A method for simultaneously purifying gastrodin, bartzol and gastrodins from gastrodia elata blume

Abstract

The application discloses a method for simultaneously purifying gastrodin, bartzol and gastrodia elata polysaccharide from gastrodia elata, which comprises the following steps: crushing, extracting, neutral or weak polar macroporous resin adsorbing, polar macroporous resin adsorbing, gradient elution, membrane separation and the like, so as to obtain gastrodia elata extracts rich in gastrodin, bartzol and gastrodia elata polysaccharide respectively. The method uses gastrodia elata as raw material, has simple extraction and separation and purification methods, uses green and non-polluted solvents, has low cost, and through the adsorption of different polar resins, gradient elution and membrane separation and the like, the gastrodia elata extracts rich in gastrodin, bartzol and gastrodia elata polysaccharide are obtained respectively, the effect of squeezing dry and taking all is achieved, the utilization rate is high, the utilization value of gastrodia elata is improved, and the method has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of natural ingredient separation and purification technology, specifically to a method for simultaneously purifying gastrodin, barisonin, and gastrodin polysaccharide from Gastrodia elata. Background Technology

[0002] Gastrodia elata, the tuberous part of the orchid Gastrodia elata, is used in traditional Chinese medicine. Its functions include calming wind and stopping spasms, suppressing liver yang, and dispelling wind and unblocking meridians. Modern pharmacological studies have shown that Gastrodia elata not only possesses pharmacological effects related to its traditional efficacy, such as anti-epileptic, anticonvulsant, and sleep improvement, but also exhibits effects in improving memory, anti-anxiety, and anti-fatigue. Gastrodin, barisonin, and Gastrodia elata polysaccharides are the three main active substances in Gastrodia elata. Gastrodin and its aglycone p-hydroxybenzyl alcohol have sedative and hypnotic effects, regulate nerves, relieve neuropathic pain, dilate cerebral blood vessels and improve cerebral circulation, enhance memory, have antioxidant effects, and have immunomodulatory effects. Barisonin has neuroprotective, cognitive function improving, antidepressant and anti-aging, antioxidant, anti-inflammatory, and antitumor effects. Gastrodia elata polysaccharides have antioxidant aging, antitumor, immunomodulatory, memory-improving, cerebral ischemia-improving, blood pressure-lowering, antibacterial, and lipid-lowering effects.

[0003] Gastrodia elata is a traditional and precious Chinese medicinal herb. It can be used as a food-medicine homologous substance for the development and application of related products. This invention simultaneously separates and purifies three main active substances from Gastrodia elata: gastrodin, barisonin, and gastrodia polysaccharide. These substances can be applied to different products according to specific needs, resulting in high utilization of Gastrodia elata, enhancing its value, and showing broad application prospects.

[0004] CN202410654032.7 discloses a method for extracting gastrodin and its application. The method involves extracting gastrodin by reflux with 70% ethanol, adsorbing it with macroporous resin, eluting it, collecting the 70% ethanol eluent, and concentrating it under reduced pressure to obtain gastrodin.

[0005] CN202410741052.8 discloses a method for extracting gastrodin, the resulting gastrodin composition, and its uses. The method involves pulverizing and enzymatically hydrolyzing gastrodin, followed by extraction, chromatographic column purification, and molecularly imprinted polymer microsphere refining to obtain pure gastrodin and prepare a gastrodin composition.

[0006] CN201410150002.9 discloses a method for preparing balisinoside reference standard, which involves extracting Gastrodia elata powder by ethanol reflux, concentrating the extract under reduced pressure and defatting it, then adsorbing and eluting it with macroporous resin using gradient elution, separating the eluent with silica gel column chromatography and purifying it by preparative liquid chromatography to obtain balisinoside reference standard.

[0007] CN202510333032.1 discloses a method for preparing and applying Gastrodia elata polysaccharide to improve memory. The method involves ultrasonically extracting Gastrodia elata powder to obtain crude Gastrodia elata polysaccharide, removing proteins to obtain protein-free crude Gastrodia elata polysaccharide, and then performing DEAE-52 cellulose column chromatography and Sephadex G-100 gel column chromatography to obtain purified Gastrodia elata polysaccharide.

[0008] CN202411802383.4 discloses a purified polysaccharide from Gastrodia elata, its preparation method and application. Gastrodia elata powder is extracted by ethanol reflux, the extract is concentrated and then precipitated with alcohol to obtain crude polysaccharide from Gastrodia elata, which is then deproteinized, dialyzed, and finally purified by column chromatography to obtain purified polysaccharide from Gastrodia elata.

[0009] Gastrodin, barisonin, and gastrodin polysaccharide are all important active ingredients in gastrodia elata, each possessing strong pharmacological effects. The above-mentioned techniques were used to prepare gastrodin, barisonin, or gastrodin polysaccharide separately, thus introducing the risk of loss of other active ingredients. Summary of the Invention

[0010] The purpose of this invention is to provide a method for simultaneously purifying gastrodin, basilin, and gastrodin polysaccharide from Gastrodia elata. The method involves adsorbing gastrodin and basilin onto resins of different polarities, followed by gradient elution to obtain purified gastrodin and basilin. The gastrodin polysaccharide is then subjected to multi-stage membrane filtration through membranes of different pore sizes to obtain homogeneous gastrodin polysaccharide. This invention simultaneously separates and purifies the three main active substances in Gastrodia elata—gastrodin, basilin, and gastrodin polysaccharide—allowing them to be applied to different products as needed. This method offers high utilization of Gastrodia elata, enhances its utilization value, and has broad application prospects.

[0011] A method for simultaneously purifying gastrodin, barisonin, and gastrodin polysaccharide from Gastrodia elata includes the following steps:

[0012] Step 1: Extraction of Gastrodia elata: Take Gastrodia elata, crush and sieve it, soak it in pure water, extract it at 70-100℃, let the extract stand and cool, and filter to clarify.

[0013] Step 2, Bartholin's glycosides resin adsorption: The filtered and clarified Gastrodia elata extract is adsorbed onto macroporous resin, and then rinsed with purified water after adsorption is complete.

[0014] Step 3, Barisonin Resin Analysis: After adsorption of barisonin, the macroporous resin was eluted with 2-5 BV of 10%-20% ethanol at a rate of 2-6 BV / h, followed by elution with 4-8 BV of 40%-70% ethanol at a rate of 2-6 BV / h. The 40%-70% ethanol eluent was collected, concentrated, and then freeze-dried to obtain the Gastrodia elata barisonin extract.

[0015] Step 4: Gastrodin Resin Adsorption: The effluent from the barisonin resin adsorption and the washing solution obtained in Step 2 are combined and then subjected to adsorption of gastrodin and p-hydroxybenzyl alcohol on a polar macroporous resin at a rate of 2-4 BV / h. After adsorption is completed, 4-10 BV of purified water is added for rinsing at a rate of 2-4 BV / h.

[0016] Step 5: Gastrodin Resin Analysis: After the gastrodin is adsorbed, the macroporous resin is eluted with 2-5 BV of 10%-30% ethanol at a rate of 2-6 BV / h, and then eluted again with 4-8 BV of 50%-70% ethanol at a rate of 2-6 BV / h. The 50%-70% ethanol eluent is collected, concentrated, and then freeze-dried to obtain the gastrodin extract.

[0017] Step 6: Purification of Gastrodia elata polysaccharide: The effluent from the gastrodin resin adsorption and the washing liquid obtained in Step 4 are combined and filtered using an 80-100 kDa ultrafiltration membrane to remove macromolecular substances. The filtrate is then filtered again using a 10-20 kDa ultrafiltration membrane to remove small molecular substances. The solution is then concentrated and freeze-dried to obtain the Gastrodia elata polysaccharide extract.

[0018] Preferably, in step one, the sieve used to sift the pulverized Gastrodia elata is 30 to 60 mesh.

[0019] Preferably, in step one, the sample is soaked in 5 to 10 times its volume of purified water for 4 to 8 hours, extracted at 70 to 100°C for 1 to 3 hours, extracted 2 to 3 times, and the extract is left to stand for at least 8 hours.

[0020] Preferably, in step two, the macroporous resin used is a non-polar or weakly polar macroporous resin.

[0021] Preferably, in step two, the resin loading rate is 2-4 BV / h, the amount of purified water used for rinsing is 4-10 BV, and the rinsing rate is 2-4 BV / h.

[0022] Preferably, in step three, the content of barisonin in the extract is ≥80%.

[0023] Preferably, the macroporous resin used in step four is a polar macroporous resin.

[0024] Preferably, in step five, the total content of gastrodin and p-hydroxybenzyl alcohol in the gastrodin extract is ≥80%.

[0025] Preferably, in step six, the polysaccharide content of the Gastrodia elata polysaccharide extract is ≥50%.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) Comprehensive utilization to enhance utilization value: Gastrodia elata is a traditional and precious Chinese medicinal material. By simultaneously separating and purifying the three active substances in Gastrodia elata, namely gastrodin, barisonin and gastrodia elata polysaccharide, the active substances in Gastrodia elata are comprehensively utilized, thereby enhancing the utilization value of Gastrodia elata.

[0028] (2) Selective adsorption: Gastrodin and basilin were separated by adsorption through macroporous resins of different polarities.

[0029] (3) Homogeneous polysaccharide: Gastrodia elata polysaccharide is filtered through membranes with different pore sizes in multiple stages to remove macromolecules and small molecules, thereby obtaining homogeneous polysaccharide. Detailed Implementation

[0030] The present invention will now be described in detail with reference to specific implementation examples, so that researchers in the field can refer to this specification to implement it.

[0031] Example 1

[0032] This embodiment provides a method for simultaneously purifying gastrodin, barisonin, and gastrodin polysaccharide from Gastrodia elata, including the following steps:

[0033] Gastrodia elata extraction: Take Gastrodia elata, crush it through a 30-mesh sieve, weigh 1000g of the sieved Gastrodia elata powder, add 5000ml of purified water and soak for 4 hours, extract at 100℃ for 1 hour, extract 3 times, let the extract stand for 12 hours, and filter and clarify it through a Buchner funnel.

[0034] Bartholin resin adsorption: The filtered and clarified Gastrodia elata extract was adsorbed onto 1000 ml of AB-8 macroporous resin at a rate of 2 BV / h. After adsorption, 10 BV of purified water was added for rinsing at a rate of 4 BV / h.

[0035] Barisoniside Resin Analysis: The macroporous resin after barisoniside adsorption was eluted with 2 BV of 20% ethanol at a rate of 6 BV / h, followed by elution with 4 BV of 70% ethanol at a rate of 6 BV / h. The 70% ethanol eluent was collected, concentrated, and freeze-dried to obtain 17.4 g of Gastrodia elata extract I (HPLC analysis showed that the contents of barisoniside A, barisoniside B, barisoniside C, and barisoniside E were 26.7%, 21.3%, 10.4%, and 24.4%, respectively, totaling 82.8%).

[0036] Gastrodin resin adsorption: The effluent from the barisonin resin adsorption and the washing solution were combined, and then 2000 ml of XPD-700 macroporous resin was added at a rate of 2 BV / h to adsorb gastrodin and p-hydroxybenzyl alcohol. After adsorption was completed, 10 BV of purified water was added and rinsed at a rate of 4 BV / h.

[0037] Step 5: Gastrodin Resin Analysis: The macroporous resin after gastrodin adsorption was eluted with 2 BV of 30% ethanol at a rate of 6 BV / h, followed by 4 BV of 70% ethanol at a rate of 6 BV / h. The 70% ethanol eluent was collected, concentrated, and freeze-dried to obtain 6.5 g of Gastrodia elata extract II (HPLC analysis showed that the contents of gastrodin and p-hydroxybenzyl alcohol were 75.8% and 8.2%, respectively, totaling 84.0%).

[0038] Step 6: Purification of Gastrodia elata polysaccharides: The effluent from the gastrodin resin adsorption and the washing liquid were combined and filtered using a 100kDa ultrafiltration membrane to remove macromolecular substances. The filtrate was then filtered again using a 10kDa ultrafiltration membrane to remove small molecules. The filtrate was then concentrated and freeze-dried to obtain 63.1g of Gastrodia elata extract III (polysaccharide content was 53.7% as determined by the sulfuric acid phenol method).

[0039] Example 2

[0040] This embodiment provides a method for simultaneously purifying gastrodin, barisonin, and gastrodin polysaccharide from Gastrodia elata, including the following steps:

[0041] Gastrodia elata extraction: Take Gastrodia elata, crush it through a 40-mesh sieve, weigh 1000g of the sieved Gastrodia elata powder, add 7500ml of purified water and soak for 6 hours, extract at 85℃ for 2 hours, extract 3 times, let the extract stand for 10 hours, and filter and clarify through a Buchner funnel.

[0042] Bartholin resin adsorption: The filtered and clarified Gastrodia elata extract was adsorbed onto 1000 ml of HPD-100 macroporous resin at a rate of 3 BV / h. After adsorption, 7 BV of purified water was added for rinsing at a rate of 3 BV / h.

[0043] Barisoniside Resin Analysis: The macroporous resin after barisoniside adsorption was eluted with 4 BV of 15% ethanol at a rate of 4 BV / h, followed by elution with 6 BV of 50% ethanol at a rate of 4 BV / h. The 50% ethanol eluent was collected, concentrated, and freeze-dried to obtain 17.1 g of Gastrodia elata extract I (HPLC analysis showed that the contents of barisoniside A, barisoniside B, barisoniside C, and barisoniside E were 27.7%, 21.7%, 11.4%, and 24.1%, respectively, totaling 84.9%).

[0044] Gastrodin resin adsorption: The effluent from the barisonin resin adsorption and the washing solution were combined, and then 2000 ml of XDA-8G macroporous resin was added at a rate of 3 BV / h to adsorb gastrodin and p-hydroxybenzyl alcohol. After adsorption was completed, 7 BV of purified water was added and rinsed at a rate of 3 BV / h.

[0045] Step 5: Gastrodin Resin Analysis: The macroporous resin after gastrodin adsorption was eluted with 4 BV of 20% ethanol at a rate of 4 BV / h, followed by elution with 6 BV of 60% ethanol at a rate of 4 BV / h. The 60% ethanol eluent was collected, concentrated, and freeze-dried to obtain 6.3 g of Gastrodia elata extract II (HPLC analysis showed that the contents of gastrodin and p-hydroxybenzyl alcohol were 78.3% and 8.5%, respectively, totaling 86.8%).

[0046] Step 6: Purification of Gastrodia elata polysaccharides: The effluent from the gastrodin resin adsorption and the washing liquid were combined and filtered using a 90kDa ultrafiltration membrane to remove macromolecular substances. The filtrate was then filtered again using a 15kDa ultrafiltration membrane to remove small molecular substances. The filtrate was then concentrated and freeze-dried to obtain 60.7g of Gastrodia elata extract III (polysaccharide content was 52.4% as determined by the sulfuric acid phenol method).

[0047] Example 3

[0048] This embodiment provides a method for simultaneously purifying gastrodin, barisonin, and gastrodin polysaccharide from Gastrodia elata, including the following steps:

[0049] Gastrodia elata extraction: Take Gastrodia elata, crush it through a 60-mesh sieve, weigh 1000g of the sieved Gastrodia elata powder, add 10000ml of purified water and soak for 8 hours, extract at 70℃ for 3 hours, extract twice, let the extract stand for 8 hours, and filter and clarify through a Buchner funnel.

[0050] Bartholin resin adsorption: The filtered and clarified Gastrodia elata extract was adsorbed onto 1000ml of X-5 macroporous resin at a rate of 4BV / h. After adsorption, 4BV of purified water was added for rinsing at a rate of 2BV / h.

[0051] Barisonin Resin Analysis: The macroporous resin after barisonin adsorption was eluted with 5 BV of 10% ethanol at a rate of 2 BV / h, followed by elution with 8 BV of 40% ethanol at a rate of 2 BV / h. The 40% ethanol eluent was collected, concentrated, and freeze-dried to obtain 16.3 g of Gastrodia elata extract I (HPLC analysis showed that the contents of barisonin A, barisonin B, barisonin C, and barisonin E were 28.6%, 22.3%, 12.5%, and 26.2%, respectively, totaling 89.6%).

[0052] Gastrodin resin adsorption: The effluent from the barisonin resin adsorption and the washing solution were combined, and then 2000 ml of LSD-001 macroporous resin was added at a rate of 4 BV / h to adsorb gastrodin and p-hydroxybenzyl alcohol. After adsorption was completed, 4 BV of purified water was added and rinsed at a rate of 2 BV / h.

[0053] Step 5: Gastrodin Resin Analysis: The macroporous resin after gastrodin adsorption was eluted with 5 BV of 10% ethanol at a rate of 2 BV / h, followed by elution with 8 BV of 50% ethanol at a rate of 2 BV / h. The 50% ethanol eluent was collected, concentrated, and freeze-dried to obtain 5.9 g of Gastrodia elata extract II (HPLC analysis showed that the contents of gastrodin and p-hydroxybenzyl alcohol were 80.5% and 9.7%, respectively, totaling 90.2%).

[0054] Step 6: Purification of Gastrodia elata polysaccharides: The effluent from the gastrodin resin adsorption and the washing liquid were combined and filtered using an 80kDa ultrafiltration membrane to remove macromolecular substances. The filtrate was then filtered again using a 20kDa ultrafiltration membrane to remove small molecular substances. The filtrate was then concentrated and freeze-dried to obtain 52.2g of Gastrodia elata extract III (polysaccharide content was 54.7% as determined by the sulfuric acid phenol method).

[0055] The above embodiments are merely illustrative examples of preferred embodiments of the present invention and do not encompass all aspects of the invention. Various modifications and refinements made by those skilled in the art without departing from the spirit and scope of the present invention are considered to fall within the protection scope of the claims of the present invention.

Claims

1. A method for simultaneously purifying gastrodin, barisonin, and gastrodin polysaccharide from Gastrodia elata, characterized in that, Includes the following steps: Step 1: Extraction of Gastrodia elata: Take Gastrodia elata, crush and sieve it, soak it in pure water, extract it at 70-100℃, let the extract stand and cool, and filter to clarify. Step 2, Bartholin's glycosides resin adsorption: The filtered and clarified Gastrodia elata extract is adsorbed onto macroporous resin, and then rinsed with purified water after adsorption is complete. Step 3, Barisonin Resin Analysis: After adsorption of barisonin, the macroporous resin was eluted with 2-5 BV of 10%-20% ethanol at a rate of 2-6 BV / h, followed by elution with 4-8 BV of 40%-70% ethanol at a rate of 2-6 BV / h. The 40%-70% ethanol eluent was collected, concentrated, and then freeze-dried to obtain the Gastrodia elata barisonin extract. Step 4, Gastrodin Resin Adsorption: The effluent from the barisonin resin adsorption and the washing solution obtained in Step 2 are combined and then the adsorption of gastrodin and p-hydroxybenzyl alcohol is carried out on a polar macroporous resin at a rate of 2-4 BV / h. After the adsorption is completed, 4-10 BV of pure water is added for rinsing at a rate of 2-4 BV / h. Step 5: Gastrodin Resin Analysis: After the gastrodin is adsorbed, the macroporous resin is eluted with 2-5 BV of 10%-30% ethanol at a rate of 2-6 BV / h, and then eluted again with 4-8 BV of 50%-70% ethanol at a rate of 2-6 BV / h. The 50%-70% ethanol eluent is collected, concentrated, and then freeze-dried to obtain the gastrodin extract. Step 6: Purification of Gastrodia elata polysaccharide: The effluent from the gastrodin resin adsorption and the washing liquid obtained in Step 4 are combined and filtered using an 80-100 kDa ultrafiltration membrane to remove macromolecular substances. The filtrate is then filtered again using a 10-20 kDa ultrafiltration membrane to remove small molecular substances. The solution is then concentrated and freeze-dried to obtain the Gastrodia elata polysaccharide extract.

2. The method according to claim 1, characterized in that, In step one, the sieve used to sift the pulverized Gastrodia elata is 30 to 60 mesh.

3. The method according to claim 1, characterized in that, In step one, add 5 to 10 times the volume of purified water and soak for 4 to 8 hours. Extract at 70 to 100°C for 1 to 3 hours, repeat 2 to 3 times, and let the extract cool for more than 8 hours.

4. The method according to claim 1, characterized in that, In step two, the macroporous resin used is a non-polar or weakly polar macroporous resin.

5. The method according to claim 1, characterized in that, In step two, the resin loading rate is 2-4 BV / h, and the amount of purified water used for rinsing is 4-10 BV at a rate of 2-4 BV / h.

6. The method according to claim 1, characterized in that, In step three, the content of barisonin in the extract is ≥80%.

7. The method according to claim 1, characterized in that, In step four, the macroporous resin used is a polar macroporous resin.

8. The method according to claim 1, characterized in that, In step five, the total content of gastrodin and p-hydroxybenzyl alcohol in the gastrodin extract is ≥80%.

9. The method according to claim 1, characterized in that, In step six, the polysaccharide content of the Gastrodia elata polysaccharide extract is ≥50%.