Pachymaran capable of delaying senescence and preparation method of pachymaran

Abstract

The invention relates to the field of medicines, in particular to pachymaran with a senescence delaying effect and a preparation method of the pachymaran. The pachymaran is a polymer of glucose, and the molecular weight is 2-200kDa; according to the preparation method, pachymaran is obtained through alkali extraction and alcohol precipitation, oxidative degradation, membrane separation and other methods. Particularly, the pachymaran is dextran composed of glucose, the main chain of the dextran is formed by connecting beta-1, 3-glucose, and a small number of branched chains exist at the O-2 site, the O-4 site and the O-6 site of the glucose of the main chain. In-vivo experiment results show that the pachymaran has a good aging delaying effect, and the pachymaran is simple in preparation method, free of toxicity and pollution, capable of being industrially produced and wide in market prospect.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a pachymaran with anti-aging effect and a preparation method thereof. Background Art

[0002] Poria cocos is the dried sclerotium of the fungus Poria cocos (Schw.) Wolf of the Polyporaceae family. Poria cocos is one of the eight treasures of traditional Chinese medicine. It is sweet, light, and flat in nature. It enters the heart, lung, spleen, and kidney meridians; it is diuretic, invigorating, and calming. It is non-toxic and has a wide range of effects. Modern pharmacological studies have shown that Poria cocos has multiple pharmacological effects such as diuresis, immunomodulation, liver protection, anti-tumor, antioxidant, anti-inflammatory, and antiviral. In recent years, with the development of medical science, many Chinese and foreign medical scholars have conducted extensive research on its effective ingredients, pharmacological effects, and mechanisms, and have made significant progress, making Poria cocos widely used in clinical Chinese medicine and people's lives. Finished medicines using Poria cocos as raw materials are also being developed continuously, showing good application prospects.

[0003] As one of its most important active ingredients, Poria cocos polysaccharide has the most in-depth research on its pharmacological activities, including anti-tumor and immunomodulatory effects. In addition to the above aspects, it also has antiviral, anti-mutation, anti-aging, anti-fatigue, anti-coagulation, hypoglycemic, hypolipidemic, and stone prevention effects.

[0004] Poria cocos itself contains a large amount of polysaccharides, but there are relatively few studies on Poria cocos polysaccharides. The main reason is that Poria cocos polysaccharides have poor water solubility, which limits their application in food and medicine. The water-soluble Poria cocos polysaccharides prepared by the present invention through multiple technical means have outstanding effects on the fruit fly model and have good application prospects and development potential. Summary of the invention

[0005] The invention provides a tuckahoe polysaccharide with anti-aging effect and a preparation method thereof. The invention uses tuckahoe as a raw material and is prepared through alkali extraction, alcohol precipitation, oxidative hydrolysis, ultrafiltration and other process steps. The main purpose is to provide a natural and safe food, cosmetic or pharmaceutical raw material for anti-aging. The tuckahoe polysaccharide of the invention can effectively delay aging. The invention is prepared from fungi with medicinal and edible properties, is safe, non-toxic and has no side effects. The invention has a simple formula, low cost and significant effect.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a Poria cocos polysaccharide with anti-aging effect, wherein the Poria cocos polysaccharide is a polymer of glucose, and the molecular weight of the Poria cocos polysaccharide is 2 to 200 kDa; the Poria cocos polysaccharide is prepared by the following steps: obtaining Poria cocos alkaloid extract, concentrating, adding an organic solvent to form a precipitate, re-dissolving after centrifugation, and adding an oxidant to degrade.

[0007] Preferably, the main chain of the Pachymaran is connected by β-1,3-glucosidic bonds.

[0008] Preferably, the pachymannan includes a branch at the O-2 position, O-4 position or O-6 position of glucose.

[0009] Preferably, the pachymaran comprises Figure 1 Substances with the structural formula shown;

[0010] In the formula, n is an integer; preferably, n is selected from 1-99.

[0011] Preferably, the molecular weight of the pachymaran is 30-50 kDa.

[0012] Another aspect of the present invention provides the application of the above-mentioned pachymaran, wherein the pachymaran is used to prepare anti-aging food, cosmetics or anti-aging drugs.

[0013] Another aspect of the present invention provides a method for preparing pachymaran with anti-aging effect, comprising the following steps:

[0014] S1: extracting Poria cocos with an alkaline aqueous solution to obtain a Poria cocos alkali extract;

[0015] S2: concentrating the tuckahoe alkaloid extract, adding an organic solvent so that the concentration of the organic solvent reaches 60-90%, and centrifuging to obtain a precipitate;

[0016] S3: adding water to redissolve the precipitate, and then adding an oxidant to degrade it to obtain a Poria cocos extract hydrolyzate;

[0017] S4: ultrafiltration of the Poria cocos extract hydrolyzate through a 3 kDa ultrafiltration membrane, collecting the retentate, concentrating and drying to obtain Poria cocos polysaccharide.

[0018] Preferably, in step S1, the water extraction temperature is 60-100°C, the alkali added to the alkaline aqueous solution is sodium hydroxide, the material ratio of Poria cocos to sodium hydroxide is 1:1 to 20:1, the number of extractions is 1-2 times, the extraction time is 0.5-3h, and the material-liquid ratio is 5:1 to 30:1.

[0019] Preferably, the organic solvent in step S2 is selected from at least one of methanol, ethanol, propanol and acetone.

[0020] Preferably, in step S3, the oxidant is selected from one of hydrogen peroxide, peracetic acid, and sodium hypochlorite, the material ratio of the oxidant to Poria cocos is 1:1 to 30:1, and the degradation time is 1-3 hours.

[0021] The beneficial effects of the present invention are as follows: the present invention provides a natural and safe food, cosmetic or pharmaceutical raw material for delaying aging; the Poria cocos polysaccharide of the present invention can effectively delay aging; the present invention is prepared from a medicinal and edible fungus, is safe, non-toxic, has no side effects, has a simple formula, is low in cost, and has a significant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A schematic diagram of the chemical structure of Pachymaran according to an embodiment of the present invention;

[0023] Figure 2 The monosaccharide composition chromatogram of Pachymaran 4 (PCP) of the embodiment of the present invention;

[0024] Figure 3 Chromatogram of standard monosaccharides;

[0025] Figure 4 Effects of different doses of Pachymaran 4 (PCP) on the lifespan of Drosophila.

[0026] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials and test strains used in the following examples are purchased from commercial channels unless otherwise specified.

[0030] Example 1

[0031] A kind of tuckahoe polysaccharide with anti-aging effect and preparation method thereof

[0032] Take 400g of Poria cocos, add 4L of H2O and 40g of sodium hydroxide, extract twice, each time for 30min, the extraction temperature is 60℃, the extracts are combined and filtered with a 50-mesh sieve, ethanol is added to the extract to make the alcohol content reach 90%, centrifuge to obtain a precipitate, add water to redissolve the precipitate, add 400g of hydrogen peroxide, degrade for 2h, the temperature is 55℃, the reaction is completed, the hydrolyzate is passed through a 3K ultrafiltration membrane, the retentate is collected, concentrated and dried, and Poria cocos polysaccharide 1 is obtained. The molecular weight of the Poria cocos polysaccharide prepared in this embodiment is in the range of 2 to 200kDa. In a preferred embodiment, the molecular weight range of Poria cocos polysaccharide can be further controlled by controlling the degradation time and adopting multi-stage ultrafiltration, for example, controlled to 30-50kDa.

[0033] Example 2

[0034] A kind of tuckahoe polysaccharide with anti-aging effect and preparation method thereof

[0035] Take 100 kg of Poria cocos, add 800 kg of H2O and 5 kg of sodium hydroxide, extract twice, each time for 60 min, the extraction temperature is 100°C, the extracts are combined and filtered with a 50-mesh sieve, ethanol is added to the extract to make the alcohol content reach 60%, centrifuge to obtain a precipitate, add water to re-dissolve the precipitate, add 500 kg of sodium hypochlorite, degrade for 2 hours, the temperature is 35°C, the reaction is completed, the hydrolyzate is passed through a 3K ultrafiltration membrane, the retentate is collected, concentrated and dried to obtain Poria cocos polysaccharide 2.

[0036] Example 3

[0037] A kind of tuckahoe polysaccharide with anti-aging effect and preparation method thereof

[0038] Take 100g of Poria cocos, add 1000g of H2O and 100g of sodium hydroxide, extract twice, each time for 50min, the extraction temperature is 80℃, the extracts are combined and filtered with a 50-mesh sieve, ethanol is added to the extract to make the alcohol content reach 70%, centrifuge to obtain a precipitate, add water to re-dissolve the precipitate, add 100g of peracetic acid, degrade for 2h, the temperature is 75℃, the reaction is completed, the hydrolyzate is passed through a 3K ultrafiltration membrane, the retentate is collected, concentrated and dried to obtain Poria cocos polysaccharide 3.

[0039] Example 4

[0040] A kind of tuckahoe polysaccharide with anti-aging effect and preparation method thereof

[0041] Take 10 kg of Poria cocos, add 100 kg of H2O and 1 kg of sodium hydroxide, extract twice, each time for 60 minutes, the extraction temperature is 100°C, the extracts are combined and filtered with a 50-mesh sieve, ethanol is added to the extract to make the alcohol content reach 65%, centrifuge to obtain a precipitate, add water to re-dissolve the precipitate, add 100 kg of hydrogen peroxide, degrade for 2 hours, the temperature is 45°C, the reaction is completed, the hydrolyzate is passed through a 3K ultrafiltration membrane, the retentate is collected, concentrated and dried, and Poria cocos polysaccharide 4 (PCP) is obtained.

[0042] In some other embodiments, the organic solvent used in the organic solvent precipitation step can be methanol, ethanol, propanol, acetone and mixtures thereof, and ethanol is preferably used in embodiments 1 to 4. The prepared Poria cocos polysaccharide can be appropriately purified to meet the requirements of food grade, cosmetic grade or pharmaceutical grade raw materials and can be used for the production of corresponding food, cosmetics or medicines.

[0043] The structure of the pachymaran 4 prepared in Example 4 was characterized, which included the following steps:

[0044] (1) The monosaccharide composition determination method is as follows:

[0045] Ion chromatography was used to determine the monosaccharide composition of Poria cocos polysaccharide. The sample was prepared into a 5 mg / mL solution, 0.5 mL was taken and placed in an ampoule, 0.5 mL of 4M TFA solution was added, and the ampoule was sealed with an alcohol lamp. After hydrolysis in an oven at 105°C for 6 hours, the ampoule was dried with a nitrogen blower, and repeated 3-5 times. The mixed standard sample was prepared into a 100 ppm solution.

[0046] Chromatographic conditions:

[0047] Chromatographic column: Dionex CarboPacTM PA1 BioLCTM 4×250mm;

[0048] Mobile phase: A: H2O, B: 10 mM NaOH;

[0049] Column temperature: 35°C;

[0050] Flow rate: 0.8 mL / min;

[0051] Injection volume: 25 μL;

[0052] Detector: Ampereometric detector;

[0053] Data collection time: 30 minutes.

[0054] The monosaccharide composition of Pachymaran 4 is shown in the figure. Pachymaran 4 is composed only of glucose.

[0055] (2) The connection method is as follows:

[0056] Methylation reaction:

[0057] Take 5 mg of the sample to be tested, add 500 μL DMSO to dissolve. Add 1 mg NaH. Add 50 μL iodomethane solution and react for 1 hour. Add 2 mL dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Take the lower dichloromethane phase and evaporate to dryness.

[0058] Hydrolysis reaction

[0059] Add 100 μL 2M TFA, react at 110°C for 90 min, and evaporate to dryness at 30°C.

[0060] Reduction reaction

[0061] Add 50 μL 2M ammonia water and 50 μL 1M NaBD4, mix well, react at room temperature for 2.5 h, add 20 μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL methanol, and blow dry with nitrogen.

[0062] Acetylation reaction

[0063] Add 250 μL of acetic anhydride, vortex mix, and react at 100°C for 2.5 hours. Add 1 mL of water and let stand for 10 minutes. Add 500 μL of dichloromethane, vortex mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Take the lower dichloromethane phase and test it on the machine.

[0064] Chromatographic conditions:

[0065] Chromatographic column: HP-5MS quartz capillary column 30m*250μm*0.25μm. Carrier gas is helium. Split ratio 100:1 injection, injection port temperature 250℃, flow rate 1mL / min, injection volume 1μL.

[0066] GC method: maintain at 50°C for 1.0 min, increase the temperature to 130°C at 50°C / min, increase the temperature to 220°C at 3°C / min, and maintain for 2 min.

[0067] MS acquisition method: quadrupole temperature: 150°C, ion source temperature: 230°C, scan type: full scan, scan rate: N=1.

[0068] The four-linked forms of Poria cocos polysaccharide are composed of β-1,3-glc, t-glc, β-1,3,6-glc, β-1,2,3-glc, and β-1,3,4-glc; thus, its chemical structure can be represented by Figure 1 The structural formula shown is used for illustration, wherein n is an integer, n is 1-99, and in a preferred embodiment, n is preferably 10-20.

[0069] Example 5

[0070] Anti-aging effect experiment

[0071] Experimental drug: Poria polysaccharide 4 (PCP).

[0072] Experimental animals: w1118 male Drosophila melanogaster (provided by Bloomington Drosophila Breeding Center (BDSC) in Indiana, USA).

[0073] Experimental methods: Fruit fly populations were raised on standard fruit fly medium at 25°C. Newly eclosing w1118 male fruit flies were collected and divided into four groups (150 in each group). The fruit flies were placed in standard medium or medium supplemented with 0.2 mg / mL, 2 mg / mL, and 6 mg / mL Poria polysaccharide 4. The fruit flies were transferred to new test tubes filled with fresh medium every 3 days, and the number of dead fruit flies was recorded. The survival curve was obtained until the last fruit fly died. In addition, the average lifespan, median lifespan, and maximum lifespan of the fruit flies were calculated.

[0074] Experimental results: To test the effect of adding pachymaran 4 on lifespan, newly emerged male fruit flies were raised on medium containing different concentrations of pachymaran 4. The results showed that compared with the control group, supplementation with 2 and 6 mg / mL pachymaran 4 significantly increased the survival rate and average lifespan of male fruit flies ( Figure 3 , Table 1; Figure 3 Middle: Survival curve data of male flies fed with different doses of PCP are presented as mean ± SD, and p values ​​are determined by logrank test; **p<0.01; ****p<0.0001). Among them, the average lifespan was extended by 7.28% and 13.83%, respectively. The results show that supplementation with PCP can delay the aging of Drosophila.

[0075] Table 1 Effects of Poria cocos polysaccharide 4 on the life span of Drosophila melanogaster

[0076]

[0077] a,p<0.05;b,p<0.001vs Control

[0078] Experimental conclusion: At the tested dose, supplementation with Poria cocos polysaccharide 4 can delay the aging of fruit flies, and with the increase of the dosage, it shows a good dose-dependent relationship.

Claims

1. A pachymaran with anti-aging effect, characterized in that: The tuckahoe polysaccharide is a polymer of glucose, and the molecular weight of the tuckahoe polysaccharide is 2 to 200 kDa; the tuckahoe polysaccharide is prepared by the following steps: obtaining tuckahoe alkaloid extract, concentrating, adding organic solvent to form precipitation, re-dissolving after centrifugation, and adding oxidant to degrade.

2. The pachymaran with anti-aging effect according to claim 1, characterized in that: The main chain of the pachymaran is connected by β-1,3-glucose glycosidic bonds.

3. The pachymaran with anti-aging effect according to claim 1, characterized in that: The pachymannan includes a branched chain at the O-2 position, O-4 position or O-6 position of glucose.

4. The pachymaran with anti-aging effect according to claim 1, characterized in that: The tuckahoe polysaccharide includes substances with the following structural formula: Wherein n is an integer.

5. The pachymaran with anti-aging effect according to claim 4, characterized in that: Wherein n is selected from 1-99.

6. The pachymaran with anti-aging effect according to claim 1, characterized in that: The molecular weight of the tuckahoe polysaccharide is 30-50 kDa.

7. An application of the pachymaran as claimed in claims 1 to 6, characterized in that: The pachymaran is used for preparing anti-aging food, cosmetics or anti-aging medicine.

8. A method for preparing pachymaran with anti-aging effect, characterized in that: The following steps are involved: S1: extracting Poria cocos with an alkaline aqueous solution to obtain a Poria cocos alkali extract; S2: concentrating the tuckahoe alkaloid extract, adding an organic solvent so that the concentration of the organic solvent reaches 60-90%, and centrifuging to obtain a precipitate; S3: adding water to redissolve the precipitate, and then adding an oxidant to degrade it to obtain a Poria cocos extract hydrolyzate; S4: ultrafiltration of the Poria cocos extract hydrolyzate through a 3 kDa ultrafiltration membrane, collecting the retentate, concentrating and drying to obtain Poria cocos polysaccharide.

9. The preparation method according to claim 8, characterized in that: In step S1, the water extraction temperature is 60-100°C, the alkali added to the alkaline aqueous solution is sodium hydroxide, the material ratio of Poria cocos to sodium hydroxide is 1:1 to 20:1, the number of extractions is 1-2 times, the extraction time is 0.5-3h, and the material-liquid ratio is 5:1 to 30:

1.

10. The preparation method according to claim 8, characterized in that: The organic solvent in step S2 is selected from at least one of methanol, ethanol, propanol and acetone.

11. The preparation method according to claim 8, characterized in that: In step S3, the oxidant is selected from one of hydrogen peroxide, peracetic acid, and sodium hypochlorite, the material ratio of the oxidant to Poria cocos is 1:1 to 30:1, and the degradation time is 1-3 hours.

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