Preparation method of rhodiololactone in rhodiola rosea

Abstract

The invention discloses a preparation method of rhodiolamin in rhodiola rosea, and belongs to the technical field of traditional Chinese medicines. According to the present invention, the alcohol extraction liquid contains the salidroside and the rhodiolamin, the alcohol extraction can extract more than 90% of the salidroside and the rhodiolamin in the rhodiola rosea medicinal material, the aqueous two-phase system is adopted to perform extraction, and the polarity of the system is closer to the polarity of the rhodiolamin, the tributyl ethyl phosphonium bromide is added to adjust the polarity of the aqueous two-phase system, so that the distribution coefficient of the aqueous two-phase system to the rhodiola rosea is larger, the content of the rhodiola rosea in the upper-layer extraction liquid is remarkably increased, and the content of the salidroside in the upper-layer extraction liquid is remarkably reduced. Then, after activated carbon adsorption, desorption, gel column chromatography and recrystallization are carried out, a salidrosin monomeric compound with the purity larger than 98% is obtained, and efficient extraction and separation of trace components in the rhodiola rosea medicinal material are achieved.

Description

Technical Field

[0001] The present invention relates to a method for preparing rhodiosin from Rhodiola rosea, belonging to the technical field of traditional Chinese medicine. Background Art

[0002] Rhodiosin is a flavonoid compound extracted from the rhizome and root of Rhodiola rosea. Flavonoid compounds have biological activities such as antioxidant, free radical scavenging, antibacterial, and blood pressure lowering.

[0003] In the prior art, there is no determination of the content of rhodiosin in Rhodiola rosea medicinal materials. Only Zhao Liang et al. used UPLC-MS / MS method to determine the content of 6 active ingredients (salidroside, rhodiosin, acteoside, actein, kaempferol, and quercetin) in Rhodiola rosea extract. Among them, the content of rhodiosin in the extract is (0.38±0.01~0.42±0.01)%.

[0004] Rhodiola rosea extract is an extract of Rhodiola rosea medicinal materials. There are two main types of Rhodiola rosea extracts in China: Rhodiola crenulata and Rhodiola rosea. Rhodiola crenulata is mainly produced in Tibet, and the main extracted component is salidroside; Rhodiola rosea is produced in Xinjiang, and the content of isomers such as rosavin is relatively high. Thus, it can be seen that the content of rhodiosin in the existing Rhodiola rosea extracts is relatively low, which also indicates that the content of rhodiosin in Rhodiola rosea medicinal materials is even lower. Therefore, there is a problem in the prior art that it is difficult to extract rhodiosin with low content from Rhodiola rosea medicinal materials. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a method for preparing rhodiosin from Rhodiola rosea. This method selects Rhodiola crenulata as the raw material to prepare the monomer compound of rhodiosin. The prepared extract can be further used as the raw material for preparing components such as salidroside.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing rhodiosin from Rhodiola rosea, comprising the following steps:

[0008] Step 1, extraction: Take the dried rhizome and root of Rhodiola rosea, crush them, and pass through a sieve of 80 - 200 meshes. Extract with 50 - 70% ethanol by hot reflux for at least 2 times. Each time, the amount of 50 - 70% ethanol used is 10 - 20 times the weight of the raw medicinal material. Extract for 1 - 3 hours each time. After extraction, combine the extraction solutions and concentrate under reduced pressure until the weight of the concentrated solution is 0.3 - 0.5 times the weight of the raw medicinal material to obtain a concentrated extraction solution.

[0009] Step 2, prepare the extraction solution: 30 - 35 wt% polyethylene glycol 6000, 15 - 18 wt% diammonium hydrogen phosphate (NH4)2HPO4, 1 - 2 wt% tributylethylphosphonium bromide [P2444 Br and the balance of water.

[0010] The preparation method is as follows: First, add 30 - 35 wt% of polyethylene glycol 6000 and 45 - 54 wt% of water into a beaker. After stirring evenly, add 15 - 18 wt% of diammonium hydrogen phosphate drop by drop while stirring. After the addition is completed, continue to add 1 - 2 wt% of tributyl ethylphosphonium bromide [P 2444 Br drop by drop while stirring. After stirring for at least 5 min, the extraction solution is obtained.

[0011] Step 3, extraction: Add the concentrated extract into the extraction solution. The volume ratio of the concentrated extract to the extraction solution is 1:5. Shake in a separatory funnel for at least 20 min. After standing for stratification, take the upper layer.

[0012] Step 4, activated carbon adsorption: Add activated carbon particles equivalent to 5 - 10 wt% of the weight of the raw drug substance to the upper layer solution. After stirring for at least 1 h, separate the activated carbon particles.

[0013] Step 5, desorption: Desorb the activated carbon particles in 95% ethanol. The number of desorption times is at least 2 times. The amount of 95% ethanol used each time is 50 - 60 times the weight of the activated carbon particles. The desorption time each time is 2 - 3 h. Combine the desorption solutions and concentrate under reduced pressure until the alcohol smell disappears to obtain a refined concentrated solution.

[0014] Step 6, gel column chromatography: After adding 2 - 3 mL of methanol to assist dissolution in the refined concentrated solution, filter, load the sample, and purify through a Sephadex LH - 20 column. The amount of Sephadex LH - 20 packing used is equivalent to 10 - 15 wt% of the weight of the raw drug substance. After loading the sample, adsorb for 1 - 2 h, elute with methanol at a flow rate of 3 - 5 mL / min. After eluting with methanol for 3 - 6 column volumes, collect the eluate, concentrate under reduced pressure at 50 - 60 °C and then dry to obtain the crude product.

[0015] Step 7, dissolve the crude product in methanol equivalent to 3 - 4 times its weight, filter, and gradually add a mixed solution of ethyl acetate and chloroform with a volume ratio of 1:1 drop by drop to the filtrate. The addition amount of the mixed solution of ethyl acetate and chloroform is 1 - 1.5 times the amount of methanol. After the addition is completed, stir for at least 5 min, stand overnight at 0 - 4 °C to precipitate white crystalline powder. After centrifugation, wash the white crystalline powder with water and dry to obtain salidroside.

[0016] The particle size range of the activated carbon particles is: 0.5 - 5 mm.

[0017] For desorption, ultrasonic, stirring, heating and other means can be used to accelerate desorption.

[0018] The ultrasonic power is 100 - 500 W.

[0019] The stirring rate is 100 - 200 rpm.

[0020] The heating temperature is 50 - 60 °C.

[0021] The diameter - height ratio of the gel column is 1:(5 - 8).

[0022] The salidroside obtained by the method for preparing salidroside from Rhodiola rosea in the present invention has a salidroside content > 98%.

[0023] The application of the salidroside of the present invention in pharmaceutical intermediates.

[0024] A pharmaceutical intermediate is obtained by preparing with the salidroside of the present invention.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The present invention provides a method for preparing salidroside from Rhodiola rosea. The ethanol extract contains salidroside and salidroside. The extraction method of the present invention can extract more than 90% of salidroside and salidroside from Rhodiola rosea medicinal materials. And in the medicinal materials, the content of salidroside is significantly greater than that of salidroside, which also shows that there is a certain difficulty in preparing trace salidroside in Rhodiola rosea. Next, the present invention uses an aqueous two - phase system for extraction. The aqueous two - phase system of the present invention is 30 - 35 wt% polyethylene glycol 6000, 15 - 18 wt% diammonium hydrogen phosphate (NH4)2HPO4, 1 - 2 wt% tributyl ethylphosphonium bromide [P 2444 Br and the balance of water. This system has a polarity closer to that of salidroside. By adding tributyl ethylphosphonium bromide to adjust the polarity of the aqueous two - phase system, the partition coefficient of salidroside is made larger, significantly increasing the content of salidroside in the upper extraction liquid and significantly reducing the content of salidroside in the upper extraction liquid. Then, after the operations of activated carbon adsorption, desorption, gel column chromatography and recrystallization in the present invention, a salidroside monomer compound with a purity greater than 98% is obtained, realizing the efficient extraction and separation of trace components in Rhodiola rosea medicinal materials. Description of the Drawings

[0027] Figure 1 It is the chromatogram of the upper extraction liquid of the present invention;

[0028] Figure 2 It is the chromatogram of the refined and concentrated liquid of the present invention;

[0029] Figure 3 It is the chromatogram of the white crystalline powder salidroside of the present invention. Detailed Embodiments

[0030] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0031] Example 1

[0032] A method for preparing salidroside from Rhodiola rosea, comprising the following steps:

[0033] Step 1, extraction: Take the dried rhizomes and roots of Rhodiola rosea, crush them, pass through a 100-mesh sieve, and extract with 60% ethanol by hot reflux twice. The amount of 60% ethanol used each time is 15 times the weight of the raw material drug, and each extraction is carried out for 2 h. After the extraction is completed, the extraction solutions are combined and concentrated under reduced pressure until the weight of the concentrated solution is 0.4 times the weight of the raw material drug to obtain a concentrated extraction solution.

[0034] Step 2, prepare the extraction solution: 35 wt% polyethylene glycol 6000, 15 wt% diammonium hydrogen phosphate (NH4)2HPO4, 1 wt% tributylethylphosphonium bromide [P 2444 Br and the balance of water.

[0035] The preparation method is as follows: First, add 35 wt% polyethylene glycol 6000 and 49 wt% water to a beaker, stir evenly, then gradually add 15 wt% diammonium hydrogen phosphate drop by drop while stirring. After the addition is completed, stir until stratified, and then continue to gradually add 1 wt% tributylethylphosphonium bromide [P 2444 Br drop by drop while stirring. After stirring for 5 min, the extraction solution is obtained.

[0036] Step 3, extraction: Add the concentrated extraction solution to the extraction solution. The volume ratio of the concentrated extraction solution to the extraction solution is 1:5. Shake in a separating funnel for 20 min, let it stand and separate into layers, and take the upper layer.

[0037] Step 4, activated carbon adsorption: Add activated carbon particles equivalent to 8 wt% of the weight of the raw material drug to the upper layer solution, stir for 1 h, and then separate the activated carbon particles. The particle size of the activated carbon particles is about 2 mm.

[0038] Step 5, desorption: Desorb the activated carbon particles in 95% ethanol. The number of desorption times is 2 times. The amount of 95% ethanol used each time is 55 times the weight of the activated carbon particles, and the desorption time each time is 2.5 h. Combine the desorption solutions and concentrate under reduced pressure until there is no alcohol smell to obtain a refined concentrated solution. During desorption, ultrasonic, stirring, heating and other means can be used to accelerate desorption. The ultrasonic power is 250 W, the stirring rate is 150 rpm, and the heating temperature is 55 °C. In this example, a method of heating at 55 °C with stirring assistance is preferably used to help desorption.

[0039] Step 6, Gel column chromatography: After adding 2.5 mL of methanol to the refined and concentrated solution to assist dissolution, filter, load the sample, and purify it through a Sephadex LH-20 column. The dosage of Sephadex LH-20 packing is equivalent to 12 wt% of the weight of the API. The diameter-height ratio of the gel column is 1:6. After loading the sample, adsorb for 1.5 h, elute with methanol at a flow rate of 4 mL / min. After eluting with 5 column volumes of methanol, collect the eluate, concentrate it under reduced pressure at 55 °C and then dry to obtain the crude product.

[0040] Step 7, Dissolve the crude product in methanol equivalent to 3 times its weight, filter, and gradually add a mixed solution of ethyl acetate and chloroform with a volume ratio of 1:1 dropwise to the filtrate. The addition amount of the mixed solution of ethyl acetate and chloroform is 1 time the amount of methanol. After adding, stir for 5 min, let it stand overnight at 4 °C to precipitate white crystalline powder. After centrifugation, wash the white crystalline powder with water and dry to obtain salidroside.

[0041] Application of the salidroside in this example in pharmaceutical intermediates.

[0042] A pharmaceutical intermediate obtained by preparing with the salidroside in this example.

[0043] The steps for HPLC content determination are as follows:

[0044] 1. Standards stock solutions of rhodioside and salidroside (1 mg / mL): Weigh appropriate amounts of the two reference standards precisely, dissolve them in methanol and dilute to the mark to prepare reference standard stock solutions with a concentration of 1 mg / mL each. Store at below -18 °C, and the validity period is 6 months.

[0045] 2. Standard series working solutions: Pipette appropriate amounts of the reference standard stock solutions and dilute them with methanol to a series of concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.20 mg / mL, and 0.50 mg / mL respectively.

[0046] 3. High performance liquid chromatograph

[0047] 4. Preparation of test solution: Weigh an appropriate amount of the homogeneous sample accurately to 0.0001 g, place it in a 25 mL volumetric flask, add 20 mL of methanol, extract ultrasonically for 30 min, cool to room temperature, make up the volume to the mark with methanol, mix well and filter through a 0.45 μm filter membrane for liquid chromatographic analysis.

[0048] The samples include the following test articles:

[0049] Sample a, 0.5 g of dried rhizome and root powder of Rhodiola rosea;

[0050] Sample b, 0.2 g of dried powder of concentrated extract;

[0051] Sample c, 0.1 g of the powder after drying the upper-layer extraction solution;

[0052] Sample d, 0.5 g of the powder after drying the lower-layer extraction solution;

[0053] Sample e, 0.5 g of the powder after drying the solution adsorbed by activated carbon;

[0054] Sample f, 0.05 g of the powder after drying the refined and concentrated solution;

[0055] Sample g, 0.02 g of the crude product powder;

[0056] Sample h, 0.2 g of white crystalline powder.

[0057] 5. Reference conditions for high performance liquid chromatography:

[0058] a) Chromatographic column: C18 chromatographic column 4.6 μm * 150 mm, 5 μm or Kromasil C18 column (250 mm × 4.6 mm, 5 μm), or those with equivalent performance;

[0059] b) Column temperature: 35 °C;

[0060] c) Detector: UV detector;

[0061] d) Detection wavelength: 275 nm;

[0062] e) Injection volume: 10 μL;

[0063] f) Flow rate: 1.0 mL / min;

[0064] g) Mobile phase: A is 0.5% acetic acid aqueous solution, B is acetonitrile. The gradient elution conditions are shown in Table 1.

[0065] Table 1 Gradient elution conditions of mobile phase

[0066]

[0067]

[0068] 6. Preparation of standard curve: Inject the standard series working solutions into the liquid chromatograph respectively, measure the peak areas of the corresponding substances, use the concentration of the substance in the standard series working solution as the abscissa, and the response value of the peak area of the substance as the ordinate to draw the standard curve.

[0069] 7. Determination of sample solution: Inject the sample solution into the liquid chromatograph to obtain the peak area, and obtain the mass concentrations of salidroside and rhodiolin in the test solution according to the standard curve. The contents of salidroside and rhodiolin in the sample are calculated according to the standard curve or the following formula.

[0070]

[0071] In the formula:

[0072] X—the value of salidroside and rhodioloside in the sample, unit: microgram per kilogram (μg / kg);

[0073] A1—the peak areas of salidroside and rhodioloside in the sample solution;

[0074] C—the concentrations of salidroside and rhodioloside in the reference substance solution;

[0075] A2—the average peak area of the reference substance solution;

[0076] V—the value of the final volume of the sample solution, unit: milliliter (mL);

[0077] m—the value of the sample mass, unit: gram (g).

[0078] 8. Sample h, white crystalline powder, rhodioloside, verifies the content by the following content determination method.

[0079] High performance liquid chromatography reference conditions:

[0080] a) Chromatographic column: C18 chromatographic column 4.6μm * 150mm, 5μm or Kromasil C18 column (250mm × 4.6mm, 5μm), or equivalent;

[0081] b) Column temperature: 35°C;

[0082] c) Detector: ultraviolet detector;

[0083] d) Detection wavelength: 280nm;

[0084] e) Injection volume: 10μL;

[0085] f) Flow rate: 1.0mL / min;

[0086] g) Mobile phase: A is 0.5% acetic acid aqueous solution, B is acetonitrile. The gradient elution conditions are shown in Table 2.

[0087] Table 2 Mobile phase gradient elution conditions for verifying the content of rhodioloside

[0088] Time / min Flow rate (mL / min) B / ％ 0.000 1.0 23.0 10.000 1.0 23.0 15.000 1.0 90.0 25.000 1.0 90.0

[0089] The content determination results of each sample are shown in Table 3 below.

[0090] Table 3 Content determination results of the samples in Example 1

[0091] Salidroside / % Rhodioloside / % Specimen a 2.32 0.15 Specimen b 15.96 1.43 Specimen c 2.84 8.78 Specimen d 10.89 0.34 Specimen e 2.55 0.87 Specimen f 0 39.52 Specimen g 0 84.46 Specimen h 0 99.98

[0092] As can be seen from Table 3, the alcohol extraction method of this example can extract more than 90% of salidroside and rhodiolin from the Rhodiola rosea L. medicinal materials. Moreover, in the medicinal materials, the content of salidroside is significantly greater than that of rhodiolin, which also indicates that there is a certain difficulty in the preparation of trace rhodiolin in Rhodiola rosea L. Next, this example uses an aqueous two-phase system for extraction. The aqueous two-phase system is 30 - 35 wt% polyethylene glycol 6000, 15 - 18 wt% diammonium hydrogen phosphate (NH4)2HPO4, 1 - 2 wt% tributylethylphosphonium bromide [P 2444 Br and the balance of water. This system has a polarity closer to that of rhodiolin. By adding tributylethylphosphonium bromide to adjust the polarity of the aqueous two-phase system to make its partition coefficient for rhodiolin larger. As Figure 1 shown, this example significantly increases the content of rhodiolin in the upper extraction solution and significantly reduces the content of salidroside in the upper extraction solution (see the data of sample c and sample d in Table 2). Then, after activated carbon adsorption, desorption, gel column chromatography and recrystallization operations, a rhodiolin monomer compound with a purity greater than 98% is obtained, realizing the efficient extraction and separation of trace components in the Rhodiola rosea L. medicinal materials. As Figure 2 shown, sample f of this example, that is, the refined concentrated solution, that is, the concentrated solution after desorption, does not contain salidroside, indicating that the combination of the aqueous two-phase system and activated carbon adsorption - desorption of the present invention can completely remove the salidroside impurities in the target extract without column passing, which greatly improves the efficiency of subsequent gel column chromatography and recrystallization. As Figure 3 shown, sample h obtained in this example is a white crystalline powder, and the content of rhodiolin is as high as 99.98%.

[0093] Example 2

[0094] A preparation method of rhodiolin in Rhodiola rosea L. includes the following steps:

[0095] Step 1, extraction: Take the dried rhizome and root of Rhodiola rosea L., after pulverization, sieve through a 80 - mesh sieve, and extract with 50% ethanol by hot reflux for 3 times. Each time, the amount of 50% ethanol used is 10 times the weight of the raw medicinal material, and each extraction is for 1 h. After extraction, combine the extraction solutions and concentrate under reduced pressure until the weight of the concentrated solution is 0.3 times the weight of the raw medicinal material to obtain a concentrated extraction solution.

[0096] Step 2, prepare the extraction solution: 30 wt% polyethylene glycol 6000, 18 wt% diammonium hydrogen phosphate (NH4)2HPO4, 2 wt% tributylethylphosphonium bromide [P 2444 Br and the balance of water.

[0097] The preparation method is as follows: First, add 30 wt% polyethylene glycol 6000 and 50 wt% water into a beaker. After stirring evenly, add 18 wt% diammonium hydrogen phosphate drop by drop while stirring. After the addition is completed and stirring causes layering, continue to add 2 wt% tributylethylphosphonium bromide [P 2444 Br drop by drop while stirring. After stirring for 8 min, the extraction solution is obtained.

[0098] Step 3, extraction: Add the concentrated extract into the extraction solution. The volume ratio of the concentrated extract to the extraction solution is 1:5. Shake in a separatory funnel for 30 min. After standing for layering, take the upper layer.

[0099] Step 4, activated carbon adsorption: Add activated carbon particles equivalent to 5 wt% of the weight of the raw drug substance to the upper layer solution and stir for 2 h, then separate the activated carbon particles. The particle size of the activated carbon particles is about 0.5 mm.

[0100] Step 5, desorption: Desorb the activated carbon particles in 95% ethanol. The number of desorption times is 3 times. The amount of 95% ethanol used each time is 50 times the weight of the activated carbon particles. The desorption time each time is 2 h. Combine the desorption solutions and concentrate under reduced pressure until there is no alcohol smell to obtain a refined concentrated solution. In this example, a combined method of heating and ultrasonic is used for desorption. The ultrasonic power is 100 W and the heating temperature is 50 °C.

[0101] Step 6, gel column chromatography: After adding 2 mL of methanol to the refined concentrated solution for solubilization, filter, load the sample, and purify it through a Sephadex LH-20 column. The amount of Sephadex LH-20 packing used is equivalent to 10 wt% of the weight of the raw drug substance. The diameter-to-height ratio of the gel column is 1:5. After loading the sample, adsorb for 1 h, elute with methanol at a flow rate of 3 mL / min. After eluting with methanol for 3 column volumes, collect the eluate, concentrate under reduced pressure at 50 °C and then dry to obtain the crude product.

[0102] Step 7, dissolve the crude product in methanol equivalent to 4 times its weight, filter, and slowly add a mixed solution of ethyl acetate and chloroform with a volume ratio of 1:1 drop by drop to the filtrate. The addition amount of the ethyl acetate and chloroform mixed solution is 1.5 times the amount of methanol. After the addition is completed, stir for 10 min, stand overnight at 0 °C to precipitate white crystalline powder. After centrifugation, wash the white crystalline powder with water and dry to obtain salidroside.

[0103] The salidroside obtained by the preparation method of salidroside in rhodiola of this example has a salidroside content of 99.47%.

[0104] Application of the salidroside of this example in pharmaceutical intermediates.

[0105] A pharmaceutical intermediate is prepared from the salidroside of this example.

[0106] Example 3

[0107] A method for preparing salidroside from Rhodiola rosea, comprising the following steps:

[0108] Step 1, extraction: Take the dried rhizomes and roots of Rhodiola rosea, pulverize them, pass through a 200-mesh sieve, and extract with 70% ethanol by hot reflux twice. The amount of 70% ethanol used each time is 20 times the weight of the raw material drug, and each extraction lasts for 3 h. After the extraction is completed, combine the extraction solutions and concentrate them under reduced pressure until the weight of the concentrated solution is 0.5 times the weight of the raw material drug to obtain a concentrated extraction solution.

[0109] Step 2, prepare the extraction liquid: 32 wt% polyethylene glycol 6000, 16 wt% diammonium hydrogen phosphate (NH4)2HPO4, 1 wt% tributyl ethyl phosphonium bromide [P 2444 Br and the balance of water.

[0110] The preparation method is as follows: First, add 32 wt% polyethylene glycol 6000 and 51 wt% water to a beaker, stir evenly, then gradually add 16 wt% diammonium hydrogen phosphate drop by drop while stirring. After the addition is completed, stir until stratified, and then continue to gradually add 1 wt% tributyl ethyl phosphonium bromide [P 2444 Br drop by drop while stirring. After stirring for 6 min, the extraction liquid is obtained.

[0111] Step 3, extraction: Add the concentrated extraction solution to the extraction liquid. The volume ratio of the concentrated extraction solution to the extraction liquid is 1:5. Shake in a separatory funnel for 20 min, let it stand for stratification, and then take the upper layer.

[0112] Step 4, activated carbon adsorption: Add activated carbon particles equivalent to 10 wt% of the weight of the raw material drug to the upper layer liquid, stir for 1 h, and then separate the activated carbon particles. The particle size of the activated carbon particles is about 5 mm.

[0113] Step 5, desorption: Desorb the activated carbon particles in 95% ethanol. The number of desorption times is 2 times. The amount of 95% ethanol used each time is 60 times the weight of the activated carbon particles, and the desorption time each time is 3 h. Combine the desorption solutions and concentrate them under reduced pressure until there is no alcohol smell to obtain a refined concentrated solution. In this example, ultrasonic desorption is used, and the ultrasonic power is 500 W.

[0114] Step 6, gel column chromatography: After adding 3 mL of methanol to the refined concentrated solution to assist dissolution, filter, load the sample, and purify it through a Sephadex LH-20 column. The amount of Sephadex LH-20 packing used is equivalent to 15 wt% of the weight of the raw material drug. The diameter-height ratio of the gel column is 1:8. After loading the sample, adsorb for 2 h, elute with methanol at a flow rate of 5 mL / min. After eluting with 6 column volumes of methanol, collect the eluate, concentrate it under reduced pressure at 60 °C and then dry it to obtain the crude product.

[0115] Step 7: The crude product is dissolved in methanol in an amount 3 to 4 times its weight, filtered, and a mixed solution of ethyl acetate and chloroform with a volume ratio of 1:1 is added dropwise to the filtrate. The addition amount of the mixed solution of ethyl acetate and chloroform is 1.2 times the amount of methanol. After the addition is completed, it is stirred for 7 min, allowed to stand overnight at 4°C, and a white crystalline powder is precipitated. After centrifugation, the white crystalline powder is washed with water and dried to obtain salidroside.

[0116] The salidroside obtained by the method for preparing salidroside from rhodiola in this example has a salidroside content of 98.99%.

[0117] Application of the salidroside in this example in pharmaceutical intermediates.

[0118] A pharmaceutical intermediate is obtained by preparing from the salidroside in this example.

[0119] Example 4

[0120] The difference between this example and Example 1 is only that:

[0121] During desorption, stirring is used for desorption, and the stirring rate is 100 rpm.

[0122] Example 5

[0123] The difference between this example and Example 1 is only that:

[0124] During desorption, stirring is used for desorption, and the stirring rate is 200 rpm.

[0125] Example 6

[0126] The difference between this example and Example 1 is only that:

[0127] During desorption, heating is used for desorption, and the heating temperature is 50°C.

[0128] Example 7

[0129] The difference between this example and Example 1 is only that:

[0130] During desorption, heating is used for desorption, and the heating temperature is 60°C.

[0131] It should be understood that, in order to streamline the present disclosure and assist in understanding one or more of the various inventive aspects, in the foregoing description of the exemplary embodiments of the present invention, the various features of the present invention are sometimes grouped together into a single embodiment or the description thereof. However, the disclosed method should not be construed as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as reflected by the claims, the inventive aspects lie in less than all the features of the previously disclosed embodiments. Thus, the claims following the detailed description hereby expressly incorporate the detailed description, where each claim itself serves as a separate embodiment of the present invention.

[0132] Although the present invention has been described in terms of a limited number of embodiments, those skilled in the art in this technical field will appreciate, upon benefit of the foregoing description, that other embodiments may be contemplated within the scope of the present invention as thus described. Additionally, it should be noted that the language used in this specification has been principally selected for readability and teaching purposes rather than for the purpose of explaining or limiting the subject matter of the present invention. Accordingly, many modifications and variations will be apparent to those of ordinary skill in this technical field without departing from the scope and spirit of the appended claims. For the scope of the present invention, the disclosure of the present invention is illustrative rather than restrictive, and the scope of the present invention is defined by the appended claims.

[0133] The foregoing are only the preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the art in this technical field, several improvements and refinements can be made without departing from the principle of the present invention, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing salidroside from Rhodiola rosea, characterized in that, It includes the following steps: Step 1, extraction: Take the dried rhizome and roots of Rhodiola rosea, crush them, sieve through a 80 - 200 mesh sieve, and extract with 50 - 70% ethanol by hot reflux for at least 2 times. The amount of 50 - 70% ethanol used each time is 10 - 20 times the weight of the raw drug. Extract for 1 - 3 h each time. After extraction, combine the extraction solutions and concentrate under reduced pressure until the weight of the concentrated solution is 0.3 - 0.5 times the weight of the raw drug to obtain a concentrated extraction solution; Step 2: Prepare the extraction solution, which includes: 30-35 wt% polyethylene glycol 6000, 15-18 wt% diammonium hydrogen phosphate (NH4)2HPO4, 1-2 wt% tributylethylphosphonium bromide 2444 Br, and the balance being water; Step 3, extraction: Add the concentrated extraction solution to the extraction liquid. The volume ratio of the concentrated extraction solution to the extraction liquid is 1:

5. Shake in a separatory funnel for at least 20 min. After standing for stratification, take the upper layer; Step 4, activated carbon adsorption: Add activated carbon particles equivalent to 5 - 10 wt% of the weight of the raw drug to the upper layer liquid. Stir for at least 1 h and then separate the activated carbon particles; Step 5, desorption: Desorb the activated carbon particles in 95% ethanol. The number of desorption times is at least 2 times. The amount of 95% ethanol used each time is 50 - 60 times the weight of the activated carbon particles. The desorption time each time is 2 - 3 h. Combine the desorption solutions and concentrate under reduced pressure until there is no alcohol smell to obtain a refined concentrated solution; Step 6, gel column chromatography: After adding 2 - 3 mL of methanol to the refined concentrated solution for solubilization, filter, load the sample, and purify through a Sephadex LH - 20 column. After loading the sample, adsorb for 1 - 2 h, elute with methanol at a flow rate of 3 - 5 mL / min. After eluting with methanol for 3 - 6 column volumes, collect the eluate, concentrate under reduced pressure at 50 - 60 °C and then dry to obtain the crude product; Step 7, dissolve the crude product in methanol equivalent to 3 - 4 times its weight, filter, and gradually add a mixed solution of ethyl acetate and chloroform with a volume ratio of 1:1 to the filtrate while stirring. The addition amount of the ethyl acetate and chloroform mixed solution is 1 - 1.5 times the amount of methanol. After adding, stir for at least 5 min, stand overnight at 0 - 4 °C to precipitate white crystalline powder. After centrifugation, wash the white crystalline powder with water and dry to obtain salidroside.

2. The preparation method of salidroside in rhodiola rosea according to claim 1, characterized in that, In Step 2, the extraction solution is prepared as follows: First, add 30 - 35 wt% polyethylene glycol 6000 and 45 - 54 wt% water into a beaker. After stirring evenly, gradually add 15 - 18 wt% diammonium hydrogen phosphate drop by drop while stirring. After the addition is complete, stir until stratified, and then continue to gradually add 1 - 2 wt% tributylethylphosphonium bromide 2444 [P]Br drop by drop while stirring. After stirring for at least 5 minutes, the extraction solution is obtained.

3. The preparation method of salidroside in rhodiola according to claim 1, characterized in that, In step 4, the particle size range of the activated carbon particles is: 0.5 - 5 mm.

4. A method for preparing salidroside from Rhodiola rosea according to claim 1, characterized in that, In step 5, during desorption, use one or several of ultrasonic, stirring, and heating to accelerate desorption.

5. The preparation method of salidroside in rhodiola rosea according to claim 4, characterized in that, The ultrasonic power is 100 - 500 W; the stirring rate is 100 - 200 rpm; the heating temperature is 50 - 60 °C.

6. The preparation method of salidroside in Rhodiola rosea according to claim 1, characterized in that, In step 6, the amount of Sephadex LH - 20 packing is equivalent to 10 - 15 wt% of the weight of the raw drug, and the diameter - height ratio of the gel column is 1:(5 - 8).

7. A method for preparing salidroside from Rhodiola rosea according to claim 1, characterized in that, In step 7, the content of salidroside > 98%.

8. Salidroside obtained by the method for preparing salidroside in Rhodiola rosea according to any one of claims 1 - 7.

9. Use of the salidroside according to claim 8 in a pharmaceutical intermediate.

10. A pharmaceutical intermediate prepared from the salidroside according to claim 8.

Images