Chrysanthemum polysaccharide preparation method and hangover alleviating and liver protecting application thereof

Abstract

The invention discloses a chrysanthemum polysaccharide preparation method and hangover-alleviating and liver-protecting application thereof, and particularly relates to the technical field of chrysanthemum polysaccharide preparation.According to the preparation method, chrysanthemum is used as a raw material, chrysanthemum polysaccharide I, chrysanthemum polysaccharide II and chrysanthemum polysaccharide III are obtained through extraction, separation and purification, and the chrysanthemum polysaccharide I, chrysanthemum polysaccharide II and chrysanthemum polysaccharide III are obtained through in-vivo evaluation of zebra fish and mice. Chrysanthemum polysaccharide II has obvious hangover-alleviating and liver-protecting effects, the chrysanthemum polysaccharide II is prepared into products which are convenient to take, including but not limited to buccal tablets, alcohol metabolism can be obviously promoted, and a better method is provided for hangover-alleviating and liver-protecting.

Description

Technical Field

[0001] The present invention relates to the technical field of chrysanthemum polysaccharide preparation, and specifically relates to a method for preparing chrysanthemum polysaccharide and its application in relieving alcohol and protecting the liver. Background Art

[0002] Chrysanthemum is a perennial herbaceous plant of the Compositae family and the Chrysanthemum genus in plant taxonomy. Chrysanthemum has the following effects: dispersing wind-heat, clearing the liver and improving eyesight, and can relieve cold symptoms caused by wind-heat, such as fever and headache; it has a regulatory effect on dizziness and headache caused by excessive liver fire; it can improve problems such as red, swollen and painful eyes or blurred vision; it can also help eliminate skin sores with redness, swelling and pain. It is commonly used to treat initial colds, persistent dizziness and headache, eye inflammation, and skin infections and suppuration.

[0003] Among them, polysaccharide is one of the effective medicinal active ingredients in chrysanthemum. At present, it is found that chrysanthemum polysaccharide has medicinal values such as antioxidant, hypoglycemic, anti-tumor, anti-inflammatory, antiviral, and promoting immunity. The activity of chrysanthemum polysaccharide in relieving alcohol and protecting the liver has not been fully explored, and there is still a broad market demand for the development of the biological activity of chrysanthemum polysaccharide. Based on this, the present invention provides a method for preparing chrysanthemum polysaccharide and its application in relieving alcohol and protecting the liver. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing chrysanthemum polysaccharide and its application in relieving alcohol and protecting the liver. Chrysanthemum polysaccharide is prepared from chrysanthemum as a raw material, and it is found that chrysanthemum polysaccharide has an obvious effect of relieving alcohol and protecting the liver. Products including but not limited to buccal tablets are made from chrysanthemum polysaccharide, which can significantly promote alcohol metabolism and provide a good method for sobering up, relieving alcohol and protecting the liver.

[0005] In order to achieve the above purpose, the present invention provides the following technical solutions: A method for preparing chrysanthemum polysaccharide, comprising the following steps:

[0006] Step 1. Extraction of crude chrysanthemum polysaccharide:

[0007] S1.1: Mix chrysanthemum with purified water and heat for extraction three times. After combining the extraction solutions, filter and concentrate.

[0008] S1.2: Centrifuge the concentrated solution, then add 95% ethanol to the supernatant and precipitate for 24 hours.

[0009] S1.3: Centrifuge the precipitated solution, retain the precipitate, dissolve the precipitate in water and then perform freeze-drying to obtain crude chrysanthemum polysaccharide.

[0010] Step 2. Separation and purification of chrysanthemum polysaccharide:

[0011] S2.1: Dissolve the crude chrysanthemum polysaccharide in pure water to obtain a polysaccharide solution. Add Sevag reagent to the polysaccharide solution, shake and mix evenly, then let it stand for stratification to remove denatured proteins.

[0012] S2.2: Repeat step S2.1 for 6 - 10 times, then collect the upper aqueous phase liquid, concentrate it by rotary evaporation and freeze-dry it to obtain chrysanthemum polysaccharide.

[0013] Preferably, dissolve the chrysanthemum polysaccharide obtained in step S2.2 in pure water, load it onto a DEME-52 ion exchange resin column, and elute it successively with pure water, 0.2% NaCl solution, and 0.4% NaCl solution. Collect the eluate in fractions and freeze-dry it to obtain chrysanthemum polysaccharide Ⅰ, chrysanthemum polysaccharide Ⅱ, and chrysanthemum polysaccharide Ⅲ.

[0014] Preferably, chrysanthemum polysaccharide Ⅱ is a homogeneous polysaccharide with an average molecular weight of 16,880, which is composed of monosaccharides such as glycine, galacturonic acid, glucose, and xylose, and the molar ratio is 4:1:19:4.

[0015] Preferably, in steps S1.1 and S2.2, the extraction liquid and the upper aqueous phase liquid are both concentrated in a rotary evaporator, and in steps S1.3 and S2.2, a vacuum freeze dryer is used for freeze-drying.

[0016] Preferably, in step S1.2, the concentrated liquid is centrifuged in a centrifuge at a centrifugal speed of 8000 r / min for 5 min; the ratio of the supernatant to 95% ethanol is 1:4.2.

[0017] Preferably, in step S1.3, the alcohol-precipitated liquid is centrifuged in a centrifuge at a centrifugal speed of 8000 r / min for 10 min.

[0018] Preferably, in step S2.1, the volume ratio of Sevag reagent to the polysaccharide solution is 1:4; the Sevag method is an effective method for removing free proteins. Add a chloroform-n-butanol mixed solution to the crude polysaccharide solution and shake it well to denature the free proteins into insoluble substances, which can be separated and removed to achieve the removal purpose. The Sevag method is relatively mild and has little impact on the polysaccharide structure, but the efficiency is low, and it needs to be repeated more than five times to achieve the ideal effect.

[0019] Make the chrysanthemum polysaccharide into a product convenient for taking, including but not limited to buccal tablets. The manufacturing steps of the chrysanthemum polysaccharide buccal tablets are as follows:

[0020] H1: Include the following raw materials by percentage: 40 - 60% of chrysanthemum polysaccharide Ⅱ, 20 - 35% of sugar alcohol, 10 - 20% of binder, 1 - 5% of flavoring agent, 0.5 - 3% of anti-sticking agent;

[0021] H2: Mix chrysanthemum polysaccharide Ⅱ with sugar alcohol and flavoring agent in a three-dimensional mixer for 30 min, then add the binder to make a soft material. Extrude the soft material through a 16-mesh sieve, and control the thickness of the wet granules at 1 - 2 mm;

[0022] H3: The wet granules are dried in a fluidized bed at a temperature of 50 - 60 °C for 30 min to make the moisture content of the wet granules less than 3%, and then sized through a 18 - mesh sieve;

[0023] H4: Add an anti - sticking agent and mix for 5 min, then use a rotary tablet press with a punch die diameter of 8 - 10 mm, adjust the pressure to a tablet hardness of 50 - 80 N, and the disintegration time limit is less than 30 min to obtain the chrysanthemum polysaccharide buccal tablets.

[0024] Preferably, the sugar alcohol is one or more of mannitol, sorbitol, erythritol, maltitol; the binder is one or more of maltodextrin, gum arabic, xanthan gum, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose; the flavoring agent is one or more of citric acid, DL - malic acid, sodium citrate; the anti - sticking agent is one or more of magnesium stearate, calcium stearate, microcrystalline cellulose.

[0025] The present invention also provides an application of chrysanthemum polysaccharide prepared by the above - mentioned preparation method in relieving alcohol intoxication and protecting the liver.

[0026] In the above technical solution, the technical effects and advantages provided by the present invention are:

[0027] The present invention uses chrysanthemum as the raw material, and through extraction, separation, and purification, chrysanthemum polysaccharide Ⅰ, chrysanthemum polysaccharide Ⅱ, and chrysanthemum polysaccharide Ⅲ are obtained. Through in - vivo evaluation of zebrafish and mice, it is found that chrysanthemum polysaccharide Ⅱ has an obvious effect of relieving alcohol intoxication and protecting the liver. Adding chrysanthemum polysaccharide Ⅱ to excipients to make products including but not limited to buccal tablets can significantly promote alcohol metabolism, providing a good method for sobering up, relieving alcohol intoxication, and protecting the liver. Description of the Drawings

[0028] Figure 1 It is a comparison chart of the movement distances of zebrafish in each group;

[0029] Figure 2 It is a comparison chart of the alcohol dehydrogenase content in the liver tissues of zebrafish in each group;

[0030] Figure 3 It is a comparison chart of the aldehyde dehydrogenase content in the liver tissues of zebrafish in each group. Detailed Embodiments

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further introduced in detail below.

[0032] I. Materials:

[0033] Huangshan Gongju chrysanthemum is selected. Other methods used are all conventional methods known to those skilled in the art unless otherwise specified. The reagents, equipment, etc. used are all commercially available products unless otherwise specified.

[0034] II. Method:

[0035] Example 1. Preparation method of chrysanthemum polysaccharide I:

[0036] It includes the following steps:

[0037] Step 1. Extraction of crude chrysanthemum polysaccharide:

[0038] S1.1: Take 50 g of Huangshan Gongju chrysanthemum and put it into a round-bottom flask, add 600 mL of purified water, heat for 3 h for extraction. Repeat the above steps three times, combine the extraction solutions, filter, and then concentrate to 390 mL in a rotary evaporator. The temperature of the rotary evaporator is controlled at 75 °C;

[0039] S1.2: Centrifuge the concentrated solution in a centrifuge at a centrifugal speed of 8000 r / min for 5 min. Then add the supernatant to 95% ethanol at a ratio of 1:4.2 and perform alcohol precipitation for 24 hours;

[0040] S1.3: Centrifuge the alcohol-precipitated solution in a centrifuge at a centrifugal speed of 8000 r / min for 10 min, retain the precipitate, dissolve the precipitate in water, and perform freeze-drying in a vacuum freeze dryer to obtain crude chrysanthemum polysaccharide;

[0041] Step 2. Separation and purification of chrysanthemum polysaccharide:

[0042] S2.1: Dissolve the crude chrysanthemum polysaccharide in pure water to obtain a polysaccharide solution. Add Sevag reagent to the polysaccharide solution at a volume ratio of 1:4, shake for 10 min to mix evenly, then let it stand for stratification to remove denatured proteins;

[0043] S2.2: Repeat step S2.1 eight times, then collect the upper aqueous phase liquid, concentrate it by rotary evaporation in a rotary evaporator, and then perform freeze-drying in a vacuum freeze dryer to obtain chrysanthemum polysaccharide. Dissolve the chrysanthemum polysaccharide in pure water, load it onto a DEME-52 ion exchange resin column, elute with pure water, collect, and perform freeze-drying in a vacuum freeze dryer to obtain chrysanthemum polysaccharide I.

[0044] Example 2. Preparation method of chrysanthemum polysaccharide II:

[0045] The difference from Example 1 is that in step S2.2, dissolve the chrysanthemum polysaccharide in pure water, load it onto a DEME-52 ion exchange resin column, elute with 0.2% NaCl solution, collect, and perform freeze-drying in a vacuum freeze dryer to obtain chrysanthemum polysaccharide II;

[0046] The remaining steps are the same as those in Example 1.

[0047] Example 3. Preparation method of chrysanthemum polysaccharide III:

[0048] Different from Example 1 and Example 2, in step S2.2 of Example 3, chrysanthemum polysaccharide was dissolved in pure water, loaded onto a DEME-52 ion exchange resin column, eluted with 0.4% NaCl solution, collected, and freeze-dried in a vacuum freeze dryer to obtain chrysanthemum polysaccharide Ⅲ;

[0049] The remaining steps were the same as those in Example 1.

[0050] III. Evaluation of the effect of chrysanthemum polysaccharide on relieving hangover and protecting the liver:

[0051] 1. Zebrafish experiment

[0052] Well-developed adult zebrafish at 3 - 4 months old were selected and evenly divided into five groups, namely the normal group, the model group, the chrysanthemum polysaccharide Ⅰ group, the chrysanthemum polysaccharide Ⅱ group, and the chrysanthemum polysaccharide Ⅲ group, with 15 fish in each group, and they were acclimated for 1 week. The zebrafish in the normal group were placed in a conventional breeding water body, while the zebrafish in the model group and the chrysanthemum polysaccharide Ⅰ, Ⅱ, and Ⅲ groups were placed in a breeding water body containing 2.0% alcohol. After 4 hours of breeding, the model group was changed to a conventional breeding water body, and the chrysanthemum polysaccharide Ⅰ, Ⅱ, and Ⅲ groups were changed to breeding water bodies containing 2.0% chrysanthemum polysaccharide Ⅰ, Ⅱ, and Ⅲ respectively.

[0053] A behavior analyzer was used to analyze the movement trajectories of zebrafish within 0.5 h, and the livers of zebrafish were taken after 0.5 h to measure alcohol dehydrogenase and aldehyde dehydrogenase. The results are as Figures 1-3 shown:

[0054] Figure 1 is a comparison chart of the movement distances of zebrafish in each group. Compared with the normal group, **p < 0.01; compared with the model group, ## p < 0.01;

[0055] Figure 2 , Figure 3 are respectively the comparison charts of the contents of alcohol dehydrogenase and aldehyde dehydrogenase in the liver tissues of zebrafish in each group. Compared with the normal group, *p < 0.05; compared with the model group, # p < 0.05.

[0056] It can be seen from Figure 1 that the zebrafish in the model group were in a "drunk" state, showing behavioral characteristics such as decreased activity ability and loss of balance. However, the total movement distances of the zebrafish in the chrysanthemum polysaccharide Ⅱ and Ⅲ groups were significantly longer than those in the model group, indicating that chrysanthemum polysaccharide Ⅱ and Ⅲ can significantly improve the activity behavior ability of "drunk" zebrafish.

[0057] It can be seen from Figure 2It can be seen that the contents of alcohol dehydrogenase and aldehyde dehydrogenase in the liver tissues of zebrafish in the model group were significantly lower than those in the normal group, indicating obvious liver damage. When chrysanthemum polysaccharide II was administered, the contents of alcohol dehydrogenase and aldehyde dehydrogenase increased significantly, indicating that chrysanthemum polysaccharide II could significantly improve the metabolism of ethanol and acetaldehyde in "drunk" zebrafish and reduce the content of alcohol in their tissues.

[0058] From the experimental results of the above zebrafish, it can be seen that chrysanthemum polysaccharide II has an obvious regulatory effect on the activity, alcohol dehydrogenase, aldehyde dehydrogenase, etc. of "drunk" zebrafish, indicating that this chrysanthemum polysaccharide II has good potential for relieving alcohol and protecting the liver.

[0059] Based on this, gel chromatography was used to analyze chrysanthemum polysaccharide II, and it was found that chrysanthemum polysaccharide II was a homogeneous polysaccharide with an average molecular weight of 16,880. After monosaccharide composition analysis, chrysanthemum polysaccharide II was composed of monosaccharides such as glycine, galacturonic acid, glucose, and xylose, and the molar ratio was 4:1:19:4.

[0060] 2. Animal experiment

[0061] Thirty-six male mice were randomly divided into a normal group, a model group, and a chrysanthemum polysaccharide II group, with 12 mice in each group. After 3 days of adaptive feeding, the mice in the normal group and the model group were intragastrically administered 0.1 mL / 10 g body weight of normal saline, and the mice in the chrysanthemum polysaccharide II group were intragastrically administered 0.1 mL / 10 g body weight of 5% chrysanthemum polysaccharide solution for 3 consecutive days. At 0.5 h after administration on the 3rd day, the rats in the model group and the chrysanthemum polysaccharide II group were intragastrically administered 0.1 mL / 10 g body weight of 56% ethanol solution.

[0062] Six mice were randomly selected from each group, and blood was collected from the eye sockets 0.5 h after the administration of the alcohol solution. The supernatant was obtained by centrifugation, and the contents of blood ethanol, alcohol dehydrogenase, and aldehyde dehydrogenase were measured. The results are shown in Table 1.

[0063] Table 1 Effects of chrysanthemum polysaccharide II on the contents of blood ethanol, alcohol dehydrogenase, and aldehyde dehydrogenase

[0064]

[0065] (Compared with the normal group, **p < 0.01; compared with the model group, ## p < 0.01)

[0066] As can be seen from Table 1, after intragastric administration of 56% ethanol solution, the content of blood ethanol in the serum of mice increased significantly, and the contents of alcohol dehydrogenase and aldehyde dehydrogenase decreased significantly; after intervention with chrysanthemum polysaccharide II solution, the content of blood ethanol decreased significantly, and the contents of alcohol dehydrogenase and aldehyde dehydrogenase increased significantly, indicating that chrysanthemum polysaccharide II has a good regulatory effect on blood ethanol, alcohol dehydrogenase, and aldehyde dehydrogenase in "drunk" mice.

[0067] Continuously observe the behavioral characteristics of the other 6 mice in each group after intragastric administration of alcohol, and record the disappearance and recovery time of the righting reflex of the mice (turn the mouse to the supine position, and if it cannot turn over within 30 s, it is determined to be drunk, that is, record the latency of drunkenness, and the recovery of the righting reflex is determined to be sobering up). The results are shown in Table 2.

[0068] Table 2 Effects of Chrysanthemum Polysaccharide Ⅱ on Sobering-up Time

[0069] Group Number of mice Number of non-intoxicated mice Intoxication time (min) Normal 6 6 / Model 6 0** 85.2±25.7** Chrysanthemum polysaccharide II 6 <![CDATA[2 ## > <![CDATA[45.7±13.6 # >

[0070] (Compared with the normal group, **p < 0.01; compared with the model group, # p < 0.05, ## p < 0.01)

[0071] As can be seen from Table 2, after intragastric administration of 56% ethanol solution, the mice got drunk, and the drunkenness time reached 85.2 min; after intervention with Chrysanthemum Polysaccharide Ⅱ solution, the number of drunken mice decreased by 33.33%, and the drunkenness time was reduced to 45.7 min, a decrease to 53.6%, indicating that Chrysanthemum Polysaccharide Ⅱ can not only reduce the drunkenness rate, but also accelerate the awakening of drunken mice.

[0072] IV. Based on the above-mentioned hangover and liver protection effects of Chrysanthemum Polysaccharide Ⅱ, Chrysanthemum Polysaccharide Ⅱ is made into a product convenient for taking, including but not limited to buccal tablets. The production steps of Chrysanthemum Polysaccharide buccal tablets are as follows:

[0073] H1: Include the following raw materials by percentage: 55% of Chrysanthemum Polysaccharide Ⅱ, 30% of sugar alcohol, 10% of binder, 3% of flavoring agent, 2% of anti-sticking agent;

[0074] The sugar alcohol is selected as erythritol, the binder is selected as maltodextrin, the flavoring agent is selected as citric acid, and the anti-sticking agent is selected as magnesium stearate;

[0075] H2: Mix Chrysanthemum Polysaccharide Ⅱ with sugar alcohol and flavoring agent in a three-dimensional mixer for 30 min, then add the binder to make a soft material. The soft material is extruded through a 16-mesh sieve, and the thickness of the wet granules is controlled at 2 mm;

[0076] H3: Dry the wet granules in a fluidized bed at a temperature of 60 °C for 30 min to make the moisture content of the wet granules less than 3%, and then screen and size them through an 18-mesh sieve;

[0077] H4: Add the anti-sticking agent and mix for 5 min, then use a rotary tablet press with a punch die diameter of 10 mm, adjust the pressure to a tablet hardness of 80 N, and the disintegration time limit is less than 30 min to obtain Chrysanthemum Polysaccharide buccal tablets.

[0078] The Chrysanthemum Polysaccharide buccal tablets prepared using Chrysanthemum Polysaccharide Ⅱ can significantly promote alcohol metabolism, providing a good method for hangover and liver protection.

[0079] Only some exemplary embodiments of the present invention have been described by way of illustration. Without doubt, for those of ordinary skill in the art, the described embodiments can be modified in various different ways without departing from the spirit and scope of the present invention. Therefore, the above description is illustrative in nature and should not be construed as a limitation on the scope of protection of the claims of the present invention.

Claims

1. A method for preparing chrysanthemum polysaccharide, characterized in that: The following steps are involved: Step 1: Extraction of crude chrysanthemum polysaccharides: S1.1: Chrysanthemum and purified water are mixed and heated for extraction three times, and the extracts are combined, filtered and concentrated; S1.2: Centrifuge the concentrate, then add 95% ethanol to the supernatant and precipitate for 24 hours; S1.3: centrifuging the alcohol precipitate, retaining the precipitate, dissolving the precipitate in water, and then freeze-drying to obtain crude chrysanthemum polysaccharide; Step 2: Separation and purification of chrysanthemum polysaccharides: S2.1: Dissolve the crude chrysanthemum polysaccharide in pure water to obtain a polysaccharide solution. Add Sevag reagent to the polysaccharide solution and shake to mix evenly. Let it stand and separate to remove denatured proteins. S2.2: Step S2.1 is repeated 6-10 times, and then the upper aqueous phase liquid is collected, concentrated by rotary evaporation, and freeze-dried to obtain chrysanthemum polysaccharide.

2. The method for preparing chrysanthemum polysaccharide according to claim 1, characterized in that: The chrysanthemum polysaccharide obtained in step S2.2 was dissolved in pure water, loaded on a DEME-52 ion exchange resin column, eluted with pure water, 0.2% NaCl solution, and 0.4% NaCl solution in sequence, collected in sections, and freeze-dried to obtain chrysanthemum polysaccharide I, chrysanthemum polysaccharide II, and chrysanthemum polysaccharide III.

3. The method for preparing chrysanthemum polysaccharide according to claim 2, characterized in that: Chrysanthemum polysaccharide II is a homogeneous polysaccharide with an average molecular weight of 16880. It is composed of glycine, galacturonic acid, glucose and xylose in a molar ratio of 4:1:19:

4.

4. The method for preparing chrysanthemum polysaccharide according to claim 2, characterized in that: In steps S1.1 and S2.2, the extract and the upper aqueous phase liquid are concentrated in a rotary evaporator, and in steps S1.3 and S2.2, a vacuum freeze dryer is used for freeze drying.

5. The method for preparing chrysanthemum polysaccharide according to claim 1, characterized in that: In step S1.2, the concentrated solution is centrifuged in a centrifuge at a speed of 8000 r / min for 5 min; the ratio of the supernatant to 95% ethanol is 1:4.

2.

6. The method for preparing chrysanthemum polysaccharide according to claim 1, characterized in that: In step S1.3, the alcohol precipitate is centrifuged in a centrifuge at a speed of 8000 r / min for 10 min.

7. The method for preparing chrysanthemum polysaccharide according to claim 1, characterized in that: In step S2.1, the volume ratio of Sevag reagent to polysaccharide solution is 1:

4.

8. A method for preparing chrysanthemum polysaccharide according to any one of claims 1 to 7, characterized in that: The specific steps for making chrysanthemum polysaccharide buccal tablets are as follows: H1: includes the following raw materials by percentage: chrysanthemum polysaccharide II 40-60%, sugar alcohol 20-35%, binder 10-20%, flavoring agent 1-5%, anti-adhesive agent 0.5-3%; H2: Mix chrysanthemum polysaccharide II, sugar alcohol and flavoring agent in a three-dimensional mixer for 30 minutes, then add a binder to make a soft material, and extrude the soft material through a 16-mesh screen to granulate, and the thickness of the wet granules is controlled at 1-2 mm; H3: The wet granules are dried in a fluidized bed at 50-60°C for 30 min to reduce the moisture content of the wet granules to less than 3%, and then sieved through an 18-mesh screen; H4: Add anti-adhesive agent and mix for 5 minutes, then use a rotary tablet press with a die diameter of 8-10 mm, adjust the pressure to a tablet hardness of 50-80N, and a disintegration time of less than 30 minutes to obtain chrysanthemum polysaccharide buccal tablets.

9. The method for preparing chrysanthemum polysaccharide according to claim 8, characterized in that: The sugar alcohol is one or more of mannitol, sorbitol, erythritol and maltitol; The binder is one or more of maltodextrin, gum arabic, xanthan gum, sodium carboxymethyl cellulose, and hydroxypropyl methylcellulose; The flavoring agent is one or more of citric acid, DL-malic acid, and sodium citrate; The anti-adhesive agent is one or more of magnesium stearate, calcium stearate, and microcrystalline cellulose.

10. Use of chrysanthemum polysaccharide prepared by the preparation method according to any one of claims 1 to 7 in sobering up and protecting the liver.

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