A method for extracting and purifying polysaccharide of green brick tea
By combining petroleum ether and anhydrous ethanol extraction with ethanol precipitation and polyamide column purification, the problems of low purity and poor bioactivity of polysaccharides extracted from green brick tea were solved, and high-purity and high-bioactivity polysaccharides were prepared.
Patent Information
- Application Number
- CN202310900057.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-21
- Publication Date
- 2026-02-03
- Estimated Expiration
- 2043-07-21
AI Technical Summary
Existing methods for extracting polysaccharides from green brick tea result in low purity and poor biological activity. Traditional methods are cumbersome and require a lot of chemical reagents, which may affect the activity of polysaccharides.
Extraction with petroleum ether and anhydrous ethanol was combined with ethanol precipitation and polyamide column purification. The crude extract of green brick tea polysaccharides was then treated with activated polyamide powder and a balanced column to reduce the use of chemical reagents and improve the purity and bioactivity of the polysaccharides.
This study achieved high-purity extraction of polysaccharides from green brick tea and simplified the process, reducing costs, decreasing the use of chemical reagents, and improving the bioactivity of polysaccharides.
Abstract
Description
Technical Field
[0001] This invention relates to the field of functional component extraction technology of green brick tea, and in particular to a method for extracting, purifying and preparing polysaccharides from green brick tea. Background Technology
[0002] Green brick tea possesses various biological activities, including lowering lipids and aiding weight loss, lowering blood sugar, and lowering blood pressure. Among these, polysaccharides are one of its effective components, exhibiting antioxidant activity and in vitro blood sugar-lowering effects. Traditional methods for extracting polysaccharides include acid-base extraction, enzyme-assisted extraction, ultrasound-assisted extraction, microwave-assisted extraction, and supercritical fluid extraction. However, acid-base extraction is cumbersome, enzyme-assisted extraction is affected by the activity of the enzymes used, ultrasound-assisted extraction may degrade soluble polysaccharides and alter their biological activity, and microwave-assisted extraction yields complex extracts and presents difficulties in subsequent separation and purification. Summary of the Invention
[0003] To address the shortcomings of existing methods for extracting polysaccharides from green brick tea, this invention provides a method for extracting, purifying, and preparing polysaccharides from green brick tea, which solves the problems of low purity and poor biological activity in existing methods. This is achieved through the following techniques.
[0004] A method for extracting, purifying, and preparing polysaccharides from green brick tea, comprising the following steps:
[0005] Take green brick tea powder and extract it sequentially with petroleum ether and anhydrous ethanol / ethanol solution to obtain green brick tea crude powder; first extract the green brick tea crude powder with water, then precipitate it with ethanol, centrifuge the precipitate and redissolve it with water to obtain green brick tea polysaccharide crude extract.
[0006] Polyamide powder was activated, and a polyamide equilibrium column was prepared. The crude extract of green brick tea polysaccharide was passed through the polyamide equilibrium column, equilibrated, eluted, and the eluent was collected, concentrated, dialyzed with water, and dried to obtain the finished product of green brick tea polysaccharide.
[0007] Preferably, the ratio of green brick tea powder to petroleum ether is 1:(5-30), the extraction temperature is 60-90℃, and the extraction time is 1-6h.
[0008] Preferably, the ratio of the green brick tea powder to the ethanol solution is 1:(5-30), the concentration of the ethanol solution is 40-99%, and the extraction time is 1-6 hours.
[0009] Preferably, when the coarse green brick tea powder is extracted with water, the ratio of coarse green brick tea powder to pure water is 1:(4-64), the extraction temperature is 40-100℃, and the extraction time does not exceed 12 hours. It should be noted that the extraction time can be either immediately after soaking in hot water followed by the next step of alcohol precipitation, or extraction can be performed after a certain period of time. The longer the extraction time, the more polysaccharides will be extracted, but to save costs, it generally does not exceed 12 hours.
[0010] Preferably, when using ethanol for precipitation, the concentration of the ethanol solution is 55-95%, and the precipitation time does not exceed 24 hours. It should be noted that a large amount of precipitate will appear immediately upon the addition of ethanol during precipitation. Therefore, the precipitation time can be either to start centrifugation immediately after the addition of ethanol or to let it stand for a period of time before centrifugation. The longer the precipitation time, the more polysaccharide precipitate will be obtained. However, in order to save costs, it generally does not exceed 24 hours.
[0011] Preferably, the method for activating polyamide powder is as follows: soaking in 3-4 BV column volumes of 95% ethanol solution overnight, filtering and washing with water until neutral; soaking in 2-3 BV column volumes of 5% NaOH solution for 4-5 hours, filtering and washing with water until neutral; soaking in 2-3 BV column volumes of 10% hydrochloric acid for 4-5 hours, filtering and washing with water until neutral.
[0012] Preferably, the method for preparing the polyamide equilibrium column is as follows: Soak degreased cotton in water and place it at the bottom of the elution column. Place washed quartz sand on top of the cotton. Suspend the activated polyamide powder in water, add it to the elution column, allow it to settle naturally, and then equilibrate using pure water.
[0013] More preferably, the flow rate of pure water used for equilibrium is 0-9 mL / min.
[0014] Preferably, the concentration of the crude polysaccharide extract from green brick tea during polyamide column equilibration is 5-20 mg / mL, and the flow rate for equilibration and elution with pure water after polyamide column equilibration is 0-9 mL / min.
[0015] Compared with the prior art, the advantages of the present invention are: the extraction and purification preparation method of green brick tea polysaccharides is simple and reduces costs; the water extraction and alcohol precipitation method uses fewer chemical reagents and has lower toxicity; and the refined green brick tea polysaccharides prepared by this method have higher purity and better biological activity. Detailed Implementation
[0016] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0017] The extraction and purification method for polysaccharides from green brick tea used in the following examples requires pre-activation and equilibration of the polyamide powder before passing the crude green brick tea polysaccharide solution through a polyamide equilibrium column. The specific method is as follows:
[0018] (1) Activation of polyamide powder: Soak in 95% ethanol overnight with 3 BV (3-4 BV) column volume, filter to remove ethanol, and wash with water until no alcohol odor is present; add 2 BV (2-3 BV is acceptable) of 5% NaOH solution and soak for 4h (4-5h is acceptable), filter and wash with water until neutral; add 2 BV (2-3 BV is acceptable) of 10% hydrochloric acid and soak for 4h (4-5h is acceptable), filter and wash with water until neutral.
[0019] (2) Equilibration of the polyamide column: Take an appropriate amount of degreased cotton, soak it, and place it at the bottom of the elution column (thickness 1-2 cm is acceptable). Take an appropriate amount of quartz sand, wash it with water, and place it on top of the degreased cotton (thickness 1-2 cm is acceptable). Add the activated polyamide powder into the elution column and let it settle naturally. After it has completely settled, connect the peristaltic pump and equilibrate with pure water at a flow rate of 6 mL / min to 2 BV.
[0020] Example 1
[0021] The extraction, purification, and preparation method of polysaccharides from green brick tea used in this embodiment is as follows:
[0022] (1) Pretreatment of green brick tea powder: Take green brick tea, crush it and sieve it through a 40-mesh sieve. Dry the tea powder to constant weight. Add petroleum ether at a material-liquid ratio of 1:5. Heat and reflux at 50°C for 6 hours. Filter the residue and dry it to constant weight.
[0023] After drying, the tea powder is added to anhydrous ethanol at a ratio of 1:5, heated and refluxed at 80°C for 1 hour, the tea residue is filtered out, and dried to constant weight to obtain coarse tea powder for green brick tea.
[0024] (2) Extraction of crude polysaccharides from green brick tea: Green brick tea powder was added to pure water at a ratio of 1:4, heated and cooled under reflux for 12 hours at 40°C, the supernatant was collected by centrifugation, concentrated by rotary evaporation, and an appropriate amount of ethanol was added to make the final concentration 95%. The ethanol was precipitated at 4°C for 24 hours, the precipitate was collected by centrifugation, redissolved and concentrated by rotary evaporation, and then freeze-dried to obtain crude polysaccharides from green brick tea.
[0025] (3) Prepare a 10 mg / mL solution of crude polysaccharide from green brick tea, add it to a polyamide equilibrium column, and balance it with 2 BV of pure water at a flow rate of 1.5 mL / min. Then elute with 2 BV of pure water. After adding the crude polysaccharide, collect the eluent, concentrate the eluent by rotary evaporation, dialyze it with pure water for 48 h after concentration, and freeze-dry it to obtain the refined polysaccharide from green brick tea. The purity was determined to be 64.8% by the phenol-sulfuric acid method.
[0026] Example 2
[0027] The extraction, purification, and preparation method of polysaccharides from green brick tea used in this embodiment is as follows:
[0028] (1) Pretreatment of green brick tea powder: Take green brick tea, crush it and sieve it through a 60-mesh sieve. Dry the tea powder to constant weight. Add petroleum ether at a material-liquid ratio of 1:10. Heat and reflux at 60°C for 3 hours. Filter the residue and dry it to constant weight.
[0029] After drying, the tea powder is added to an 80% ethanol solution at a ratio of 1:10, heated and refluxed at 80°C for 2 hours, filtered to remove tea residue, and dried to constant weight to obtain coarse tea powder for green brick tea.
[0030] (2) Extraction of crude polysaccharides from green brick tea: Green brick tea powder was added to pure water at a ratio of 1:16, heated and cooled under reflux for 4 hours at 60°C, the supernatant was collected by centrifugation, concentrated by rotary evaporation, and an appropriate amount of ethanol was added to make the final concentration 75%. The ethanol was precipitated at 4°C for 12 hours, the precipitate was collected by centrifugation, reconstituted, concentrated by rotary evaporation, and freeze-dried to obtain crude polysaccharides from green brick tea.
[0031] (3) Prepare a 10 mg / mL solution of crude polysaccharide from green brick tea, add it to a polyamide equilibrium column, and equilibrate it with 2 BV of pure water at a flow rate of 4.5 mL / min. Then elute with 2 BV of pure water. After adding the crude polysaccharide, collect the eluent, concentrate the eluent by rotary evaporation, dialyze it with pure water for 48 h after concentration, and freeze-dry it to obtain the refined polysaccharide from green brick tea. The purity was calculated to be 69.3%.
[0032] Example 3
[0033] The extraction, purification, and preparation method of polysaccharides from green brick tea used in this embodiment is as follows:
[0034] (1) Pretreatment of green brick tea powder: Take green brick tea, crush it and sieve it through a 60-mesh sieve. Dry the tea powder to constant weight. Add petroleum ether at a material-liquid ratio of 1:15. Heat and reflux at 70°C for 3 hours. Filter the residue and dry it to constant weight.
[0035] After drying, the tea powder is added to an 80% ethanol solution at a ratio of 1:15. The mixture is heated and refluxed at 80°C for 2 hours. The tea residue is then filtered and dried to constant weight to obtain coarse tea powder for green brick tea.
[0036] (2) Extraction of crude polysaccharides from green brick tea: Green brick tea powder was added to pure water at a ratio of 1:32, heated and cooled under reflux for 8 hours at 60°C, the supernatant was collected by centrifugation, concentrated by rotary evaporation, and an appropriate amount of ethanol was added to make the final concentration 75%. The ethanol was precipitated at 4°C for 12 hours, the precipitate was collected by centrifugation, redissolved and concentrated by rotary evaporation, and then freeze-dried to obtain crude polysaccharides from green brick tea.
[0037] (3) Prepare a 10 mg / mL solution of crude polysaccharide from green brick tea, add it to a polyamide equilibrium column, and equilibrate it with 2 BV of pure water at a flow rate of 4.5 mL / min. Then elute with 2 BV of pure water. After adding the crude polysaccharide, collect the eluent, concentrate the eluent by rotary evaporation, dialyze it with pure water for 48 h after concentration, and freeze-dry it to obtain the refined polysaccharide from green brick tea. The purity was calculated to be 73.1%.
[0038] Example 4
[0039] The extraction, purification, and preparation method of polysaccharides from green brick tea used in this embodiment is as follows:
[0040] (1) Pretreatment of green brick tea powder: Take green brick tea, crush it and sieve it through an 80-mesh sieve. Dry the tea powder to constant weight. Add petroleum ether at a material-liquid ratio of 1:20. Heat and reflux at 80°C for 3 hours. Filter the residue and dry it to constant weight.
[0041] After drying, the tea powder is added to a 60% ethanol solution at a ratio of 1:20, heated and refluxed at 80°C for 4 hours, the tea residue is filtered out, and dried to constant weight to obtain coarse tea powder for green brick tea.
[0042] (2) Extraction of crude polysaccharides from green brick tea: Green brick tea powder was added to pure water at a ratio of 1:32, heated and cooled under reflux for 4 hours at 80°C, the supernatant was collected by centrifugation, concentrated by rotary evaporation, and an appropriate amount of ethanol was added to make the final concentration 55%. The ethanol was precipitated at 4°C for 6 hours, the precipitate was collected by centrifugation, redissolved and concentrated by rotary evaporation, and then freeze-dried to obtain crude polysaccharides from green brick tea.
[0043] (3) Take the crude polysaccharide of green brick tea and prepare a solution of 15 mg / mL. Add it to a polyamide equilibrium column and balance it with 2 BV of pure water at a flow rate of 6 mL / min. Then elute with 2 BV of pure water. After adding the crude polysaccharide, collect the eluent and concentrate it by rotary evaporation. After concentration, dialyze with pure water for 48 h and freeze dry to obtain the refined polysaccharide of green brick tea. The purity is calculated to be 71.4%.
[0044] Example 5
[0045] The extraction, purification, and preparation method of polysaccharides from green brick tea used in this embodiment is as follows:
[0046] (1) Pretreatment of green brick tea powder: Take green brick tea, crush it and sieve it through a 100-mesh sieve. Dry the tea powder to constant weight, add petroleum ether at a material-liquid ratio of 1:30, heat and reflux at 90℃ for 1 hour, filter out the sediment, and dry to constant weight.
[0047] After drying, the tea powder is added to a 60% ethanol solution at a ratio of 1:30, heated and refluxed at 80°C for 6 hours, the tea residue is filtered out, and dried to constant weight to obtain coarse tea powder for green brick tea.
[0048] (2) Extraction of crude polysaccharides from green brick tea: Green brick tea powder was added to pure water at a ratio of 1:64, heated and cooled under reflux for 2 hours at 100℃, the supernatant was collected by centrifugation, concentrated by rotary evaporation, and an appropriate amount of ethanol was added to make the final concentration 55%. The ethanol was precipitated at 4℃ for 3 hours, the precipitate was collected by centrifugation, redissolved and concentrated by rotary evaporation, and then freeze-dried to obtain crude polysaccharides from green brick tea.
[0049] (3) Prepare a solution of crude polysaccharide from green brick tea at a concentration of 20 mg / mL, add it to a polyamide equilibrium column, and equilibrate it with 2 BV of pure water at a flow rate of 9 mL / min. Then elute with 2 BV of pure water. After adding the crude polysaccharide, collect the eluent, concentrate the eluent by rotary evaporation, dialyze it with pure water for 48 h after concentration, and freeze-dry it to obtain the refined polysaccharide from green brick tea. The purity was calculated to be 70.8%.
[0050] Comparative Example
[0051] The extraction, purification, and preparation method of polysaccharides from green brick tea used in this embodiment is as follows:
[0052] (1) Take an appropriate amount of tea powder, dry and grind it, pass it through a 60-mesh standard sieve, add 85% ethanol and soak for 24 hours, dry it and store it in the dark for later use.
[0053] (2) Accurately weigh 10g of tea sample, add an appropriate amount of distilled water at a material-to-liquid ratio of 1:30g / mL, bathe in a 75℃ water bath for 2h, filter the tea soup and evaporate and concentrate it to 75mL; add Sevage reagent (chloroform and n-butanol volume ratio 5:1) to remove protein.
[0054] (3) Adjust the pH of the tea soup to pH=7.0 with ammonia water, add 20% hydrogen peroxide to decolorize until the tea soup fades, and keep warm for 2 hours;
[0055] (4) Continue to concentrate to 1 / 2 of the original tea soup volume, then add anhydrous ethanol to a final concentration of 80% to precipitate the tea polysaccharides.
[0056] (5) The sample solution was placed in a refrigerator at 4°C and allowed to stand overnight. The precipitate was centrifuged, redissolved, dialyzed, concentrated by distillation, and freeze-dried to obtain refined tea polysaccharide. The purity of the polysaccharide was calculated to be 58.6%.
[0057] Compared with the control example, the method provided by the present invention uses fewer chemical reagents, has lower toxicity, is simpler, and increases the polysaccharide content while ensuring yield.
[0058] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. A method for extracting, purifying, and preparing polysaccharides from green brick tea, characterized in that, Includes the following steps: Take green brick tea powder and extract it sequentially with petroleum ether and anhydrous ethanol / ethanol solution to obtain green brick tea crude powder; first extract the green brick tea crude powder with water, then precipitate it with ethanol, centrifuge the precipitate and redissolve it with water to obtain green brick tea polysaccharide crude extract. Polyamide powder was activated, and a polyamide equilibrium column was prepared. The crude extract of green brick tea polysaccharide was passed through the polyamide equilibrium column, equilibrated with 2 BV of pure water, eluted with 2 BV of pure water, the eluent was collected, concentrated, dialyzed with water, and dried to obtain the finished product of green brick tea polysaccharide.
2. The method for extracting, purifying, and preparing polysaccharides from green brick tea according to claim 1, characterized in that, The ratio of green brick tea powder to petroleum ether is 1:(5-30), the extraction temperature is 60-90℃, and the extraction time is 1-6h.
3. The method for extracting, purifying, and preparing polysaccharides from green brick tea according to claim 1, characterized in that, The ratio of green brick tea powder to ethanol solution is 1:(5-30), the concentration of ethanol solution is 40-99%, and the extraction time is 1-6 hours.
4. The method for extracting, purifying, and preparing polysaccharides from green brick tea according to claim 1, characterized in that, When the coarse powder of green brick tea is extracted with water, the ratio of coarse powder of green brick tea to pure water is 1:(4-64), the extraction temperature is 40-100℃, and the extraction time does not exceed 12 hours.
5. The method for extracting, purifying, and preparing polysaccharides from green brick tea according to claim 1, characterized in that, When using ethanol precipitation, the concentration of the ethanol solution is 55-95%, and the precipitation time does not exceed 24 hours.
6. The method for extracting, purifying, and preparing polysaccharides from green brick tea according to claim 1, characterized in that, The method for activating polyamide powder is as follows: soak it overnight in 3-4 BV column volume of 95% ethanol solution, filter it, and wash it with water until it is neutral. Add 2-3 BV column volumes of 5% NaOH solution and soak for 4-5 hours, then filter and wash with water until neutral. Add 2-3 BV column volumes of 10% hydrochloric acid and soak for 4-5 hours. Filter and wash with water until neutral.
7. The method for extracting, purifying, and preparing polysaccharides from green brick tea according to claim 1, characterized in that, The method for preparing a polyamide equilibrium column is as follows: Soak degreased cotton in water and place it at the bottom of the elution column. Take washed quartz sand and place it on the degreased cotton. Suspend the activated polyamide powder in water and add it into the elution column. After natural sedimentation, use pure water for equilibrium.
8. The method for extracting, purifying, and preparing polysaccharides from green brick tea according to claim 7, characterized in that, The flow rate for using pure water for equilibrium is 0-9 mL / min.
9. The method for extracting, purifying, and preparing polysaccharides from green brick tea according to claim 1, characterized in that, The concentration of crude polysaccharide extract from green brick tea during polyamide equilibration was 5-20 mg / mL, and the flow rate of pure water equilibration and elution was 0-9 mL / min.