Method for extracting ginsenosides Rb1 and Rd from pseudo-ginseng

Abstract

The invention discloses a method for extracting ginsenosides Rb1 and Rd from pseudo-ginseng. The method comprises the following steps: step 1, extracting raw materials; step 2, recovering ethanol; step 3, preparing crude extraction column loading liquid; step 4, crude extraction; step 5, re-dissolving; step 6, separating a single component; step 7, refining a single component of ginsenoside Rb1; and step 8, refining a single component of ginsenoside Rd. According to the method disclosed by the invention, the non-polar macroporous adsorption resin and the moderately polar macroporous adsorption resin are combined for use, gradient elution is performed, and a high-content and batch-level single-component product can be obtained; on the basis, a high-content product is further obtained through the steps of crystallization and chromatography. The method disclosed by the invention is easy to operate and implement, can be repeatedly used and is relatively high in yield.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification of plant extracts, and in particular to a method for extracting ginsenosides Rb1 and Rd from Panax notoginseng. Background Art

[0002] Ginsenosides are composed of two parts: an aglycone and a glycosyl group. Based on the differences in the aglycone's core skeleton, they can be divided into dammarane-type tetracyclic triterpenoid saponins and oleanolic acid-type pentacyclic triterpenoid saponins. Dammarane-type ginsenosides are further divided into protopanaxadiol saponins (PPD) and protopanaxatriol saponins (PPT) based on the differences in the glycosyl substituents at C-3, C-6, and C-20. The main saponins in Panax notoginseng include notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd; all five of these saponins are dammarane-type.

[0003] Ginsenoside Rb1 is a monomeric saponin of the protopanaxadiol type, widely found in plants such as ginseng, American ginseng, and Panax notoginseng. It is a major saponin in the genus Panax. In addition to its excellent pharmacological activity in cardiovascular and cerebrovascular diseases, recent studies have shown that ginsenoside Rb can activate RNA polymerase, promote the synthesis of RNA, DNA, and proteins in nerve cells, repair damaged nerve cells, promote nerve cell regeneration, protect cerebral cortex cells, enhance thinking ability, and improve memory. Furthermore, it has a significant anti-cancer effect.

[0004] Ginsenoside Rd has fewer side chain sugars and is less polar, making it easily absorbed by the human gastrointestinal tract. This results in higher biological activity and the ability to specifically block receptor-dependent calcium channels. It possesses unique effects not found in other monomeric ginsenosides, including renal protection, immune regulation, inhibition of HeLa cell growth, induction of COX22 production, and radiation protection. Compared to other monomeric ginsenosides, Rd also exhibits stronger analgesic and neuroprotective effects, making it a promising candidate drug. Therefore, the extraction of each individual component is becoming increasingly important.

[0005] Chinese patent CN 1334267A discloses a "method for preparing Panax notoginseng total glycosides". The process steps include crushing the Panax notoginseng raw material → extraction → concentration → clarification → desugaring with macroporous adsorption resin → decolorization with ion exchange resin → concentration → spray drying → finished product Panax notoginseng total glycosides.

[0006] Chinese patent CN1414011A discloses a "method for extracting notoginseng saponins". The main process is: crushing of the notoginseng raw material → extraction → dealcoholization → dilution → adsorption with a strong polar macroporous adsorption resin → analysis → concentration → drying → finished product of notoginseng total saponins. Although the above preparation method obtains a notoginseng total saponin product with a certain purity through clarification, chromatography, and decolorization, it does not study a single component.

[0007] Chinese patent CN03127664.4 discloses a "method for the isolation of ginsenoside Rb1 in high yields." Using silica gel column chromatography, the patent obtained notoginsenoside Rb1 with a purity of 95% and a yield of 20%. However, this method has a low yield and a low throughput, making large-scale production difficult.

[0008] Chinese patent CN200710178212.9 discloses a "method for producing ginsenosides". This patent uses a non-polar macroporous adsorption resin and organic solvent recrystallization method to obtain a product with a 90% Rb1 content. The content of the product obtained by this method is not high, the data is too general, and the specific yield of the experiment is not mentioned.

[0009] Chinese patent CN201810146308.5 discloses a "method for automatically controlling the separation and purification of ginsenoside Rd". This patent uses DOS filler chromatography to separate ginsenosides Rg1, Rb1 and Rd. However, this method has too small a processing capacity and can only remain in the laboratory stage.

[0010] The current market has not yet found a mature process for large-scale production of single-component ginsenoside Rb1 and ginsenoside Rd with high yield and high content. Based on the above reasons, the present invention is proposed. Summary of the Invention

[0011] In view of this, the present invention provides a method for extracting ginsenosides Rb1 and Rd from Panax notoginseng.

[0012] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0013] The present invention discloses a method for extracting ginsenosides Rb1 and Rd from Panax notoginseng, comprising the following steps:

[0014] Step 1: Raw material extraction

[0015] Adding the powder of Panax notoginseng medicinal material to the ethanol solution and performing hot reflux extraction, filtering, extracting, and combining the filtrate;

[0016] Step 2: Ethanol recovery

[0017] The treated filtrate obtained in step 1 is subjected to reduced pressure distillation to recover ethanol to obtain a dry product;

[0018] Step 3: Prepare crude column liquid

[0019] Add purified water to the dried product obtained in step 2, stir, and filter to obtain a crude supernatant;

[0020] Step 4: Rough Extraction

[0021] The liquid obtained in step 3 is measured and passed through a non-polar macroporous adsorption resin. After adsorption is completed, the mixture is washed with water, pre-lysed, and lysed. The lysate is dealcoholized and dried to obtain a crude product of Panax notoginseng total glycosides.

[0022] Step 5: Reconstitution

[0023] Add the crude notoginsenoside obtained in step 4 into deionized water and dissolve it completely under stirring to obtain a liquid, which is then set aside for later use;

[0024] Step 6: Single component separation

[0025] The liquid obtained in step 5 is passed through a medium-polarity macroporous adsorption resin, and then washed, pre-lyzed, and gradient-lyzed to obtain a high-content ginsenoside Rb1 component product and a high-content ginsenoside Rd component product, respectively;

[0026] Step 7: Single component purification of ginsenoside Rb1

[0027] Recrystallize the high-content ginsenoside Rb1 component product obtained in step 6 with an organic solvent to obtain the final product;

[0028] Step 8: Single component purification of ginsenoside Rd

[0029] The high-content ginsenoside Rd component product obtained in step 6 is loaded onto a dextran resin for chromatography experiments.

[0030] Preferably, in step 1, the temperature of the hot reflux extraction is 60-80° C., and the reflux time is 1-2 h;

[0031] The material-liquid ratio of Panax notoginseng medicinal material powder to ethanol solution is 1:3-10.

[0032] Preferably, in step 2, the mass ratio of ethanol to water in the ethanol solution is 1-5:1;

[0033] The total content of five saponins in the obtained product is 25-35%, wherein the content of ginsenoside Rb1 is 6-11%, and the content of ginsenoside Rd is 2-4%.

[0034] Preferably, in step 3, the stirring time is 1-3 hours, the mass ratio of the obtained dry product to deionized water is 10-20:100, and the total glycoside concentration of the prepared liquid is 2-4%.

[0035] Preferably, in step 4, the amount of resin loaded is based on 60-80 g / L of total glycosides, the water washing amount is 2-3 BV of the resin volume, the pre-solution agent is a 10-30% alcohol solution, the pre-solution amount is 1-3 BV, the desolvant is a 50-80% alcohol solution, and the desolvation amount is 2-5 BV;

[0036] The total content of five saponins in the obtained solid product is 65-75%, wherein the content of ginsenoside Rb1 is 50-60%, and the content of ginsenoside Rd is 10-20%. The product yield is 95-98%.

[0037] Preferably, in step 5, the mass ratio of the crude notoginsenoside obtained in step 4 to deionized water is 10-20:100; and the total glycoside concentration of the prepared solution in step 5 is 15-20%.

[0038] Preferably, in step 6, the amount of resin loaded is based on 60-80 g / L of total glycosides, the water wash volume is 2-3 BV of the resin volume, the pre-solvent is a 10-30% alcohol solution, the pre-solvent volume is 1-3 BV, the first part of the resolving agent is a 40-60% alcohol solution, the resolving volume is 2-5 BV, and the second part of the resolving agent is a 60-80% alcohol solution, the resolving volume is 2-5 BV;

[0039] The total content of the five saponins in the first part of the solid product obtained by analysis is 80-85%, of which the content of ginsenoside Rb1 is 70-80%, and the yield is 85-90%. The total content of the five saponins in the second part of the solid product obtained by analysis is 70-80%, the content of ginsenoside Rd is 60-70%, and the yield is 85-90%;

[0040] Preferably, the resin is a medium-polar resin of the methyl acrylate type.

[0041] Preferably, in step 7, the solvent used for recrystallization is a water and acetone system, or an ethanol aqueous solution and an ethyl acetate, isobutanol solution; and the content of ginsenoside Rb1 is above 98%.

[0042] Preferably, in step 8, the resin aspect ratio is 30-40, the resin mobile phase is 5-95% ethanol, the column load is 10 g / L, the flow rate is 0.2-2 BV / h; and the ginsenoside Rd is 95%.

[0043] Compared with the prior art, the present invention has achieved the following technical effects:

[0044] (1) The present invention selects a non-polar resin and a medium-polar resin for use in combination, and each resin is subjected to gradient analysis, batch purification, and multiple processes are cross-used to achieve a batch-level production process suitable for large-scale production, and obtain a single-component product with high yield and high content. The entire process is simple and easy to operate;

[0045] (2) The present invention first uses a non-polar resin to preliminarily separate the five saponin components, and uses a low concentration of organic solvent to separate the notoginsenoside R1 and ginsenoside Re, Rg1 components and some impurities by taking advantage of the fact that the molecular polarity of notoginsenoside R1 and ginsenoside Re, Rg1 is greater than that of ginsenoside Rb1, Rd; then, a high concentration of organic solvent is used to resolve the ginsenoside Rb1 and Rd components, and a crude product can be obtained after dealcoholization; ginsenoside Rb1 belongs to the ginsenoside Rd type saponin, which contains four sugar groups, and ginsenoside Rb1 belongs to the ginsenoside Rd type saponin, which contains three sugar groups, so the two tend to be medium polar molecules, and the molecular polarity of Rb1 is greater than the molecular polarity of Rd. Because of their polarity, a medium-polarity resin was selected and gradient elution was used to further purify the two. After obtaining a product with a ginsenoside Rb1 content of more than 70%, further purification with a resin separation medium reduced efficiency and increased cost. Based on the different polarities and solubilities of ginsenoside Rb1 and other components, organic solvent recrystallization was performed to obtain a single-component product with a high content of ginsenoside Rb1. After obtaining a product with a ginsenoside Rd content of more than 60%, it was difficult to purify it with a resin separation medium because the impurities and the effective substance were extremely similar. Solvent crystallization was also not ideal. Therefore, dextran chromatography medium was selected for further chromatographic separation to obtain a chromatographic-grade high-content ginsenoside Rd product. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the liquid phase spectrum of the product prepared in step ② of Example 1 of the present invention;

[0047] Figure 2 This is the liquid phase spectrum of the product prepared in step ④ of Example 1 of the present invention;

[0048] Figure 3 and Figure 4 This is the liquid phase spectrum of the product prepared in step ⑥ of Example 1 of the present invention;

[0049] Figure 5 This is the liquid phase spectrum of the product prepared in step ⑦ of Example 1 of the present invention;

[0050] Figure 6 This is the liquid phase spectrum of the product prepared in step ⑧ of Example 1 of the present invention. DETAILED DESCRIPTION

[0051] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0052] The volume of the non-polar macroporous adsorption resin column used in the embodiment of the present invention is 30 mL, the volume of the intermediate polar macroporous adsorption resin column is 30 mL, and the volume of the dextran chromatography resin is 100 mL.

[0053] The HPLC detection method is:

[0054] Chromatographic column: octadecylsilane column, 4.6×250 mm, 5 μm;

[0055] Mobile phase: acetonitrile (A) and water (B)

[0056] Gradient elution program:

[0057]

[0058] Flow rate: 1.5 mL / min; detection wavelength: 203 nm; column temperature: 25°C; injection volume: 20 uL.

[0059] Example 1:

[0060] A method for extracting ginsenosides Rb1 and Rd from Panax notoginseng, comprising the following steps:

[0061] Step 1: Raw material extraction

[0062] Weigh 1 kg of Panax notoginseng powder and place it in a round-bottom flask. First, add 8 L of 75% ethanol solution at 70°C and reflux for 2 h. Filter and repeat the same operation. Add 6 L of ethanol for the second time and 4 L of ethanol for the third time. Extract three times and combine the filtrates.

[0063] Step 2: Ethanol recovery

[0064] The treated filtrate obtained in step 1 is subjected to reduced pressure distillation at a certain temperature to recover ethanol to obtain a dry product;

[0065] The total content of the five saponins in the obtained solid product was 30.31%, of which the content of ginsenoside Rb1 was 10.11% and the content of ginsenoside Rd was 3.35%;

[0066] Step 3: Prepare crude column liquid

[0067] Weigh 7 g of the dried product obtained in step 3, add 200 mL of purified water, stir for 1 h to dissolve the material, and filter to obtain the crude supernatant;

[0068] Step 4: Rough extraction

[0069] The liquid obtained in step 3 was loaded onto a non-polar macroporous adsorption resin DM30 at an adsorption flow rate of 2 BV / h. After adsorption, the mixture was washed with water for 2 BV at the same flow rate. The pre-solvent was 15% isopropanol for 3 BV at a flow rate of 1.0 BV / h, and the desolvent was 70% ethanol for 3 BV at a flow rate of 0.8 BV / h. The desolvent was dealcoholized and dried to obtain a crude product of Panax notoginseng total glycosides.

[0070] The total content of the five saponins in the obtained solid product was 71%, of which the content of ginsenoside Rb1 was 51.33% and the content of ginsenoside Rd was 15.02%. The product yield was 95%;

[0071] Step 5: Reconstitution

[0072] Weigh 3 g of the crude notoginsenoside obtained in step 4, add 90 mL of deionized water, and dissolve completely under stirring to obtain a liquid, which is set aside for later use;

[0073] Step 6: Single component separation

[0074] The feed liquid in step 5 was passed through a medium-polarity macroporous adsorption resin LK1300S at a flow rate of 2BV / h, washed with water for 1BV, and the flow rate remained unchanged. The pre-solvent was 10% methanol for 3BV at a flow rate of 2.0BV / h, the first part of the desolvent was 40% methanol for 2.5BV at a flow rate of 0.8BV / h, and the second part of the desolvent was 60% methanol for 4BV at a flow rate of 0.8BV / h to obtain a high-content ginsenoside Rb1 component product and a high-content ginsenoside Rd component product, respectively;

[0075] The total content of the five saponins in the first part of the solid product obtained by analysis was 82.33%, of which the content of ginsenoside Rb1 was 71.27%, and the yield was 86%. The total content of the five saponins in the second part of the solid product obtained by analysis was 75%, of which the content of ginsenoside Rd was 63.66%, and the yield was 90%.

[0076] Step 7: Single component purification of ginsenoside Rb1

[0077] Weigh 2.0 g of the high-content ginsenoside Rb1 component product obtained in step 6, add 10 ml of water, heat to dissolve, and after dissolution, place in a 4°C refrigerator to cool, then add 80 ml of acetone solution dropwise at a slow rate. After the addition is complete, place in a 4°C refrigerator to store at low temperature for 2 hours, filter, and repeat crystallization twice. The final product has a ginsenoside Rb1 content of 98.66%;

[0078] Step 8: Single component purification of ginsenoside Rd

[0079] Weigh 1.5 g of the high-content ginsenoside Rd component product obtained in step 6, add 10 ml of 3% ethanol aqueous solution, stir to dissolve, and wait for loading onto the column dextran resin for chromatography experiments.

[0080] The resin was Biophdex LH-20 dextran resin with a height-to-diameter ratio of 30-40. The mobile phase was 35% ethanol, the column volume was 5 ml, and the flow rate was 0.5 BV / h. The final product had a ginsenoside Rd of 95.72%.

[0081] Example 2:

[0082] A method for extracting ginsenosides Rb1 and Rd from Panax notoginseng, comprising the following steps:

[0083] Step 1: Raw material extraction

[0084] Weigh 1 kg of Panax notoginseng powder and place it in a round-bottom flask. First, add 9 L of 75% ethanol solution at 70°C and reflux for 2 h. Filter and repeat the same operation. Add 7 L of ethanol for the second time and 5 L of ethanol for the third time. Extract three times and combine the filtrates.

[0085] Step 2: Ethanol recovery

[0086] The treated filtrate obtained in step 1 is subjected to reduced pressure distillation at a certain temperature to recover ethanol to obtain a dry product;

[0087] The total content of the five saponins in the obtained solid product was 31.35%, of which the content of ginsenoside Rb1 was 10.78% and the content of ginsenoside Rd was 3.86%.

[0088] Step 3: Prepare crude column liquid

[0089] Weigh 8 g of the dried product obtained in step 2, add 240 mL of purified water, stir for 1 h to dissolve the material, and filter to obtain the crude supernatant;

[0090] Step 4: Rough extraction

[0091] The liquid obtained in step 3 was loaded onto a non-polar macroporous adsorption resin DM30 at an adsorption flow rate of 2.5 BV / h. After adsorption, the mixture was washed with water for 3 BV at the same flow rate. The pre-solvent was 20% methanol for 2.5 BV at a flow rate of 1.0 BV / h, and the desolvent was 50% ethanol for 3 BV at a flow rate of 0.8 BV / h. The desolvent was dealcoholized and dried to obtain a crude product of Panax notoginseng total glycosides.

[0092] The total content of the five saponins in the obtained solid product was 71%, of which the content of ginsenoside Rb1 was 50.68%, the content of ginsenoside Rd was 15.55%, and the product yield was 97%;

[0093] Step 5: Reconstitution

[0094] Weigh 2.5 g of the crude notoginsenoside obtained in step 4, add 75 mL of deionized water, and dissolve completely under stirring to obtain a liquid, which is set aside for later use;

[0095] Step 6: Single component separation

[0096] The feed liquid in step 5 was passed through a medium-polarity macroporous adsorption resin LK17 at a flow rate of 1.5 BV / h, washed with water for 1 BV, and the flow rate remained unchanged. The pre-solvent was 10% methanol for 3 BV at a flow rate of 2.0 BV / h, the first part of the desolvent was 50% methanol for 2.0 BV at a flow rate of 0.5 BV / h, and the second part of the desolvent was 75% methanol for 3 BV at a flow rate of 0.8 BV / h to obtain a high-content ginsenoside Rb1 component product and a high-content ginsenoside Rd component product, respectively;

[0097] The total content of the five saponins in the first part of the solid product obtained by analysis was 83.67%, of which the content of ginsenoside Rb1 was 70.33%, and the yield was 88%. The total content of the five saponins in the second part of the solid product obtained by analysis was 73%, of which the content of ginsenoside Rd was 61.35%, and the yield was 88.86%.

[0098] Step 7: Single component purification of ginsenoside Rb1

[0099] Weigh 2.5 g of the high-content ginsenoside Rb1 component product obtained in step 6, add 10 ml of a 15% ethanol aqueous solution, heat to dissolve, and after dissolution, place in a 4°C refrigerator to cool, then add 100 ml of isobutanol solution dropwise at a slow rate. After the addition is complete, place in a 4°C refrigerator for 2 hours, filter, and repeat crystallization twice. The final product has a ginsenoside Rb1 content of 99.10%;

[0100] Step 8: Single component purification of ginsenoside Rd

[0101] Weigh 2.0 g of the high-content ginsenoside Rd component product obtained in step 6, add 10 ml of 10% ethanol aqueous solution, stir to dissolve, and wait for loading onto the column dextran resin for chromatography experiments.

[0102] The resin was Biophdex LH-20 dextran resin with a height-to-diameter ratio of 30-40. The mobile phase was 90% methanol, the column volume was 2 ml, and the flow rate was 1.0 BV / h. The final product had a ginsenoside Rd of 96.33%.

[0103] Example 3:

[0104] A method for extracting ginsenosides Rb1 and Rd from Panax notoginseng, comprising the following steps:

[0105] Step 1: Raw material extraction

[0106] Weigh 1 kg of Panax notoginseng powder and place it in a round-bottom flask. First, add 10 L of 75% ethanol solution at 70°C and reflux for 2 h. Filter and repeat the same operation. Add 6 L of ethanol for the second time and 2 L of ethanol for the third time. Extract three times and combine the filtrates.

[0107] Step 2: Ethanol recovery

[0108] The treated filtrate obtained in step 1 is subjected to reduced pressure distillation at a certain temperature to recover ethanol to obtain a dry product;

[0109] The total content of the five saponins in the obtained solid product was 30.59%, of which the content of ginsenoside Rb1 was 9.88% and the content of ginsenoside Rd was 4.01%.

[0110] Step 3: Prepare crude column liquid

[0111] Weigh 7 g of the dried product obtained in step 2, add 210 mL of purified water, stir for 1 h to dissolve the material, and filter to obtain the crude supernatant;

[0112] Step 4: Rough Extraction

[0113] The liquid obtained in step 3 was loaded onto a non-polar macroporous adsorption resin DM101 at an adsorption flow rate of 2 BV / h. After adsorption, the mixture was washed with water for 2 BV at the same flow rate. The pre-solvent was 15% isopropanol for 3 BV at a flow rate of 1.0 BV / h, and the desolvent was 70% ethanol for 3 BV at a flow rate of 0.8 BV / h. The desolvent was dealcoholized and dried to obtain a crude product of Panax notoginseng total glycosides.

[0114] The total content of the five saponins in the obtained analyzed solid product was 71%, among which the content of ginsenoside Rb1 was 51.33%, the content of ginsenoside Rd was 15.02%, and the product yield was 95%.

[0115] Step 5: Reconstitution

[0116] Weigh 3 g of the crude notoginsenoside obtained in step 4, add 90 mL of deionized water, and dissolve completely under stirring to obtain a liquid, which is set aside for later use;

[0117] Step 6: Single component separation

[0118] The feed liquid in step 5 was passed through a medium-polarity macroporous adsorption resin LK37 at a flow rate of 2BV / h, washed with water for 3BV, and the flow rate remained unchanged. The pre-solvent was 20% ethanol 1.5BV at a flow rate of 1.5BV / h, the first part of the desolvent was 40% ethanol 4.0BV at a flow rate of 1.2BV / h, and the second part of the desolvent was 80% methanol 2.5BV at a flow rate of 0.5BV / h to obtain a high-content ginsenoside Rb1 component product and a high-content ginsenoside Rd component product, respectively;

[0119] The total content of the five saponins in the first part of the solid product obtained by analysis was 78.88%, of which the content of ginsenoside Rb1 was 70.02%, and the yield was 91.36%. The total content of the five saponins in the second part of the solid product obtained by analysis was 73.55%, of which the content of ginsenoside Rd was 65.63%, and the yield was 86.79%;

[0120] Step 7: Single component purification of ginsenoside Rb1

[0121] Weigh 2.0 g of the high-content ginsenoside Rb1 component product obtained in step 6, add 10 ml of a 20% ethanol aqueous solution, heat to dissolve, and after dissolution, place in a 4°C refrigerator to cool, then add 90 ml of ethyl acetate solution dropwise at a slow rate. After the addition is complete, place in a 4°C refrigerator to store at low temperature for 2 hours, filter, and repeat crystallization three times. The final product has a ginsenoside Rb1 content of 99.01%;

[0122] Step 8: Single component purification of ginsenoside Rd

[0123] Weigh 1.5 g of the high-content ginsenoside Rd component product obtained in step 6, add 10 ml of 90% ethanol aqueous solution, stir to dissolve, and wait for loading onto the column dextran resin for chromatography experiments.

[0124] The resin was Biophdex LH-20 dextran resin with a height-to-diameter ratio of 30-40. The mobile phase was 90% ethanol, the column volume was 3 ml, and the flow rate was 1.0 BV / h. The final product had a ginsenoside Rd of 95.92%.

[0125] Example 4:

[0126] A method for extracting ginsenosides Rb1 and Rd from Panax notoginseng, comprising the following steps:

[0127] Step 1: Raw material extraction

[0128] Weigh 1 kg of Panax notoginseng powder and place it in a round-bottom flask. First, add 8 L of 75% ethanol solution at 70°C and reflux for 2 h. Filter and repeat the same operation. Add 8 L of ethanol for the second time and 4 L of ethanol for the third time. Extract three times and combine the filtrates.

[0129] Step 2: Ethanol recovery

[0130] The treated filtrate obtained in step 1 is subjected to reduced pressure distillation at a certain temperature to recover ethanol to obtain a dry product;

[0131] The total content of the five saponins in the obtained solid product was 32.33%, of which the content of ginsenoside Rb1 was 11.36% and the content of ginsenoside Rd was 4.59%.

[0132] Step 3: Prepare crude column liquid

[0133] Weigh 10 g of the dried product obtained in step 2, add 260 mL of purified water, stir for 1 h to dissolve the material, and filter to obtain the crude supernatant;

[0134] Step 4: Rough Extraction

[0135] The liquid obtained in step 3 was loaded onto a non-polar macroporous adsorption resin DM101 at an adsorption flow rate of 3 BV / h. After adsorption, the mixture was washed with water for 3 BV at the same flow rate. The pre-solvent was 25% ethanol for 3 BV at a flow rate of 1.0 BV / h, and the desolvent was 70% ethanol for 3 BV at a flow rate of 0.8 BV / h. The desolvent was dealcoholized and dried to obtain a crude product of Panax notoginseng total glycosides.

[0136] The total content of the five saponins in the obtained analyzed solid product was 69.27%, of which the content of ginsenoside Rb1 was 50.28%, the content of ginsenoside Rd was 16.21%, and the product yield was 89%.

[0137] Step 5: Reconstitution

[0138] Weigh 3.0 g of the crude notoginsenoside obtained in step 4, add 90 mL of deionized water, and dissolve completely under stirring to obtain a feed solution, which is set aside for later use;

[0139] Step 6: Single component separation

[0140] The feed liquid in step 5 was passed through a medium-polarity macroporous adsorption resin LK37 at a flow rate of 2.5 BV / h, washed with water at 3.5 BV, and the flow rate remained unchanged. The pre-solvent was 20% ethanol 1.5 BV at a flow rate of 1.5 BV / h, the first part of the desorbent was 45% ethanol 4.0 BV at a flow rate of 1.0 BV / h, and the second part of the desorbent was 60% methanol 3.0 BV at a flow rate of 0.5 BV / h to obtain a high-content ginsenoside Rb1 component product and a high-content ginsenoside Rd component product, respectively;

[0141] The total amount of the five saponins in the first part of the solid product obtained by analysis was 81.36%, among which the content of ginsenoside Rb1 was 72.67%, and the yield was 88.39%. The total amount of the five saponins in the second part of the solid product obtained by analysis was 75.66%, among which the content of ginsenoside Rd was 68.36%, and the yield was 90.85%.

[0142] Step 7: Single component purification of ginsenoside Rb1

[0143] Weigh 4.0 g of the high-content ginsenoside Rb1 component product obtained in step 6, add 10 ml of water, heat to dissolve, and after dissolution, place in a 4°C refrigerator to cool, then add 120 ml of acetone solution dropwise at a slow rate. After the addition is complete, place in a 4°C refrigerator to store at low temperature for 2 hours, filter, and repeat crystallization three times. The final product has a ginsenoside Rb1 content of 99.53%;

[0144] Step 8: Single component purification of ginsenoside Rd

[0145] Weigh 3.5 g of the high-content ginsenoside Rd component product obtained in step 6, add 10 ml of 90% ethanol aqueous solution, stir to dissolve, and wait for loading onto the column dextran resin for chromatography experiments.

[0146] The resin was Biophdex LH-20 dextran resin with a height-to-diameter ratio of 30-40. The mobile phase was 75% ethanol, the column volume was 5 ml, and the flow rate was 1.0 BV / h. The final product had a ginsenoside Rd of 90.37%.

[0147] The above description is merely a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for extracting ginsenosides Rb1 and Rd from Panax notoginseng, characterized in that: The steps include: Step 1: Raw material extraction Adding the powder of Panax notoginseng medicinal material to the ethanol solution and performing hot reflux extraction, filtering, extracting, and combining the filtrate; Step 2: Ethanol recovery The treated filtrate obtained in step 1 is subjected to reduced pressure distillation to recover ethanol to obtain a dry product; Step 3: Prepare crude column liquid Add purified water to the dried product obtained in step 2, stir, and filter to obtain a crude supernatant; Step 4: Rough extraction The liquid obtained in step 3 is measured and passed through a non-polar macroporous adsorption resin. After adsorption is completed, the mixture is washed with water, pre-lysed, and lysed. The lysate is dealcoholized and dried to obtain a crude product of Panax notoginseng total glycosides. Step 5: Reconstitution Add the crude notoginsenoside obtained in step 4 into deionized water and dissolve it completely under stirring to obtain a liquid, which is then set aside for later use; Step 6: Single component separation The liquid obtained in step 5 is passed through a medium-polarity macroporous adsorption resin, and then washed, pre-lyzed, and gradient-lyzed to obtain a high-content ginsenoside Rb1 component product and a high-content ginsenoside Rd component product, respectively; Step 7: Single component purification of ginsenoside Rb1 Recrystallize the high-content ginsenoside Rb1 component product obtained in step 6 with an organic solvent to obtain the final product; Step 8: Single component purification of ginsenoside Rd The high-content ginsenoside Rd component product obtained in step 6 is loaded onto a dextran resin for chromatography experiments.

2. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 1, characterized in that: In step 1, the temperature of the hot reflux extraction is 60-80°C, and the reflux time is 1-2h; The material-liquid ratio of Panax notoginseng medicinal material powder to ethanol solution is 1:3-10.

3. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 1, characterized in that: In step 2, the mass ratio of ethanol to water in the ethanol solution is 1-5:1; The total content of five saponins in the obtained product is 25-35%, wherein the content of ginsenoside Rb1 is 6-11%, and the content of ginsenoside Rd is 2-4%.

4. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 1, characterized in that: In step 3, the stirring time is 1-3 hours, the mass ratio of the obtained dry product to deionized water is 10-20:100, and the total glycoside concentration of the prepared liquid is 2-4%.

5. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 1, characterized in that: In step 4, the amount of resin loaded is based on 60-80 g / L of total glycosides, the water wash volume is 2-3 BV of the resin volume, the pre-solution agent is a 10-30% alcohol solution, the pre-solution volume is 1-3 BV, the desolvant is a 50-80% alcohol solution, and the desolvation volume is 2-5 BV; The total content of five saponins in the obtained solid product is 65-75%, wherein the content of ginsenoside Rb1 is 50-60%, and the content of ginsenoside Rd is 10-20%. The product yield is 95-98%.

6. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 1, characterized in that: In the step 5, the mass ratio of the crude notoginsenoside obtained in the step 4 to deionized water is 10-20:100; and the total glycoside concentration of the prepared solution in the step 5 is 15-20%.

7. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 1, characterized in that: In the step 6, the resin column amount is based on 60-80 g / L of total glycosides, the water wash volume is 2-3 BV of the resin volume, the pre-solvent is a 10-30% alcohol solution, the pre-solvent volume is 1-3 BV, the first part of the resolving agent is a 40-60% alcohol solution, the resolving volume is 2-5 BV, and the second part of the resolving agent is a 60-80% alcohol solution, and the resolving volume is 2-5 BV; The total content of the five saponins in the first part of the solid product obtained by analysis is 80-85%, among which the content of ginsenoside Rb1 is 70-80%, and the yield is 85-90%. The total content of the five saponins in the second part of the solid product obtained by analysis is 70-80%, the content of ginsenoside Rd is 60-70%, and the yield is 85-90%.

8. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 7, characterized in that: The resin is a methyl acrylate medium-polar resin.

9. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 1, characterized in that: In step 7, the solvent used for recrystallization is a water and acetone system, or an ethanol aqueous solution and an ethyl acetate, isobutanol solution; the content of ginsenoside Rb1 is above 98%.

10. The method for extracting ginsenosides Rb1 and Rd from Panax notoginseng according to claim 1, characterized in that: In step 8, the resin height-to-diameter ratio is 30-40, the resin mobile phase is 5-95% ethanol, the column load is 10 g / L, the flow rate is 0.2-2 BV / h; and the ginsenoside Rd is 95%.

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