Method for preparing pinoresinol-beta-D-glucoside from zornia gibbosa tubers

Abstract

The invention relates to a method for extracting, separating and preparing pinoresinol-beta-D-glucoside from tubers of zornia gibbosa. The method comprises the following specific steps: adding an ethanol-water solution into a zornia gibbosa tuber for extraction, concentrating an extracting solution, adding ethanol for alcohol precipitation, enriching supernate through macroporous adsorption resin, decolorizing through an ion resin material, and crystallizing and recrystallizing a decolorizing solution to obtain the high-purity pinoresinol-beta-D-glucoside (the purity is greater than 97%). The process flow provided by the invention is simple, the transfer rate is high, and large-scale preparation and industrial production can be stably and repeatedly realized; the process is green and environment-friendly, only ethanol is used as a solvent and can be recycled, the cost is reduced, and the environmental pollution is small.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a preparation method for extracting and preparing high-purity pinoresinol-β-D-glucoside from Boschniakia himalaica Hook. f. et Thoms. Background Art

[0002] Boschniakia himalaica Hook. f. et Thoms, also known as Qianjinzhui, is a plant of the genus Boschniakia in the Orobanchaceae family. It is distributed in Yunnan, Tibet, Shaanxi, Sichuan, Hubei and other places. It grows in shady forests or shrubs in alpine mountainous areas and often parasitizes on the roots of plants of the genus Rhododendron. It has the effects of dispelling wind and activating collaterals, regulating qi and strengthening the stomach. Its dried tuber has been included as a medicinal material, namely Qianjinzhui, in the "Yunnan Chinese Medicinal Materials Standard" (2005 edition). Qianjinzhui is one of the main raw materials of Qiancao Naomaitong mixture, and pinoresinol-β-D-glucoside is one of the main index components in the mixture. The main treatment function of this mixture is promoting blood circulation to remove blood stasis, resolving phlegm and activating collaterals. It is used to treat stroke, middle meridian, hemiplegia, deviation of the mouth and eyes, and speech disorders caused by phlegm and blood stasis obstruction.

[0003] Boschniakia himalaica mainly contains phenylpropanoid glycosides and lignan components, among which the lignan content is relatively high, accounting for about 3% - 10% of the total components. Pinoresinol- β -β-D-glucoside, as the main component of lignans, has a content of up to 2% - 8%. Pinoresinol- β -β-D-glucoside is also known as (+)-pinoresinol- β -D-pyranoglucoside, (+)-pinoresinol-4-O-glucoside. The invention patent with the publication number CN119258083 A discloses the application of pinoresinol-4-O-glucoside in the preparation of drugs for treating allergic asthma diseases, showing the prospect of developing new anti-asthma drugs. The patent with the publication number CN 119174776 A discloses the application in the preparation of drugs for treating and / or preventing renal fibrosis, providing a new direction for treating and / or preventing 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. Its structural formula and carbon spectrum comparison information are shown in Figure 1And Table 2, the carbon spectrum information is consistent with that of Compound 6 reported in the literature by MARIKO CHIBA et al. (13C NMR analysis of symplocosin and (+)-epipinoresinol glucoside Phytochemistry 1980, Vd. 19. pp. 335-336). In 1978, Chiba, M. et al. (Chiba, M., Hisada, S. and Nishibe, S. (1978) Shoyakugaku Zasshi 32, 194.) first isolated pinoresinol-β-D-glucoside from Forsythia suspensa. Subsequently, scholars also successively discovered pinoresinol-β-D-glucoside from plants of the genus Cistanche in Orobanchaceae. Pan Yingni (Research on the Chemical Constituents and Biological Activities of Fresh Cistanche tubulosa [D]. Shenyang Pharmaceutical University, 2011.), Jin Yinping (Study on the Chemical Constituents of Xylanche himalaica [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 the Chemical Constituents of Xylanche himalaica [J] Acta Botanica Sinica, 1992, 34(11):878-882), etc. all isolated pinoresinol-β-D-glucoside when separating the chemical constituents of different plants, but all used laboratory-scale separation and purification methods, and the yield and purity of pinoresinol-β-D-glucoside were not reported. This method conducts research on the preparation process of pinoresinol-β-D-glucopyranoside in Xylanche himalaica. Its preparation process is simple, easy to implement, environmentally friendly, has a high transfer rate, can achieve large-scale preparation and industrial production, provides feasibility for its development as a new drug, and has broad future application and market prospects. Summary of the Invention

[0005] The present invention aims to provide a method for extracting, separating and preparing pinoresinol-β-D-glucoside from Xylanche himalaica. After the tubers of Xylanche himalaica are coarsely pulverized, an ethanol-aqueous solution is added for extraction, and the extract is enriched by a macroporous adsorption resin column and decolorized by an ion resin column to obtain a crude product of pinoresinol-β-D-glucoside. The crude product is prepared into high-purity pinoresinol-β-D-glucoside (purity greater than 97%) through crystallization and recrystallization. The process of the present invention is simple, feasible, can stably obtain high-purity pinoresinol-β-D-glucose, has a short production cycle, can achieve large-scale preparation and industrial production. The solvents used are easy to recover, can be reused, have a low cost, and cause little environmental pollution.

[0006] 1. The present invention relates to a method for extracting and separating and preparing pinoresinol-β-D-glucoside from Boschniakia rossica, comprising the following steps:

[0007] 1) Coarsely pulverize the tubers of Boschniakia rossica, extract with an ethanol-aqueous solution, with a material-liquid ratio of 1:3 to 1:30, extract 1 to 5 times, each extraction for 30 min to 180 min, concentrate the extract to a material-liquid ratio of 1:0.5 to 1:6 (w / v), and set aside;

[0008] 2) Add ethanol to the concentrated solution obtained in step 1) for alcohol precipitation, with an alcohol precipitation concentration of 60% to 80%, let stand for 3 h to 16 h, perform solid-liquid separation, and recover ethanol from the supernatant to obtain a concentrated solution;

[0009] 3) Pass the concentrated solution obtained in step 2) through a macroporous adsorption resin column, first wash with 4 to 10 times the column volume of water, then elute with an ethanol aqueous solution with a concentration of 30% to 80%, collect the ethanol eluate, and concentrate to a material-liquid ratio of 1:0.5 to 1:10 (w / v);

[0010] 4) Pass the concentrated solution obtained in step 3) through an ion resin column, elute with an ethanol aqueous solution with a concentration of 30% to 80% and 4 to 10 times the column volume, collect the ethanol eluate, concentrate, and dry to obtain a crude product of pinoresinol-β-D-glucoside, and set aside;

[0011] 5) Dissolve the crude product obtained in step 4) with an ethanol-aqueous solution, let stand for crystallization, wash the crystals with water until white, then perform recrystallization with an ethanol-aqueous solution again, dry, grind finely, to obtain high-purity pinoresinol-β-D-glucoside.

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

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

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

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

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

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

[0018] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; acetonitrile∶0.1% formic acid = 18∶82; the detector is a DAD detector, the detection wavelength is 230 nm, the flow rate is 1.0 ml / min, and the column temperature is 30 °C. The number of theoretical plates calculated based on the pinoside peak should be not 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 100 ml volumetric flask, dissolve it with chromatographic methanol and dilute to the scale to prepare a solution containing 0.2 mg per 1 ml, that is, obtain.

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

[0021] Determination method: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine, that is, obtain. Description of the drawings

[0022] The following further describes in detail the specific embodiments of the present invention in conjunction with the drawings. In the drawings:

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

[0024] Figure 2 is the 1H NMR spectrum of pinoresinol-β-D-glucoside reference substance;

[0025] Figure 3 is the 13C NMR spectrum of pinoresinol-β-D-glucoside reference substance;

[0026] Figure 4 is the high-resolution mass spectrum of pinoresinol-β-D-glucoside reference substance;

[0027] Figure 5 is the HPLC detection result diagram of pinoresinol-β-D-glucoside obtained by the preparation method provided in Example 1;

[0028] Figure 6 is the HPLC detection result diagram of pinoresinol-β-D-glucoside obtained by the preparation method provided in Example 2;

[0029] Figure 7 is the HPLC detection result diagram of pinoresinol-β-D-glucoside obtained by the preparation method provided in Example 3;

[0030] Figure 8 The HPLC detection result diagram of pinoresinol-β-D-glucoside obtained by the preparation method provided for Example 4;

[0031] Figure 9 The HPLC detection result diagram of pinoresinol-β-D-glucoside obtained by the preparation method provided for Example 5; Detailed implementation manners

[0032] The present invention will be further described in detail below in conjunction with embodiments. Those skilled in the art can appropriately modify relevant parameters according to specific actual equipment by referring to the content of the present invention. The present invention has described pinoresinol-β-D-glucoside in relatively detail. Relevant personnel can make changes or appropriate alterations and combinations to the method described in the present invention without departing from the content and scope of the present invention to implement and apply the method of the present invention. However, the protection scope of the present invention is not limited to the content described. Unless otherwise specified, the methods in the embodiments are all conventional methods, and the reagents used are all conventional commercially available reagents or reagents prepared according to conventional methods. The information summaries of each embodiment are shown in Table 1, and the comparison of the carbon spectrum data of pinoresinol-β-D-glucoside is shown in Table 2.

[0033]

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

[0035]

[0036] Example 1

[0037] 1) Take 30 kg of the tubers of Pedicularis rhinanthoides Schrenk var. tientschanica (Maxim.) W. T. Wang & Tsai, pulverize, add 15 times the amount of 50% ethanol aqueous solution of the medicinal material for percolation extraction, collect the percolate, concentrate to a material-liquid ratio of 1:3 (w / v), and set aside;

[0038] 2) Add ethanol to the concentrated solution obtained in step 1) for alcohol precipitation, the alcohol precipitation concentration is 70%, let stand for 3 h, filter, collect the filtrate and recover ethanol to obtain a concentrated solution, and set aside.

[0039] 3) Pass the concentrated solution obtained in step 2) through a macroporous adsorption resin column, elute with 4 times the column volume of aqueous solution, discard; then elute with 40% ethanol solution, collect the ethanol eluate, and concentrate the 40% ethanol eluate to a material-liquid ratio of 1:3, and set aside;

[0040] 4) Load the concentrated solution obtained in step 3) onto an ion resin column, elute with 10 times the column volume of 40% ethanol solution, collect the 40% ethanol eluate, and concentrate to obtain the crude product of pinoresinol-β-D-glucoside, and set aside;

[0041] 5) The crude product obtained in step 4) was dissolved in water, allowed to stand for crystallization, and the crystals were washed with water until white, and then recrystallized with an ethanol-aqueous solution, dried, and ground to obtain 0.987 kg of high-purity pinoresinol-β-D-glucoside. After HPLC detection, the purity was 97.00% and the water content was 2.62%.

[0042] Example 2

[0043] 1) Take 30 kg of the tubers of Pedicularis viridiflora, coarsely crush them, soak them in 10 times the amount of 10% ethanol aqueous solution for 2 h, transfer them to a stainless steel column for percolation extraction, and concentrate the extract to a material-liquid ratio of 1:4 for standby;

[0044] 2) Ethanol was added to the concentrated solution obtained in step 1) for ethanol precipitation, the ethanol precipitation concentration was 60%, allowed to stand for 3 h, filtered, and the supernatant was recycled with ethanol to obtain a concentrated solution for standby.

[0045] 3) The concentrated solution obtained in step 2) was loaded onto a macroporous adsorption resin column, eluted with 5 times the column volume of 10% ethanol solution, and discarded; then eluted with 40% ethanol solution, and the ethanol eluate was collected. The 40% ethanol eluate was concentrated to a material-liquid ratio of 1:3 for standby;

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

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

[0048] Example 3

[0049] 1) Take 30 kg of the tubers of Pedicularis viridiflora, coarsely crush them, reflux and extract with 5 times the amount of aqueous solution for 5 times, each extraction for 60 min, filter the extract, and concentrate it to a material-liquid ratio of 1:3 for standby;

[0050] 2) Ethanol was added to the concentrated solution obtained in step 1) for ethanol precipitation, the ethanol precipitation concentration was 60%, allowed to stand for 6 h, filtered, and the supernatant was recycled with ethanol to obtain a concentrated solution for standby.

[0051] 3) The concentrated solution obtained in step 2) was loaded onto a macroporous adsorption resin column, eluted with 5 times the column volume of 10% ethanol solution, and discarded; then eluted with 60% ethanol solution, and the ethanol eluate was collected. The 60% ethanol eluate was concentrated to a material-liquid ratio of 1:4 for standby;

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

[0053] 5) The crude product obtained in step 4) is dissolved in 30% ethanol aqueous solution, allowed to stand for crystallization. After the crystals are washed with water until white, they are recrystallized with ethanol-aqueous solution again, dried, and ground to obtain 1.014 kg of high-purity pinoresinol-β-D-glucoside. Detected by HPLC, the purity is 96.95% and the moisture content is 2.60%.

[0054] Example 4

[0055] 1) Take 30 kg of the tubers of Aphyllorchis thomsoni, coarsely crush them, add 10 times of 30% ethanol solution and reflux for extraction for 3 times, each extraction for 90 min. The extract is filtered and concentrated to a solid-liquid ratio of 1:4 for standby.

[0056] 2) Ethanol is added to the concentrated solution obtained in step 1) for alcohol precipitation, with an alcohol precipitation concentration of 80%. It is allowed to stand for 10 h, filtered, and the supernatant is recovered of ethanol to obtain a concentrated solution for standby.

[0057] 3) The concentrated solution obtained in step 2) is loaded onto a macroporous adsorption resin column and eluted with 6 column volumes of 20% ethanol solution and discarded; then eluted with 70% ethanol solution, and the ethanol eluate is collected. The 70% ethanol eluate is concentrated to a solid-liquid ratio of 1:6 for standby.

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

[0059] 5) The crude product obtained in step 4) is dissolved in 40% ethanol aqueous solution, allowed to stand for crystallization. After the crystals are washed with water until white, they are recrystallized with ethanol-aqueous solution again, dried, and ground to obtain 1.026 kg of high-purity pinoresinol-β-D-glucoside. Detected by HPLC, the purity is 97.68% and the moisture content is 2.81%.

[0060] Example 5

[0061] 1) Take 30 kg of the tubers of Aphyllorchis thomsoni, add 20 times of 60% ethanol solution and warm soak for extraction for 1 time, with an extraction temperature of 80 °C and extraction for 150 min. The extract is filtered and concentrated to a solid-liquid ratio of 1:5 for standby.

[0062] 2) Ethanol is added to the concentrated solution obtained in step 1) for alcohol precipitation, with an alcohol precipitation concentration of 80%. It is allowed to stand for 16 h, filtered, and the supernatant is recovered of ethanol to obtain a concentrated solution for standby.

[0063] 3) The concentrated solution obtained in step 2) is loaded onto a macroporous adsorption resin column, eluted with a 20% ethanol solution 6 times the column volume, and discarded; then eluted with a 70% ethanol solution, and the ethanol eluate is collected. The 70% ethanol eluate is concentrated to a feed liquid ratio of 1:5 and reserved for use;

[0064] 4) The concentrated solution obtained in step 3) is loaded onto an ion resin column, eluted with a 70% ethanol solution 3 times the column volume, and the 70% ethanol eluate is collected. The crude product of pinoresinol-β-D-glucoside is obtained by concentration and reserved for use;

[0065] 5) The crude product obtained in step 4) is dissolved in a 50% ethanol aqueous solution, allowed to stand for crystallization. After the crystals are washed with water until white, recrystallized again with an ethanol-aqueous solution, dried, and ground to obtain 0.994 kg of high-purity pinoresinol-β-D-glucoside. After HPLC detection, the purity is 97.09% and the moisture content is 2.56%.

Claims

1. A method for preparing pinoresinol-β-D-glucoside from the tubers of Boschniakia rossica, characterized in that It includes the following steps: 1) Coarsely crush the tubers of Boschniakia rossica, extract with an ethanol-aqueous solution, with a material-liquid ratio of 1:3 to 1:30, extract 1 to 5 times, each extraction for 30 min to 180 min, and concentrate the extract to a material-liquid ratio of 1:0.5 to 1:6 (w / v) for standby; 2) Add ethanol to the concentrated solution obtained in step 1) for alcohol precipitation, with an alcohol precipitation concentration of 60% to 80%, stand for 3 h to 16 h, separate the solid and liquid, and recover ethanol from the supernatant to obtain a concentrated solution; 3) Pass the concentrated solution obtained in step 2) through a macroporous adsorption resin column, first wash with 4 to 10 times the column volume of water, and then elute with an ethanol aqueous solution with a concentration of 30% to 80%, collect the ethanol eluate, and concentrate to a material-liquid ratio of 1:0.5 to 1:10 (w / v); 4) Pass the concentrated solution obtained in step 3) through an ion resin column, elute with an ethanol aqueous solution with a concentration of 30% to 80% and a volume of 4 to 10 times the column volume, collect the ethanol eluate, concentrate, and dry to obtain the crude product of pinoresinol-β-D-glucoside for standby; 5) Dissolve the crude product obtained in step 4) with an ethanol-aqueous solution, stand for crystallization, wash the crystals with water until white, then recrystallize with an ethanol-aqueous solution again, dry, grind finely to obtain high-purity pinoresinol-β-D-glucoside.

2. The method for preparing pinoresinol-β-D-glucoside from the tubers of Xylanche himalaica according to claim 1, characterized in that, The extraction method in step 1) is one of cold maceration, percolation, warm maceration or reflux.

3. A method for preparing pinoresinol-β-D-glucoside from the tubers of Xylanche himalaica, as claimed in claim 1, wherein The type of macroporous adsorption resin described in step 3) is one of X-5, HPD-100, HPD-700, D-101, AB-8, DM-301 or a combination of two of them.

4. A method for preparing pinoresinol-β-D-glucoside from the tubers of Xylanche himalaica according to claim 1, characterized in that, The ion resin described in step 4) is one of D318, D280, D951, D941, D201 or a combination of two of them.

5. A method for preparing pinoresinol-β-D-glucoside from the tubers of Xylanche himalaica according to claim 1, characterized in that, The solvents for crystallization and recrystallization of the crude product in step 5) are water or an ethanol aqueous solution with a concentration ≤ 50%, and the standing temperature is 0 °C to 40 °C.

6. The method for preparing pinoresinol-β-D-glucoside from the tubers of Xylanche himalaica according to claim 1, characterized in that, The drying method of the crystals in step 5) includes one of freeze drying, vacuum drying, spray drying or drying in an oven, and the drying temperature is controlled below 90 °C.

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