WO2006076841A1 - Heteropolysaccharides de tremelle, leurs produits d’extraction, procede de preparation et utilisations de ceux-ci - Google Patents

Heteropolysaccharides de tremelle, leurs produits d’extraction, procede de preparation et utilisations de ceux-ci Download PDF

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WO2006076841A1
WO2006076841A1 PCT/CN2005/001459 CN2005001459W WO2006076841A1 WO 2006076841 A1 WO2006076841 A1 WO 2006076841A1 CN 2005001459 W CN2005001459 W CN 2005001459W WO 2006076841 A1 WO2006076841 A1 WO 2006076841A1
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tremella
heteropolysaccharide
extract
ethanol
preparation
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PCT/CN2005/001459
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Chinese (zh)
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Guiyun Liu
Bin Luo
Feng Luo
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Shanghai Wenda Biotech Inc.
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Publication of WO2006076841A1 publication Critical patent/WO2006076841A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the invention relates to a white fungus heteropolysaccharide and a preparation method and use thereof.
  • Tremella fuciformis Berk also known as white fungus, mulberry ("Qing Yi Lu")
  • Tremellales is Tremellales, Tremellacease, Tremella.
  • Tremella fuciformis is a heteropolysaccharide extracted from Tremella or microbial fermentation broth. Since ancient times, China has called white fungus high-grade supplements and medicines, and the best for medicine and food, it is the court's finest tonic food. However, its composition and structure and corresponding functions are rarely reported. Summary of the invention
  • One of the objects of the present invention is to provide a Tremella heteropolysaccharide.
  • the Tremella heteropolysaccharide has a structure of ⁇ -(1-3)mannose as the main chain, mainly composed of 70-93% by weight of neutral total sugar, 6-28% of glucuronic acid, 0.1-3% by weight.
  • the binding protein composition; the average molecular weight of the Tremella heteropolysaccharide is 85-1.6 million Daltons, wherein more than 90% by weight of the molecular weight is greater than 6000 Daltons.
  • the neutral total sugar mainly comprises the following content of glycosyl: 35-60% mannose, 10-25% fucose, 8-18% xylose and 5-15% glucose, the above content is the silver ear heteropolysaccharide Weight meter.
  • the side chain of the tremella heteropolysaccharide has a plurality of different combinations of changes, one of which has been determined by X-ray diffraction as a structural unit represented by the following structural formula I, which has a molecular weight of 2578.5 or 2724.7.
  • the sugar structure is composed of ⁇ -(1-3)mannose (Manp) as the main chain skeleton, and two ⁇ -(1-2) xylose (Xylp) and three ⁇ -glucose are attached to the two carbon atoms.
  • Another object of the present invention is to provide a white fungus heteropolysaccharide extract having a percentage (WAV) of each component of the Tremella heteropolysaccharide extract: 80-98% of the Tremella heteropolysaccharide of the present invention, and 0.1-3.3 of free protein %, ash 0.5-8%.
  • the Tremella heteropolysaccharide extract has an average molecular weight of from 7 to 1.4 million Daltons, wherein the molecular weight of more than 6000 Daltons is from 75 to 98% by weight.
  • Tremella fuciformis extract of the present invention Some other characteristics and detection methods of the Tremella fuciformis extract of the present invention are briefly described as follows: Brief description of the test items and detection methods Result Viscosity (preparation of 0.5% aqueous solution of Tremella polysaccharide extract, constant temperature 25 0.6-1.8 pa.s
  • Appearance White or light grayish white powder or granules Another object of the present invention is to provide a method for preparing a silver fungus heteropolysaccharide extract of the present invention, which comprises the following steps:
  • the raw material of the white fungus fruit body is a dry raw material of the tremella fruit body, and the dried tremella fruit material is soaked, expanded and cleaned with water (including a yellow-brown base), 20-100 times each time, and multiple times of extraction. Not more than 200 times by weight of the dried fermented fruit body material amount of water is stirred and extracted at 80-100 ° C to obtain a crude extract.
  • the raw materials of the white fungus fruit body may also be raw materials of fresh white fungus fruit body, fresh frozen fungus fruit raw material or frozen fresh white fungus fruit raw material, and the raw material of the white fungus fruit body is removed to the base of the yellow-brown and cleaned, and each time is 2 -15 times, multiple extractions, no more than 30 times (by weight) of the amount of the raw material of the fungus fruit body is stirred at 80-100 Torr to obtain a crude extract.
  • step 1) the raw material of the tremella fruit body is stirred with a single extraction of water at 80-100 Torr for 1-4 hours to obtain a crude extract; or it may be extracted multiple times, and the obtained extract is combined to obtain a crude extract, more
  • the total extraction time is > 1 hour, 5.5 hours.
  • Step 2 The obtained filtrate may be further concentrated if it is not viscous to obtain a concentrate, and the obtained concentrate may be used in the step 3).
  • step 3 the filtrate obtained in the step 2) is stirred while being added to the ethanol.
  • the precipitate obtained in step 3 can be dehydrated by dehydration with 95-100 v/v% ethanol.
  • the drying may be various conventional drying methods below 80 ° C, generally -40 ° C ⁇ 8 (TC vacuum drying, hot drying, hot air drying or freeze drying; preferably 40 ⁇ 80 ° C Vacuum drying, hot drying or hot air drying, or -40 ⁇ freeze drying.
  • the above preparation method is convenient to use, and is convenient to store and transport raw materials; the dried white fungus is soaked in water and expanded, and the cells of the white fungus are fully sucked up by water soaking, which is beneficial to the above.
  • the heteropolysaccharide in the Tremella fuciformis naturally exudes, which not only extracts the conditions, but also shortens the extraction time; and adds 20-100 times (multiple extractions of total water does not exceed 200 times) in the water extraction of the dry material, which is the dry white fungus.
  • the optimum conditions for the exudation and dissolution of the solid heteropolysaccharide in the extract although the test shows that a small amount of the product of the invention can be obtained by adding water less than 20 times that of the dry material, but the extract is very viscous, which causes inconvenience to the operation.
  • the product yield is also very low; at the same time, although the test also shows that the addition of more than 100 times (multiple extraction of more than 200 times the total amount of water) can also obtain the product of the invention in the water extraction of the dry material, but the proportion of the heteropolysaccharide in the extract Too low will greatly increase energy consumption or greatly increase the amount of alcohol used in precipitation;
  • fresh tremella fruit body is used as raw material, the damage of Tremella fupopolysaccharide in the drying process can be avoided, the water soaking step is omitted, and the production efficiency is improved.
  • the use of fresh-keeping refrigerated tremella fruit body as raw material facilitates the storage of raw materials and Short-distance transportation can prevent the deterioration of fresh white fungus in a short period of time; if the low-temperature frozen fresh white fungus fruit body is used as raw material, it can facilitate long-term storage and long-distance transportation of raw materials, and at the same time, due to low temperature quick freezing, some Tremella cell wall rupture will occur.
  • the polysaccharide is more likely to ooze out, and the extraction efficiency can be improved; the water extraction of the fresh material is carried out by adding 2 times to 15 times (the total water volume is not more than 30 times), which is exuded and dissolved in the extract of the fresh white fungus fruit polysaccharide.
  • the test shows that a small amount of the product of the invention can be obtained by adding less than 2 times the water extraction of the fresh material, but the extract is very viscous, which causes inconvenience to the operation and the product yield is low;
  • the test also showed that the addition of more than 15 times (multiple extractions of total water more than 30 times) can also be obtained from the water extraction of fresh materials.
  • the product of the invention is a macromolecular polysaccharide, which is heated and extracted (80 ° C -10 (TC water temperature extraction), can not only dissolve the macromolecular polysaccharide rapidly, shorten the extraction time, and can degenerate the free hybrid protein in the white fungus. Precipitation increases the purity of the product.
  • the extraction time is the key to obtaining an average molecular weight of 850,000 or more of Tremella fuciformis. Because the extraction time of single heating and stirring is less than 1h, the average molecular weight of the obtained Tremella heteropolysaccharide will be less than 850,000 (even if it is available, the yield is very low); the extraction time of single heating and stirring is more than 4h, due to the continuous heating extraction.
  • the macromolecular Tremella heteropolysaccharide is cleaved (the average molecular weight will be less than 850,000, 1.7% of the aqueous solution of the invention will have a viscosity of less than 20 pa.s), resulting in the inability to obtain macromolecular polysaccharides (or macromolecular polysaccharides having an average molecular weight of more than 850,000).
  • the yield is very low, it is not suitable for large-scale production); the total extraction time is more than 5.5 hours, and the obtained white fungus polysaccharide is extracted after 5.5 hours.
  • the average molecular weight will be less than 850,000 (even if available, the yield is very low, which will affect the extraction efficiency).
  • Uniform stirring can make the filtrate or its concentrate and ethanol mix and react well.
  • the final concentration of ethanol in the mixed liquid is below 50%, the polysaccharide can not be precipitated (or the yield is very low, which is not suitable for large-scale production);
  • the final concentration of ethanol is more than 85%, the excessive use of alcohol is not suitable for large-scale production, and it is also not conducive to controlling impurities or small molecules in the polysaccharide precipitate, which affects product purity.
  • a further object of the present invention is to provide a process for the preparation of another extract of the Tremella fuciformis of the present invention, which comprises the steps of:
  • the filtration in the step 1) may be a conventional method, such as plate and frame filtration, and a filter aid such as diatomaceous earth may be added during the filtration.
  • the white fungus fermentation liquid obtained by microbial fermentation is used as a raw material, so that the source and process of the raw material of the tremella polysaccharide are more stable, the dependence on the natural or cultured Tremella resources is reduced, and the drying process of the white fungus fruit body is avoided.
  • Destruction omitting the step of soaking the dried tremella fruit body in water, omitting the step of heating extraction; fermenting the fermentation system of the white fungus obtained by microbial fermentation, heating 80 ° C -10 (TC, 0.5 h -2 h, can kill
  • the mycelium and microorganisms in the fermentation liquid of Tremella fuciformis can denaturing and precipitate the free hybrid protein in the fermentation broth, which is beneficial to improve the purity of the product; conventional filtration (can add diatomaceous earth and other filter aids), discard the filter residue, and finally clear the solution.
  • a further object of the present invention is to provide a process for the preparation of the Tremella heteropolysaccharide of the present invention which comprises the step of further purifying the extract obtained by the above preparation method.
  • the purification was carried out by the Sevag method and the dialysis method.
  • other polysaccharide purification methods such as chromatographic separation methods, can also produce the Tremella heteropolysaccharide of the present invention.
  • Another object of the present invention is to provide the use of the Tremella heteropolysaccharide of the present invention and an extract thereof.
  • the Tremella fuciformis polysaccharide and the extract thereof have the functions of moisturizing, lubricating, scavenging free radicals and anti-aging and other anti-oxidation, anti-allergic, thickening and stabilizing system, and the performance and state of the Tremella heteropolysaccharide and its extract are stable; Widely used in cosmetics, personal care products, daily chemicals, food, health foods and pharmaceuticals.
  • Figure 1 is an infrared spectrum of the Tremella fuciformis polysaccharide of the present invention.
  • Fig. 2 is a diagram showing the identification of proteins in the silver fungus heteropolysaccharide of the present invention by gel column chromatography.
  • Fig. 3 is a diagram showing the identification of the ⁇ -elimination reaction of the protein in the Tremella fuciformis polysaccharide of the present invention.
  • Figure 4 is a graph showing the ability of the Tremella fuciformis polysaccharide to inhibit oxygen free radicals according to the present invention.
  • Figure 5 is a graph showing the relationship between Tremella fuciformis and SOD activity in skin cells.
  • Figure 6 is a comparison of the moisturizing ability of the Tremella fuciformis and the control sample.
  • Figure ⁇ is a comparison of the drinking lubricity of Tremella fuciformis and control samples.
  • Figure 8 is a comparison of the smear lubrication of the Tremella fuciformis and the control sample.
  • Figure 9 is a graph showing the effect of suppressing itching of Tremella fuciformis.
  • Figure 10 is a graph showing the effect of suppressing allergic spots by Tremella fuciformis. detailed description
  • the dried tremella body is soaked in water, expanded, and then washed to the yellow-brown base and cleaned; the washed raw material is placed in an extraction tank, 20 times of dry water is added, and the mixture is stirred at 80 ° C for 1 hour. Then, it is filtered, and the supernatant finally passes through a filter with a pore size of Ol- ⁇ ; under uniform stirring, the filtrate and ethanol are thoroughly mixed, and the final concentration of ethanol in the mixed liquid is 50%, the precipitate is collected, and dehydrated with 95% ethanol. Decolorization, collection of precipitate; 80 ° C hot air drying, pulverization. Product yield 1.5% (based on dry tremella weight).
  • Example 2 The dried tremella body is soaked in water, expanded, and then removed to the yellow-brown base and cleaned; the washed raw material is placed in an extraction tank, 40 times of dry water is added, and the mixture is stirred at 90 ° C water temperature. 2h, and collect the extract; the raw material is repeatedly extracted again for 2h, and then the extract is filtered, and the clear liquid finally passes through a filter with a pore diameter of 0.1-1 ⁇ m; under the uniform stirring, the filtrate and the ethanol are thoroughly mixed to make the mixed liquid The final concentration of ethanol was 70%, dehydrated with 95% ethanol, and the precipitate was collected; dried at 40 ° C in vacuum and pulverized. Product yield 15% (based on dry tremella weight).
  • the fresh white fungus that has been frozen at a low temperature is thawed, and then the yellow-brown base is removed and cleaned; the washed raw material is placed in an extraction tank, and the water of the fresh material is added twice, and the mixture is stirred at a water temperature of 80 ° C for 1 hour. Then, the solution is finally passed through a filter having a pore size of Ol- ⁇ ; under uniform stirring, the filtrate and the ethanol are thoroughly mixed, so that the final concentration of the ethanol in the mixed liquid is 50%, the precipitate is collected, and the 95% ethanol is dehydrated and decolored. The precipitate was collected; freeze-dried at 40 ° C and pulverized. Product yield 0.2% (based on the weight of fresh white fungus).
  • the white fungus fermentation broth obtained by microbial fermentation is heated at 80 ° C for 2 h, filtered, diatomaceous earth is added with filter aid, the filter residue is discarded, and the supernatant is finally passed through a filter having a pore diameter of 0.1-1 ⁇ ; under uniform stirring, the filtrate is filtered.
  • the liquid and the ethanol were thoroughly mixed so that the final concentration of the ethanol in the mixed liquid was 50%, the precipitate was collected, dehydrated by dehydration with 95% ethanol, and the precipitate was collected; dried at 80 ° C by hot air, and pulverized. Product yield 0.25% (based on the weight of the fermentation broth).
  • the white fungus fermentation broth obtained by microbial fermentation is warmed, 0.5h, conventionally filtered, and the filter residue is discarded.
  • the supernatant finally passes through a filter with a pore size of Ol- ⁇ ; under uniform agitation, the filtrate and ethanol are thoroughly mixed to make a mixture.
  • the final concentration of ethanol in the liquid was 85%, the precipitate was collected, dehydrated by dehydration with 100% ethanol, and the precipitate was collected; freeze-dried at 40 ° C, and pulverized.
  • Product yield 0.55% (based on the weight of the fermentation broth).
  • the white fungus fermentation liquid obtained by microbial fermentation is heated at 90 ° C for 1.5 h, added with a filter aid, and the filter residue is discarded.
  • the clear liquid finally passes through a filter having a pore size of Ol- ⁇ ; under uniform stirring, the filtrate and ethanol are fully stirred.
  • the mixture was mixed so that the final concentration of ethanol in the mixed liquid was 70%, the precipitate was collected, dehydrated by dehydration with 100% ethanol, and the precipitate was collected; vacuum dried at 60 ° C, Smash.
  • the product yield was 0.45% (based on the weight of the fermentation broth).
  • the supernatant after centrifugation was mixed with the above-mentioned chloroform-n-butanol mixed solution at a ratio of 3:1 (volume ratio), and centrifuged (4000 r/min, 30 minutes) with a centrifuge, and the supernatant was collected. Repeat the above operation and repeat the treatment until the OD value of the water phase at 280 mn (using the 752 UV grating spectrophotometer, Shanghai Precision Scientific Instrument Factory) is no longer reduced.
  • the aqueous phase was concentrated under reduced pressure to a third, and the concentrate was thoroughly mixed with 3 times (volume ratio) of 95% ethanol in an aqueous phase, and centrifuged at 4000 r/min for 30 minutes to obtain a precipitate.
  • the precipitate is dissolved in 100 times by weight of deionized water, centrifuged, and the insoluble matter is removed.
  • the supernatant is placed in a 30 cm long dialysis bag (the dialysis bag can pass through 5000 molecular weight or less) for dialysis (the two ends are tied and hoisted)
  • the deionized water bottle was rinsed with deionized water for 12 hours), and 5000 molecular weight or higher substances were collected.
  • the dialyzed supernatant was thoroughly mixed with 3 times by volume of pure ethanol, and centrifuged again at 4000 r/min for 30 minutes to obtain a precipitate.
  • the precipitate was dehydrated with absolute ethanol and dried under vacuum at 60 ° C to obtain the pure form of the present invention.
  • Tremella heteropolysaccharide The precipitate was dehydrated with absolute ethanol and dried under vacuum at 60 ° C to obtain the pure form of the present invention.
  • Tremella heteropolysaccharides in Example 12 means 11 Tremella heteropolysaccharides obtained by purifying each of Examples 1-11, respectively.
  • Tremella heteropolysaccharide and Tremella heteropolysaccharide extract were subjected to thin layer chromatography.
  • both the white fungus heteropolysaccharide and the white fungus heteropolysaccharide extract contained mannose, fucose, xylose, glucose, N-acetylglucosamine, glucuronic acid, arabinose and galactose.
  • Tremella fuciformis variant neutral sugars account for the weight percentage of total Tremella heteropolysaccharides
  • Tremella fuciformis contains mannose, fucose, xylose and glucose, and its percentage (W/W) is mannose 35 %-60%, fucose 10%-25%, xylose 8 %-18%, glucose 5%-15%.
  • N-acetylglucosamine, arabinose and galactose could not be detected by gas chromatography, but the above thin layer chromatography showed that the tauber heteropolysaccharide also contained traces or traces of N-acetylglucosamine, arabinose and half. lactose.
  • Tremella heteropolysaccharide contains gluconic acid, and the proportion by weight is 6%-28%.
  • Tremella heteropolysaccharide extract contains glucuronic acid, and the proportion by weight is 6%-26%.
  • Tremella fuciformis polysaccharides were identified by infrared spectroscopy (instrument model: magna F TIR-750, manufacturer: Nicolet magna IR, USA) to identify the characteristic peaks of Tremella fuciformis.
  • Infrared spectroscopy conditions potassium bromide tableting; sample scanning times: 32 times; background scanning times: 32 times; resolution: 4; sample yield: 2; mirror speed: 0.6329; aperture: 100.00; detector: DTGSKBR; : IR.
  • a dextran Dextran (Model: T500, Pharmacia) column (2.5 cm X 35 cm) was first equilibrated with a 0.1 M NaCl aqueous solution, and then 100 ⁇ g of Tremella heteropolysaccharide was dissolved in 200 times by weight of an aqueous solution of 0.1 M NaCl. Installed on the top of the column, rinse the column with O.lM NaCl aqueous solution at a flow rate of 15 ml per hour, partially collect 5 ml per tube, identify by thin layer method, combine the same fraction, take one fraction for dialysis, and collect molecular weight at 5000.
  • the dialyzed supernatant is collected and mixed with 4 times by weight of ethanol, and the precipitate is collected by centrifugation, eluted twice with pure ethanol, and dried to obtain a heteropolysaccharide combination in the white fungus heteropolysaccharide, and the sample is dissolved.
  • the solution was treated with 75% aqueous methanol, eluted twice with pure methanol, and naturally dried to obtain a crystal of Tremella heteropolysaccharide.
  • the structure of the crystal was identified by X-ray diffraction (device model: RASA-7R, manufacturer: Nippon Ryokan Co., Ltd.). The results of thin layer chromatography, infrared spectroscopy, and gas chromatography were combined to identify the structure.
  • a combination of the Tremella heteropolysaccharide comprises the structural unit represented by the above structural formula I, which has a molecular weight of 2578.5 or 2724.7.
  • the protein in Tremella fuciformis is a binding protein, and the proportion (W/W) is 0.1%-3 %.
  • the total protein weight percentage in the extract of Tremella fuciformis The detection results of the total protein content of the above-mentioned Tremella heteropolysaccharide extract were respectively subtracted from the binding protein in the Tremella fuciformis polysaccharide obtained by the purification of Example 12, and the calculation formula was as follows: A-A1 - A2 XB
  • A the percentage of free protein in each of the white fungus heteropolysaccharide extracts
  • A1 total protein weight percentage of each of the white fungus heteropolysaccharide extracts
  • A2 the weight percentage of binding protein in each of the white fungus heteropolysaccharides
  • the content of free protein in the extract of Tremella fuciformis polysaccharides is as follows: Conclusion: The free protein in the extract of Tremella fuciformis is 0.1% by weight.
  • Tremella fuciformis is between 850,000 and 1.6 million Daltons;
  • the average molecular weight of the Tremella fuciformis extract is between 750,000 and 1.4 million Daltons.
  • Detector Waters2410 differential detector, Waters2487 dual wavelength UV detector
  • the weight ratio of molecular weight greater than 6000 Daltons in Tremella fuciformis is 90%-100%, and the weight ratio of molecular weight less than 6000 Daltons is less than 10%.
  • the weight ratio of the material with molecular weight greater than 6000 Dalton in the extract of Tremella fuciformis is 75%-98%, and the weight ratio of the material with molecular weight less than 6000 Dalton is 2-25%.
  • Tremella fuciformis and Tremella fuciformis extract aqueous solution were prepared separately and tested by pH meter.
  • a 0.5% (by weight) aqueous solution of Tremella fuciformis and Tremella fuciformis extract was prepared at a constant temperature of 25 ° C, and the viscosity was measured by a rotary viscometer (Model: NDJ-1, No. 3 rotor, Shanghai Precision Scientific Instrument Factory).
  • Tremella heteropolysaccharide and Tremella fuciformis extract aqueous solution were prepared at a constant temperature of 25 ° C.
  • the viscosity was measured by a rotary viscometer (Model: NDJ-1 Shanghai Precision Scientific Instrument Factory), and the state was observed. .
  • METHODS 0.5% by weight of Tremella fuciformis and Tremella fuciformis extract aqueous solution were prepared, and water was used as a control.
  • the 752 UV grating spectrophotometer (Shanghai Precision Scientific Instrument Factory) was used to detect at 400 nm transmittance.
  • Tremella fuciformis and Tremella fuciformis extract were weighed separately and dissolved in 100ml of various solutions. Stir at high speed. After the Tremella polysaccharide and Tremella polysaccharide extract were dissolved or evenly dispersed, the solution was centrifuged.
  • the Tremella heteropolysaccharide and the Tremella heteropolysaccharide extract of the present invention can be dissolved in water, an aqueous solution containing soluble protein or dairy products, and a low concentration organic solvent (ethanol, propylene glycol, butylene glycol, glycerin, etc.), while the Tremella heteropolysaccharide and Tremella heteropolysaccharide extract can be dissolved or well dispersed in emulsion (soft phospholipids, oils, etc.);
  • Tremella fuciformis and Tremella polysaccharide extracts of the present invention have the functions of scavenging free radicals and anti-aging, such as anti-oxidation, moisturizing, lubricating, anti-allergic and irritating, thickening and stabilizing systems, and are considered to be related to their structure. :
  • the Tremella fupopolysaccharide contains glucuronic acid glycosyl group, and its percentage (W/W) is high (6%-28%), the hydrogen atom of the hydroxyl structure in glucuronic acid can be combined with free radicals. Thus, peroxidic free radicals can be removed.
  • the macromolecular Tremella heteropolysaccharide has excellent hydrophilic film-forming property, it can block the damage and invasion of the biofilm such as skin by the external environment. Therefore, the Tremella fuciformis has the functions of scavenging free radicals and anti-aging.
  • Tremella fupopolysaccharide has a structural property of an average molecular weight of 85-1.6 million Daltons and a molecular weight of more than 6000 Daltons of 90%-100%, and also has a physical characteristic of viscosity enthalpy determined by molecular weight characteristics ( 0.5%).
  • the viscosity of the aqueous solution can reach 0.7-2pa.s; the viscosity of 2% aqueous solution is greater than 20pa.s, and the solution is in a gel state), so it also has obvious functions in the following aspects -
  • Tremella heteropolysaccharide Because it is a macromolecular substance, it can form a hydrophilic membrane on the surface of the object, and at the same time, because the structure of the Tremella heteropolysaccharide is not easily decomposed (having heat resistance and weak acid-base characteristics), it can be in human body and in vitro. (For example: skin, oral cavity) to form a protective film to resist and block the invasion of various types of sensitizing stimuli, so Tremella fuciformis has anti-allergic and stimulating effects;
  • the Tremella fuciformis can thicken, and the solubility and stability tests also show that this feature also has a significant effect on the stability system.
  • Tremella fuciformis and Tremella fuciformis extracts have wide application fields and reliable performance, and can be widely used in various fields. The following is explained by way of an effect embodiment.
  • Example 2 The experimental sample was obtained by the method of Example 2, and the silver ear heteropolysaccharide obtained by the purification of Example 12 (gluconic acid content 6.4%) was used.
  • Tremella heteropolysaccharide was mixed with distilled water to a concentration of 1.0%, 0.5%, and 0.1%. Using distilled water as a reference control, an active oxygen determination kit was used to generate an off amount according to the Fenton reaction. Capacity determination.
  • Tremella heteropolysaccharide could significantly inhibit the production of oxygen free radicals in Fenton reaction, and the ability to inhibit oxygen free radicals with the increase of the concentration of polysaccharides in Tremella fuciformis.
  • the enhanced trend indicates that Tremella fuciformis has strong antioxidant capacity.
  • Tremella fuciformis polysaccharides Experiment on anti-oxidative aging function of skin cells by Tremella fuciformis polysaccharides. The aim was to observe the effect of Tremella fuciformis on the anti-oxidative aging function of skin cells.
  • the experimental sample was prepared by the method of Example 2, and the Tremella heteropolysaccharide obtained by the purification of Example 12 (gluconic acid content 6.4%) was used.
  • the silver fungus heteropolysaccharide was separately prepared into keratinocyte culture solution and fibroblast culture medium to a concentration of 0.5%, 0.1% and 0.05% of the test application liquid, and then filtered and sterilized, and dispensed. Store at 4 ° C for later use.
  • SOD activity After the primary cultured skin keratinocytes and fibroblasts reach the sub-fusion state, the culture medium is replaced with the silver-containing ear polysaccharides by 0.5%, 0.1% and 0.05%. Application of the culture fluid. After 24 hours of culture, the SOD activity was measured using a SOD assay kit.
  • MDA malondialdehyde
  • Tremella heteropolysaccharide dose grouping (%) keratinocytes fibroblasts
  • the experimental sample was obtained from the Tremella heteropolysaccharide (average molecular weight of 850,000 Daltons) obtained by the purification of Example 3 and having a lower average molecular weight, and purified by Example 12.
  • the Tremella fuciformis was mixed with distilled water to prepare a concentration of 1.0%, and the same concentration of hyaluronic acid and glycerol were used as reference controls.
  • the water loss rate (%) at different times was measured under the conditions of a temperature of 25 Torr and a relative humidity of 34%.
  • Tremella heteropolysaccharide group water-based
  • the 1.0% Tremella heteropolysaccharide also had a higher moisturizing ability than 1.0% glycerol when exposed to the test environment for a long period of time, indicating that the Tremella fuciformis of the present invention is an excellent moisturizer.
  • the experimental sample was obtained from the Tremella heteropolysaccharide (average molecular weight of 850,000 Daltons) obtained by the purification of Example 3 and having a lower average molecular weight, and purified by Example 12.
  • Method 1 The white fungus heteropolysaccharide, honey and maltodextrin were separately formulated into 0.1%, 0.3%, 0.5%, 1% application liquid with distilled water, and water was used as a reference control (slip index is 0) .
  • slip index is 0
  • 40 volunteers (20 males and 20 females) were subjected to a drinking blind test to record the lubrication feeling during drinking (no lubricity index is 0, slightly lubricity is index 1, strong) Lubrication is index 2, strong lubrication is index 3), the statistics are shown in the table below and Figure 7.
  • the lubricious feeling of the aqueous solution of Tremella fuciformis is better than that of the same concentration of honey and maltodextrin aqueous solution. It indicates that the silver fungus heteropolysaccharide can significantly enhance the lubrication of the solution.
  • Method 2 Mixing Tremella polysaccharides, glycerin and silicone oil into 0.1%, 0.3%, 0.5%, 1% application liquid (silicone oil emulsion) with distilled water, using water as a reference control (lubrication index) Is 0).
  • lubrication index a reference control
  • 40 volunteers (20 males and 20 females) were subjected to a hand-back blind test, and the lubrication feeling at the time of application was recorded.
  • the non-lubricating index was 0, and the slightly lubricious feeling was index 1.
  • Strong lubrication is index 2
  • strong lubrication refers to The number 3), the statistics are shown in the table below and Figure 8.
  • the lubrication is significantly better than the same concentration of glycerin and silicone oil emulsion. The test shows that the Tremella heteropolysaccharide can enhance the application of the solution.
  • Tremella heteropolysaccharide has anti-allergic and stimulating function '
  • the experimental sample was obtained from the Tremella heteropolysaccharide (average molecular weight of 850,000 Daltons) obtained by the purification of Example 3 and having a lower average molecular weight, and purified by Example 12.
  • a blind test was conducted from 24 volunteers (12 males and 12 females), each of which received 10 ml of the sensitizing stimulator SLS (sodium lauryl sulfate), respectively, with 0.3% (w/w) aqueous solution of Tremella heteropolysaccharide 100 ml and distilled water (control) 100ml, add 1ml of the above mixture to the filter paper, and then attach it to the inner skin of the arm with aluminum buckle, and observe for 30 hours (every 2 hours/time). Record the diameter of each allergic spot and ask for itchy skin. And the feeling of pain, and record the index, take the average to draw a 30-hour curve (as shown in Figures 9, 10).
  • SLS sodium lauryl sulfate
  • Itching sensation index no feeling is 0, slightly itching is 1 , itching is 2, itching is 3, slightly pain is 4, mild pain is 5, tingling or burning is 6;
  • Allergic spots have an aluminum buckle diameter of 100% and no redness of 0%.
  • Tremella fuciformis has a protective effect on the skin, which can obviously resist and block the invasion of sensitizing stimuli, and has certain functions of inhibiting pain and swelling. Therefore, Tremella fuciformis has anti-allergic and anti-irritant effects.

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Abstract

La présente invention décrit des hétéropolysaccharides de trémelle, dans lesquels le α-(1-3)mannose représente la chaîne structurelle principale. Les hétéropolysaccharides de trémelle mentionnés ci-dessus sont principalement constitués de 70 à 93 % en poids de saccharides totalement neutres, de 6 à 28 % en poids d'acide glucuronique et de 0,1 à 3 % en poids de protéine conjuguée. La masse moléculaire moyenne des hétéropolysaccharides de trémelle est de 850 000 à 1 600 000 Daltons, le pourcentage de la portion de plus de 6000 Daltons représentant plus de 90 % en poids. La présente invention décrit également leurs produits d’extraction et le procédé de préparation de ceux-ci. Les hétéropolysaccharides de trémelle et leurs produits d’extraction selon la présente invention ont de nombreuses propriétés telles que le maintien de la peau et des muqueuses humides et douces, l’augmentation d’une sensation glissante au toucher, l’anti-oxydation, l’anti-anaphylaxie, l’épaississement, la stabilisation, etc.
PCT/CN2005/001459 2005-01-18 2005-09-12 Heteropolysaccharides de tremelle, leurs produits d’extraction, procede de preparation et utilisations de ceux-ci WO2006076841A1 (fr)

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