are used as eluents in sequence to remove impurities;

[0020] (3) acid-alcohol desorption: after the impurity removal, acetic acid-ethanol mixed aqueous solution is used as an eluent to continue desorption and elution, the eluate is collected, concentrated and dried to obtain the extract.

[0021] Preferably, the mass ratio of calcium hydroxide to Aconiti Lateralis Radix Praeparata in step (1) is 1:20-30.

[0022] Preferably, the amount of water added for the first time in step (1) is 8-16 times the mass of Aconiti Radix, and the amount of water added for the second time is 6-12 times the mass of Aconiti Radix.

[0023] Preferably, the mass-volume ratio of resin to medicinal material in the resin column in step (2) is 5-6 kg:1-2 L.

[0024] Preferably, the single use amount of the eluent in step (2) is 2-8 times the column volume.

[0025] Preferably, the concentration of acetic acid in the acetic acid-ethanol mixed aqueous solution in step (3) is 0.5-1% w / w, and the concentration of ethanol is 10-15% v / v.

[0026] Preferably, the use amount of the acetic acid-ethanol mixed aqueous solution in step (3) is 4-8 times the column volume.

[0027] Another object of the present application is to provide an application of total protoalkaloids rich in neoprotoalkaloids, and the total protoalkaloids are used to prepare a medicine for relieving chronic inflammatory pain.

[0028] Preferably, the medicine dosage form is any one of a tablet, a capsule, a granule, an oral solution, a gel, a spray or a plaster.

[0029] Preferably, the chronic inflammatory pain includes rheumatoid arthritis, neuropathic pain or postoperative chronic inflammatory pain.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] This invention provides a method for extracting total aconitine rich in neoacin and its application in drugs for chronic inflammatory pain. The method first involves extracting aconite root by decocting it in alkaline water, followed by resin purification. During purification, water, NaOH solution, water, and dilute ethanol solution are used sequentially as eluents. Acid-alcohol desorption is then performed. By adjusting the concentration and amount of eluents used in the purification and desorption processes, this invention increases the content of neoacin. The extract can be used to prepare drugs for chronic inflammatory pain, improving its analgesic effect. Attached Figure Description

[0032] Figure 1 The HPLC chromatogram of extract A from Example 1 is shown below.

[0033] Figure 2 The HPLC chromatogram of extract B from Comparative Example 1 is shown.

[0034] Figure 3 The results of the heat-induced paw retraction latency in mice in Experiment 4;

[0035] Figure 4 The mechanical pain threshold of the mice in Experiment 4 was 50%.

[0036] Figure 5 The concentration of TNF-α in the mouse spinal cord was determined by ELISA in Experiment 5.

[0037] Figure 6 The concentration of IL-6 in the mouse spinal cord was determined by ELISA in Experiment 5.

[0038] Figure 7 The concentration of IL-1β in the mouse spinal cord was determined by ELISA in Experiment 5;

[0039] Figure 8 The expression of SP in the mouse spinal cord was measured by ELISA in Experiment 5.

[0040] Figure 9 The expression of CGRP in the mouse spinal cord was measured by ELISA in Experiment 5. Detailed Implementation

[0041] This invention provides a method for extracting total alkaloids rich in neoaconitine, the steps of which are as follows:

[0042] (1) Alkaline decoction: Take Aconitum carmichaelii root and crush it (crushing is a conventional method in this field, crush Aconitum carmichaelii root to less than 2 mm), add calcium hydroxide and 8-16 times the weight of Aconitum carmichaelii root in water and decoct for 1-2 hours, then filter; add 6-12 times the weight of Aconitum carmichaelii root in water to the dregs and decoct for 0.5-1 hours, then combine the filtrates; the mass ratio of calcium hydroxide to Aconitum carmichaelii root slices is 1:20-30;

[0043] (2) Resin purification: The filtrate was loaded onto an AB-8 macroporous resin column (the mass-volume ratio of resin to medicinal material was 5-6 kg: 1-2 L), and eluted with water (2-8 times the column volume), NaOH solution (0.1-1% w / w), water, and ethanol solution (5-7.5% v / v) as eluents to remove impurities.

[0044] (3) Acid-ethanol desorption: After the impurity removal is completed, continue to use 4-8 times the column volume of acetic acid-ethanol mixed aqueous solution as the eluent for desorption and elution. Collect the eluent, concentrate and dry it to obtain the extract. The concentration of acetic acid in the acetic acid-ethanol mixed aqueous solution is 0.5-1% w / w and the concentration of ethanol is 10-15% v / v.

[0045] The total alkaloids rich in neoaconitine extracted in this invention are used to prepare drugs for relieving chronic inflammatory pain; the drug dosage form is any one of tablets, capsules, granules, oral liquids, gels, sprays or plasters; the chronic inflammatory pain includes rheumatoid arthritis, neuropathic pain or postoperative chronic inflammatory pain.

[0046] The present invention will be further described below with reference to the embodiments.

[0047] Example 1

[0048] A method for extracting total alkaloids rich in neoaconitine, comprising the following steps:

[0049] (1) Alkaline decoction: Take 6 kg of raw Aconitum carmichaelii root, crush it, add 240 g of calcium hydroxide and 12 times the weight of Aconitum carmichaelii root in water, decoct at 100℃ for 1 hour, and filter; add 10 times the weight of Aconitum carmichaelii root in water to the dregs and decoct for 0.5 hours, and combine the filtrates;

[0050] (2) Resin impurity removal: The filtrate was loaded onto an AB-8 macroporous resin column (resin volume 2L), and eluted with 8 column volumes of water, 2 column volumes of 0.5% w / w NaOH solution, 4 column volumes of water, and 4 column volumes of 5% v / v ethanol solution as eluents to remove impurities.

[0051] (3) Acid-ethanol desorption: After the impurity removal is completed, continue to use 6 column volumes of acetic acid-ethanol mixed aqueous solution as the eluent for desorption and elution. Collect the eluent, concentrate under reduced pressure and dry under vacuum to obtain brown total alkaloid rich in neoaconitine (denoted as extract A); the concentration of acetic acid in the acetic acid-ethanol mixed aqueous solution is 0.5% w / w and the concentration of ethanol is 15% v / v.

[0052] Extract A weighed 56.4 g and had a calculated yield of 0.94%.

[0053] Extract A prepared in Example 1 was analyzed by HPLC:

[0054] Instruments: Waterse2695 high performance liquid chromatograph (with PDA detector), ELSD6000 evaporative light scattering detector; Chromatographic conditions: Column: Hedera ODS-2C18 (4.6 mm × 25 mm, 5 μm); eluent: acetonitrile (A) - 0.2% trifluoroacetic acid (B); gradient elution as shown in Table 1; flow rate: 1 mL / min; gas flow rate of evaporative light scattering detector: 3 L / min; drift tube temperature: 110 °C.

[0055] Table 1

[0056] Time (min) Mobile phase A (%) Mobile phase B (%) 0 8 92 40 18 82

[0057] The specific detection method is as follows: Dissolve 5 mg of extract A in 10 ml of methanol, filter, inject the sample, and detect the result. Figure 1 , Figure 1 The peak at 11.0 min is aconitine, the peak at 17.3 min is neoaconitine, the peak at 22.5 min contains aconitine, and the peak at 33.8 min is aconitine.

[0058] from Figure 1 It can be seen that the neoaconitine peak was the highest, with a neoaconitine content of 16.7%. Further testing was performed according to the detection method for aconite in the pharmacopoeia, using the same instrument and chromatographic column as above. Acetonitrile was used as mobile phase A, and 0.2% glacial acetic acid solution (adjusted to pH 6.20 with triethylamine) was used as mobile phase B. Gradient elution was performed according to Table 2, and the detection wavelength was 235 nm. No diester aconitines (aconitine, hypoaconitine, and neoaconitine) or monoester aconitines (benzoylneopaconitine, benzoylaconitine, and benzoylhypoaconitine) were detected (detection limit ≤ 0.001%).

[0059] Table 2

[0060] Time (min) Mobile phase A (%) Mobile phase B (%) 0～44 21→31 79→69 44～65 31→35 69→65 65～70 35 65

[0061] Comparative Example 1

[0062] (1) Alkaline decoction: Take 6 kg of Aconitum carmichaelii root, crush it, add 240 g of calcium hydroxide and 12 times the weight of Aconitum carmichaelii root in water, decoct at 100℃ for 1 hour, and filter; add 10 times the weight of Aconitum carmichaelii root in water to the dregs and decoct for 0.5 hours, and combine the filtrates;

[0063] (2) Resin purification: The filtrate was loaded onto an AB-8 macroporous resin column (2L of resin). The column was eluted sequentially with 8 column volumes of water, 2 column volumes of 0.5% w / w NaOH solution, 4 column volumes of water, 4 column volumes of 15% w / w ethanol solution, and 6 column volumes of 0.1% acetic acid aqueous solution.

[0064] (3) Acid-ethanol desorption: After the impurity removal is completed, continue to use 6 column volumes of acetic acid-ethanol mixed aqueous solution as the eluent for desorption and elution. Collect the eluent, concentrate under reduced pressure and dry under vacuum to obtain brown total alkaloid containing neoaconitine (denoted as extract B); the concentration of acetic acid in the acetic acid-ethanol mixed aqueous solution is 0.5% w / w and the concentration of ethanol is 15% v / v.

[0065] Extract B weighed 27.1 g, with a calculated yield of 0.45%.

[0066] Extract B was analyzed by HPLC, and the chromatogram is shown below. Figure 2 , Figure 2 The highest peak was aconitine, followed by aconitine and 15-hydroxyaconitine; the content of neoaconitine was 1.22%. The content of neoaconitine in this extract was extremely low.

[0067] Comparative Example 1 used the same raw materials and processing procedures as Example 1. The only difference was that the ethanol concentration for impurity removal and elution in Comparative Example 1 was 15%, and acid elution was added. The extract yielded 27.1g (yield 0.45%), and the content of neoacin was only 1.22%. Therefore, it can be concluded that high-concentration ethanol elution leads to the loss of effective ingredients and a decrease in yield (down to about half).

[0068] Experiment 1: Acetic acid writhing test for analgesia

[0069] Experimental animals: Male ICR mice, weighing 18-22g. Animal housing environment: room temperature 22℃, relative humidity around 50%, free access to water and food, regular changing of bedding and cages. The experiment complied with GLP standards, animal ethics numbers 202305A011 and 202310A026; the experimental drug was extract A (total alkaloids rich in neoaconitine) from Example 1.

[0070] ICR mice were randomly divided into 8 groups of 10 mice each, based on body weight: a blank control group and groups treated with different concentrations of extract A. The dosages for the treatment groups were 1, 2, 4, 8, 16, 32, and 64 mg / kg, respectively. Except for the blank control group, which received an equal volume of physiological saline by gavage daily, the other groups received extract A by gavage once daily for 7 consecutive days. On the seventh day, unopened glacial acetic acid was mixed with physiological saline to prepare a 0.6% glacial acetic acid solution, which was prepared and used immediately.

[0071] One hour after the end of gavage in each group of mice on the seventh day, 0.6% glacial acetic acid solution was injected intraperitoneally 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 extract A on acetic acid-induced pain was calculated using the following formula (1):

[0072] (1)

[0073] 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 3.

[0074] Table 3. Effects of extract A on mouse acetic acid writhing test ( (n=10)

[0075]

[0076] In Table 3, compared with the control group, ;

[0077] The results showed that, compared with the blank control group, the number of writhing movements in mice decreased after treatment with different concentrations of extract A, indicating that extract A has a certain effect on relieving acetic acid-induced pain in mice. Among them, the number of writhing movements in mice treated with extract A at 1, 2, and 4 mg / kg was significantly reduced compared with the blank control group (P < 0.01). However, the analgesic effect decreased with higher concentrations. Therefore, it was finally decided to set 1, 2, and 4 mg / kg of aconitine as low, medium, and high dose groups to study its therapeutic effect on CFA-induced chronic inflammatory pain in mice.

[0078] Experiment 2: Comparative Experiment on the Analgesic Activity of Neoaconitine and Other Main Components in the Extract

[0079] The experimental animals were the same as in Experiment 1. ICR mice were randomly divided into 5 groups according to body weight: blank group, neoaconitine group, aconitine group, aconitine group, and aconitine group, with 10 mice in each group. The dosage of drugs was 0.2 mg / kg for neoaconitine, and 3.2 mg / kg for aconitine, aconitine, and aconitine.

[0080] The aforementioned neoaconitine, aconitine, aconitine, and aconitine are all raw materials with a purity of 96%.

[0081] Except for the control group, which received an equal volume of physiological saline via gavage daily, the other groups were administered neoaconitumine and various aconitine components via gavage once daily for 7 consecutive days. On the seventh day, an appropriate amount of unopened glacial acetic acid was taken and mixed with physiological saline to prepare a 0.6% glacial acetic acid solution, which was prepared and used immediately.

[0082] 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 indentation) of each mouse within 15 minutes was observed and recorded. The pain inhibition rate of different doses of Aconitum carmichaelii alkaloids on acetic acid-induced pain was calculated (same as Experiment 1).

[0083] 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 4.

[0084] Table 4. Effects of different drugs on the acetic acid writhing test in mice. (n=10)

[0085]

[0086] In Table 4, compared with the control group, ;

[0087] The results showed that neoaconitine, along with aconitine and acetaminophen at effective doses, reduced the number of writhing movements in mice. Acetaminophen had no significant effect, possibly due to its C20 structure. Neoaconitine, at 1 / 16th the dose of the other doses, exhibited the highest pain inhibition rate. Although there was no statistically significant difference between neoaconitine and aconitine or acetaminophen, its pain inhibition rate was superior, indicating the highest activity. Therefore, the extract from the control literature lost the highly active ingredient (neoaconitine) during preparation.

[0088] Experiment 3: Comparative Experiment of Acetic Acid Writhing Test for Analgesia of Different Extracts

[0089] The experimental animals were the same as in Experiment 1. ICR mice were randomly divided into 6 groups according to body weight (including a blank control group, extract A group, and different doses of extract B groups), with 10 mice in each group. The dose of extract A was 2 mg / kg, and the doses of extract B were 2, 4, 8, and 12 mg / kg. Except for the blank control group, which was given an equal volume of physiological saline by gavage daily, the other groups were given extract A and different doses of extract B 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.

[0090] 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, and the number of writhing movements of each mouse within 15 minutes was observed and recorded.

[0091] The number of times mice in each group twisted (lifted buttocks, extended hind limbs, and tucked in abdomen) was observed and recorded, and the pain inhibition rate of different doses of Aconitum carmichaelii alkaloids on acetic acid-induced pain was calculated (the calculation method is the same as in Experiment 1).

[0092] 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 5.

[0093] Table 5. Effects of different extracts on the acetic acid writhing test in mice. (n=8)

[0094]

[0095] In Table 5, compared with the control group, Compared with the control extract at the same dose (2 mg / kg), ### P < 0.001.

[0096] The results showed that both extract A and different doses of extract B reduced the number of writhing movements in mice. However, the extract A group was significantly more effective than the extract B group at the same dose, which was statistically significant, indicating that the effective dose of extract A was lower. Although the extract A group (2 mg / kg) with a dose 6 times lower was not statistically significant compared with the highest dose group of extract B (12 mg / kg), extract A had a higher percentage of pain inhibition.

[0097] Therefore, the effective dose of extract A is significantly reduced, by about 6 times, making it more promising for treating chronic inflammatory pain than extract B.

[0098] Experiment 4: Effects of extract A on CFA-induced chronic inflammatory pain in mice

[0099] The experimental animals were the same as in Experiment 1. Mice were randomly divided into four groups according to body weight: Con group (negative control group), CFA group (model group), Nar group (positive drug group), and low, medium, and high dose groups of extract A, with 10 mice in each group. CFA (10 μl) was slowly injected subcutaneously into the left posterior paw of the mice to induce a chronic inflammatory pain model. An equal volume of physiological saline was injected into the left posterior paw of the control group. Two days after modeling, naproxen (naproxen tablets: Nanjing Baijingyu Pharmaceutical Co., Ltd., 221104; dosage: 75 mg / kg) and extract A (1, 2, and 4 mg / kg) were administered to the experimental group mice. The Con group and CFA group were given the same volume 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 tests were completed on the last day, all mice were sacrificed and their samples were collected for further testing.

[0100] 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.

[0101] Mice were acclimatized to the test environment for 30 minutes prior to the hot plate test. Mice were placed on a hot / cold plate analgesia meter (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 behaviors (withdrawal of hind paws or licking / stomping), was recorded. Each test was repeated three times, and the average value was taken. The effect of extract A on the mechanical and thermal pain thresholds in mice with chronic inflammatory pain is as follows: Figure 3-4 , Figure 3 This refers to the heat-induced paw retraction latency period in mice. Figure 4 The mechanical pain threshold for mice is 50%.

[0102] Figure 3 and Figure 4 In comparison with the Con group, ####P<0.001; compared with the CFA group, , .

[0103] from Figure 3It can be seen that there was no significant difference in the baseline pain threshold among the groups of mice (P>0.05); compared with the blank control group, the mechanical pain threshold of mice after subcutaneous injection of CFA in the foot of the mouse decreased significantly within 14 days (P<0.0001) and remained persistent; compared with the model group, mice treated with different doses of extract A showed that it significantly increased the mechanical pain threshold of mice and remained persistent (P<0.01); the results of thermal pain threshold are as follows. Figure 4 The results showed that on day 0, there was no significant difference in the thermal withdrawal threshold among the groups of mice (P > 0.05). However, after subcutaneous injection of CFA into the left hind paw of the mice, the thermal withdrawal threshold decreased significantly (P < 0.0001). But after gavage administration of extract A, the situation was significantly improved. Different doses of extract A could significantly increase the thermal withdrawal threshold of mice subcutaneously injected with CFA (P < 0.01). Therefore, the above experimental results indicate that extract A can significantly improve the pain status of mice with CFA-induced chronic inflammatory pain, and its efficacy is comparable to that of naproxen 75 mg / kg.

[0104] Experiment 5: Anti-inflammatory effects and mechanisms of extract A

[0105] After euthanizing the mice in Experiment 4, 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.

[0106] (1) Effects of extract A on TNF-α, IL-1β and IL-6 in the spinal cord of mice with chronic inflammatory pain

[0107] During the course of spinal cord inflammation, TNF-α, IL-1β, and IL-6 secreted by activated immune cells (such as macrophages, microglia, and astrocytes) can not only trigger... These pro-inflammatory factors can enhance the expression levels of pain-related genes by stimulating signaling pathways and increasing the sensitivity of pain receptors by stimulating transient receptor potential (TRP) channels (such as TRPV1). These pro-inflammatory factors can not only drive inflammatory responses and activate pain transmission pathways, but also play an important role in the regulation of neuroinflammation. They are key factors in the occurrence and persistence of inflammatory pain. Their interactions form a stable and complex inflammatory network, which further amplifies pain signals.

[0108] The concentrations of TNF-α, IL-1β, and IL-6 in the spinal cord of mice were quantitatively analyzed by ELISA to explore whether aconitine-containing compounds could alleviate pain by reducing the levels of pro-inflammatory factors. Figure 5-7As shown, compared with the Con group mice, the concentrations of TNF-α, IL-1β and IL-6 in the spinal cord of CFA mice were significantly upregulated (P < 0.001), and the concentrations of TNF-α, IL-1β and IL-6 were significantly reduced after different doses of aconitine intervention (P < 0.05).

[0109] Effects of extract A on pro-inflammatory factors in the spinal cord of mice with CFA-induced inflammatory pain, such as Figure 5-7 . Figure 5 To determine the concentration of TNF-α in the spinal cord of mice using ELISA; Figure 6 To determine the concentration of IL-6 in the spinal cord of mice using ELISA; Figure 7 To determine the concentration of IL-1β in the spinal cord of mice using ELISA; Figure 5-7 middle( (n=6), compared with the Con group, ###P<0.001, compared with the CFA group, 0.05.

[0110] (2) Effects of extract A on CGRP and substance P in the spinal cord of mice with chronic inflammatory pain

[0111] Human sensory nerve endings synthesize and secrete various important polypeptides, among which calcitonin gene-related peptide (CGRP) and substance P (SP) are particularly crucial at the genetic and physiological levels. Both not only dilate blood vessels and enhance microvascular permeability but also exacerbate neurogenic inflammatory processes and trigger corresponding receptors to amplify pain transmission. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of CGRP and SP in the spinal cord of mice in each group. The results are shown in the figure. Compared with the con group, the expression levels of CGRP and SP in the spinal cord of mice in the CFA group were significantly increased (P<0.001). After treatment with different concentrations of aconitine, the content of CGRP and SP in the spinal cord of mice was significantly decreased (P<0.01). These results indicate that extract A significantly inhibits the transmission of pain information and pain sensitivity.

[0112] Effects of extract A on CGRP and substance P in the spinal cord of mice with CFA-induced inflammatory pain: Figure 8-9 . Figure 8 The expression of SP in the mouse spinal cord was determined by ELISA; Figure 9 To determine the expression of CGRP in the spinal cord of mice using ELISA; Figure 8-9 middle( (n=6), compared with the Con group, ###P<0.001, compared with the CFA group: .

Claims

1. A method for extracting total alkaloids rich in neoaconitine, characterized in that, Includes the following steps: (1) Alkaline decoction: Take the root of Aconitum carmichaelii, crush it, 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 combine the filtrates; (2) Resin purification: The filtrate was loaded onto an AB-8 macroporous resin column and eluted sequentially with water, NaOH solution with a concentration of 0.1-1% w / w, water, and ethanol solution with a concentration of 5-7.5% v / v. (3) Acid-alcohol desorption: After the impurity removal is completed, continue to use an acetic acid-ethanol mixed aqueous solution as the eluent for desorption and elution. Collect the eluent, concentrate and dry it to obtain the extract.

2. The method for extracting total alkaloids rich in neoaconitine according to claim 1, characterized in that, The mass ratio of calcium hydroxide to aconite slices in step (1) is 1:20-30.

3. The method for extracting total alkaloids rich in neoaconitine according to claim 1, characterized in that, Step (1) The amount of water added for the first time is 8-16 times the weight of the Aconitum carmichaelii root; the amount of water added for the second time is 6-12 times the weight of the Aconitum carmichaelii root.

4. The method for extracting total alkaloids rich in neoaconitine according to claim 1, characterized in that, In step (2), the mass-volume ratio of resin to medicinal material in the resin column is 5-6 kg: 1-2 L.

5. The method for extracting total alkaloids rich in neoaconitine according to claim 1, characterized in that, The amount of elution solution used in step (2) is 2-8 times the column volume per use.

6. The method for extracting total alkaloids rich in neoaconitine according to claim 1, characterized in that, In step (3), the concentration of acetic acid in the acetic acid-ethanol mixed aqueous solution is 0.5-1% w / w, and the concentration of ethanol is 10-15% v / v.

7. The method for extracting total alkaloids rich in neoaconitine according to claim 1, characterized in that, The amount of the acetic acid-ethanol mixed aqueous solution used in step (3) is 4-8 times the column volume.

8. An application of a total alkaloid rich in neoaconitine, characterized in that, The total alkaloids were used to prepare drugs for relieving chronic inflammatory pain.

9. The application of the total alkaloids rich in neoaconitine according to claim 8, characterized in that, The drug dosage form is any one of tablets, capsules, granules, oral liquids, gels, sprays, or plasters.

10. The application of the total alkaloids rich in neoaconitine according to claim 8, characterized in that, The chronic inflammatory pain includes rheumatoid arthritis, neuropathic pain, or postoperative chronic inflammatory pain.

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