Method for rapidly preparing 3, 29-dibenzoyl snakegourd triol

Abstract

The invention discloses a method for rapidly preparing 3, 29-dibenzoyl snakegourd kernel triol, which is characterized in that in a high-speed counter-current chromatography separation process, a two-phase solvent system adopts n-hexane and acetonitrile in a ratio of 1: 1, 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 the counter-current chromatography separation column, the rotating speed of a main machine is 800r / min, the rotating speed of the main machine is 800r / min, and the rotating speed of the main machine is 800r / min. The method comprises the following steps of: collecting target components by using preparative high performance liquid chromatography, selecting octadecylsilane chemically bonded silica as a main filling material, selecting methanol-water in a mobile phase ratio of 97: 3, eluting at a flow rate of 3 mL / min, and preparing the 3, 29-dibenzoyl karounitriol compound under the conditions that the flow rate of the mobile phase is 2 mL / min and the separation temperature is 30 DEG C, the method has the advantages of low cost, simple process, high purity, rapidness in separation and purification, and easiness in preparation of high-purity traditional Chinese medicine chemical reference substances.

Description

Technical Field

[0001] The invention relates to a method for extracting and separating a quality marker of trichosanthes seeds, namely 3,29-dibenzoyl trichosanthes trichosanthes, from trichosanthes seeds, and belongs to the field of traditional Chinese medicine chemistry. Background Art

[0002] Trichosanthes seeds are the dried and mature seeds of the plant Trichosanthes kirilowii Maxim in the Cucurbitaceae family. Usually, the mature fruits of Trichosanthes kirilowii are collected in autumn, and the mature seeds are taken, washed, and dried to obtain Trichosanthes seeds. The nature of Trichosanthes seeds is sweet and has a cold nature. It belongs to the lung, stomach, and large intestine meridians. Trichosanthes seeds have the effects of clearing heat, dissolving phlegm, eliminating dryness and heat, and lubricating the intestines. They are often used to treat coughs caused by lung heat and constipation caused by dry intestines. Trichosanthes seeds are a product with both medicinal and edible value. They have the effects of lowering blood sugar and blood lipids, anti-oxidation, and can improve cardiovascular diseases, resist bacteria, and inhibit tumor growth. From the perspective of material basis research, Trichosanthes seeds contain a variety of functional ingredients such as protein, phenylpropanoids, terpenes, sterols, sugars, and oils. 3,29-Dibenzoyltriol is the main active ingredient of Trichosanthes fruit, which has a certain affinity to lung and stomach tissues. This property is consistent with the meridian effect of Trichosanthes fruit, and the lung and stomach are also its main targets, providing a material basis for the treatment of diseases of the lung and gastric systems.

[0003] The Chinese Pharmacopoeia has clear regulations on the quality of Fructus Trichosanthis, especially the content of 3,29-dibenzoyl trichosanthis, which must be no less than 0.08%. The separation and preparation process of 3,29-dibenzoyl trichosanthis has been described in detail in existing literature. This process is mainly completed by high performance liquid chromatography technology, but this process faces many challenges, such as complex workload, long separation cycle, large amount of solvent consumption and serious sample loss. Therefore, it is particularly important to explore an efficient and simple separation method.

[0004] The present invention reports a novel extraction and purification technology for effective components of traditional Chinese medicine, which is based on high-speed countercurrent chromatography (HSCCC), 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; it can significantly improve the sample recovery effect. HSCCC is based on a two-layer incompatible layered solvent system, using multi-layer spiral pipes for high-speed planetary centrifugation, thereby successfully separating samples in an incompatible two-phase solvent system.

[0005] In this technology, high-speed countercurrent chromatography and preparative high-performance liquid chromatography are integrated to quickly prepare 3,29-dibenzoyltriol, a quality marker of Trichosanthes fruit. Summary of the invention

[0006] The invention provides a simple and practical method for separating and purifying 3,29-dibenzoyl trichosanthes trichosanthes, 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 rapidly preparing 3,29-dibenzoyl trichosanthes trichosanthes trifoliate comprises separating and purifying the organic solvent extract of the fruit of trichosanthes by high-speed countercurrent chromatography and high-performance liquid chromatography, collecting each fraction, and then concentrating under reduced pressure to evaporate the solvent to obtain a 3,29-dibenzoyl trichosanthes trifoliate reference substance.

[0009] Preferably, the organic solvent used for extraction is petroleum ether, gasoline, hexane, or dichloromethane with a boiling range of 60-90°C, the solid-liquid ratio is 1:8-1:20, the extraction time is 30-120 minutes, and the extraction method used is ultrasonic vibration extraction.

[0010] Preferably, the separation is a two-step separation method, wherein the first step uses high-speed countercurrent chromatography technology and the second step uses preparative high-performance liquid chromatography technology.

[0011] Preferably, in the high-speed countercurrent chromatography technique, the two-phase solvent system is hexane-acetonitrile volume ratio 1:1 or hexane-90% ethanol 1:1, the countercurrent chromatography host speed is 700rpm-1000rpm, the countercurrent extraction speed is 2mL / min, and the detection wavelength is 254nm.

[0012] Preferably, preparative high performance liquid chromatography technology is used to achieve further separation operations, and the preparative chromatography conditions are as follows: the chromatographic column used is C18 filler, the column length is 250mm, the diameter is 10.0mm, the filler is 5μm, the column temperature is 30°C, the mobile phase is methanol and water volume ratio of 90:10 to 97:3, the flow rate is 2-3.0mL / min, the detection wavelength is 230nm, and the injection volume is 50-100μL.

[0013] Preferably, after the target components are separated and collected, the solvent needs to be evaporated under reduced pressure, and the required temperature is 40-60°C.

[0014] The beneficial effects of the present invention are:

[0015] 1. Screening suitable organic solvents to extract the active ingredients of Trichosanthes fruit. Because the target ingredients have low polarity, they are easily extracted by low-polarity organic solvents, such as hexane, petroleum ether, gasoline, etc. Considering the extraction cost and extraction rate, petroleum ether was selected for extraction.

[0016] 2. Ultrasonic low-temperature extraction has high efficiency and little damage to heat-sensitive components. The ultrasonic extraction method is selected in the present invention. The specific method is as follows: crush the seeds of Trichosanthes kirilowii, weigh a certain amount of Trichosanthes kirilowii powder, ultrasonically extract for 30 minutes with 10 times the amount of petroleum ether, filter to obtain an extract, and then concentrate under reduced pressure on a rotary evaporator to recover the solvent to obtain an oily extract.

[0017] 3. The process is not complicated. Prepare the sample, take the oily extract, and disperse it in equal volumes of the separation system solvent hexane-acetonitrile as the sample solution. Prepare the countercurrent chromatography solvent system, take equal volumes of hexane and acetonitrile, shake and mix in a separatory funnel, release the lower phase and the 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, and then start to detect and collect the target components. Collect each chromatographic peak, and detect the purity of each peak by high-performance liquid chromatography. The target compound is not pure after a high-speed countercurrent chromatography separation, and further high-performance liquid chromatography separation is required. The sample obtained by high-speed countercurrent chromatography separation was concentrated under reduced pressure and then dissolved in methanol for preparative high-performance liquid chromatography separation to finally obtain a high-purity product. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The chemical structure of the compound 3,29-dibenzoyl trichosanthes triol studied in the present invention is:

[0019] Figure 2 is the standard curve of compound 3,29-dibenzoyltriol;

[0020] Figure 3 is the distribution coefficient under different solvent systems in the embodiments of this patent;

[0021] Figure 4 It is the effect of mobile phase flow rate on the retention rate of high-speed countercurrent chromatography stationary phase;

[0022] Figure 5 It is the effect of the main engine speed on the retention rate of the stationary phase of high-speed countercurrent chromatography;

[0023] Figure 6 It is a single countercurrent chromatography separation chromatogram;

[0024] Figure 7 This is the high-speed countercurrent chromatography separation diagram for two consecutive times with different injection volumes;

[0025] Figure 8It is the result of countercurrent chromatography, preparative liquid chromatography and HPLC spectrum of Trichosanthes kirilowii seed oil. DETAILED DESCRIPTION

[0026] Example 1: Extraction of Trichosanthes kirilowii oil

[0027] 50g of Fructus Trichosanthis was dried and crushed, and then placed in a 1000mL stoppered conical flask. 500mL of n-hexane was poured into the conical flask and sealed. Extraction was performed using an ultrasonic cleaning device with a power of 200W and a frequency of 40kHz, and the extraction time was 30min. The extract was cooled, shaken, filtered, and filtrated to obtain a filtrate. The filtrate was concentrated and the solvent was recovered using a RE-52AA rotary evaporator. Then 200mL of the recovered n-hexane was subjected to secondary ultrasonic extraction and filtered, and the solvent was recovered by concentrating under reduced pressure. 8.9g of Fructus Trichosanthis oil was obtained, and the extraction rate was 17.8%.

[0028] Example 2: Selection of solvent for high-speed countercurrent chromatography separation

[0029] In this experiment, the solvent systems of n-hexane-acetonitrile, n-hexane-ethyl acetate-acetonitrile, n-hexane-ethyl acetate-methanol-water, and hexane-methanol were used for the experiment. After mixing in a certain proportion and shaking well, the stratification phenomenon of the two phases was observed. 10mL of the solution was taken from each of the upper and lower layers, and then 10μL of Trichosanthes oil was added in a static state, mixed, stirred, and stratified. Finally, 1mL of each of the upper and lower layers was taken for HPLC analysis to calculate the distribution coefficient K. The results are shown in Figure 3 The K value of the separation system with a hexane-acetonitrile volume ratio of 1:1 is 1.1, which is suitable for countercurrent chromatography.

[0030] Example 3: Effect of mobile phase flow rate on stationary phase retention

[0031] During the separation process of high-speed countercurrent chromatography, the separation effect is not only affected by the solvent system, but also by the separation conditions. In this study, the effect of different flow rates on the separation effect was observed. When the forward speed of the high-speed countercurrent chromatography main engine was 800r / min, the stationary phase retention rate (Sf) was calculated under the conditions of flow rates of 2, 3, 4, and 5mL / min, as shown in the following figure: Figure 4 As shown in the figure, as the flow rate gradually increases, the static retention rate without injection shows a decreasing trend. The retention amount of the stationary phase is reduced, resulting in a decrease in separation efficiency. This is because different separation components may affect each other during the separation process, affecting the purity of the separated compounds. Therefore, a flow rate of 2mL / min is used for separation.

[0032] Example 4: Effect of rotation speed on stationary phase retention

[0033] The main engine speed is an important factor affecting the separation time and resolution of high-speed countercurrent chromatography. Hexane and acetonitrile are used as the two-phase solvent system, in which the upper layer is the stationary phase and the lower layer is the mobile phase. The main engine speed ranges from 0 to 900 r / min. Under the premise of keeping other conditions unchanged, the speed is increased from 500 r / min to 900 r / min. -1 At the beginning, the rotation speed is gradually increased and the retention rate (Sf) of the stationary phase is calculated, such as Figure 5 As shown in the figure, within a certain range, as the main engine speed increases, the retention rate of the stationary phase gradually increases, and the separation effect is significant; however, too high a speed may cause the solvent system to emulsify, which will cause a large amount of stationary phase that has been injected to be lost, thus having a negative impact on the separation effect. The main engine speed is determined to be 800r / min, and its retention rate is as high as 88.7%. The results are shown in the figure. Figure 5 shown.

[0034] Example 5: Single injection high speed countercurrent chromatography separation

[0035] The solvent system used is a two-phase solvent system composed of n-hexane and acetonitrile in a ratio of 1:1, with the upper layer as the stationary phase and the lower layer as the mobile phase. The separation conditions are determined by adjusting the injection volume, flow rate and speed test parameters. Take 500 mg of Trichosanthes kirilowii oil sample, mix and layer it in a ratio of 1:1 between n-hexane and acetonitrile, and take 3 mL of dissolved and dispersed sample in each layer. High-speed countercurrent chromatography separation is carried out under the separation conditions of a flow rate of 2 mL / min and a main engine speed of 800 r / min. Under this condition, the total outflow of the stationary phase reaches 121.8 mL. The instrument has a total column volume of 500 mL, and its stationary phase retention rate reaches 75.6%. See the corresponding chromatographic analysis diagram. Figure 6 The trichosanthes seed oil can be successfully separated into three components, I, II, and III, within 160 minutes.

[0036] Example 6: High-speed countercurrent chromatography with two consecutive injections

[0037] Similar to other chromatographic separation techniques, HSCLC can achieve continuous injection when the stationary phase has little loss. Through continuous injection, not only the time required to pump the stationary phase and the equilibration time before injection are saved, but also the amount of solvent required for the stationary phase is reduced. Figure 7 The separation diagram of two consecutive injections of Trichosanthes seed oil is shown. It can be found that the two injections have relatively little effect on the peak morphology and resolution. The peak value of the second injection with a volume of 1000 mg is significantly higher than that of the first injection, and the separation effect has not decreased significantly.

[0038] Example 7: Second step of separation, i.e. further purification by preparative liquid chromatography

[0039] The chromatographic peak components IV and VI obtained by continuous injection separation of high-speed countercurrent chromatography were collected and concentrated, and then analyzed by HPLC. The results showed that the compounds in component IV were relatively complex, and the content of 3,29-dibenzoyl trichosanthes triol was relatively low, so it was not worth further analysis. In component VI, Figure 8 As shown: the chromatogram has two significant peaks, and their retention times are 20.37min and 22.87min, respectively. The content of 3,29-dibenzoyl trichosanthes triol is relatively high. Its peak wavelength has maximum absorption at 230nm, which is consistent with the ultraviolet absorption characteristics of 3,29-dibenzoyl trichosanthes triol. After HPLC analysis, the concentrated component VI was collected and then subjected to secondary separation by preparative HPLC. The preparation conditions are as follows: the chromatographic column is YMC-Pack ODS-A (250×10.0mm lDS-5μm, 12nm); column temperature: 30℃; mobile phase: methanol to water volume ratio 97:3; flow rate: 3.0mL / min; detection wavelength: 230nm; injection volume 100μL. Detection time 25min. After the separated components were concentrated under reduced pressure, 1.7 mg of a compound with a purity of 95.7% was obtained. The HPLC retention time of the compound was consistent with that of the 3,29-dibenzoyl trichosanthes triol reference substance, and its chemical structure was further identified by nuclear magnetic resonance spectroscopy. The content of the other compound was low and was not identified.

Claims

1. A method for rapidly preparing 3,29-dibenzoyl trichosanthes trichosanthes triol, characterized in that: The organic solvent extract of the fruit of trichosanthes 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 3,29-dibenzoyl trichosanthes trichosanthes trichosanthes reference substance.

2. The method for rapidly preparing 3,29-dibenzoyl trichosanthes trichosanthes triol according to claim 1, characterized in that: The organic solvents used for extraction are petroleum ether, gasoline, hexane and dichloromethane with a boiling range of 60-90°C, the solid-liquid ratio is 1:8-1:20, the extraction time is 30-120 minutes, and the extraction method adopted is ultrasonic vibration extraction.

3. The method for rapidly preparing 3,29-dibenzoyl trichosanthes trichosanthes triol according to claim 1, characterized in that: The separation adopted is a two-step separation method, in which the first step uses high-speed countercurrent chromatography technology and the second step uses preparative high-performance liquid chromatography technology.

4. The method for rapidly preparing 3,29-dibenzoyl trichosanthes trichosanthes triol according to claim 3, characterized in that: In the high-speed countercurrent chromatography technology, the two-phase solvent system is hexane-acetonitrile volume ratio 1:1 or hexane-90% ethanol 1:1, the countercurrent chromatography host speed is 700rpm-1000rpm, the countercurrent extraction speed is 2mL / min, and the detection wavelength is 254nm.

5. The method for rapidly preparing 3,29-dibenzoyl trichosanthes triol according to claim 3, characterized in that: Preparative high performance liquid chromatography technology was used to achieve further separation operations. The preparative chromatographic conditions were as follows: the chromatographic column used was C18 filler, with a column length of 250 mm, a diameter of 10.0 mm, a filler of 5 μm, a column temperature of 30°C, a mobile phase of methanol and water in a volume ratio of 90:10 to 97:3, a flow rate of 2-3.0 mL / min, a detection wavelength of 230 nm, and an injection volume of 50-100 μL.

6. The method for rapidly preparing 3,29-dibenzoyl trichosanthes trichosanthes triol according to claim 4 or 5, characterized in that: After the target components are separated and collected, the solvent needs to be evaporated under reduced pressure, and the required temperature is 40-60°C.

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