Method for preparing polygonatum polysaccharide gel tea through ionizing radiation
Polygonatum polysaccharide gel tea was prepared through ionizing radiation technology, and the hydrogel slow-release layer was used to stably release Polygonatum oligosaccharide, which solved the problem of short effect time of Polygonatum tea, and achieved the long-term refreshing and fatigue relief effect of Polygonatum tea. Environmentally friendly process and low cost.
Patent Information
- Application Number
- CN202411407952.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2024-12-17
AI Technical Summary
The existing Polygonatum tea has a short time to refresh the mind and relieve fatigue, and the Polygonatum polysaccharide cannot be continuously released, which cannot meet the long-term health care effect needs.
Polygonatum polysaccharide gel tea is prepared by ionizing radiation technology. The hydrogel is used as a sustained release layer to stably and continuously release Polygonatum oligosaccharide, prolonging the effective time of refreshing and relieving fatigue.
It achieves the continuous rich taste of Polygonatum tea and the continuous release of Polygonatum polysaccharide, extends the time to refresh and relieve fatigue, and has environmentally friendly technology, low cost and high production efficiency.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of health food and functional beverage, and in particular relates to a method for preparing polygonatum polysaccharide gel tea by ionizing radiation, and in particular, a new method for preparing polygonatum polysaccharide gel sustained-release tea with high biological activity by using ionizing radiation as the main preparation method, which is environmentally friendly and efficient. Background Art
[0002] Polygonatum sibiricum is the dried rhizome of Polygonatum sibiricum, Polygonatum multiflorum or Polygonatum yunnanensis, a perennial herbaceous plant of the genus Polygonatum in the family Liliaceae. China is rich in Polygonatum sibiricum resources. It is recorded in the medical classics "Shenxian Zhicao Jing" and "Compendium of Materia Medica" that "Polygonatum sibiricum can relieve the stomach and replenish qi, regulate the five internal organs, make muscles full, strengthen bones, prevent aging for many years, turn white into black, and regenerate teeth after they fall out." Modern research shows that Polygonatum sibiricum mainly contains polysaccharides, steroidal saponins, anthraquinones, alkaloids and amino acids and other functional ingredients, which have antioxidant, refreshing, fatigue relief, anti-inflammatory, lipid-lowering and arteriosclerosis prevention, blood sugar lowering, memory enhancement and improvement, anti-tumor, immune regulation and anti-virus effects. Polysaccharide is one of the main active ingredients of Polygonatum sibiricum. Modern medical research shows that polysaccharides have many health benefits such as inhibiting osteoporosis, enhancing memory, improving immunity, anti-virus, anti-aging and blood sugar lowering.
[0003] Polygonatum sibiricum is a traditional Chinese medicinal material that is both a medicine and food. Its rhizome is fleshy and contains a large amount of polysaccharides and other nutrients. Eating it raw and stewing it can not only satisfy hunger, but also has the health-care effect of warming and nourishing, which is very beneficial to the body. People in mountainous areas often eat Polygonatum sibiricum as a vegetable, and use seedlings to stir-fry or make soup. In addition to traditional consumption, there are some research reports on deep-processed products of Polygonatum sibiricum, such as Polygonatum sibiricum oral liquid, Polygonatum sibiricum orange juice compound beverage, Polygonatum sibiricum enzyme, Polygonatum sibiricum tonic wine, Polygonatum sibiricum porridge, Polygonatum sibiricum cake, Polygonatum sibiricum paste, Polygonatum sibiricum fermented products, etc. Polygonatum sibiricum tea is a solid beverage tea that can be dissolved in water, with the advantages of good solubility, easy storage, and convenient use. In order to solve the problem of tea portability while ensuring the taste of tea, Polygonatum sibiricum polysaccharide gel sustained-release tea can keep the taste of tea strong over time within a certain period of time, and can make the effective ingredient Polygonatum sibiricum polysaccharide in Polygonatum sibiricum continuously released. First, it can prolong the effect time of Polygonatum sibiricum tea in refreshing and relieving fatigue, and secondly, it can ensure that the concentration fluctuation of Polygonatum sibiricum in the body is relatively stable. At the same time, due to the processing of raw materials and irradiation preparation technology, there are no aromatics, pigments and other ingredients. Under the premise of ensuring the taste of tea, it is also healthier and more environmentally friendly. The efficacy of Polygonatum sibiricum can adapt to the requirements of life for food convenience, safety, nutrition and health care, meet the needs of contemporary consumption, and have extremely high development prospects. Developing instant tea with medicinal and edible medicinal materials not only meets people's pursuit of health, but also changes the traditional way of eating Chinese medicinal materials. It is a new way to develop the application of Chinese medicinal materials.
[0004] Polygonatum sibiricum polysaccharide has a large molecular weight, large volume, and poor water solubility, which is not conducive to the absorption of organisms. In recent years, many studies have found that the molecular weight of Polygonatum sibiricum polysaccharide is closely related to biological activity. In comparison, low molecular weight Polygonatum sibiricum polysaccharide, namely Polygonatum sibiricum oligosaccharide, has higher biological activity and relatively enhanced pharmacological effects. Polygonatum sibiricum polysaccharide with a large molecular weight can change its molecular weight through degradation to improve its biological activity. To this end, this technology uses Polygonatum sibiricum as raw material and ionizing radiation technology to produce Polygonatum sibiricum polysaccharide gel tea with high biological activity and sustainable sustained release, providing a technical basis for the deep processing of Polygonatum sibiricum.
[0005] Chinese patent CN107432355A discloses a kind of instant polygonatum tea and its preparation method. The preparation method of instant polygonatum tea is simple, fresh lotus leaves are used each time when steaming, which can better eliminate the bitter taste and throat irritation of polygonatum tea, enhance the lotus leaf aroma of polygonatum tea and the fat decomposition, constipation elimination and diuresis effect of polygonatum tea; adding honey to fresh polygonatum before steaming can better eliminate the bitter taste and throat irritation of polygonatum tea and improve the poor taste of polygonatum. However, the main effect of polygonatum tea is to refresh and relieve fatigue, which requires the continuous release of polygonatum polysaccharide to achieve the effect of maintaining refreshing, but instant tea cannot achieve this effect.
[0006] Chinese patent CN110800837A discloses a processing technology for a ready-to-drink polygonatum tea beverage, which is made from fresh polygonatum and tea leaves as main raw materials, so that the beverage has health care functions, and the polygonatum is compounded with tea leaves, astragalus, Chinese yam, and Poria cocos, so that the numbing effect of the polygonatum itself is completely eliminated; fresh polygonatum is directly used as the raw material, and the "steaming and drying" process is simplified, so that the production process is simple and fast, and the production efficiency is greatly improved; a colloid homogenizing device is used to homogenize the polygonatum concentrated juice, so that the polygonatum concentrated juice is more fully ground and has a good homogenizing effect; a circulating pump extracts the polygonatum concentrated juice containing solid particles and returns it to the mixing box through a reflux pipe for further grinding and homogenization, so that the solid particles in the polygonatum concentrated juice are extremely small, thereby improving the quality of the polygonatum concentrated juice; a filter plate is installed inside the filter tube, so that the space occupied by the equipment is reduced and the production cost is reduced. However, ready-to-drink Polygonatum sibiricum tea beverages have the same problem as instant Polygonatum sibiricum tea, that is, the Polygonatum sibiricum polysaccharides in Polygonatum sibiricum tea cannot be released continuously, resulting in only temporary effects of refreshing and relieving fatigue, and the effect of sustained release cannot be achieved.
[0007] In the field of health food and functional beverage technology of polygonatum tea, the health drink of polygonatum instant tea series is more common at present. As described in the above patent, although polygonatum instant tea can achieve the effect of refreshing and relieving fatigue, the effect time is short and the problem of short duration of tea taste is also prominent. In order to overcome the above problems of polygonatum instant tea, polygonatum polysaccharide gel sustained-release tea came into being. First, hydrogel is a three-dimensional network or interpenetrating network between liquid and solid, which is a hydrophilic polymer gel that can significantly swell in water but cannot dissolve in water. As a tea bag, it starts to swell in water, and the tea leaves in the gel tea bag can contact with water, and the polygonatum polysaccharide is released stably and continuously, so that the effect of refreshing and relieving fatigue can be sustained. Secondly, in the ionizing radiation preparation process, the reaction process does not need to add any substance toxic to the human body, is pollution-free, and the reaction conditions are mild. It can be carried out at room temperature, and the industrial production efficiency is high, the cost is extremely low, and the production controllability is good.
[0008] The problem to be solved by the present invention is to disclose a method that can make the tea taste good and continue to be strong over time, and can continuously release the effective component of polygonatum sibiricum polysaccharide in polygonatum sibiricum, so as to prolong the effect time of polygonatum sibiricum tea, such as refreshing and relieving fatigue. At the same time, a method for preparing polygonatum sibiricum polysaccharide gel sustained-release tea with high biological activity by ionizing radiation is also provided, which is highly efficient, environmentally friendly and pollution-free, and has extremely low production cost, so as to overcome the above-mentioned defects of the prior art.
[0009] After searching, there is no patent application in China that is identical to the present invention. Summary of the invention
[0010] The purpose of the present invention is to provide a method for preparing polygonatum polysaccharide gel tea by ionizing radiation in view of various problems existing in the existing polygonatum tea technology such as short duration of refreshing and fatigue relieving effects. The technical problem to be solved by the present invention is how to prepare polygonatum polysaccharide gel sustained-release tea by ionizing radiation method.
[0011] The purpose of the present invention can be achieved through the following technical solutions: a method for preparing polygonatum polysaccharide gel tea by ionizing radiation, characterized in that the polygonatum polysaccharide gel sustained-release tea uses hydrogel as a sustained-release layer, contacts with water, and the hydrogel swells, and the tea leaves in the hydrogel layer can contact with water, stably and continuously releasing polygonatum oligosaccharides, thereby achieving the purpose of sustained effects of refreshing and relieving fatigue. The product of the invention has simple process, good stability, and can be released for a long time. The preparation method of the invention has short operation time and low cost. A sustainable sustained-release polygonatum polysaccharide gel tea and its ionizing radiation preparation method, comprising the following steps:
[0012] ① Weigh an appropriate amount of sliced and freeze-dried polygonatum, crush and sieve to obtain polygonatum powder.
[0013] ② The polygonatum powder is placed under a dose rate of 20 kGy / pass and a radiation absorption dose of 0-320 kGy for ionizing radiation extraction. The irradiated polygonatum powder is added with water with a solid-liquid ratio of 1:10-1:30, and reflux extraction is performed 1-5 times. The extraction time for each time is 1-3 hours. The extraction temperature is 70-110°C, and the mixture is filtered to obtain the crude polygonatum polysaccharide.
[0014] ③ Separate and purify the crude polygonatum polysaccharide to obtain polygonatum polysaccharide.
[0015] ④ The obtained polygonatum sibiricum polysaccharide is subjected to ionizing radiation degradation, and after radiation, it is passed through an ultrafiltration system, rotary evaporation, alcohol precipitation, centrifugation, and freeze-drying to obtain polygonatum sibiricum oligosaccharide.
[0016] ⑤ Weigh a certain amount of green tea, crush it, sieve it, and get green tea powder. Add water and reflux and extract it twice, each time for 2 hours, filter it, and collect the filtrate.
[0017] ⑥ The green tea filtrate is clarified using a ceramic membrane.
[0018] ⑦ The filtrate after green tea clarification was concentrated under reduced pressure at 60°C.
[0019] ⑧ The concentrated green tea extract is placed in a vacuum freezer for drying to obtain green tea powder for later use.
[0020] ⑨ Add water to polyvinyl alcohol (PVA) and place in a water bath and stir. Add sodium carboxymethyl starch (CMS) to water and stir. After the polyvinyl alcohol is completely dissolved, add the completely dissolved sodium carboxymethyl starch solution and stir until uniform.
[0021] ⑩ Pour the evenly stirred hydrogel into the mold, add dried green tea and polygonatum oligosaccharide, and quickly cycle freeze-drying to shape.
[0022] The products are packed into boxes and stored in warehouses to obtain finished products.
[0023] Furthermore, the raw material of Polygonatum sibiricum is crushed and sieved using 30 mesh before irradiation to increase the surface area of Polygonatum sibiricum, thereby increasing its radiation contact surface and the contact area with water during extraction, which is beneficial to the dissolution of the effective ingredient Polygonatum sibiricum polysaccharide; at the same time, it also avoids excessive crushing that is not conducive to subsequent solid-liquid separation.
[0024] Furthermore, the separation and purification method uses a DEAE-52 chromatography column for separation and purification.
[0025] Further, the filtrate was concentrated under reduced pressure at 60°C, and after concentration, 4 times the volume of anhydrous ethanol solution was slowly added, and refrigerated at 4°C overnight.
[0026] Furthermore, the impregnated polygonatum polysaccharide is packaged in PE bags, sealed, and subjected to electron beam radiation degradation at 80 to 320 kGy.
[0027] Furthermore, the average molecular weight of the polygonatum oligosaccharide is about 200kDa.
[0028] Furthermore, the product was concentrated to 1 / 5 volume by rotary evaporation under reduced pressure at 50°C through an ultrafiltration system with a molecular cutoff of 200 kDa, and 4 times the volume of anhydrous ethanol was added to precipitate the polysaccharide. The product was placed in a refrigerator at 4°C overnight, and the precipitated flocs were centrifuged at 4000 r / min for 15 min. The precipitate was collected and vacuum freeze-dried to obtain Polygonatum sibiricum oligosaccharide.
[0029] Furthermore, the green tea filtrate is clarified using a 0.1-0.5 μm ceramic membrane.
[0030] Further, polyvinyl alcohol (PVA) is added with water (polyvinyl alcohol: water = 1:8-1:10) and placed in a water bath for heating and stirring. Sodium carboxymethyl starch (CMS) is added with water (sodium carboxymethyl starch: water = 1:15-1:18) and stirred. After the polyvinyl alcohol is completely dissolved, the completely dissolved sodium carboxymethyl starch solution is added thereto, cooled and stirred until uniform.
[0031] Furthermore, the evenly stirred hydrogel is poured into a mold, and dried green tea and polygonatum oligosaccharide (in a ratio of 9:1 to 10:1) are added, and freeze-drying is performed in a rapid cycle to shape the hydrogel.
[0032] Compared with the prior art, this method has the following advantages:
[0033] 1. Polygonatum sibiricum polysaccharide gel sustained-release tea can make the tea taste excellent and continuously rich over time, and can continuously release the effective ingredient Polygonatum sibiricum polysaccharide in Polygonatum sibiricum, thereby prolonging the effect of Polygonatum sibiricum tea in refreshing and relieving fatigue.
[0034] 2. The entire extraction process does not require the addition of any substances that are toxic to the human body, and is safe and environmentally friendly.
[0035] 3. The extraction process has mild reaction conditions and can be carried out at room temperature.
[0036] 4. The production process of Polygonatum sibiricum polysaccharide gel sustained-release tea saves costs.
[0037] 5. Low molecular weight Polygonatum oligosaccharide is more effective in refreshing and relieving fatigue. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 Response surface analysis of PSCP extraction
[0039] Figure 2Figure (a) is the elution curve of Polygonatum cyrtonema polysaccharide; Figure 2 Figure (b) is the extraction rate curve; Figure 2 Figure (c) is a comparison of the properties of Polygonatum sibiricum polysaccharide in different states.
[0040] Figure 3 It is the effect of radiation absorption dose on the molecular weight of PSO prepared by PSP degradation.
[0041] Figure 4 It is the effect of PSO on the activity of E.coli.
[0042] Figure 5 (a) shows the effect of PSO on CAL-27 cell death detected by fluorescence staining; Figure 5 Figure (b) is the statistical analysis of cell viability detected by fluorescence staining; Figure 5 (c) is the flow cytometry analysis of the effect of PSO on CAL-27 cell death; Figure 5 Panel (d) is the statistical analysis of flow cytometry.
[0043] Figure 6 (a) shows the effect of PSP, PSO-1, PSO-2 and PSO-3 on H2O2-induced CAL-27 cell death detected by fluorescence staining; Figure 6 (b) is a statistical analysis of the percentage of live cells labeled by fluorescent staining assay; Figure 6 (c) is a statistical analysis of the percentage of dead cells marked by fluorescent staining.
[0044] Figure 7 (a) in the figure is the evaluation of ROS levels of CAL-27 cells treated with different methods using DCFH-DA staining; Figure 7 Figure (b) is a statistical analysis of the relative fluorescence intensity of ROS levels detected by DCFH-DA staining. DETAILED DESCRIPTION
[0045] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.
[0046] Example 1: Weigh 1000g of Polygonatum sibiricum, wash and drain, cut into slices, freeze-dry for 1-2 days, crush, pass through a 30-mesh sieve, and obtain Polygonatum sibiricum powder. Take 600g of the crushed and sieved Polygonatum sibiricum powder, pack it in PE bags and seal it, and extract it with electron beam radiation. The irradiation dose is 0kGy and the dose rate is 20kGy / pass. The irradiated Polygonatum sibiricum powder is added with water with a solid-liquid ratio of 1:10 and refluxed for extraction once, each time for 1h, and filtered. The filtrate is concentrated under reduced pressure at 60°C, and after concentration, 4 times the volume of anhydrous ethanol solution is slowly added, and refrigerated at 4°C overnight. Centrifuge, discard the supernatant, wash the precipitate with 90% ethanol 3 times, freeze-dry, and obtain Polygonatum sibiricum crude polysaccharide. The obtained Polygonatum sibiricum crude polysaccharide is separated and purified using a DEAE-52 chromatography column, and then concentrated, precipitated with alcohol, centrifuged, freeze-dried, and dried to obtain Polygonatum sibiricum polysaccharide. Take an appropriate amount of Polygonatum sibiricum polysaccharide, pack it in a bag and seal it, and then use a 1MeV electron accelerator to degrade it by radiation, select a dose rate of 20kGy / pass, and a total radiation absorption dose of 80kGy. After radiation, pass through an ultrafiltration system with a molecular cutoff of 200kDa, and concentrate it to 1 / 5 volume by reduced pressure rotary evaporation at 50°C. Add 4 times the volume of anhydrous ethanol to precipitate the polysaccharide, place it in a 4°C refrigerator and stand overnight. The precipitated flocculent precipitates at 4000r / min and centrifuges for 15min. After collecting the precipitate, vacuum freeze-dry it to obtain Polygonatum sibiricum oligosaccharide. Weigh 100g of green tea, crush it, sieve it, and get green tea powder. Add 15 times water to the green tea powder and reflux extract it twice, each time for 2h, filter it, and collect the filtrate. The green tea filtrate is clarified using a 0.1μm ceramic membrane and concentrated under reduced pressure at 60°C. The concentrated green tea extract is placed in a vacuum freezer for drying to obtain green tea powder for standby use. Add 16g water to 2g of polyvinyl alcohol (PVA) and place in a water bath for heating and stirring. Add 1.2g of sodium carboxymethyl starch (CMS) to 18g of water and stir. After the polyvinyl alcohol is completely dissolved, add the completely dissolved sodium carboxymethyl starch solution, cool and stir until uniform. Pour the uniformly stirred hydrogel into a mold, add dried green tea and polygonatum oligosaccharides (ratio of 9:1), and freeze-dry and shape. Pack into boxes and store in warehouses to obtain the finished product.
[0047] Example 2: Weigh 1000g of Polygonatum sibiricum, wash and drain, cut into thin slices, freeze-dry for 1-2 days, crush, pass through a 30-mesh sieve, and obtain Polygonatum sibiricum powder. Take 600g of the crushed and sieved Polygonatum sibiricum powder, pack it in PE bags and seal it, and extract it by electron beam radiation. The irradiation dose is 240kGy and the dose rate is 20kGy / pass. The irradiated Polygonatum sibiricum powder is added with water with a solid-liquid ratio of 1:20 and refluxed for 2 times, each time for 1.5h, and filtered. The filtrate is concentrated under reduced pressure at 60°C, and after concentration, 4 times the volume of anhydrous ethanol solution is slowly added, and refrigerated at 4°C overnight. Centrifuge, discard the supernatant, wash the precipitate with 90% ethanol 3 times, freeze-dry, and obtain Polygonatum sibiricum crude polysaccharide. The obtained Polygonatum sibiricum crude polysaccharide is separated and purified using a DEAE-52 chromatography column, and then concentrated, alcohol precipitated, centrifuged, freeze-dried, and dried to obtain Polygonatum sibiricum polysaccharide. Take an appropriate amount of Polygonatum sibiricum polysaccharide, pack it in a bag and seal it, and then use a 1MeV electron accelerator to degrade it by radiation, select a dose rate of 20kGy / pass, and a total radiation absorption dose of 80kGy. After radiation, pass through an ultrafiltration system with a molecular cutoff of 200kDa, and concentrate it to 1 / 5 volume by rotary evaporation under reduced pressure at 50°C. Add 4 times the volume of anhydrous ethanol to precipitate the polysaccharide, place it in a refrigerator at 4°C and let it stand overnight. The precipitated flocculent precipitates at 4000r / min and centrifuges for 15min. After collecting the precipitate, vacuum freeze-dry it to obtain Polygonatum sibiricum oligosaccharide. Weigh 100g of green tea, crush it, sieve it, and get green tea powder. Add 15 times water to the green tea powder and reflux extract it twice, each time for 2h, filter it, and collect the filtrate. The green tea filtrate is clarified using a 0.5μm ceramic membrane. The clarified filtrate of green tea is concentrated under reduced pressure at 60°C. The concentrated green tea extract is placed in a vacuum freezer for drying to obtain green tea powder for standby use. Add 2g of polyvinyl alcohol (PVA) to 18g of water and place in a water bath for heating and stirring. Add 1.2g of sodium carboxymethyl starch (CMS) to 19.8g of water and stir. After the polyvinyl alcohol is completely dissolved, add the completely dissolved sodium carboxymethyl starch solution, cool and stir until uniform. Pour the uniformly stirred hydrogel into a mold, add dried green tea and polygonatum oligosaccharides (ratio of 9.7:1), and freeze-dry and shape. Pack into boxes and store in warehouses to obtain the finished product.
[0048] Example 3: Weigh 1000g of Polygonatum sibiricum, wash and drain, cut into thin slices, cut the fibrous root sample into pieces, freeze-dry for 1-2 days, crush, and pass through a 30-mesh sieve to obtain Polygonatum sibiricum powder. Take 600g of the crushed and sieved Polygonatum sibiricum powder, pack it in PE bags and seal it, and extract it with electron beam radiation. The irradiation dose is 320kGy and the dose rate is 20kGy / pass. The irradiated Polygonatum sibiricum powder is added with water with a solid-liquid ratio of 1:30 and refluxed for 5 times, each time for 3h, and filtered. The filtrate is concentrated under reduced pressure at 60°C, and after concentration, 4 times the volume of anhydrous ethanol solution is slowly added, and refrigerated at 4°C overnight. Centrifuge, discard the supernatant, wash the precipitate with 90% ethanol 3 times, and freeze-dry to obtain Polygonatum sibiricum crude polysaccharide. The obtained Polygonatum sibiricum crude polysaccharide is separated and purified using a DEAE-52 chromatography column, and then concentrated, precipitated with alcohol, centrifuged, freeze-dried, and dried to obtain Polygonatum sibiricum polysaccharide. Take an appropriate amount of Polygonatum sibiricum polysaccharide, pack it in a bag and seal it, and then use a 1MeV electron accelerator to degrade it by radiation, select a dose rate of 20kGy / pass, and a total radiation absorption dose of 80kGy. After radiation, pass through an ultrafiltration system with a molecular cutoff of 200kDa, and concentrate it to 1 / 5 volume by rotary evaporation under reduced pressure at 50°C. Add 4 times the volume of anhydrous ethanol to precipitate the polysaccharide, place it in a refrigerator at 4°C and let it stand overnight. The precipitated flocculent precipitates at 4000r / min and centrifuges for 15min. After collecting the precipitate, vacuum freeze-dry it to obtain Polygonatum sibiricum oligosaccharide. Weigh 100g of green tea, crush it, sieve it, and get green tea powder. Add 15 times water to the green tea powder and reflux extract it twice, each time for 2h, filter it, and collect the filtrate. The green tea filtrate is clarified using a 0.5μm ceramic membrane. The clarified filtrate of green tea is concentrated under reduced pressure at 60°C. The concentrated green tea extract is placed in a vacuum freezer for drying to obtain green tea powder for standby use. Add 20g water to 2g of polyvinyl alcohol (PVA) and place in a water bath for heating and stirring. Add 1.2g of sodium carboxymethyl starch (CMS) to 21.6g of water and stir. After the polyvinyl alcohol is completely dissolved, add the completely dissolved sodium carboxymethyl starch solution, cool and stir until uniform. Pour the uniformly stirred hydrogel into a mold, add dried green tea and polygonatum oligosaccharides (ratio of 10:1), and freeze-dry and shape. Pack into boxes and store in warehouses to obtain the finished product.
[0049] Combined with the attached drawings for analysis and explanation:
[0050] Figure 1This is the response surface analysis of PSCP extraction. (a) Response surface experiment to optimize PSCP extraction process; (b) Regression model variance analysis; (c) Error analysis statistics of fitting equation; (d) Optimal extraction process conditions. Note: *: p<0.05, **: p<0.01. (*: represents statistical difference; **: represents significant statistical difference). It can be found from the figure that three single factors that have a greater impact on radiation extraction of PSCP, namely extraction times, extraction temperature and radiation absorption dose, are studied by response surface experiment. Through response surface curve analysis, it is determined that the optimal process conditions for radiation extraction of PSCP are extraction times 2 times, extraction temperature 93℃, radiation absorption dose 240kGy, and the theoretical optimal PSCP yield is 32.40%. The extraction rate is higher than that of traditional processes.
[0051] Figure 2 (a) Elution curve of Polygonatum sibiricum polysaccharide; (b) Extraction rate; (c) Comparison of properties of Polygonatum sibiricum polysaccharide in different states. From the figure, it can be found that through separation and purification, the 240kGy sample with the highest crude polysaccharide extraction rate also has the highest PSP extraction rate.
[0052] Figure 3 This is the effect of radiation absorption dose on the molecular weight of PSO prepared by PSP degradation. (a) Determination of the molecular weight of PSO by GPC; (b) Quantitative analysis of molecular weight and peak area. It can be found from the figure that with the increase of radiation absorption dose, the relative molecular mass is getting smaller and smaller. The molecular weights are 674kDa (0kGy), 409kDa (80kGy), 322kDa (160kGy), 201kDa (240kGy), and 100kDa (320kGy), respectively. However, as the molecular weight gradually decreases, the peak area accounts for an increasingly large proportion, indicating that the molecular weight is also becoming more and more concentrated.
[0053] Figure 4 The effect of PSO on the activity of E.coli. (a) SEM image of E.coli in the control group; (b) SEM image of E.coli after adding PSO for 60 minutes; (c) UV analysis of the activity of E.coli. It can be seen from the figure that as the culture time of PSO and E.coli increases, the activity of E.coli becomes better and better. The main reason is that PSO can provide the energy needed for the growth and reproduction of E.coli. E.coli usually obtains the required carbon source by attaching to the surface of PSO and decomposing polysaccharides, which is beneficial to its growth.
[0054] Figure 5(a) Fluorescence staining method to detect the effect of PSO on CAL-27 cell death; (b) Statistical analysis of cell viability detected by fluorescence staining method; (c) Flow cytometry analysis of the effect of PSO on CAL-27 cell death; (d) Statistical analysis of flow cytometry. It can be seen from the figure that as the concentration of PSO (201kDa) solution increases, the activity of CAL-27 cells gradually improves, reaching 107% at 200mg / mL. The apoptotic cells decreased from the original 5.15% to 3.25%. The results of fluorescence staining method show that PSO has good biological activity on CAL-27 cells and can promote the growth of CAL-27 cells.
[0055] Figure 6 (a) Fluorescence staining method was used to detect the effects of PSP, PSO-1, PSO-2 and PSO-3 on H2O2-induced CAL-27 cell death; (b) Statistical analysis of the percentage of live cells marked by fluorescence staining method; (c) Statistical analysis of the percentage of dead cells marked by fluorescence staining method. Note: *: p<0.05, ***: p<0.001 (*: represents statistical difference; ***: represents extremely significant statistical difference). The 6-well plates were blank group, control group, PSO-1 (409kDa), PSO-2 (201kDa), and PSO-3 (100kDa). The blank group was added with culture medium, and the control group and experimental group were added with H2O2 culture medium solution, and AM and PI, Hoechst 33342 were added respectively, and then observed and photographed under an inverted fluorescence microscope. From the figure, it can be found that after adding polysaccharide samples in the presence of H2O2, H2O2 reduced cell apoptosis caused by oxidative stress, and played an antioxidant role. Among them, the proportion of apoptotic cells of PSO-2 was the lowest, indicating that PSO-2, i.e. PSO with a molecular weight of 201 kDa, had the strongest antioxidant effect.
[0056] Figure 7(a) Evaluation of ROS levels of CAL-27 cells treated with different methods using DCFH-DA staining; (b) Statistical analysis of the relative fluorescence intensity of ROS levels detected by DCFH-DA staining. Note: *: p<0.05, ***: p<0.001 (*: represents statistical difference; ***: represents extremely significant statistical difference). The 6-well plates were blank group, control group, PSO-1 (409kDa), PSO-2 (201kDa), and PSO-3 (100kDa). The blank group was added with culture medium, and the control group and experimental group were added with H2O2 culture medium solution. An appropriate amount of antioxidant reagent (DCFH-DA stain) was added to the 6-well plate. Then the cells were observed and photographed under an inverted fluorescence microscope. From the figure, we can find that the fluorescence intensity of PSP is 76.67%, PSO-1 is 64.33%, PSO-2 is 37.00%, and PSO-3 is 51.67%, which is between 83.67% of the Control group and 12.33% of the blank group, indicating that PSP and PSO can play an antioxidant role, and PSO-2 has the strongest antioxidant capacity.
[0057] In summary, the radiation extraction method was first used, and the optimal radiation extraction process was established by single factor and response surface methodology to obtain crude polygonatum polysaccharide. Subsequently, a DEAE-52 chromatography column was used for separation and purification to obtain polygonatum polysaccharide (PSP). And the radiation degradation method was used to degrade polygonatum oligosaccharide (PSO). Then, the biological activity experiment proved that PSO has good biocompatibility. Finally, the in vitro activity experiment and the intracellular ROS scavenging ability test experiment proved that PSO has a good ROS scavenging ability, which is stronger than PSP, and the best one is PSO-2 (201kDa). Based on the above research, it is proved that PSO-2 (201kDa) obtained by electron beam radiation extraction degradation cycle combined technology can be used in the field of ROS scavenging, and has the effects of refreshing and relieving fatigue.
[0058] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.
Claims
1. A method for preparing polygonatum polysaccharide gel tea by ionizing radiation, characterized in that: The following steps are involved: 1) Weigh an appropriate amount of sliced and freeze-dried polygonatum, crush and sieve to obtain polygonatum powder; 2) The polygonatum powder is placed under a dose rate of 20 kGy / pass and a radiation absorption dose of 0-320 kGy, and ionizing radiation extraction is performed. The irradiated polygonatum powder is added with water with a solid-liquid ratio of 1:10-1:30, and reflux extraction is performed 1-5 times, each extraction time is 1-3 hours, and the extraction temperature is 70-110° C., and filtered to obtain a crude polygonatum polysaccharide; 3) separating and purifying the crude polygonatum polysaccharide to obtain polygonatum polysaccharide; 4) subjecting the obtained polygonatum sibiricum polysaccharide to ionizing radiation degradation, and after irradiation, passing through an ultrafiltration system, rotary evaporation, alcohol precipitation, centrifugation, and freeze drying to obtain polygonatum sibiricum oligosaccharide; 5) Weigh a certain amount of green tea, crush it, sieve it, and obtain green tea powder; add water and reflux and extract it twice, each time for 2 hours, filter it, and collect the filtrate; 6) Green tea filtrate is clarified using ceramic membrane; 7) The clarified filtrate of green tea was concentrated under reduced pressure at 60°C; 8) The concentrated green tea extract is placed in a vacuum freezer for drying to obtain green tea powder for later use; 9) Add polyvinyl alcohol (PVA) to water and stir in a water bath. Add sodium carboxymethyl starch (CMS) to water and stir; after the polyvinyl alcohol is completely dissolved, add the completely dissolved sodium carboxymethyl starch solution and stir until uniform; 10) Pour the uniformly stirred hydrogel into a mold, add dried green tea and polygonatum oligosaccharide, and rapidly cycle freeze-drying and shaping; 11) Pack into boxes, store in warehouse, and get the finished product.
2. The method for preparing polygonatum polysaccharide gel tea by ionizing radiation according to claim 1, characterized in that: In step 4), after obtaining the polygonatum sibiricum polysaccharide, it is packed and sealed, and subjected to radiation degradation using a 1MeV electron accelerator, with a selected dose rate of 20 kGy / pass and a total radiation absorption dose of 80-320 kGy.
3. The method for preparing polygonatum polysaccharide gel tea by ionizing radiation according to claim 1, characterized in that: In step 4), the average molecular weight of the polygonatum oligosaccharide is about 200 kDa, and an ultrafiltration system with a molecular cutoff of 200 kDa is selected.
4. The method for preparing polygonatum polysaccharide gel tea by ionizing radiation according to claim 1, characterized in that: In step 4), alcohol precipitation is performed by rotary evaporation under reduced pressure at 50° C. to concentrate to 1 / 5 of the volume, adding 4 times the volume of anhydrous ethanol to precipitate the polysaccharide, and placing in a refrigerator at 4° C. to stand overnight.
5. The method for preparing polygonatum polysaccharide gel tea by ionizing radiation according to claim 1, characterized in that: In step 6), the green tea filtrate is clarified using a 0.1-0.5 μm ceramic membrane.
6. The method for preparing polygonatum polysaccharide gel tea by ionizing radiation according to claim 1, characterized in that: In step 9), the ratio of polyvinyl alcohol (PVA) to water is polyvinyl alcohol: water = 1:8 to 1:10, and the ratio of sodium carboxymethyl starch (CMS) to water is sodium carboxymethyl starch: water = 1:15 to 1:
18.
7. The method for preparing polygonatum polysaccharide gel tea by ionizing radiation according to claim 1, characterized in that: In step 10), the ratio of the added dried green tea to polygonatum oligosaccharide is 9:1 to 10:1.