A method for preparing pinoresinol-β-D-glucoside from tubers of Herba Lycopodii

Abstract

The present invention relates to a method for extracting, separating and preparing pinoresinol-β-D-glucoside from tubers of dianthus sinensis. The specific steps of the method are: extracting the tubers of dianthus sinensis with an ethanol-water solution, concentrating the extract and adding ethanol for alcohol precipitation, taking the supernatant and enriching it with a macroporous adsorption resin and decolorizing it with an ionic resin material, and then crystallizing and recrystallizing the decolorized liquid to obtain high-purity pinoresinol-β-D-glucoside (purity greater than 97%). The process provided by the present invention is simple, has a high transfer rate, and can stably and repeatedly achieve large-scale preparation and industrial production; the process is green and environmentally friendly, uses only ethanol as a solvent, can be recycled and reused, reduces costs and has little pollution to the environment.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to a method for extracting and preparing high-purity pinoresinol-β-D-glucoside from sedge grass. Background Art

[0002] Boschniakia himalaica (Hook. f. et Thoms), also known as "Qian Jin Zhui," is a member of the genus Boswelliae in the family Orobanchaceae. It is found in Yunnan, Tibet, Shaanxi, Sichuan, and Hubei. It grows in the shade of trees or thickets in high-altitude mountainous areas, often parasitizing the roots of Rhododendron plants. It has the effects of dispelling wind, activating the collaterals, regulating qi, and strengthening the stomach. Its dried tubers are listed as a medicinal herb, "Qian Jin Zhui," in the "Yunnan Province Standard for Traditional Chinese Medicine" (2005 edition). Qian Jin Zhui is one of the main ingredients in the Qiancao Naomaitong mixture, of which pinoresinol-β-D-glucoside is a key ingredient. This mixture primarily promotes blood circulation, dissipates blood stasis, and resolves phlegm to activate the collaterals. It is used to treat stroke caused by phlegm and blood stasis blocking the collaterals, meridian problems, hemiplegia, facial paralysis, and difficulty speaking.

[0003] The main components of the plant are phenylethanoid glycosides and lignans, among which the content of lignans is relatively high, accounting for about 3% to 10% of the total components. β -D-glucoside is the main component of lignans, and its content can reach 2% to 8%. β -D-glucoside is also known as (+) pinoresinol- β -D-pyranoside, (+) pinoresinol-4-O-glucoside. Patent publication number CN119258083 A discloses the use of pinoresinol-4-O-glucoside in the preparation of a drug for treating allergic asthma, suggesting the potential for developing new anti-asthma drugs. Patent publication number CN 119174776 A discloses its use in the preparation of a drug for treating and / or preventing renal fibrosis, providing a new direction for the treatment and / or prevention of renal fibrosis.

[0004] Pinoresinol-β-D-glucoside, English name: pinoresinol-β-D-glucoside, molecular formula C 26 H 32 O 11 , CAS: 69251-96-3, and their structural formula and carbon spectrum comparison information are shown in Figure 1As shown in Table 2, the carbon spectrum information is consistent with compound 6 reported in the literature by MARIKO CHIBA et al. (13C NMR analysis of symplocosin and (+)-epipinoresinol glucosidePhytochemistry 1980, Vd. 19. pp. 335-336.). In 1978, Chiba, M. et al. (Chiba, M., Hisada, S. and Nishibe, S. (1978) ShoyakugakuZasshi 32, 194.) first isolated pinoresinol-β-D-glucoside from Forsythia suspensa. Subsequently, scholars also discovered pinoresinol-β-D-glucoside from plants of the genus Cistanche in the family Orobanchaceae. Pan Yingni (Study on the chemical composition and biological activity of fresh products of Cistanche tubulosa [D]. Shenyang Pharmaceutical University, 2011.), Jin Yinping (Study on the chemical composition of Dingzuocaea [J]. Central South Pharmacy, 2008, 6 (1): 43-45.), Li Hui LlU et al. (A New Lignan from Boschniakia himalaica [J] Chinese Chemical Letters, 2004, 15 (1): 43–45.), Chen Yupeng (Study on Chemical Constituents of Dingzuocae [J] Acta Botanica Sinica, 1992, 34 (11): 878-882), etc. have isolated pinoresinol-β-D-glucoside when isolating chemical components from different plants. However, the methods adopted were all laboratory-scale separation and purification methods, and the yield and purity of pinoresinol-β-D-glucoside have not been reported. This method is used to study the preparation process of pinoresinol-β-D-pyranoglucoside from Dingzuocae. The preparation process is simple, environmentally friendly, and has a high transfer rate. It can be prepared in large quantities and industrialized, making it feasible for new drug development and has broad future application and market prospects. Summary of the Invention

[0005] The present invention provides a method for extracting, separating, and preparing pinoresinol-β-D-glucoside from sedge. After coarsely crushing sedge tubers, an ethanol-water solution is added for extraction. The extract is then enriched on a macroporous adsorption resin column and decolorized on an ionic resin column to obtain a crude pinoresinol-β-D-glucoside product. The crude product is then crystallized and recrystallized to produce high-purity pinoresinol-β-D-glucoside (purity greater than 97%). The process is simple and feasible, capable of consistently obtaining high-purity pinoresinol-β-D-glucoside, with a short production cycle, enabling large-scale preparation and industrial production. The solvent used is easily recyclable and reusable, resulting in low cost and minimal environmental pollution.

[0006] 1. The present invention relates to a method for extracting, separating and preparing pinoresinol-β-D-glucoside from dwarf ...

[0007] 1) Crush the tubers of the herbaceous plant and extract them with an ethanol-water solution at a solid-liquid ratio of 1:3-1:30. Perform 1-5 extractions, each for 30-180 minutes. Concentrate the extract to a solid-liquid ratio of 1:0.5-1:6 (w / v) and set aside.

[0008] 2) adding ethanol to the concentrated solution obtained in step 1) for alcohol precipitation at an alcohol precipitation concentration of 60% to 80%, standing for 3 hours to 16 hours, separating the solid and liquid, and recovering ethanol from the supernatant to obtain a concentrated solution;

[0009] 3) Passing the concentrated solution obtained in step 2) through a macroporous adsorption resin column, eluting first with 4 to 10 column volumes of water, and then eluting with a 30% to 80% ethanol aqueous solution, collecting the ethanol eluate, and concentrating it to a solid-liquid ratio of 1:0.5 to 1:10 (w / v);

[0010] 4) Passing the concentrated solution obtained in step 3) through an ion resin column and eluting with 4 to 10 column volumes of a 30% to 80% ethanol aqueous solution, collecting the ethanol eluate, concentrating it, and drying it to obtain a crude pinoresinol-β-D-glucoside product for later use;

[0011] 5) The crude product obtained in step 4) is dissolved in an ethanol-water solution, allowed to stand and crystallize, the crystals are washed with water until white, and then recrystallized again from an ethanol-water solution, dried, and ground to obtain high-purity pinoresinol-β-D-glucoside.

[0012] 2. Preferably, the extraction method in step 1) is one of cold soaking, percolation, warm soaking or reflux.

[0013] 3. Preferably, the macroporous adsorption resin in step 3) is one or a combination of X-5, HPD-100, HPD-700, D-101, AB-8, and DM-301.

[0014] 4. Preferably, the ion resin in step 4) is one or a combination of D318, D280, D951, D941, and D201.

[0015] 5. Preferably, the solvent for the crystallization and recrystallization of the crude product in step 5) is water or an ethanol aqueous solution with a concentration of ≤50%, and the standing temperature is 0°C to 40°C.

[0016] 6. Preferably, the drying method of the crystals in step 5) includes freeze drying, reduced pressure drying, spray drying or oven drying, and the drying temperature is controlled below 90°C.

[0017] 7. In step 5), high-purity pinoresinol-β-D-glucoside is obtained, and its content is greater than 97% as determined by high-performance liquid chromatography. The HPLC detection method includes:

[0018] Chromatographic conditions and system suitability tests were conducted using octadecylsilane bonded silica gel as the filler; the column mixture was acetonitrile:0.1% formic acid = 18:82; the detector was a DAD detector, the detection wavelength was 230 nm, the flow rate was 1.0 ml / min, and the column temperature was 30°C. The number of theoretical plates calculated based on the pinoresin peak should be no less than 3000.

[0019] Preparation of reference solution: Take about 20 mg of pinoresinol-β-D-glucoside reference substance, accurately weigh it, place it in a 100ml volumetric flask, add chromatographic methanol to dissolve it and dilute it to the scale to make a solution containing 0.2 mg per 1ml.

[0020] Preparation of test solution: Take about 20 mg of the product, accurately weigh it, place it in a 100ml volumetric flask, add chromatographic methanol to dissolve it and dilute it to the scale to make a solution containing 0.2 mg per 1ml.

[0021] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The specific embodiments of the present invention are described in further detail below with reference to the accompanying drawings, in which:

[0023] Figure 1 is the chemical structural formula of pinoresinol-β-D-glucoside;

[0024] Figure 2 It is the hydrogen spectrum of pinoresinol-β-D-glucoside reference substance;

[0025] Figure 3 This is the carbon spectrum of pinoresinol-β-D-glucoside reference substance;

[0026] Figure 4 High-resolution mass spectrometry of pinoresinol-β-D-glucoside reference;

[0027] Figure 5 This is a graph showing the HPLC test results of pinoresinol-β-D-glucoside obtained by the preparation method provided in Example 1;

[0028] Figure 6 This is a graph showing the HPLC test results of pinoresinol-β-D-glucoside obtained by the preparation method provided in Example 2;

[0029] Figure 7 This is a graph showing the HPLC test results of pinoresinol-β-D-glucoside obtained by the preparation method provided in Example 3;

[0030] Figure 8 This is a graph showing the HPLC test results of pinoresinol-β-D-glucoside obtained by the preparation method provided in Example 4;

[0031] Figure 9 This is a graph showing the HPLC test results of pinoresinol-β-D-glucoside obtained by the preparation method provided in Example 5; DETAILED DESCRIPTION

[0032] Below in conjunction with embodiment, the present invention is described in further detail, and those skilled in the art can realize by drawing lessons from the present invention content, suitably improving relevant parameters according to specific actual equipment. The present invention has been described in more detail to pinoresinol-β-D-glucoside, and relevant personnel can change or appropriately change and combine the method of the present invention without departing from the content of the present invention and the scope, to realize and apply the inventive method. However, protection scope of the present invention is not limited to the content, and in the embodiment, method is conventional method unless otherwise specified, and reagent is conventional commercially available reagent or reagent prepared according to a conventional method unless otherwise specified, and each embodiment information is summarized in Table 1, and pinoresinol-β-D-glucoside carbon spectrum data comparison is shown in Table 2.

[0033]

[0034] Note: The moisture content of the material is 6.62%.

[0035]

[0036] Example 1

[0037] 1) Grind 30 kg of tubers of Herba Dioscoreae, add 15 times the amount of the herbal material in a 50% ethanol aqueous solution for percolation extraction, collect the percolate, and concentrate to a solid-liquid ratio of 1:3 (w / v) for later use;

[0038] 2) Add ethanol to the concentrated solution obtained in step 1) for alcohol precipitation, with the alcohol precipitation concentration being 70%. Let it stand for 3 hours, filter, and collect the filtrate to recover the ethanol to obtain a concentrated solution for later use.

[0039] 3) The concentrated solution obtained in step 2) was passed through a macroporous adsorption resin column, eluted with 4 column volumes of aqueous solution, and discarded; then eluted with 40% ethanol solution, the alcohol eluate was collected, and the 40% ethanol eluate was concentrated to a solid-liquid ratio of 1:3 for later use;

[0040] 4) The concentrated solution obtained in step 3) was loaded onto an ion-binding resin column and eluted with 10 column volumes of a 40% ethanol solution. The 40% ethanol eluate was collected and concentrated to obtain a crude product of pinoresinol-β-D-glucoside for later use;

[0041] 5) Step 4) The crude product was dissolved in water and allowed to crystallize. The crystals were washed with water until white and then recrystallized again from an ethanol-water solution. The solution was dried and ground to obtain 0.987 kg of high-purity pinoresinol-β-D-glucoside. HPLC analysis showed a purity of 97.00% and a moisture content of 2.62%.

[0042] Example 2

[0043] 1) Take 30 kg of tubers of Trichoderma lucidum, coarsely crush them, soak them in 10% ethanol water (10 times the volume) for 2 hours, transfer them to a stainless steel column for percolation extraction, and concentrate the extract to a solid-liquid ratio of 1:4, and set aside.

[0044] 2) The concentrated solution obtained in step 1) was precipitated with ethanol at a concentration of 60%, and allowed to stand for 3 h. The solution was filtered and the ethanol was recovered from the supernatant to obtain a concentrated solution for later use.

[0045] 3) The concentrated solution obtained in step 2) was applied to a macroporous adsorption resin column, eluted with 5 column volumes of 10% ethanol solution, and discarded; then eluted with 40% ethanol solution, the alcohol eluate was collected, and the 40% ethanol eluate was concentrated to a solid-liquid ratio of 1:3 for later use;

[0046] 4) The concentrated solution obtained in step 3) was loaded onto an ion-binding resin column and eluted with 8 column volumes of 40% ethanol solution. The 40% ethanol eluate was collected and concentrated to obtain the crude product of pinoresinol-β-D-glucoside for later use;

[0047] 5) Step 4) The crude product obtained was dissolved in a 30% ethanol-water solution, the crystals were washed with water until white, and then recrystallized again from an ethanol-water solution, dried, and ground to obtain 1.013 kg of high-purity pinoresinol-β-D-glucoside. HPLC analysis showed a purity of 97.30% and a moisture content of 2.35%.

[0048] Example 3

[0049] 1) Take 30 kg of tubers of Herba Dioscoreae, coarsely crush them, add 5 times the amount of aqueous solution and reflux extract, extract 5 times, each extraction time is 60 minutes, filter the extract, concentrate to the solid-liquid ratio of 1:3, and set aside;

[0050] 2) The concentrated solution obtained in step 1) was precipitated with ethanol at a concentration of 60%, and the solution was allowed to stand for 6 hours. The solution was filtered and the ethanol was recovered from the supernatant to obtain a concentrated solution for later use.

[0051] 3) The concentrated solution obtained in step 2) was applied to a macroporous adsorption resin column, eluted with 5 column volumes of 10% ethanol solution, and discarded; then eluted with 60% ethanol solution, the alcohol eluate was collected, and the 60% ethanol eluate was concentrated to a solid-liquid ratio of 1:4 for later use;

[0052] 4) The concentrated solution obtained in step 3) was loaded onto an ion-binding resin column and eluted with 8 column volumes of a 50% ethanol solution. The 50% ethanol eluate was collected and concentrated to obtain a crude product of pinoresinol-β-D-glucoside for later use;

[0053] 5) Step 4) The crude product obtained was dissolved in a 30% ethanol-water solution and allowed to crystallize. The crystals were washed with water until white and then recrystallized again from an ethanol-water solution. The product was dried and ground to obtain 1.014 kg of high-purity pinoresinol-β-D-glucoside. HPLC analysis showed a purity of 96.95% and a moisture content of 2.60%.

[0054] Example 4

[0055] 1) Take 30 kg of tubers of Herba Dioscoreae, coarsely crush them, and extract them by reflux with 10 times 30% ethanol solution. Extract three times, each time for 90 minutes. Filter the extract and concentrate it to a solid-liquid ratio of 1:4. Set aside.

[0056] 2) The concentrated solution obtained in step 1) was precipitated with ethanol to a concentration of 80%, and the solution was allowed to stand for 10 h. The solution was filtered and the ethanol was recovered from the supernatant to obtain a concentrated solution for later use.

[0057] 3) The concentrated solution obtained in step 2) was applied to a macroporous adsorption resin column, eluted with 6 columns of 20% ethanol solution, and discarded; then eluted with 70% ethanol solution, the alcohol eluate was collected, and the 70% ethanol eluate was concentrated to a solid-liquid ratio of 1:6 for later use;

[0058] 4) The concentrated solution obtained in step 3) was loaded onto an ion-binding resin column and eluted with 6 column volumes of 60% ethanol solution. The 60% ethanol eluate was collected and concentrated to obtain the crude product of pinoresinol-β-D-glucoside for later use;

[0059] 5) Step 4) The crude product obtained was dissolved in 40% ethanol-water solution and allowed to stand for crystallization. The crystals were washed with water until white and then recrystallized again from ethanol-water solution, dried, and ground to obtain 1.026 kg of high-purity pinoresinol-β-D-glucoside. HPLC analysis showed a purity of 97.68% and a moisture content of 2.81%.

[0060] Example 5

[0061] 1) Take 30 kg of tubers of Herba Dioscoreae, add 20 times 60% ethanol solution and extract it at warm temperature, extract once at 80°C for 150 min, filter the extract, and concentrate it to a solid-liquid ratio of 1:5, and set aside;

[0062] 2) The concentrated solution obtained in step 1) was precipitated with ethanol to a concentration of 80%, and the solution was allowed to stand for 16 hours. The solution was filtered and the ethanol was recovered from the supernatant to obtain a concentrated solution for later use.

[0063] 3) The concentrated solution obtained in step 2) was applied to a macroporous adsorption resin column, eluted with 6-fold column-length 20% ethanol solution, and discarded; then eluted with 70% ethanol solution, the alcohol eluate was collected, and the 70% ethanol eluate was concentrated to a solid-liquid ratio of 1:5 for later use;

[0064] 4) The concentrated solution obtained in step 3) was loaded onto an ion-binding resin column and eluted with 3 column volumes of 70% ethanol solution. The 70% ethanol eluate was collected and concentrated to obtain the crude product of pinoresinol-β-D-glucoside for later use;

[0065] 5) Step 4) The crude product obtained was dissolved in 50% ethanol-water solution and allowed to stand for crystallization. The crystals were washed with water until white and then recrystallized again from ethanol-water solution, dried, and ground to obtain 0.994 kg of high-purity pinoresinol-β-D-glucoside. HPLC analysis showed a purity of 97.09% and a moisture content of 2.56%.

Claims

1. A method for preparing pinoresinol-β-D-glucoside from tubers of Herba Lycopodii, characterized in that The following steps are involved: 1) Crush the tubers of the herbaceous plant and extract them with an ethanol-water solution at a solid-liquid ratio of 1:3 to 1:

30. Extract 1 to 5 times, each extraction lasting 30 to 180 minutes. Concentrate the extract to a solid-liquid ratio of 1:0.5 to 1:6 (w / v) and set aside. 2) adding ethanol to the concentrated solution obtained in step 1) for alcohol precipitation, with the alcohol precipitation concentration being 60% to 80%, standing for 3 hours to 16 hours, separating the solid and liquid, and recovering ethanol from the supernatant to obtain a concentrated solution; 3) passing the concentrated solution obtained in step 2) through a macroporous adsorption resin column, first eluting with 4 to 10 column volumes of water, then eluting with a 30% to 80% ethanol aqueous solution, collecting the ethanol eluate, and concentrating it to a solid-liquid ratio (w / v) of 1:0.5 to 1:10; 4) passing the concentrated solution obtained in step 3) through an ion resin column and eluting with 4 to 10 column volumes of a 30% to 80% ethanol aqueous solution, collecting the ethanol eluate, concentrating, and drying to obtain a crude pinoresinol-β-D-glucoside product for later use; 5) The crude product obtained in step 4) was allowed to stand and crystallize with water or 30% ethanol aqueous solution. The crystals were washed with water until white, and then recrystallized again with 30% ethanol aqueous solution, dried, and ground to obtain high-purity pinoresinol-β-D-glucoside.

2. The method for preparing pinoresinol-β-D-glucoside from tubers of Herba Lycopodii according to claim 1, wherein: The extraction method in step 1) is one of percolation, warm immersion or reflux.

3. The method for preparing pinoresinol-β-D-glucoside from tubers of Herba Lycopodii according to claim 1, wherein: The macroporous adsorption resin column in step 3) is one or a combination of two of X-5, HPD-100, HPD-700, D-101, AB-8, and DM-301.

4. The method for preparing pinoresinol-β-D-glucoside from tubers of Herba Lycopodii according to claim 1, wherein: The ion resin in step 4) is one of D318, D280, D951, D941, and D201, or a combination of two of them.

5. The method for preparing pinoresinol-β-D-glucoside from tubers of Herba Lycopodii according to claim 1, wherein: The drying method of the crystals in step 5) includes freeze drying, reduced pressure drying, spray drying or oven drying, and the drying temperature is controlled below 90°C.

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