Method for preparing chimonanthine

Abstract

The invention discloses a method for preparing chimonanthine from chimonanthus praecox seeds. In the high-speed counter-current chromatography separation process, the two-phase solvent system adopts hexane-ethyl acetate-methanol-water with the volume ratio of 3: 5: 3: 5, the upper layer is used as a stationary phase to be pumped into a counter-current chromatography separation column, the lower layer is used as a mobile phase to be pumped into a high-speed counter-current chromatograph for counter-current extraction separation, the rotating speed of a main machine is 800r / min, the flow velocity of the mobile phase is 2mL / min, the column temperature is 30 DEG C, and the volume ratio of hexane to ethyl acetate to methanol to water is 3: 5: 3: 5; and collecting the target component, carrying out reduced pressure distillation to remove the solvent to obtain chimonanthine with the purity of 84.6%, and further recrystallizing to obtain a reference substance with the purity of 96.5%. The method has the advantages of low cost, simple process, high purity, quick separation and purification and easiness in preparation.

Description

Technical Field

[0001] The invention relates to a method for extracting and separating a quality marker of wintersweet seeds, namely, sargentodoxine, and belongs to the field of traditional Chinese medicine chemistry. Background Art

[0002] Chimonanthine is an alkaloid extracted from wintersweet seeds, which has the following uses. Antibacterial and anti-inflammatory: It has a certain inhibitory effect on a variety of bacteria and viruses, such as common respiratory tract pneumococci, Staphylococcus aureus, and beta-hemolytic streptococci. It has potential value in the treatment of respiratory infections and other diseases, and can help reduce inflammatory reactions. Antitussive and antiasthmatic: It can relax the bronchi, relieve bronchospasm, and reduce mucus secretion through its effects on respiratory smooth muscles, thereby relieving cough, asthma, and expectoration, and is helpful for relieving symptoms of diseases such as asthma and chronic bronchitis. In addition, chimonanthine also has a certain antioxidant effect, which can scavenge free radicals in the body and reduce oxidative damage. It may be of certain significance for the prevention and treatment of some diseases related to oxidative stress, such as cardiovascular diseases and neurodegenerative diseases; it also has certain activity in analgesia, and may be used as an adjuvant treatment for mild to moderate pain.

[0003] In the agricultural field, cyperine can be used as a biological pesticide: cyperine has the effect of repelling and inhibiting the growth and development of some common crop pests, such as aphids and red spiders, and can be developed into a biological pesticide to reduce the use of chemical pesticides and reduce environmental pollution. At the same time, it has a certain protective effect on crops and improves crop yield and quality.

[0004] In the chemical industry, salvia azedarach alkaloids can be used as a cosmetic raw material: based on its antioxidant and other properties, it can be added to cosmetics to help reduce the damage of free radicals to the skin and delay skin aging. It may also have a certain antibacterial effect, helping to maintain skin health and reduce skin problems such as acne.

[0005] The present invention reports a novel countercurrent extraction technology, which is based on high-speed countercurrent chromatography (HSCCC), which is a cutting-edge separation and preparation method with strong sample carrying capacity and good repeatability. It does not require solid substances as stationary phases, thus avoiding the problem of sample adsorption loss caused by solid carriers; the sample recovery effect can be significantly improved. HSCCC is based on a two-layer incompatible layered solvent system, and uses multi-layer spiral pipes for high-speed planetary centrifugation, thereby successfully separating samples in an incompatible two-phase solvent system. The present invention provides a convenient method for preparing high-purity syringine. Summary of the invention

[0006] The invention provides a simple and practical method for separating and purifying silybum alkaloids, which has the characteristics of low cost, simple process and high purity.

[0007] In order to achieve the above object, the technical method adopted by the present invention comprises the following steps:

[0008] Preferably, a method for preparing silybum alkaloids comprises using high-speed countercurrent chromatography to separate and purify the organic solvent extract of silybum alkaloids seeds, collecting each fraction, and then concentrating under reduced pressure to evaporate the solvent, and further recrystallizing to obtain a silybum alkaloids reference substance.

[0009] Preferably, the extraction adopts a two-step solvent method, in which the organic solvent used in the first step of degreasing is petroleum ether, gasoline, hexane, and dichloromethane with a boiling range of 60-90°C, a solid-liquid ratio of 1:8-1:20, an extraction time of 12-24 hours, and the extraction method adopted is room temperature cold soaking. The second step is an alcohol extraction method, using methanol or ethanol for soaking at room temperature, a solid-liquid ratio of 1:8-1:15, an extraction time of 12-24 hours, and 1-3 extraction times, and the extract is concentrated under reduced pressure to obtain a sample.

[0010] Preferably, the high-speed countercurrent chromatography technology is adopted, the two-phase solvent system is hexane-ethyl acetate-methanol-water with a volume ratio of 3:5:3:5, the countercurrent chromatography host speed is 700rpm-1000rpm, the countercurrent extraction speed is 2mL / min, and the detection wavelength is 254nm.

[0011] Preferably, the elution time of the separated and collected target component is 170-190 minutes after injection, and the target component needs to be evaporated under reduced pressure to remove the solvent, and the required temperature is 40-60°C.

[0012] The beneficial effects of the present invention are:

[0013] 1. Screening for suitable organic solvent degreasing. Because the target component has medium polarity and is easily interfered by low-polarity components and difficult to purify, organic solvent degreasing is used, such as hexane, petroleum ether, gasoline, etc. Considering the extraction cost and extraction rate, petroleum ether is selected for degreasing.

[0014] 2. Low-temperature extraction facilitates the preservation of active ingredients from being destroyed, and the room temperature cold soaking method is selected in the present invention.

[0015] 3. The process is stable and easy to operate. Prepare the countercurrent chromatography solvent system, take hexane-ethyl acetate-methanol-water with a volume ratio of 3:5:3:5, shake and mix in a separatory funnel, release the lower phase and upper phase respectively after stratification, and degas in ultrasound for 15 minutes. Pump the upper phase into the high-speed countercurrent chromatography separation tube as the stationary phase. After the stationary phase fills the entire separation column, start the main engine to rotate the separation solenoid at high speed, and set the column oven temperature to a constant value. Subsequently, pump the lower phase as the mobile phase at a certain flow rate, turn on the ultraviolet detector to record the baseline. After the two phases reach equilibrium, that is, the mobile phase flows out of the end of the separation column, start sampling, and then start sampling, and detect and collect the target components. Collect each chromatographic peak, and detect the purity of each peak by high-performance liquid chromatography. The purity of the target compound is not high after one high-speed countercurrent chromatography separation, and further recrystallization is required to finally obtain a high-purity product. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the chemical structure of the compound silybumine studied in the present invention;

[0017] Figure 2 is the HPLC chromatogram of the second step extract;

[0018] Figure 3 It is the HPLC spectrum of the target compound obtained by high-speed countercurrent chromatography;

[0019] Figure 4 This is the mass spectrum of silybumine. DETAILED DESCRIPTION

[0020] Example 1: Defatting and alcohol extraction of Chimonanthus chinensis seeds.

[0021] 100g of wintersweet seeds were dried and crushed, and then placed in a 1000mL stoppered conical flask. 900mL of petroleum ether was poured into the conical flask and sealed. After cold soaking at room temperature for 24 hours in the dark, the filtrate was filtered, and the filtrate was concentrated under reduced pressure with a RE-52AA rotary evaporator to recover the solvent. Then, 900mL of the recovered petroleum ether was cold soaked for 24 hours for secondary defatting. The defatted wintersweet seed powder was soaked and extracted with 900mL of methanol at room temperature for 24 hours to obtain a methanol extract, which was concentrated under reduced pressure to recover methanol. After repeated extraction three times, 14.5g of extract was obtained, with an extraction rate of 14.5%.

[0022] Example 2: High speed countercurrent chromatography separation.

[0023] A two-phase solvent system consisting of a solvent system of hexane-ethyl acetate-methanol-water in a ratio of 3:5:3:5 was used. 300mL, 500mL, 300mL, and 500mL were added respectively and the mixture was naturally layered. The upper and lower layers were separated and ultrasonically degassed for 15min. The upper layer was the stationary phase and the lower layer was the mobile phase. 319mg of wintersweet seed extract sample was dissolved in 10mL of the upper and lower layers of the above solvent system as the injection liquid. The upper layer was pumped in at a flow rate of 30mL / min as the stationary phase. The main engine was turned forward at a speed of 800r / min, and the column oven temperature was set to 30℃. Subsequently, the mobile phase was pumped in at a flow rate of 2mL / min. The total outflow of the stationary phase reached 160mL and reached equilibrium, and the retention ratio of the stationary phase was 68%. Subsequently, 10mL of the sample solution was injected. After the injection was completed, the UV detector was turned on to detect and record at a wavelength of 254nm. The first peak appeared 65min after the injection, and each peak was collected and analyzed by high performance liquid chromatography. The component flowing out from 170-190min was confirmed to be silybendazole after analysis. After the separated components were concentrated under reduced pressure, 14.6mg of a compound with a purity of 84.6% was obtained, and further recrystallization gave 9.5mg of a compound with a purity of 96.5%. The mass spectrum of the compound was consistent with that of silybendazole, and its chemical structure was further identified as silybendazole by nuclear magnetic resonance spectroscopy.

Claims

1. A method for preparing silybum alkaloids, characterized in that: The organic solvent extract of wintersweet seeds was separated and purified by high-speed countercurrent chromatography and high-performance liquid chromatography, and each fraction was collected, and then concentrated under reduced pressure to evaporate the solvent to obtain a silybumine reference substance.

2. The method for preparing silybum alkaloids according to claim 1, wherein: Two-step extraction is adopted: the first step is degreasing, the organic solvent used for degreasing is one of petroleum ether, gasoline and hexane with a boiling range of 60-90°C, the solid-liquid ratio is 1:8-1:20, the extraction time is 12-24h, the number of extractions is 1-3 times, and the extraction method adopted is soaking at room temperature; The second step is characterized by: using methanol or ethanol to soak at room temperature, the solid-liquid ratio is 1:8-1:15, the extraction time is 12-24h, the number of extractions is 1-3 times, and the extract is concentrated under reduced pressure to obtain the sample.

3. The method for preparing silybum alkaloids according to claim 2, wherein: The two-phase solvent system of high-speed countercurrent chromatography is hexane-ethyl acetate-methanol-water in a volume ratio of 3:5:3:5, the countercurrent chromatography host speed is 700rpm-1000rpm, the countercurrent extraction speed is 2mL / min, and the detection wavelength is 254nm.

4. The method for preparing silybum alkaloids according to claim 3, wherein: The fractions from 170 to 190 min were collected and concentrated to dryness under reduced pressure.

5. The method for preparing silybum alkaloids according to any one of claims 2 to 4, characterized in that The prepared and separated samples were dissolved in methanol or ethanol for recrystallization to improve the purity.

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