Method for preparing fritillaria thunbergii miq. extract

Abstract

The application relates to a preparation method of a fritillaria cirrhosa bulbus extract. Step one: drying and crushing fritillaria cirrhosa bulbus to obtain fritillaria cirrhosa bulbus powder, taking the fritillaria cirrhosa bulbus powder and a cyclodextrin powder to stir-fry a mixture one; step two: adopting ethanol normal-temperature reflux extraction on the mixture one to separate supernatant one and filter residue; step three: recovering and drying the filter residue in step two, taking the dried filter residue and the cyclodextrin powder to stir-fry a mixture two; step four: adopting ethanol and organic acid normal-temperature reflux extraction on the mixture two; step five: combining the supernatant one and supernatant two, adding into a macroporous adsorption resin impurity removal device, then adding ethanol into the macroporous adsorption resin impurity removal device until the eluent is colorless; concentrating and drying to obtain the fritillaria cirrhosa bulbus extract. The application can obviously improve the extraction efficiency of total alkaloids of fritillaria cirrhosa bulbus and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the technical field of natural product extraction, and in particular to a method for preparing Fritillaria cirrhosa extract. Background Technology

[0002] Fritillaria cirrhosa D. Don, commonly known as Sichuan fritillary bulb, is a perennial herb belonging to the genus Fritillaria in the family Liliaceae. The plant can reach up to 50 cm in height. Leaves are opposite, linear to linear-lanceolate. Flowers are usually solitary, purple to yellowish-green, each with leaf-like bracts that are narrow and elongated. Anthers are nearly basally attached. The capsule has narrow wings along its long ridges. Sichuan fritillary bulb typically grows in forests, under thickets, in grasslands, riverbanks, valleys, and other wetlands or rock crevices. It is a valuable traditional Chinese medicine for moistening the lungs and relieving coughs, with a long history of use. The main active ingredients in Sichuan fritillary bulb are alkaloids, among which Fritillaria cirrhosa has antitussive and anti-ulcer effects. Alcohol extraction is commonly used to extract alkaloids from Fritillaria cirrhosa, but the extraction rate remains low. For example, the invention patent CN114917296A, entitled "An Extract of Total Alcoholic Acids from Fritillaria cirrhosa, Preparation Method and Application," discloses a method for preparing total alkaloids from Fritillaria cirrhosa. This method involves reflux extraction with ethanol 2-3 times, filtration, concentration, drying, and purification of the filtrate to obtain the total alkaloids. The purification method includes dissolving the alkaloids in 2-4% hydrochloric acid, filtering to remove insoluble matter, adjusting the pH to 7-10 with saturated sodium hydroxide solution, so that 1 ml of the solution contains 0.2-2.8 g of raw medicinal material. The pH-adjusted solution is then... The sample solution was loaded onto the prepared macroporous adsorption resin (AB-8 macroporous adsorption resin, D101 macroporous adsorption resin, HPD100 macroporous adsorption resin, H103 macroporous adsorption resin), and the loading was repeated. After loading, gradient elution was performed at a flow rate of 1-4 BV / h according to the following steps: (1) Elute with water for 8-12 BV; (2) Elute with 30% ethanol for 6-8 BV; (3) Elute with 90% ethanol for 8-12 BV; (4) Elute with anhydrous ethanol for 3-8 BV; (5) Collect the fractions eluted with 90% ethanol and anhydrous ethanol; concentrate the collected fractions by rotary evaporation and dry them. The total alkaloids of Fritillaria cirrhosa were obtained. The total alkaloid content was >35%. Although the purity of the total alkaloids of Fritillaria cirrhosa was improved by improving the purification process, the extraction rate of the total alkaloids of Fritillaria cirrhosa could not be improved. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing Fritillaria cirrhosa extract, which can significantly improve the extraction efficiency of total alkaloids from Fritillaria cirrhosa. By utilizing resin adsorption with different gradient polarities and employing a special process, the preparation of Fritillaria cirrhosa extract is highly efficient and residue-free, and the total alkaloids in the Fritillaria cirrhosa extract have high purity.

[0004] To achieve the above objectives, this invention discloses a method for preparing Fritillaria cirrhosa extract, comprising:

[0005] Step 1: Dry and pulverize the bulbs of Fritillaria cirrhosa and pass them through a 60-120 mesh sieve to obtain Fritillaria cirrhosa powder. Take Fritillaria cirrhosa powder and cyclodextrin powder at a mass ratio of 120-200:1 and stir-fry for 3-8 minutes to obtain mixture 1.

[0006] Step 2: Extract mixture 1 by reflux at room temperature with 60-75% ethanol. The ratio of mixture 1 to 60-75% ethanol is 1:6-12 g / mL. Separate the supernatant and filter residue.

[0007] Step 3: Dry the filter residue from Step 2, and stir-fry the dried filter residue and cyclodextrin powder at a mass ratio of 80-120:2-5 for 2-4 minutes to obtain Mixture 2;

[0008] Step 4: Mixture 2 is extracted by reflux at room temperature using ethanol with a volume concentration of 75-95% and organic acid. The ratio of mixture 2 to 75-95% ethanol is 1:8-10, and the volume ratio of 75-95% ethanol to organic acid is 200-300:1. Supernatant 2 is obtained after separation.

[0009] Step 5: Combine supernatant one and supernatant two and add them to the macroporous adsorption resin impurity removal device. Then add ethanol to the macroporous adsorption resin impurity removal device until the eluent is colorless. Concentrate and dry to obtain Fritillaria cirrhosa extract.

[0010] Preferably, the reflux extraction time in step two is 120–240 min.

[0011] Preferably, the drying temperature in step three is 25–35°C.

[0012] Preferably, the reflux extraction time in step four is 60–120 min.

[0013] Preferably, the organic acid in step four is a mixture of lauric acid and octanoic acid, wherein the volume ratio of lauric acid to octanoic acid is 1:2.

[0014] Preferably, the volume ratio of the ethanol aqueous solution to the macroporous adsorption resin in step five is 3 to 4:1.

[0015] Preferably, the macroporous adsorption resin impurity removal device includes:

[0016] The outer casing has a liquid inlet at the top and a liquid outlet at the bottom.

[0017] The first adsorption cylinder is located in the upper middle part of the outer shell and includes multiple interconnected cylinders with progressively increasing diameters, with the topmost cylinder connected to the liquid inlet.

[0018] The second adsorption cylinder is located below the first adsorption cylinder and is connected to the first adsorption cylinder. It includes multiple cylinders with successively decreasing diameters, and the bottommost cylinder is connected to the liquid outlet.

[0019] The first adsorption cylinder contains medium polar resin in each section from top to bottom, with the filling density gradually increasing. The second adsorption cylinder contains polar resin in each section from top to bottom, with a uniform density.

[0020] Preferably, the bottom of the first adsorption cylinder is provided with a buffer layer, and the buffer layer contains a mixed resin.

[0021] Preferably, the first adsorption cylinder and the second adsorption cylinder are connected by a connecting cylinder, and the connecting cylinder is filled with a mixed resin with the same filling density as the bottom cylinder of the first adsorption cylinder. The mixed resin includes a medium polar resin and a polar resin with a mass ratio of 4 to 6:2.

[0022] The beneficial effects of this invention are:

[0023] 1. The preparation method of Fritillaria cirrhosa extract provided by the present invention involves the precipitation of polysaccharide cyclodextrin combined with alkaloids into macromolecules during the first extraction process, and the precipitation of polysaccharide and organic acid combined with alkaloids into macromolecules during the second extraction process. This greatly improves the extraction rate of total alkaloids in Fritillaria cirrhosa. The method is simple to operate, stable, and has good application prospects.

[0024] 2. The macroporous adsorption resin purification equipment used in this invention can effectively enrich the total alkaloids of Fritillaria cirrhosa. It has the advantages of high removal rate, fast purification speed and high efficiency, resulting in high purity of total alkaloids in Fritillaria cirrhosa extract.

[0025] 3. The macroporous adsorption resin purification device used in this invention has resins with gradient polarity, which significantly increases the content of fritillaria, the main alkaloid component in the extract after purification. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the pore adsorption resin impurity removal device according to one technical solution of the present invention. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments:

[0028] Example 1

[0029] Step 1: Dry and pulverize the bulbs of Fritillaria cirrhosa from Aba, Sichuan, and pass them through a 60-mesh sieve to obtain Fritillaria cirrhosa powder. Mix Fritillaria cirrhosa powder and cyclodextrin powder at a mass ratio of 120:1 and stir-fry for 3 minutes to obtain mixture 1.

[0030] Step 2: Extract mixture 1 by reflux with 60% ethanol at room temperature for 120 min. The ratio of mixture 1 to 60% ethanol is 1:6 g / mL. Separate the supernatant and filter residue.

[0031] Step 3: Dry the filter residue from Step 2 at 25°C, and stir-fry the dried filter residue and cyclodextrin powder at a mass ratio of 80:2 for 2 minutes to obtain Mixture 2.

[0032] Step 4: Extract mixture 2 by reflux at room temperature using 75% ethanol and organic acid for 60 min. The ratio of mixture 2 to 75% ethanol is 1:8, and the volume ratio of 75% ethanol to organic acid is 200:1. The organic acid is a mixture of lauric acid and octanoic acid, and the volume ratio of lauric acid to octanoic acid is 1:2. Obtain the supernatant 2.

[0033] Step 5: Combine supernatant 1 and supernatant 2 and add them to the macroporous adsorption resin impurity removal device. Then add water to the macroporous adsorption resin impurity removal device and control the flow rate to SV6. Wash with water until the eluent is colorless. Concentrate and dry to obtain Fritillaria cirrhosa extract ①.

[0034] like Figure 1 As shown, the macroporous resin impurity removal equipment includes:

[0035] The outer casing 6 has a liquid inlet 1 at the top and a liquid outlet 5 at the bottom;

[0036] The first adsorption cylinder is located in the upper middle part of the outer shell 6 and includes multiple sections of cylinder with progressively increasing diameters. The topmost cylinder is connected to the liquid inlet 1.

[0037] The second adsorption cylinder is located below the first adsorption cylinder and is connected to the first adsorption cylinder. It includes multiple cylinders with successively decreasing diameters, and the bottommost cylinder is connected to the liquid outlet 5.

[0038] The first adsorption cylinder contains medium polar resin in each section from top to bottom, with the filling density gradually increasing. The second adsorption cylinder contains polar resin in each section from top to bottom, with a uniform density.

[0039] The first adsorption cylinder comprises, from top to bottom, a first cylinder 21, a second cylinder 22, and a third cylinder 23. The second adsorption cylinder comprises, from top to bottom, a fourth cylinder 43, a fifth cylinder 42, and a sixth cylinder 41. The first and second adsorption cylinders are connected by a connecting cylinder 3. The connecting cylinder 3 contains a mixed resin with the same filling density as the bottom cylinder of the first adsorption cylinder. The mixed resin includes a neutral polar resin and a polar resin in a mass ratio of 4 to 6:2. The first cylinder 21, the second cylinder 22, and the third cylinder 23 contain a neutral polar resin, which is a polyacrylate polymer. The filling density of the neutral polar resin gradually increases from the first cylinder 21 to the third cylinder 23. The fourth cylinder 43, the fifth cylinder 42, and the sixth cylinder 41 contain a polar resin, which is acrylamide. The filling density of the polar resin is uniform from the fourth cylinder 43 to the sixth cylinder 41. Supernatant 1 and Supernatant 2 pass sequentially through the first cylinder 21 to the sixth cylinder 41. Other impurities are removed in the neutral resin section, and polysaccharides are removed in the polar resin section, resulting in alkaloids with high purity.

[0040] Comparative Example 1

[0041] Step 1: Dry and pulverize the bulbs of Fritillaria cirrhosa from Aba, Sichuan, and pass them through a 60-mesh sieve to obtain Fritillaria cirrhosa powder;

[0042] Step 2: Extract Fritillaria cirrhosa powder by reflux at room temperature with 60% ethanol for 120 min. The ratio of Fritillaria cirrhosa powder to 60% ethanol is 1:6 g / mL. Separate the supernatant and filter residue.

[0043] Step 3: Dry the filter residue from Step 2 at 25°C;

[0044] Step 4: Extract the dried filter residue by reflux with 75% ethanol at room temperature for 60 minutes. The ratio of dried filter residue to 75% ethanol is 1:8. The supernatant is then separated.

[0045] Step 5: Combine supernatant one and supernatant two and add them to the macroporous adsorption resin impurity removal device described in Example 1. Then add water to the macroporous adsorption resin impurity removal device described in Example 1, control the flow rate to SV6, and wash with water until the eluent is colorless; concentrate and dry to obtain Fritillaria cirrhosa extract ②.

[0046] Comparative Example 2

[0047] Step 1: Dry and pulverize the bulbs of Fritillaria cirrhosa from Aba, Sichuan, and pass them through a 60-mesh sieve to obtain Fritillaria cirrhosa powder. Mix Fritillaria cirrhosa powder and cyclodextrin powder at a mass ratio of 120:1 and stir-fry for 3 minutes to obtain mixture 1.

[0048] Step 2: Extract mixture 1 by reflux with 60% ethanol at room temperature for 120 min. The ratio of mixture 1 to 60% ethanol is 1:6 g / mL. Separate the supernatant and filter residue.

[0049] Step 3: Dry the filter residue from Step 2 at 25°C;

[0050] Step 4: Extract the dried filter residue by reflux with 75% ethanol at room temperature for 60 minutes. The ratio of dried filter residue to 75% ethanol is 1:8. The supernatant is then separated.

[0051] Step 5: Combine supernatant one and supernatant two and add them to the macroporous adsorption resin impurity removal device described in Example 1. Then add water to the macroporous adsorption resin impurity removal device described in Example 1, control the flow rate to SV6, and wash with water until the eluent is colorless; concentrate and dry to obtain Fritillaria cirrhosa extract ③.

[0052] Comparative Example 3

[0053] Step 1: Dry and pulverize the bulbs of Fritillaria cirrhosa from Aba, Sichuan, and pass them through a 60-mesh sieve to obtain Fritillaria cirrhosa powder. Mix Fritillaria cirrhosa powder and cyclodextrin powder at a mass ratio of 120:1 and stir-fry for 3 minutes to obtain mixture 1.

[0054] Step 2: Extract mixture 1 by reflux with 60% ethanol at room temperature for 120 min. The ratio of mixture 1 to 60% ethanol is 1:6 g / mL. Separate the supernatant and filter residue.

[0055] Step 3: The filter residue from Step 2 is dried at 25°C. The dried filter residue and cyclodextrin powder are mixed and stir-fried for 2 minutes at a mass ratio of 80:2 to obtain Mixture 2.

[0056] Step 4: Extract mixture 2 by reflux with 75% ethanol at room temperature for 60 min. The ratio of mixture 2 to 75% ethanol is 1:8. The supernatant 2 is then separated.

[0057] Step 5: Combine supernatant 1 and supernatant 2 and add them to the macroporous adsorption resin impurity removal device of Example 1. Then add water to the macroporous adsorption resin impurity removal device of Example 1, control the flow rate to SV6, and wash with water until the eluent is colorless; concentrate and dry to obtain Fritillaria cirrhosa extract ④.

[0058] Comparative Example 4

[0059] Step 1: Dry and pulverize the bulbs of Fritillaria cirrhosa from Aba, Sichuan, and pass them through a 60-mesh sieve to obtain Fritillaria cirrhosa powder. Mix Fritillaria cirrhosa powder and cyclodextrin powder at a mass ratio of 120:1 and stir-fry for 3 minutes to obtain mixture 1.

[0060] Step 2: Extract mixture 1 by reflux with 60% ethanol at room temperature for 120 min. The ratio of mixture 1 to 60% ethanol is 1:6 g / mL. Separate the supernatant and filter residue.

[0061] Step 3: The filter residue from Step 2 is dried at 25°C. The dried filter residue and cyclodextrin powder are mixed and stir-fried for 2 minutes at a mass ratio of 80:2 to obtain Mixture 2.

[0062] Step 4: Extract mixture 2 by reflux at room temperature using 75% ethanol and organic acid for 60 min. The ratio of mixture 2 to 75% ethanol is 1:8, and the volume ratio of 75% ethanol to organic acid is 200:1. The organic acid is a mixture of lauric acid and octanoic acid, and the volume ratio of lauric acid to octanoic acid is 1:2. Obtain the supernatant 2.

[0063] Step 5: Combine supernatant 1 and supernatant 2 and add them to an AB-8 macroporous adsorption resin column. Then add water to the AB-8 macroporous adsorption resin column, control the flow rate to SV6, and wash with water until the eluent is colorless. Concentrate and dry to obtain Fritillaria cirrhosa extract ⑤.

[0064] The method for measuring the total alkaloid concentration is as follows:

[0065] Preparation of reference solution: Accurately weigh 4.58 mg of cibeline into a 50 mL volumetric flask, add chloroform to dilute to volume, and prepare a reference stock solution with a concentration of 0.0916 mg / mL. Store at 4 °C for later use.

[0066] Preparation of test solution: Take appropriate amounts of Fritillaria cirrhosa extract from Examples 1 and Comparative Examples 1-3 respectively, accurately weigh the above powder, add concentrated ammonia solution and seal for 1 h, add methanol-chloroform (1:4) mixed solution, mix well, reflux in water bath at 80℃ for 2 h, filter, wash the residue with methanol-chloroform (1:4) mixed solution, add the washing solution to a 25 mL volumetric flask, add methanol-chloroform (1:4) mixed solution to the mark, shake well, and store at 4℃ for later use.

[0067] Plotting the standard curve:

[0068] The absorbance of different concentrations of cibeline reference standard was determined by ultraviolet-visible spectrophotometry. The regression equation was obtained by using the least squares method with absorbance value (A) as the ordinate and reference standard mass (X) as the abscissa: A = 3.4532X - 0.0276 (r = 0.9986).

[0069] The RSD in the repeatability test met the requirements, indicating that the method has good reproducibility.

[0070] The concentration measurement method for Fritillaria cirrhosa is as follows:

[0071] Preparation of the reference solution: Accurately weigh an appropriate amount of fritillaria cirrhosa reference standard into a 50 mL volumetric flask, add methanol to make up to the volume, support the fritillaria cirrhosa reference standard stock solution, and store it at 4℃ for later use.

[0072] Preparation of test solution: Take appropriate amounts of the Fritillaria cirrhosa extract obtained in Example 1 and Comparative Examples 1-3 respectively, add concentrated ammonia solution and seal for 1 hour, add methanol-chloroform (1:4) mixed solution, mix well, reflux in water bath at 80℃ for 2 hours, filter, wash the residue with methanol-chloroform (1:4) mixed solution, add methanol-chloroform (1:4) mixed solution to the mark, shake well, and store at 4℃ for later use.

[0073] Plotting the standard curve:

[0074] The absorbance of different concentrations of fritillaria cirrhosa reference standard was determined by ultraviolet-visible spectrophotometry. The regression equations were obtained using the least squares method, with absorbance value (A) as the ordinate and reference standard mass (X) as the abscissa:

[0075] Fritillaria cirrhosa: A1 = 1.0639X1 + 6.4732, r1 = 0.9987;

[0076] The RSDs met the requirements in the repeatability experiments, indicating that the method has good reproducibility.

[0077] Further calculations were made of the total alkaloids (calculated as cibene) and the extraction rate of fritillaria cirrhosa, and the results are shown in Table 1. The purity of the total alkaloids (calculated as cibene) in Fritillaria cirrhosa extracts ① to ⑤ was determined, and the results are shown in Table 2.

[0078] Table 1. Extraction rates of each component in Example 1 and Comparative Examples 1-3

[0079]

[0080] Table 2 Purity of total alkaloids in Fritillaria cirrhosa extracts ①~⑤

[0081] Group Extract ① Extract ② Extract ③ Extract ④ Extract ⑤ purity% 42.53 27.96 31.63 36.50 31.84

[0082] As can be seen from the data in Table 1, the preparation method of Fritillaria cirrhosa extract provided by the present invention used in Example 1 is as follows: In the first extraction, Fritillaria cirrhosa powder and cyclodextrin are stir-fried and then extracted with ethanol; in the second extraction, the filter residue and cyclodextrin are stir-fried and then extracted with a mixed solvent of ethanol and organic acid to obtain Fritillaria cirrhosa extract ①. Comparative Example 1 uses the traditional ethanol extraction method to extract Fritillaria cirrhosa extract ② twice. In Comparative Example 2, Fritillaria cirrhosa powder and cyclodextrin are stir-fried and then extracted in the first extraction, and traditional ethanol extraction is used in the second extraction to obtain Fritillaria cirrhosa extract ③. In Comparative Example 3, Fritillaria cirrhosa powder and cyclodextrin are stir-fried and then extracted in the first extraction, and the filter residue and cyclodextrin are stir-fried and then extracted with ethanol to obtain Fritillaria cirrhosa extract ④. As can be seen from the data in Table 1, the extraction rates of total alkaloids (calculated as citrinin) and Fritillaria cirrhosa extract in Example 1 are much higher than those in Comparative Example 1. The method for preparing Fritillaria cirrhosa extract provided by this invention achieves twice the extraction rates of total alkaloids and fritillaria cylindrica compared to the traditional two-stage ethanol extraction method. Compared to Comparative Example 2, although the extraction rates of total alkaloids and fritillaria cylindrica were improved by stir-frying Fritillaria cirrhosa powder with cyclodextrin and then extracting with ethanol in the first extraction, they were still significantly lower than those of Example 1. Compared to Comparative Example 3, the extraction rates of total alkaloids and fritillaria cylindrica were improved by stir-frying Fritillaria cirrhosa powder with cyclodextrin and then extracting with ethanol in the second extraction, and the extraction rates of total alkaloids and fritillaria cylindrica were improved compared to Comparative Example 2, but still lower than those of Example 1. This is because in the second extraction, the filter residue of Example 1 was stir-fried with cyclodextrin and then extracted with a mixed solvent of ethanol and organic acid, forming a eutectic system, which greatly improved the extraction rates of total alkaloids and fritillaria cylindrica.

[0083] As can be seen from Table 2, the only difference between Comparative Example 4 and Example 1 is that Comparative Example 4 uses a traditional AB-8 macroporous resin column for adsorption and purification, while Example 1 uses the gradient polarity macroporous adsorption resin purification equipment provided in this application. With the same extraction method, the purity of the total alkaloids in the Fritillaria cirrhosa extract obtained in Example 1 is much higher than that in the Fritillaria cirrhosa extract of Comparative Example 4. This proves that the macroporous adsorption resin purification equipment provided in this application can effectively improve the purity of the total alkaloids in the Fritillaria cirrhosa extract.

[0084] Example 2

[0085] The present invention also includes a method for further optimizing the macroporous adsorption resin impurity removal device. A buffer layer is provided at the bottom of the bottom of the first adsorption cylinder, that is, a buffer layer 24 is provided at the bottom of the third cylinder 23. The buffer layer 24 contains a mixed resin, which includes a medium polar resin and a polar resin in a mass ratio of 4 to 6:2. Another buffer layer is provided at the bottom of the top of the second adsorption cylinder, that is, another buffer layer 44 is provided at the top of the fourth cylinder 43. The other buffer layer 44 contains a mixed resin, which includes a medium polar resin and a polar resin in a mass ratio of 8 to 10:1.

[0086] Example 3

[0087] Step 1: Dry and pulverize the bulbs of Fritillaria cirrhosa from Kangding, Sichuan, and pass them through a 120-mesh sieve to obtain Fritillaria cirrhosa powder. Mix Fritillaria cirrhosa powder and cyclodextrin powder at a mass ratio of 200:1 and stir-fry for 8 minutes to obtain mixture 1.

[0088] Step 2: Extract mixture 1 by reflux with 70% ethanol at room temperature for 180 min. The ratio of mixture 1 to 70% ethanol is 1:12 g / mL. Separate the supernatant and filter residue.

[0089] Step 3: The filter residue from Step 2 is dried at 30°C. The dried filter residue and cyclodextrin powder are mixed and stir-fried for 4 minutes at a mass ratio of 120:5 to obtain Mixture 2.

[0090] Step 4: Mixture 2 is extracted by reflux at room temperature using 95% ethanol and organic acid for 80 min. The ratio of mixture 2 to 95% ethanol is 1:10, and the volume ratio of 95% ethanol to organic acid is 300:1. The organic acid is a mixture of lauric acid and octanoic acid, and the volume ratio of lauric acid to octanoic acid is 1:2. The supernatant 2 is then separated.

[0091] Step 5: Combine supernatant 1 and supernatant 2, first determine the concentration of total alkaloids and fritillaria syrup, then add them to the macroporous adsorption resin purification device described in Example 1, then add water to the macroporous adsorption resin purification device, control the flow rate to SV4, wash with water until the eluent is colorless; concentrate and dry to obtain Fritillaria cirrhosa extract ⑥.

[0092] Example 4

[0093] Step 1: Dry and pulverize the bulbs of Fritillaria cirrhosa from Chengdu, Sichuan, and pass them through a 100-mesh sieve to obtain Fritillaria cirrhosa powder. Mix Fritillaria cirrhosa powder and cyclodextrin powder at a mass ratio of 150:1 and stir-fry for 5 minutes to obtain mixture 1.

[0094] Step 2: Extract mixture 1 by reflux with 75% ethanol at room temperature for 240 min. The ratio of mixture 1 to 75% ethanol is 1:10 g / mL. Separate the supernatant and filter residue.

[0095] Step 3: The filter residue from Step 2 is dried at 35°C. The dried filter residue and cyclodextrin powder are mixed and stir-fried for 3 minutes at a mass ratio of 100:2 to obtain Mixture 2.

[0096] Step 4: Mixture 2 is extracted by reflux at room temperature using 85% ethanol and organic acid for 120 min. The ratio of mixture 2 to 85% ethanol is 1:9, and the volume ratio of 85% ethanol to organic acid is 250:1. The organic acid is a mixture of lauric acid and octanoic acid, and the volume ratio of lauric acid to octanoic acid is 1:2. The supernatant 2 is then separated.

[0097] Step 5: Combine supernatant 1 and supernatant 2, first determine the concentration of total alkaloids and fritillaria syrup, then add them to the macroporous adsorption resin purification device described in Example 1, then add water to the macroporous adsorption resin purification device, control the flow rate to SV5, wash with water until the eluent is colorless; concentrate, dry, and obtain Fritillaria cirrhosa extract ⑦.

[0098] The total alkaloid extraction rates of Fritillaria cirrhosa extracts ⑥ and ⑦ were both higher than 0.8 mg / g, and the fritillaria cirrhosa extract rate was higher than 0.98 mg / g, which significantly exceeded the total alkaloid extraction rates of Fritillaria cirrhosa extracts obtained in comparative examples 1 to 3. The purity of the total alkaloids of Fritillaria cirrhosa extracts ⑥ and ⑦ was also higher than 40%.

[0099] In summary, according to the present invention, the present invention has at least the following beneficial effects:

[0100] 1. The preparation method of Fritillaria cirrhosa extract provided by the present invention involves the precipitation of polysaccharide cyclodextrin combined with alkaloids into macromolecules during the first extraction process, and the precipitation of polysaccharide and organic acid combined with alkaloids into macromolecules during the second extraction process. This greatly improves the extraction rate of total alkaloids in Fritillaria cirrhosa. The method is simple to operate, stable, and has good application prospects.

[0101] 2. The macroporous adsorption resin purification equipment used in this invention can effectively enrich the total alkaloids of Fritillaria cirrhosa. It has the advantages of high removal rate, fast purification speed and high efficiency, resulting in high purity of total alkaloids in Fritillaria cirrhosa extract.

[0102] 3. The macroporous adsorption resin purification device used in this invention has resins with gradient polarity, which significantly increases the content of fritillaria, the main alkaloid component in the extract after purification.

[0103] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for preparing Fritillaria cirrhosa extract, characterized in that the steps include... include: Step 1: Dry the bulbs of Fritillaria cirrhosa, pulverize them and pass them through a 60-120 mesh sieve to obtain Fritillaria cirrhosa powder. Take Fritillaria cirrhosa powder and cyclodextrin powder at a mass ratio of 120-200:1 and stir-fry for 3-8 minutes to obtain mixture 1. Step 2: Extract mixture 1 by reflux at room temperature with 60-75% ethanol. The ratio of mixture 1 to 60-75% ethanol is 1:6-12 g / mL. Separate the supernatant and filter residue. Step 3: Dry the filter residue from Step 2, and stir-fry the dried filter residue and cyclodextrin powder at a mass ratio of 80~120:2~5 for 2~4 minutes to obtain Mixture 2; Step 4: Mixture 2 is extracted by reflux at room temperature using ethanol with a volume concentration of 75-95% and organic acid. The ratio of mixture 2 to 75-95% ethanol is 1:8-10, and the volume ratio of 75-95% ethanol to organic acid is 200-300:

1. Supernatant 2 is obtained. Step 5: Combine supernatant 1 and supernatant 2 and add them to the macroporous adsorption resin impurity removal device. Then add ethanol to the macroporous adsorption resin impurity removal device until the eluent is colorless. Concentrate and dry to obtain Fritillaria cirrhosa extract. In step four, the organic acid is a mixture of lauric acid and octanoic acid, with a volume ratio of lauric acid to octanoic acid of 1:

2.

2. The method for preparing Fritillaria cirrhosa extract according to claim 1, characterized in that, In step two, the reflux extraction time is 120~240 min.

3. The method for preparing Fritillaria cirrhosa extract according to claim 1, characterized in that, The drying temperature in step three is 25~35℃.

4. The method for preparing Fritillaria cirrhosa extract according to claim 1, characterized in that, The reflux extraction time in step four is 60~120 min.

5. The method for preparing Fritillaria cirrhosa extract according to claim 1, characterized in that, In step five, the volume ratio of the ethanol aqueous solution to the macroporous adsorption resin is 3-4:

1.

6. The method for preparing Fritillaria cirrhosa extract according to claim 1, characterized in that, The macroporous adsorption resin impurity removal equipment includes: The outer casing has a liquid inlet at the top and a liquid outlet at the bottom. The first adsorption cylinder is located in the upper middle part of the outer shell and includes multiple interconnected cylinders with progressively increasing diameters, with the topmost cylinder connected to the liquid inlet. The second adsorption cylinder is located below the first adsorption cylinder and is connected to the first adsorption cylinder. It includes multiple cylinders with successively decreasing diameters, and the bottommost cylinder is connected to the liquid outlet. The first adsorption cylinder contains medium polar resin in each section from top to bottom, with the filling density gradually increasing. The second adsorption cylinder contains polar resin in each section from top to bottom, with a uniform density.

7. The method for preparing Fritillaria cirrhosa extract according to claim 6, characterized in that, The bottom of the first adsorption cylinder is provided with a buffer layer, and the buffer layer contains mixed resin.

8. The method for preparing Fritillaria cirrhosa extract according to claim 7, characterized in that, The first adsorption cylinder and the second adsorption cylinder are connected by a connecting cylinder. The connecting cylinder contains a mixed resin with the same filling density as the bottom cylinder of the first adsorption cylinder. The mixed resin includes a medium polar resin and a polar resin with a mass ratio of 4 to 6:2.

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