Preparation method and use of total alkaloids of Fritillaria cirrhosa

Abstract

The invention belongs to the field of extraction technology of Ilex fringeii, discloses a kind of preparation method and use of Ilex fringeii total alkaloids, the method is:Take Ilex fringeii dried bulb and crush, sieve, 60 80% ethanol solution reflux extraction, filter, concentrate to dry paste;Use 1 3% HCl solution to dissolve, filter, filtrate is repeated on cation exchange resin;Sequentially eluted with deionized water and 50 70% NaCl ethanol solution, collect ethanol eluent and concentrate to dry paste;Add absolute ethanol, adjust pH to weak alkaline rear hot reflux extraction, filter the extract and be concentrated under reduced pressure to no alcohol taste;Add 1 3% HCl to dissolve completely, adjust pH to 8 9, repeatedly load over macroporous resin;Sequentially eluted with deionized water, ethanol solution of different volume concentrations;Collect target eluent decompression concentration, dry and obtain Ilex fringeii total alkaloid extract.Ilex fringeii total alkaloids of the present invention can be used for developing medicines for the treatment of chronic respiratory diseases, including: chronic obstructive pneumonia, chronic pharyngitis, pulmonary fibrosis.

Description

Technical Field

[0001] The invention relates to the technical field of fritillaria cirrhosa extraction, and in particular to a preparation method and application of fritillaria cirrhosa total alkaloids. Background Art

[0002] Fritillariae Pallidiflorae Bulbus is the dried bulb of Fritillaria walujewii Regel (also known as Fritillaria pallidiflora Schrenk), a plant of the Liliaceae family. Officially listed in the 2020 edition of the Chinese Pharmacopoeia, it is primarily found in the Ili, Tacheng, and Bortala regions of the northern Tianshan Mountains in Xinjiang. It is a valuable and authentic local medicinal material in Xinjiang, known for its high yield, high alkaloid content, and resistance to pests and diseases. Fritillaria cirrhosa, a Sichuan native, is one of the most commonly used traditional Chinese medicines for treating lung diseases. However, due to the scarcity and high price of wild Fritillaria cirrhosa resources, other Fritillaria resources should be rationally developed and utilized to expand their medicinal potential.

[0003] At present, large-scale artificial cultivation of Fritillaria cirrhosa has been achieved, and there is a large-scale planting base in Moheer Township, Yili, Xinjiang. Compared with Fritillaria cirrhosa, Fritillaria cirrhosa has abundant resources and relatively low prices. Fritillaria cirrhosa contains a variety of chemical components such as alkaloids, saponins, terpenes, flavonoids, polysaccharides, coumarins, fatty acid glycerides, amino acids, etc., among which alkaloids are its main active ingredients. However, the content of alkaloids in Fritillaria cirrhosa medicinal materials is extremely low. The 2020 edition of the "Chinese Pharmacopoeia" stipulates that the content determination of Fritillaria cirrhosa uses citrapine glycosides and citrapine as quality control indicators, and the total amount must not be less than 0.070%, so it is very important to enrich and purify it.

[0004] Modern research indicates that total alkaloids from Fritillaria cirrhosa have antitussive, expectorant, anti-asthmatic, anti-inflammatory, antibacterial, and acetylcholinesterase inhibitory activities. At equivalent doses, they are superior to total alkaloids from Fritillaria cirrhosa in antitussive and expectorant effects. Previous research in our laboratory has confirmed that total alkaloids from Fritillaria cirrhosa can slow the progression of pulmonary fibrosis. However, limited research exists on the preparation of high-purity total alkaloids from Fritillaria cirrhosa. There is still considerable room for improvement in the preparation of high-purity, high-yield, low-cost, and well-defined total alkaloids from Fritillaria cirrhosa. Furthermore, no pharmacodynamic and mechanistic studies have been conducted on their effects on pulmonary fibrosis and chronic obstructive pulmonary disease. Summary of the Invention

[0005] The present invention aims to provide a method for preparing total alkaloids of Fritillaria cirrhosa with high yield, high purity and clear composition, and to explore the pharmacodynamics and mechanism of action of the composition prepared by this method in delaying pulmonary fibrosis and chronic obstructive pulmonary disease, so as to provide a theoretical basis for the subsequent development of new drugs for anti-pulmonary fibrosis and research on their targets.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing total alkaloids from Fritillaria cirrhosa, comprising the following steps:

[0008] S1. Preparation of crude extract A

[0009] An appropriate amount of dried bulbs of Fritillaria cirrhosae are crushed, sieved, and extracted with a 60-80% ethanol solution by reflux. The extract is filtered and concentrated under reduced pressure to a dry paste to obtain a crude extract A of total alkaloids of Fritillaria cirrhosae.

[0010] S2. Initial purification with ion exchange resin

[0011] The crude extract A of total alkaloids from Ilex fuji was dissolved in a 1-3% HCl solution, filtered, and the filtrate was repeatedly loaded on a cation exchange resin until the effluent became noticeably lighter, and statically adsorbed for 1-2 hours; the extract was eluted with deionized water until the eluate showed a negative molish reaction; the extract was then eluted with an ethanol solution containing NaCl at a volume concentration of 50-70% until the eluate showed a negative alkaloid precipitation reaction, and the eluate was collected and concentrated to dryness under reduced pressure to obtain a crude extract B of total alkaloids from Ilex fuji;

[0012] S3, desalination

[0013] The crude extract B of total alkaloids from Ilex fumitorii was extracted with anhydrous ethanol under hot reflux, and a saturated NaOH solution was added to the solution before each extraction to adjust the pH to 8-9. The extract was filtered and concentrated under reduced pressure until there was no alcohol smell, thereby obtaining a crude extract C of total alkaloids from Ilex fumitorii.

[0014] S4. Macroporous resin purification

[0015] The crude extract C of total alkaloids from Bulbus Fritillariae is completely dissolved by adding 1-3% HCl, and the solution is adjusted to pH=8-9 with saturated NaOH solution. The sample is then repeatedly loaded on a macroporous resin until the effluent becomes significantly lighter, and static adsorption is performed for 1-2 hours; first, deionized water is used to elute until the molish reaction of the eluate is negative; then, a first ethanol solution, a second ethanol solution and a third ethanol solution of different volume concentrations are used for gradient elution, the first ethanol solution is used to elute until the molish reaction of the eluate is negative, and the second ethanol solution and the third ethanol solution are used to elute until the alkaloid precipitation reaction of the eluate is negative; the eluates of the second ethanol solution and the third ethanol solution are combined, concentrated under reduced pressure and dried to obtain the total alkaloid extract of Bulbus Fritillariae.

[0016] According to some embodiments of the present invention, in step S1, the amount of ethanol used is 8-12 times the weight of Fritillaria cirrhosa, and the hot reflux extraction is performed 2-3 times, with each extraction taking 3-4 hours.

[0017] According to some embodiments of the present invention, in step S2, the volume concentration of NaCl is 4-6%.

[0018] According to some embodiments of the present invention, in step S2 and step S4, the elution flow rate is 2-4 BV / h.

[0019] According to some embodiments of the present invention, in step S2, the cation exchange resin is a 001x4 type cation exchange resin.

[0020] According to some embodiments of the present invention, in step S3, the amount of ethanol used is 3-5 times the weight of the crude extract A of total alkaloids from Fritillaria cirrhosa, and the hot reflux extraction is performed 2-3 times, with each extraction lasting 1-2 hours.

[0021] According to some embodiments of the present invention, in step S4, the volume concentrations of the first ethanol solution, the second ethanol solution, and the third ethanol solution are 20-30%, 80-90%, and 99.5%-99.9%, respectively.

[0022] According to some embodiments of the present invention, in step S4, the macroporous adsorption resin is HPD722 macroporous adsorption resin.

[0023] The present invention also provides a method for preparing total alkaloids of Ilex citrifolia, and the obtained total alkaloid extract of Ilex citrifolia is used as a medicine for preparing a medicine for treating chronic respiratory diseases.

[0024] Preferably, the chronic respiratory diseases include: chronic obstructive pulmonary disease, chronic pharyngitis, and pulmonary fibrosis.

[0025] The preparation method and use of the total alkaloid extract of Fritillaria cirrhosa according to the present invention have at least the following advantages:

[0026] The present invention uses a combination of ion exchange resin and macroporous adsorption resin to purify the total alkaloid components in Fritillaria cirrhosa, and the purity of the prepared total alkaloids is significantly higher than that of single resin purification. In addition, by adopting high-concentration ethanol hot reflux desalination followed by macroporous adsorption resin water washing and desalination, the operation method is simple, and the problem of incomplete desalination by ethanol reflux, percolation or ultrasonic desalination alone in other preparation processes is solved.

[0027] The total alkaloids prepared by the present invention contain an alkaloid component (cibeline) with good therapeutic effects on chronic obstructive pulmonary disease, inflammation and oxidative stress, and this component has the highest purity among the total alkaloids, which can provide a new solution for the treatment of chronic respiratory system diseases.

[0028] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the total ion chromatogram of 9 alkaloid standards under MRM mode in the embodiment of the present invention;

[0030] Figure 2 This is a chromatogram of the HPLC-ELSD analysis of the total alkaloid extract of Fritillaria cirrhosa in Example 3 of the present invention;

[0031] Figure 3 The types and quantities of compounds contained in the total alkaloid extract of Fritillaria cirrhosa in Example 3 of the present invention;

[0032] Figure 4 Survival rate and weight change of rats in the experimental examples of the present invention (A: analysis of survival rate of each group during the rat experiment; B: weight change of each group during the rat experiment; C: analysis of weight of each group on day 21 of the rat experiment; D: analysis of weight change of rat BFP-H group and Model group during the experiment (ns: no significant difference; compared with Sham group: #P<0.05, ##P<0.01, ###P<0.005, ####P<0.001; compared with Model group: *P<0.05, **P<0.01, ***P<0.005, ****P<0.001));

[0033] Figure 5 Lung conditions and lung coefficients of rats in the test examples of the present invention (A: lung coefficient of rats; B: kidney coefficient of rats (ns: no significant difference; compared with the Sham group: #P<0.05, ##P<0.01, ###P<0.005, ####P<0.001; compared with the Model group: *P<0.05, **P<0.01, ***P<0.005, ****P<0.001));

[0034] Figure 6 The survival rate and body weight of rats in the experimental examples of the present invention are shown, and the body weight changes of mice in each group are shown (A: changes in body weight of mice in each group from 8 to 12 weeks after administration; B: body weight of mice in each group at 12 weeks (N=20, ####P<0.0001 vs. control group; *P<0.05, ****P<0.0001 vs. model group; ΔΔP<0.01, ΔΔΔP<0.001 vs. dexamethasone group));

[0035] Figure 7These are the lung function-related indicators of mice in each group in the experimental examples of the present invention (A: functional residual capacity of mice in each group; B: static compliance of mice in each group; C: forced expiratory volume in 100 ms / forced vital capacity of mice in each group; D: airway resistance index of mice in each group; E: peak expiratory flow rate of mice in each group; (N=6, #P<0.05, ##P<0.01, ###P<0.001vs. control group; *P<0.05, **P<0.01, ***P<0.001vs. model group)). DETAILED DESCRIPTION

[0036] To illustrate the technical content, achieved objectives and effects of the present invention in detail, the following description is given in conjunction with the embodiments and accompanying drawings.

[0037] Resin pretreatment

[0038] 001x4 cation exchange resin: Soak an appropriate amount of resin in deionized water until fully expanded, then rinse with deionized water until the eluate is clear. Wet-pack the resin into a column using a diameter-to-height ratio of 1:3-1:6. Soak in 5-8% HCl solution for 1-2 hours, then rinse with deionized water until the eluate is neutral. Then, soak in 4-6% NaOH solution for 1-2 hours, then rinse with deionized water until the eluate is neutral. Finally, soak in 5-8% HCl solution for 1-2 hours, then rinse with deionized water until the eluate is neutral.

[0039] HPD722 macroporous adsorption resin: Soak an appropriate amount of resin in a high-concentration ethanol solution until fully expanded. Rinse with deionized water until the eluate is clear, colorless, and free of ethanol residue. Wet-pack the resin into a column using a diameter-to-height ratio of 1:3-1:6. Soak in 5-8% HCl solution for 1-2 hours, then rinse with deionized water until the eluate is neutral. Soak in 4-6% NaOH solution for 1-2 hours, then rinse with deionized water until the eluate is neutral. Rinse with 85-95% ethanol solution until the eluate remains turbid even after adding water. Finally, rinse with deionized water until the eluate is free of ethanol residue. Remove any bubbles and set aside.

[0040] Embodiment 1 of the present invention is: a method for preparing total alkaloids from Fritillaria cirrhosa, comprising the following steps:

[0041] S1. Preparation of crude extract A

[0042] The dried bulbs of Fritillaria cirrhosa were crushed and sieved, and extracted twice with a 60% ethanol solution with a solid-liquid ratio of 1:8 under reflux, each extraction for 3 hours. The extract was filtered and concentrated under reduced pressure to a dry paste to obtain a crude extract A of total alkaloids from Fritillaria cirrhosa.

[0043] S2. Initial purification with ion exchange resin

[0044] The crude extract A of total alkaloids from Fritillaria cirrhosa was dissolved in 1% HCl solution and filtered. The filtrate was repeatedly loaded on 001x4 type cation exchange resin until the effluent became significantly lighter and statically adsorbed for 1 hour; eluted with 6BV of deionized water at a flow rate of 2BV / h until the molish reaction of the eluate was negative; then eluted with 8BV of 50% ethanol solution containing 4% NaCl at a flow rate of 2BV / h until the alkaloid precipitation reaction of the eluate was negative, the 50% ethanol eluate was collected, and concentrated under reduced pressure to a dry paste to obtain a crude extract B of total alkaloids from Fritillaria cirrhosa.

[0045] S3, desalination

[0046] First, anhydrous ethanol solution was added to the crude extract B of total alkaloids from Ilex fumitorii at a solid-liquid ratio of 1:3. Saturated NaOH solution was added to the resulting suspension to adjust the pH to 8-9 while stirring continuously. The suspension was subjected to hot reflux extraction twice, each time for 1 hour. The extract was filtered and concentrated under reduced pressure until there was no alcohol taste, thereby obtaining a crude extract C of total alkaloids from Ilex fumitorii.

[0047] S4. Macroporous resin purification

[0048] The crude extract C of total alkaloids from Fritillaria cirrhosa was completely dissolved in 1% HCl, and the solution was adjusted to pH 8 with saturated NaOH solution. The sample was then repeatedly loaded on HPD722 macroporous adsorption resin until the effluent became significantly lighter, and static adsorption was performed for 1 hour. First, 4BV of deionized water was used for elution at a flow rate of 2BV / h until the eluate showed a negative molish reaction. Then, 4BV of 20% ethanol solution was used for elution at a flow rate of 2BV / h until the eluate showed a negative molish reaction. Then, 8BV of 80% ethanol solution was used for elution at a flow rate of 2BV / h until the eluate showed a negative alkaloid precipitation reaction. Finally, 3BV of anhydrous ethanol solution was used for elution at a flow rate of 2BV / h until the eluate showed a negative alkaloid precipitation reaction. The eluates with a volume concentration of 80% ethanol and anhydrous ethanol were combined, concentrated under reduced pressure, and dried to obtain the total alkaloid extract A from Fritillaria cirrhosa.

[0049] The second embodiment of the present invention is: a method for preparing total alkaloids of Fritillaria cirrhosa, comprising the following steps:

[0050] S1. Preparation of crude extract A

[0051] An appropriate amount of dried bulbs of Fritillaria cirrhosa was crushed and sieved, and extracted with 80% ethanol solution at a solid-liquid ratio of 1:12 by volume for three times under reflux, each extraction for 4 hours. The extract was filtered and concentrated under reduced pressure to a dry paste to obtain a crude extract A of total alkaloids from Fritillaria cirrhosa.

[0052] S2. Initial purification with ion exchange resin

[0053] The crude extract A of total alkaloids from Ilex fumitorii was dissolved in 3% HCl solution and filtered. The filtrate was repeatedly loaded on 001x4 type cation exchange resin until the effluent became significantly lighter and statically adsorbed for 2 hours. The crude extract was eluted with 12BV of deionized water at a flow rate of 4BV / h until the molish reaction of the eluate was negative. The crude extract was then eluted with 12BV of 70% ethanol solution containing 6% NaCl at a flow rate of 4BV / h until the alkaloid precipitation reaction of the eluate was negative. The 70% ethanol eluate was collected and concentrated under reduced pressure to a dry paste to obtain the crude extract B of total alkaloids from Ilex fumitorii.

[0054] S3, desalination

[0055] First, anhydrous ethanol solution was added to the crude extract B of total alkaloids from Ilex fumitorii at a solid-liquid ratio of 1:5. Saturated NaOH solution was added to the resulting suspension to adjust the pH to 9 while stirring continuously. The suspension was extracted under hot reflux for 3 times, each time for 2 hours. The extract was filtered and concentrated under reduced pressure until there was no alcohol taste, thereby obtaining a crude extract C of total alkaloids from Ilex fumitorii.

[0056] S4. Macroporous resin purification

[0057] The crude extract C of total alkaloids from Fritillaria cirrhosa was completely dissolved in 3% HCl, and the solution was adjusted to pH 9 with saturated NaOH solution. The sample was then repeatedly loaded on HPD722 macroporous adsorption resin until the effluent became significantly lighter and statically adsorbed for 2 hours. First, 6BV of deionized water was used for elution at a flow rate of 4BV / h until the eluent molish reaction was negative. Then, 6BV of 30% ethanol solution was used for elution at a flow rate of 4BV / h until the eluent molish reaction was negative. Then, 12BV of 90% ethanol solution was used for elution at a flow rate of 4BV / h until the eluent alkaloid precipitation reaction was negative. Finally, 5BV of anhydrous ethanol solution was used for elution at a flow rate of 4BV / h until the eluent alkaloid precipitation reaction was negative. The eluents with a volume concentration of 90% ethanol and anhydrous ethanol were combined, concentrated under reduced pressure, and dried to obtain the total alkaloid extract B from Fritillaria cirrhosa.

[0058] Embodiment 3 of the present invention is: a method for preparing total alkaloids from Fritillaria cirrhosa, comprising the following steps:

[0059] S1. Preparation of crude extract A

[0060] Take an appropriate amount of dried bulbs of Fritillaria cirrhosa, crush them, sieve them, and extract them three times with 75% ethanol solution by volume at a solid-liquid ratio of 1:10 under hot reflux, each extraction time for 4 hours. After filtering the extract, concentrate it under reduced pressure to a dry paste to obtain a crude extract A of total alkaloids from Fritillaria cirrhosa.

[0061] S2. Initial purification with ion exchange resin

[0062] The crude extract A of total alkaloids from Fritillaria cirrhosa was dissolved in 2% HCl solution and filtered. The filtrate was repeatedly loaded on 001x4 type cation exchange resin until the effluent became significantly lighter and statically adsorbed for 2 hours; eluted with 9BV of deionized water at a flow rate of 3BV / h until the molish reaction of the eluate was negative; then eluted with 10BV of 60% ethanol solution containing 5% NaCl at a flow rate of 3BV / h until the alkaloid precipitation reaction of the eluate was negative, the 60% ethanol eluate was collected, and concentrated under reduced pressure to a dry paste to obtain a crude extract B of total alkaloids from Fritillaria cirrhosa.

[0063] S3, desalination

[0064] First, anhydrous ethanol solution was added to the crude extract B of total alkaloids from Fritillaria cirrhosa at a solid-liquid ratio of 1:4. Saturated NaOH solution was added to the resulting suspension to adjust the pH to 8 while stirring continuously. The suspension was subjected to hot reflux extraction for 3 times, each extraction for 2 hours. The extract was filtered and concentrated under reduced pressure until there was no alcohol taste, thereby obtaining a crude extract C of total alkaloids from Fritillaria cirrhosa.

[0065] S4. Macroporous resin purification

[0066] The crude extract C of total alkaloids from Fritillaria cirrhosa was completely dissolved in 2% HCl, and the pH of the solution was adjusted to 8 with saturated NaOH solution. The sample was then repeatedly loaded on HPD722 macroporous adsorption resin until the effluent became significantly lighter and statically adsorbed for 2 hours. First, 5BV of deionized water was used for elution at a flow rate of 3BV / h until the molish reaction of the eluent was negative. Then, 5BV of 25% ethanol solution was used for elution at a flow rate of 3BV / h until the molish reaction of the eluent was negative. Then, 10BV of 85% ethanol solution was used for elution at a flow rate of 3BV / h until the alkaloid precipitation reaction of the eluent was negative. Finally, 4BV of anhydrous ethanol solution was used for elution at a flow rate of 3BV / h until the alkaloid precipitation reaction of the eluent was negative. The eluents with a volume concentration of 85% ethanol and anhydrous ethanol were combined, concentrated under reduced pressure, and dried to obtain the total alkaloid extract C from Fritillaria cirrhosa.

[0067] Comparative Example 1 of the present invention is: a method for preparing total alkaloids from Bulbus Fritillariae, comprising the following steps: except for not having steps S2 and S3, other parameters are the same as those in Example 3, to obtain total alkaloid extract D from Bulbus Fritillariae.

[0068] Comparative Example 2 of the present invention is: a method for preparing total alkaloids from Bulbus Fritillariae, comprising the following steps: except for not having step S4, other parameters are the same as those in Example 3, to obtain total alkaloid extract E from Bulbus Fritillariae.

[0069] Comparative Example 3 of the present invention: A method for preparing total alkaloids from Fritillaria cirrhosa, comprising the following steps: except that step S4 in which 5BV of ethanol solution with a volume concentration of 25% is used for elution at a flow rate of 3BV / h until the molish reaction of the eluate is negative is not performed, other parameters are the same as those in Example 3 to obtain a total alkaloid extract F from Fritillaria cirrhosa.

[0070] With reference to the method of "Content Determination" under the Fritillaria cirrhosa of the "Chinese Pharmacopoeia" (Part 1 of the 2020 edition), the total alkaloids in the Fritillaria cirrhosa total alkaloid extracts obtained in the above embodiments and comparative examples were determined using an ultraviolet spectrophotometer, and the content of total alkaloids in the prepared Fritillaria cirrhosa total alkaloids was calculated. The test data results are shown in Table 1.

[0071] The components of the total alkaloid extracts of Fritillaria cirrhosa obtained in the above examples and comparative examples were analyzed using HPLC-ELSD technology:

[0072] The chromatographic conditions for HPLC-ELSD analysis were as follows:

[0073] The chromatographic column was an Agilent Eclipse Plus C18 column with a particle size of 5 μM, a diameter of 4.6 mm, and a column length of 250 mm. Column temperature: 35 °C.

[0074] Mobile phase: acetonitrile (A), 0.03% diethylamine (B), 0–10 min 30% A, 10–35 min 30% A-60% A, 35–45 min 60% A, 45–65 min 60% A-90% A, 65–75 min 90% A, 75–80 min 90% A-30% A, 80–90 min 30% A.

[0075] Flow rate: 0.1 mL / min, standard injection volume 10 μL, sample injection volume 25 μL.

[0076] By comparing the retention time (RT) of the standard and the example, it was determined that the drug contained 9 alkaloids: xibei pine, fangbei suan, fangbei xin, dehydroebedin alkaloid, xibei suan glycoside, subei suan ... Figure 1 ).

[0077] The components of the total alkaloid extracts of Fritillaria cirrhosa obtained in the above examples and comparative examples were analyzed using HPLC-MS / MS technology:

[0078] The chromatographic conditions for HPLC-MS / MS analysis were as follows:

[0079] The chromatographic column is Agilent ZORBAX SB-C18 Rapid Resolution HD 100x2.1(mm)1.8micron.

[0080] The mobile phase was: 0.1% formic acid (A), acetonitrile (B), 0–0.5 min 15% B, 0.5–3.5 min 15% B-45% B, 3.5–4.5 min 45% B-50% B, 4.5–6 min 50% B-95% B, 6–8 min 95% B, 8–8.1 min 95% B-15% B, 8.1–11 min 15% B.

[0081] Flow rate: 0.3 mL / min, injection volume: 1 μL.

[0082] The mass spectrometry conditions were as follows: ESI source; positive ion mode monitoring mode, spray voltage set to 5500 V; ion source temperature was 450°C.

[0083] Atomizing gas (Gas1) 50.0 psi, heating gas (Gas2) 50.0 psi, curtain gas (CUR) 35.0 psi, and nitrogen was introduced throughout the process.

[0084] The scanning mode adopted multiple reaction monitoring (MRM); the collision gas (CAD) pressure was 9.0; and the Q1 and Q3 resolutions were both UNIT.

[0085] A standard curve was constructed using the reference substance concentration as the abscissa (x) and the peak area as the ordinate (y). Linear regression analysis was performed to obtain the standard curve equation. The relative contents of the nine alkaloids in the drug were quantitatively analyzed using the external standard curve method. The test data are shown in Table 2.

[0086] Table 1 Detection results of total alkaloids in the total alkaloid extracts of Fritillaria cirrhosa in various examples and comparative examples

[0087] name Total alkaloid content (%) Transfer rate (%) Example 1: Total alkaloid extract A of Fritillaria cirrhosa 63.21 55.35 Example 2: Total alkaloid extract B of Fritillaria cirrhosa 65.48 63.46 Example 3: Total alkaloid extract C of Fritillaria cirrhosa 64.89 78.31 Comparative Example 1: Total Alkaloid Extract of Fritillaria cirrhosa D 31.76 48.21 Comparative Example 2: Total Alkaloid Extract E from Fritillaria cirrhosa 32.56 53.21 Comparative Example 3: Total Alkaloid Extract of Fritillaria cirrhosa F 49.21 57.34

[0088] Table 2 Detection results of alkaloid components in the total alkaloid extracts of Fritillaria thunbergii in each embodiment and comparative example

[0089]

[0090] Note: The “—” in the table indicates that the content was below the detection limit and was not detected.

[0091] Comparing the third embodiment with the first comparative example, it can be seen that the transfer rate and purity of the total alkaloids in the total alkaloid extract of Ilex fumitoriae in the first comparative example are significantly reduced, mainly because: the ion exchange resin method for purifying the total alkaloids of Ilex fumitoriae is to separate the alkaloid components from the impurities such as polysaccharides, flavonoids, organic acids, saponins, starch, etc. through ion exchange reaction to achieve the purpose of purifying the total alkaloids. When the acidic sample solution passes through the cation exchange resin, the positively charged alkaloid ions are adsorbed on the resin, while other negatively charged and uncharged impurities (polysaccharides, flavonoids, organic acids, saponins, starch, etc.) cannot be adsorbed. The impurities remaining on the resin can be removed by eluting with deionized water, and then eluted with a 60% ethanol solution containing 5% NaCl. The ions in the saline ethanol solution can exchange and elute the alkaloids adsorbed on the resin, and the ethanol solution also has good solubility for lipophilic alkaloids; and after the ion exchange resin The dry paste obtained by the initial enrichment and purification of lipids contains a large amount of NaCl. In order to obtain high-purity total alkaloids, the dry paste needs to be desalted. Since NaCl is extremely soluble in anhydrous ethanol, and the crude extract purified by cationic resin is added to anhydrous ethanol solution, the resulting suspension is acidic and the alkaloids are in ionic form. Saturated NaOH solution is added to the solution to adjust the pH to 8 and continuously stirred to release the alkaloid components, which not only can better desalinate, but also can make the alkaloids in the solution more conducive to adsorption by macroporous resin.

[0092] Comparing Example three and Comparative Example two, it can be seen that in Comparative Example 3, the yield and the purity of total alkaloids in the Bulbus Fritillariae Cibotii total alkaloid extract are significantly reduced, mainly because: because NaCl can also be slightly soluble in ethanol at high temperatures, the Bulbus Fritillariae Cibotii total alkaloid crude extract obtained still contains a small amount of NaCl, in order to further remove NaCl and large polar impurities, macroporous adsorption resin is adopted to further purify it. After weakly alkaline loading solution is loaded on macroporous adsorption resin, alkaloid components are adsorbed on resin, and deionized water and low concentration of ethanol are adopted to carry out elution successively, and the impurities such as residual NaCl and the larger polysaccharides, flavonoids, saponins of polarity can be removed in the sample. Most of the alkaloid components adsorbed on the resin can be eluted by adopting 85% ethanol to carry out elution, but the alkaloids of some lipophilicity still cannot be eluted, so it is finally adopted absolute ethanol to carry out gradient elution completely.

[0093] By comparing Example 3 with Comparative Example 3, it can be seen that the transfer rate, purity and relative content of fritillary alkaloids in the total alkaloid extract of fritillary bud in Comparative Example 4 are significantly reduced, mainly because: impurities with greater polarity are easily soluble in low concentration ethanol. Therefore, elution with low concentration ethanol can remove impurities such as polysaccharides, flavonoids, organic acids, starch, saponins and the like with greater polarity in the sample.

[0094] Test example:

[0095] 1. Pharmacodynamics test on delaying pulmonary fibrosis using the total alkaloid extract C of Fritillaria cirrhosa in Example 3

[0096] 1. Impact on rat physiological condition and survival rate

[0097] After adaptive feeding, SD rats were randomly divided into 12 groups: sham group, model group, positive control group (PFD, 150 mg / Kg / d), low-dose group (BFP-L, 15 mg / Kg / d), and high-dose group (BFP-H, 45 mg / Kg / d), for a total of 6 groups. Tracheotomy was performed and 4 mg / mL BLM solution was instilled to establish the PF model. 24 hours after modeling, oral administration was performed once a day at a fixed time for 21 days. The experimental results are shown in the figure. Figure 4 .

[0098] 2. Rat lung condition and lung coefficient

[0099] Pulmonary fibrosis can alter the normal physiological morphology of lung tissue, so the efficacy of the drug can be preliminarily evaluated by visually observing the condition of the rat's lung tissue. Normal lung tissue is highly elastic, has a normal structure, a pink color, and a smooth surface. After modeling, the rat's lung tissue developed dark patches of tissue damage due to the disease, with reduced or even absent tissue elasticity, a grayish-pale tissue color, and a rough surface. Abnormal adhesions appeared between the rat's lung lobes. Observation revealed that the number and area of ​​dark patches of tissue damage in the rat's lungs decreased after drug administration. The lung tissue in the BFP-H group was closest to the state of the Sham group, with a pink color and greater elasticity, slight hemorrhages, but no obvious dark patches. Furthermore, the BFP-L group had better lung tissue elasticity, surface smoothness, and dark area than the PFD group. This study compared and analyzed the lung coefficient data of rats in each group on the 21st day of the experiment. It was found that after the surgical modeling, the lung coefficient of the Model group increased significantly. After drug administration, the lung coefficients of rats in each group decreased to varying degrees. Among them, the lung coefficient of the BFP-H group showed the most significant downward trend, which was significantly different from that of the Model group (P<0.01). The experimental results are shown in Figure 5 .

[0100] 2. In vivo pharmacodynamics test on delaying chronic obstructive pulmonary disease using the total alkaloid extract C of Fritillaria cirrhosa in Example 3

[0101] 1. Model establishment and grouped drug administration

[0102] Balb / c mice (8-week-old, male, 18-22 g) were housed in an SPF animal facility at 25°C with a 12-h light-dark cycle. Water and food were provided as needed. Mice were divided into six groups, 20 in each: control, cigarette smoke (CS)-induced COPD, COPD plus dexamethasone (1 mg / kg / day), COPD plus low-dose (20 mg / kg / day) medication, COPD plus medium-dose (40 mg / kg / day) medication, and COPD plus high-dose (60 mg / kg / day) medication. Mice were placed in a 70 × 40 × 25 cm fumigation chamber and exposed to five filter cigarettes (11 mg tar / cigarette; 1.1 mg nicotine / cigarette) for 1 hour twice daily, with a 4-hour interval between CS exposures. CS induction lasted 6 days per week for 12 weeks. Control animals were exposed to room air for 12 weeks. At 9-12 weeks, the treated groups were gavaged with the corresponding concentrations of drug solution (dissolved in 2% Tween-80) and dexamethasone (dissolved in sterile saline) 30 minutes before CS exposure. The control and COPD groups were gavaged with 2% Tween-80. The weight of each group of mice was weighed and recorded weekly. Throughout the COPD modeling process, the mice's mental state, temperament, and general appearance were closely observed.

[0103] During the animal experiment, no mice in the control group died, their fur was smooth, their breathing was regular, and their activity was normal. Mice in the COPD model group had dull, yellowish fur, some hair loss, irritability, a tendency to pile up when smoking, fatigue, curling up, sweating, rapid breathing, and polydipsia and polyuria. Weight changes were compared between 8 and 12 weeks after drug administration. The weight of mice in the control group gradually increased with the feeding cycle, stabilizing at approximately 28 g after 8 weeks. Compared with the control group, the weight of mice in the COPD model group increased more slowly. Following drug administration, weight gain accelerated in all drug groups compared with the COPD model group. At the end of the 12th week of the experiment, the weight of mice in the COPD model group was significantly lower than that in the control group (P < 0.0001). The weight of mice in the dexamethasone group was significantly higher than that in the COPD model group (P < 0.05), but lower than that in all drug-treated groups. Compared with the COPD model group, weight increased to varying degrees in all drug-treated groups (P < 0.0001), with the highest effect observed in the high-dose group. The above results showed that the drug could reverse the trend of CS-induced weight loss in mice, and was more effective in the medium and high dose groups than in the dexamethasone group. Figure 6 .

[0104] 2. Pulmonary function test

[0105] After 12 weeks of CS exposure, 6 mice in each group were selected for lung function testing. Mice were anesthetized by intraperitoneal injection of 1% pentobarbital (60 mg / kg). The mice were fixed in a supine position, the trachea was cut open and intubated, and then placed in a computer-connected forced pulmonary operation system ( PFT system (PFT system, NC, USA). The following pulmonary function data were measured: functional residual capacity (FRC), static compliance (Cchord), forced vital capacity (FVC), forced expiratory volume in 100 ms (FEV100), peak expiratory flow (PEF), and airway resistance (RI). Pulmonary function is a key criterion for assessing COPD, effectively reflecting the patient's lung function status and a key indicator for the diagnosis and treatment of COPD. Compared with the control group, the COPD model group had significantly increased FRC, Cchord, and RI (P < 0.05, P < 0.001, P < 0.001), while FEV100 / FVC and PEF were significantly decreased (P < 0.001, P < 0.01). These data further confirmed the reliability of the COPD model. Compared with the COPD model group, the dexamethasone group had significantly decreased FRC and Cchord (P < 0.05, P < 0.01), and a significantly increased FEV100 / FVC (P < 0.001). Compared with the model group, all drug administration groups showed a trend of improving the above indicators, and the trend was dose-dependent. Overall, the high-dose group had a better effect on improving the lung function decline of mice induced by CS than the dexamethasone group. The experimental results are shown in Figure 7 .

[0106] The experimental examples show that the total alkaloid extract of Fritillaria cirrhosa can be used to prepare medicines for the treatment of chronic respiratory diseases (chronic obstructive pneumonia, chronic pharyngitis or pulmonary fibrosis); this is mainly because: component analysis research found that the content of Fritillaria cirrhosa in the total alkaloids accounts for more than 50% of the total alkaloids. In addition, previous studies have confirmed that Fritillaria cirrhosa has good therapeutic effects on chronic obstructive pulmonary disease, anti-inflammatory and antioxidant activities, and one of the important pathogenic factors of chronic respiratory diseases is persistent inflammatory response. Therefore, it is believed that the total alkaloid extract of Fritillaria cirrhosa can be used to prepare medicines for the treatment of chronic respiratory diseases.

[0107] The above is only an embodiment of the present invention, and common knowledge such as the specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A method for preparing a medicament for treating chronic respiratory diseases using a total alkaloid extract of Fritillaria cirrhosae Bulbs, characterized in that: The chronic respiratory disease is chronic obstructive pneumonia or pulmonary fibrosis; The preparation method of the total alkaloid extract of Fritillaria cirrhosa comprises the following steps: S1. Preparation of crude extract A The dried bulbs of Fritillaria cirrhosa are crushed and sieved, and then reflux-extracted with a 60-80% ethanol solution containing 8-12 times the volume of ethanol as much as the weight of the Fritillaria cirrhosa at a solid-liquid ratio of 1:8-12. The extract is filtered and concentrated under reduced pressure to a dry paste to obtain a crude extract A of total alkaloids from Fritillaria cirrhosa. S2. Initial purification with ion exchange resin The crude extract A of total alkaloids from Ilex fuji was dissolved in a 1-3% HCl solution, filtered, and the filtrate was repeatedly loaded on a 001x4 type cation exchange resin until the effluent became significantly lighter, and static adsorption was performed for 1-2 hours; the extract was eluted with deionized water until the eluate showed a negative molish reaction; the extract was then eluted with an ethanol solution containing 4-6% NaCl and 50-70% ethanol by volume until the eluate showed a negative alkaloid precipitation reaction, and the eluate was collected and concentrated to dryness under reduced pressure to obtain a crude extract B of total alkaloids from Ilex fuji; S3, desalination The crude extract B of total alkaloids from Ilex fumitorii was extracted with anhydrous ethanol under hot reflux, and a saturated NaOH solution was added to the solution before each extraction to adjust the pH to 8-9. The extract was filtered and concentrated under reduced pressure until there was no alcohol smell, thereby obtaining a crude extract C of total alkaloids from Ilex fumitorii. S4. Macroporous resin purification The crude extract C of total alkaloids from Bulbus Fritillariae is completely dissolved by adding 1-3% HCl, and the solution is adjusted to pH 8-9 with a saturated NaOH solution. The sample is then repeatedly loaded on a HPD722 macroporous resin until the effluent becomes significantly lighter, and static adsorption is performed for 1-2 hours. The extract is first eluted with deionized water until the eluate shows a negative molish reaction. The extract is then gradient eluted with a first ethanol solution with an ethanol volume concentration of 20-30%, a second ethanol solution with an ethanol volume concentration of 80-90%, and a third ethanol solution with an ethanol volume concentration of 99.5%-99.9%, the extract being eluted with the first ethanol solution until the eluate shows a negative molish reaction, and the extracts are then eluted with the second and third ethanol solutions until the eluate shows a negative alkaloid precipitation reaction. The eluates from the second and third ethanol solutions are combined, concentrated under reduced pressure, and dried to obtain the total alkaloid extract from Bulbus Fritillariae.

2. The use according to claim 1, characterized in that In step S1, the hot reflux extraction is performed 2-3 times, and each extraction lasts 3-4 hours.

3. The use according to claim 1, characterized in that In step S2 and step S4, the elution flow rate is 2-4 BV / h.

4. The use according to claim 1, characterized in that In step S3, the amount of ethanol used is 3-5 times the weight of the crude extract A of total alkaloids from Fritillaria cirrhosa, and the extraction is performed under reflux for 2-3 times, with each extraction lasting 1-2 hours.

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