Method for extracting and purifying alkaloids in datura stramonium

Abstract

The present application relates to a kind of extraction and purification method of alkaloids in Datura stramonium, which comprises hydrochloric acid solution soaking, filter residue and filtrate are obtained by filtering, ammonia water test solution is used to adjust pH value, shake extraction, remove solvent, obtain Datura stramonium alkaloid extract, set chromatographic column elution procedure, elution is carried out, and the purity of alkaloids in Datura stramonium is obtained by collecting fraction detection calculation.The extraction step of the present application is simple, ethanol impregnation is reduced, can avoid that tannin, pigment and other components in Datura stramonium enter alkaloid extract in large quantities, can reduce the difficulty of subsequent purification, improve the life of purification medium, and save cost;And the reverse phase chromatography used in the purification part of the present application reduces the use of organic phase, is environmentally friendly, saves cost;While reverse phase chromatography has better separation effect, and the purity of prepared hyoscyamine and scopolamine is higher.

Description

Technical Field

[0001] This invention relates to a method for extracting and purifying alkaloids, and particularly to a method for extracting and purifying alkaloids from Datura stramonium. Background Technology

[0002] Datura metel L., a plant in the Solanaceae family, is primarily composed of hyoscyamine alkaloids, mainly including hyoscyamine (atropine, used for antispasmodics, analgesics, antidote to organophosphate poisoning, and mydriasis), scopolamine (sedative and anesthetic), hyoscyamine alkaloids, and N-norhyoscyamine. The Chinese Pharmacopoeia uses atropine sulfate and scopolamine hydrobromide as indicator components.

[0003] There are many methods for extracting and purifying alkaloids such as scopolamine from Datura stramonium. Patent publication number CN106083871A discloses a method for extracting and purifying scopolamine from Datura stramonium. The operation steps are as follows: dry Datura stramonium is crushed, extracted with ethanol solution at constant temperature, and the supernatant is centrifuged to obtain an extract; the extract is filtered through a ceramic membrane, concentrated by nanofiltration membrane, and the ethanol is recovered to obtain a mixture; the mixture is dissolved in dilute hydrochloric acid, filtered, and the filtrate is collected; the filtrate is extracted three times with chloroform, and the aqueous solution is retained; the pH of the aqueous phase is adjusted by adding NaOH solution, and then extracted four times with chloroform to obtain the chloroform solution; the chloroform solution is dried with anhydrous Na2SO4, and the chloroform is recovered to obtain total alkaloids; the total alkaloids are separated by alkaline alumina column chromatography, and the chloroform is recovered to obtain scopolamine.

[0004] Patent publication number CN108593793B discloses a method for separating and purifying scopolamine using coordination column chromatography. The method includes: Step 1: Preparation of crude scopolamine extract. Dried Datura stramonium flowers are pulverized using a pulverizer and passed through a 40-mesh sieve to obtain Datura stramonium powder. The obtained Datura stramonium powder is added to a mixture of ammonia and dichloromethane for ultrasonic extraction. The mixture is filtered, and the residue is extracted twice. The extracts are combined and dried to obtain crude scopolamine extract. Step 2: Preparation of coordination chromatography column. Packing material and a coordinating agent are ground in a sample pulverizer to obtain powder with a particle size of 5-15 μm. Ethyl acetate is then added, and the mixture is thoroughly mixed before being packed into a 4.6 mm × 250 mm chromatographic column. The column is allowed to stand for 1 day to allow the coordinating ions to fully coordinate and combine with the packing material, thus obtaining the coordination chromatography column. Step 3: Equilibrate the coordination chromatography column. Wash the coordination chromatography column obtained in Step 2 with ethyl acetate-methanol-concentrated ammonia solution until no coordination ion reaction occurs. Step 4: Chromatographic separation: Redissolve the crude scopolamine extract in ethyl acetate, filter to remove insoluble matter, and add it to the coordination chromatography column. After the sample is completely adsorbed, elute with ethyl acetate-methanol-concentrated ammonia solution. Collect the eluent in fractions and use HPLC to detect the scopolamine content in each fraction.

[0005] The above methods suffer from cumbersome steps, low purification efficiency, and low yield. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems in the prior art and provide a method for extracting and purifying alkaloids from Datura stramonium.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting and purifying alkaloids from Datura stramonium, comprising the following steps:

[0008] S1: Take dried Datura stramonium flowers, weigh them accurately, add hydrochloric acid solution, and soak for several hours;

[0009] S2: Filter to obtain filter residue and filtrate. Wash the filter residue and filter with hydrochloric acid solution to obtain washing liquid. Combine the filtrate and washing liquid.

[0010] S3: Adjust the pH of the S2 filtrate and washing solution to 9-11 with concentrated ammonia solution, then extract with chloroform by shaking, and combine the chloroform solutions;

[0011] S4: The combined chloroform liquid is evaporated under reduced pressure to remove the chloroform solvent, and the Datura stramonium alkaloid extract is obtained. Then the extract is dissolved in a small amount of methanol aqueous solution.

[0012] S5: Take a reversed-phase chromatographic column, set the column elution program, inject the dissolved extract into the column, elute, collect the fraction, and calculate the purity of the alkaloids in Datura stramonium; the chromatographic column packing material is Silicaone Prep-10C18, and the column size is 20mm I.D. x 250mm.

[0013] The method for extracting and purifying alkaloids from Datura stramonium according to the present invention, wherein the concentration of hydrochloric acid solution in steps S1 and S2 is 2 mol / L.

[0014] The method for extracting and purifying alkaloids from Datura stramonium according to the present invention includes step S1 of soaking for 24 hours.

[0015] The method for extracting and purifying alkaloids from Datura stramonium according to the present invention includes step S3, in which the alkaloids are extracted three times by shaking with chloroform.

[0016] The elution procedure described in the above scheme is as follows: 0-15 minutes, phase A accounts for 100% and phase B accounts for 0%; 16-45 minutes, phase A accounts for 95% and phase B accounts for 5%; 46-66 minutes, phase A accounts for 90% and phase B accounts for 10%; 67-100 minutes, phase A accounts for 10% and phase B accounts for 90%.

[0017] In the above elution procedure, phase A is purified water and phase B is methanol, and the proportions are by volume.

[0018] The method for extracting and purifying alkaloids from Datura stramonium to achieve the purpose of this invention may further include the following steps:

[0019] S1: Take dried Datura stramonium flowers, weigh them accurately, and soak them in concentrated ammonia and chloroform for several hours.

[0020] S2: Filter by suction filtration, wash with a small amount of chloroform, and combine the filtrate and washings;

[0021] S3: The combined filtrate and washings are evaporated under reduced pressure to remove the chloroform solvent, yielding Datura stramonium alkaloid extract, which is then dissolved in a small amount of methanol aqueous solution.

[0022] S4: Take a reversed-phase chromatographic column, set the column elution program, inject the dissolved extract into the column, use methanol and water as the mobile phase for elution, collect the fraction and calculate the purity of the alkaloids in Datura stramonium. The chromatographic column packing material is Silicaone Prep-10C18, and the column size is 20mm I.D. x 250mm.

[0023] The above-mentioned method for extracting and purifying alkaloids from Datura stramonium involves soaking for 24 hours in step S1.

[0024] The elution procedure is as follows: at 0 minutes, phase A accounts for 100% and phase B accounts for 0%; at 120 minutes, phase A accounts for 0% and phase B accounts for 100%; in the above elution procedure, phase A is purified water and phase B is methanol, and the percentages are volume ratios.

[0025] The present invention provides two methods for the extraction and purification of alkaloids from Datura stramonium, wherein the elution flow rate during the elution process is 10 ml / min.

[0026] This invention has positive effects:

[0027] (1) The extraction steps of this invention are simple, reduce ethanol soaking, avoid large amounts of tannins, pigments and other components in Datura stramonium entering the alkaloid extract, reduce the difficulty of subsequent purification, improve the lifespan of the purification medium, and save costs.

[0028] (2) The purification part of this invention uses reversed-phase chromatography. This method mainly uses methanol and water as the mobile phase, which reduces the use of organic phase, is environmentally friendly, and saves costs. At the same time, reversed-phase chromatography has a better separation effect, and the scopolamine and hyoscyamine prepared are of higher purity. Attached Figure Description

[0029] Figure 1 This is a standard reference spectrum obtained from an embodiment of the present invention;

[0030] Figure 2 This is the detection spectrum of the Datura stramonium extract in Example 1 of the present invention;

[0031] Figure 3 This is the detection spectrum of the Datura stramonium extract in Example 2 of the present invention;

[0032] Figure 4 This is the elution spectrum of the extract mixture from Example 1 of the present invention;

[0033] Figure 5 This is the detection chromatogram of the fraction obtained from the purified extract mixture of Example 1 of the present invention after 2-24 min.

[0034] Figure 6 This is the detection chromatogram of the fraction obtained after purification of the extract mixture in Example 1 of this invention at 25-46 min.

[0035] Figure 7 This is the elution spectrum of the extract mixture from Example 2 of the present invention;

[0036] Figure 8 This is the detection chromatogram of the fraction obtained after purification of the extract mixture in Example 2 of this invention, taken 15-25 minutes later.

[0037] Figure 9 This is the detection chromatogram of the fraction obtained after purification of the extract mixture in Example 2 of this invention at 92-100 min. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below through embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] This invention provides an embodiment of the detection method for extracting certain components:

[0040] Chromatographic column: HPLCONE 5C18A 4.6mm ID x 250mm

[0041] Mobile phase: acetonitrile-30 mmol ammonium acetate solution (containing 0.02% triethylamine and 0.3% tetrahydrofuran, pH adjusted to 6.0 with glacial acetic acid) (10:90, V / V), detection wavelength: 216 nm, flow rate: 1.0 mL / min, column temperature: 30 °C.

[0042] Standard reference spectra are attached to the instruction manual. Figure 1 :

[0043] Camphor alkaloid hydrobromide: 0.54 mg / ml;

[0044] Racemic scopolamine: 0.54 mg / ml;

[0045] Scopolamine: 0.54 mg / ml;

[0046] Hyoscyamine: 0.54 mg / ml.

[0047] (Example 1)

[0048] Extraction: Accurately weigh 100 g of dried Datura stramonium and add 500 mL of 2 mol / L hydrochloric acid solution. Soak for 24 h, filter, wash the residue and filter with 2 mol / L hydrochloric acid, combine the filtrate and washings, adjust the pH to 10 with concentrated ammonia solution, and extract three times with chloroform by shaking. Combine the chloroform extracts (total volume 350 mL), take 4 mL, evaporate to dryness, add 2 mL of mobile phase for detection, and inject 5 μL. See the attached chromatogram in the instruction manual. Figure 2 .

[0049] The content was calculated using the external standard method.

[0050] (+) Scopolamine: small amount

[0051] Scopolamine: 145mg

[0052] Hyoscyamine: 52.9 mg.

[0053] Purification section: Combine the extracts, rotary evaporate under reduced pressure to remove chloroform solvent, and dissolve in a small amount of methanol aqueous solution.

[0054] Chromatographic packing material: Silicaone Prep-10C18, column dimensions: 20mm I.D. x 250mm

[0055] Detection wavelength: 216 nm, elution flow rate: 10 ml / min

[0056] Mobile phase A: purified water, Mobile phase B: methanol

[0057] Elution procedure: Time Phase A Phase B 0 100 0 15 100 0 16 95 5 45 95 5 46 90 10 66 90 10 67 10 90 100 10 90

[0066] Inject the dissolved sample into the chromatographic column and elute according to the above procedure, where the time is in minutes, and the volume ratio of phase A to phase B is [volume ratio]. See the attached instruction manual for the elution chromatogram. Figure 4 ;

[0067] Collect the fractions from 2 min to 24 min for analysis; the analysis chromatograms are attached and can be found in the instruction manual. Figure 5 Scopolamine was prepared with a purity of 86.54%.

[0068] Collect the fraction from 25 min to 46 min for analysis; the detection chromatogram is attached and can be found in the instruction manual. Figure 6 The purity of hyoscyamine prepared was 97.839%.

[0069] (Example 2)

[0070] Extraction: Accurately weigh 100g of dried Datura stramonium, add 100ml of concentrated ammonia and 400ml of chloroform, soak for 24 hours, filter, wash with a small amount of chloroform, combine the filtrate and washings (total volume 510ml), take 4ml and evaporate to dryness, add 2ml of mobile phase for detection, inject 5ul. See the attached instruction manual for the detection chromatogram. Figure 3 .

[0071] The content was calculated using the external standard method.

[0072] (+) Scopolamine: small amount

[0073] Scopolamine: 121.8 mg

[0074] Hyoscyamine: 12.6 mg

[0075] Purification section:

[0076] Combine the extracts, evaporate under reduced pressure using a rotary evaporator to remove the chloroform solvent, and dissolve in a small amount of methanol-water solution.

[0077] Chromatographic packing material: Silicaone Prep-10C18.

[0078] Column dimensions: 20mm I.D. x 250mm

[0079] Detection wavelength: 216 nm, elution flow rate: 10 ml / min

[0080] Mobile phase A: purified water, Mobile phase B: methanol

[0081] Elution procedure: Time Phase A Phase B 0 100 0 120 0 100

[0084] Inject the dissolved sample into the chromatographic column and elute according to the above procedure, where the time is in minutes, and the volume ratio of phase A to phase B is [volume ratio]. See the attached instruction manual for the elution chromatogram. Figure 7 ;

[0085] Collect the fraction from 15 min to 25 min for analysis; the detection spectrum is attached and can be found in the instruction manual. Figure 8 Scopolamine was prepared with a purity of 82.71%.

[0086] Collect the fraction from 92 min to 100 min for analysis; the analysis spectrum is attached and can be found in the instruction manual. Figure 9 The purity of hyoscyamine prepared was 94.73%.

[0087] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting and purifying alkaloids from Datura stramonium, characterized in that... Includes the following steps: S1: Take dried Datura stramonium flowers, weigh them accurately, add hydrochloric acid solution, and soak for several hours; S2: Filter to obtain filter residue and filtrate. Wash the filter residue and filter with hydrochloric acid solution to obtain washing liquid. Combine the filtrate and washing liquid. S3: Adjust the pH of the S2 filtrate and washing solution to 9-11 with concentrated ammonia solution, then extract with chloroform by shaking, and combine the chloroform solutions; S4: The combined chloroform liquid is evaporated under reduced pressure to remove the chloroform solvent, and the Datura stramonium alkaloid extract is obtained. Then the extract is dissolved in a small amount of methanol aqueous solution. S5: Take a reversed-phase chromatographic column, set the column elution program, inject the dissolved extract into the column, use methanol and water as the mobile phase for elution, collect the fractions and calculate the purity of the alkaloids in Datura stramonium.

2. The method for extracting and purifying alkaloids from Datura stramonium according to claim 1, characterized in that: The concentration of the hydrochloric acid solution in steps S1 and S2 is 2 mol / L.

3. The method for extracting and purifying alkaloids from Datura stramonium according to claim 1, characterized in that: The step S1 involves soaking for 24 hours.

4. The method for extracting and purifying alkaloids from Datura stramonium according to claim 1, characterized in that: In step S3, the sample is extracted three times by shaking with chloroform.

5. The method for extracting and purifying alkaloids from Datura stramonium according to claim 1, characterized in that: The elution procedure is as follows: 0-15 minutes, phase A accounts for 100% and phase B accounts for 0%; 16-45 minutes, phase A accounts for 95% and phase B accounts for 5%; 46-66 minutes, phase A accounts for 90% and phase B accounts for 10%; 67-100 minutes, phase A accounts for 10% and phase B accounts for 90%. In the above elution procedure, phase A is purified water and phase B is methanol, and the proportions are by volume.

6. A method for extracting and purifying alkaloids from Datura stramonium, characterized in that... Includes the following steps: S1: Take dried Datura stramonium flowers, weigh them accurately, and soak them in concentrated ammonia and chloroform for several hours. S2: Filter by suction filtration, wash with a small amount of chloroform, and combine the filtrate and washings; S3: The combined filtrate and washings are concentrated by rotary evaporation under reduced pressure to remove the chloroform solvent, yielding Datura stramonium alkaloid extract, which is then dissolved in a small amount of methanol aqueous solution. S4: Take a reversed-phase chromatographic column, set the column elution program, inject the dissolved extract into the column, use methanol and water as the mobile phase for elution, collect the fractions and calculate the purity of the alkaloids in Datura stramonium.

7. The method for extracting and purifying alkaloids from Datura stramonium according to claim 6, characterized in that: The step S1 involves soaking for 24 hours.

8. The method for extracting and purifying alkaloids from Datura stramonium according to claim 6, characterized in that: The elution procedure is as follows: at 0 minutes, phase A accounts for 100% and phase B accounts for 0%; at 120 minutes, phase A accounts for 0% and phase B accounts for 100%; in the above elution procedure, phase A is purified water and phase B is methanol, and the percentages are volume ratios.

9. The method for extracting and purifying alkaloids from Datura stramonium according to claim 1 or 6, characterized in that: The elution flow rate during the elution process is 10 ml / min.

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