WO2022241973A1 - 一种托酚酮-壳聚糖衍生物及其制备方法和应用 - Google Patents

一种托酚酮-壳聚糖衍生物及其制备方法和应用 Download PDF

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WO2022241973A1
WO2022241973A1 PCT/CN2021/117058 CN2021117058W WO2022241973A1 WO 2022241973 A1 WO2022241973 A1 WO 2022241973A1 CN 2021117058 W CN2021117058 W CN 2021117058W WO 2022241973 A1 WO2022241973 A1 WO 2022241973A1
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tropolone
chitosan
chitosan derivative
alkyl
preparation
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French (fr)
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巫世贵
敖卓玲
张璟
穆博
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珠海市自然之旅生物技术有限公司
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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
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3562—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/736—Chitin; Chitosan; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of antibacterial substances, and in particular relates to a tropolone-chitosan derivative and a preparation method and application thereof.
  • Chitin is a natural polymer compound, which is formed by connecting N-acetyl-D-glucosamine and D-glucosamine through ⁇ -(1-4) glycosidic bonds. It can be partially or completely deacetylated to obtain the commonly used chitosan. Most chitosan on the market is obtained from shrimp and crab shells through a series of treatments (acid washing or alkali washing). It is non-toxic and tasteless, and has been widely used in food, medicine, cosmetics and other industries with excellent moisturizing properties Sex, immunomodulatory activity, antibacterial and other advantages. However, chitosan also has some limitations.
  • chitosan is insoluble in water and can only be dissolved under acidic conditions;
  • the positive charge carried is easy to flocculate with the anion raw materials in the system, resulting in poor stability of the system.
  • Chitosan contains amino groups and hydroxyl groups, and can be chemically modified under relatively mild reaction conditions.
  • water-soluble chitosan derivatives such as carboxymethyl chitosan
  • the antibacterial properties of the modified chitosan derivatives are often poor, resulting in a better antibacterial effect, usually need to add a higher concentration of chitosan derivatives, which is not conducive to Applications of chitosan derivatives.
  • the existing chitosan derivatives have poor stability to high temperature (for example, 120° C.), and the antibacterial performance of chitosan derivatives treated at high temperature decreases significantly.
  • the present invention aims to solve at least one of the technical problems in the above-mentioned prior art. For this reason, the present invention proposes a kind of tropolone-chitosan derivative and its preparation method and application, described tropolone-chitosan derivative has good antibacterial property, especially through high temperature (for example greater than 140 °C , specifically have 150 °C, 180 °C) processed tropolone-chitosan derivatives, still have good antibacterial properties, as seen the tropolone-chitosan derivatives of the present invention have good stability to temperature .
  • high temperature for example greater than 140 °C , specifically have 150 °C, 180 °C
  • the present invention uses chitosan or chitosan derivatives, and tropolone or substances containing tropolone structural units as reaction raw materials, and these two types of reactants react to obtain a Compound (also can be referred to as a kind of salt), the compound described in the present invention has good antibacterial property, especially has good high temperature resistance stability (such as 150 °C, 180 °C), good solubility, low preparation cost, very Facilitate the application of the compound.
  • a Compound also can be referred to as a kind of salt
  • the compound described in the present invention has good antibacterial property, especially has good high temperature resistance stability (such as 150 °C, 180 °C), good solubility, low preparation cost, very Facilitate the application of the compound.
  • the first aspect of the present invention provides a tropolone-chitosan derivative.
  • tropolone-chitosan derivative the structural formula of described tropolone-chitosan derivative is as shown in formula (1):
  • R represents at least one of -H, hydroxyl-containing alkyl or carboxyl-containing alkyl
  • R represents at least one of -H, hydroxyl - containing alkyl, carboxyl-containing alkyl or carbonyl-containing alkyl;
  • R 3 , R 4 , R 5 , R 6 , and R 7 each independently represent at least one of -H, hydroxyl, carboxyl, alkenyl, substituted alkyl or unsubstituted alkyl;
  • the x and y independently represent positive integers.
  • the substituted alkyl groups independently represented by R 3 , R 4 , R 5 , R 6 , and R 7 include alkylcarbonyl, alkoxy, alkyl ester, and alkylhydroxy, such as -OCH 3 , -COCH 3 .
  • the R 1 is at least one selected from -H, -CH 2 COOH, -CH 2 CH 2 OH or -CH 2 CH 2 (OH)CH 3 .
  • the R 2 is at least one selected from -H, -COCH 3 , CH 2 COOH or CH 2 CH 2 (OH)CH 3 .
  • the R 3 is at least one selected from -H, -OH or -CH(CH 3 ) 2 .
  • the R 4 is at least one selected from -H, -CH(CH 3 ) 2 , -CH ⁇ CH 2 CH 3 , -COCH 3 or -COOH.
  • the R 6 is at least one selected from -H or -OH.
  • the R 7 is at least one selected from -H or -OCH 3 .
  • the value range of x is 1-50000; further preferably, the value range of x is 1-46300.
  • the value range of y is 1-50000; further preferably, the value range of y is 1-46300.
  • the raw material components for preparing the tropolone-chitosan derivatives include chitosan or chitosan derivatives, and tropolone or substances containing tropolone structural units.
  • the deacetylation degree of the chitosan derivative is between 60%-100%, preferably between 80%-99%, and the molecular weight of the chitosan derivative can be between 100-10000000Da.
  • the lower the amino substitution degree of the chitosan derivative the better, preferably less than 10%. Because the lower the degree of amino substitution, more amino groups can be released, and the chitosan derivative can react with tropolone or a substance containing tropolone structural unit, thereby enhancing the antibacterial property of the final product.
  • the degree of substitution of hydroxyl groups at the 3rd and 6th positions of the chitosan derivative can be 1-100%, preferably 60-100%. This helps to increase the water solubility of chitosan derivatives.
  • the chitosan derivative is selected from at least one of carboxymethyl chitosan, carboxyethyl chitosan, carboxyethyl chitosan, potassium salt of chitosan or sodium salt of chitosan kind.
  • the substance containing tropolone structural unit is selected from the group consisting of ⁇ -thujen, ⁇ -thujen, ⁇ -thujen, thujol, 4-acetyl-tropolone, preconditions, 3,6-Dihydroxy-5-oxo-1,3,6-cycloheptatriene-1-carboxylic acid, ⁇ -axocenol, ⁇ -thukiol, iso-porphyrin or mannitol (or D - at least one of mannitol).
  • the second aspect of the present invention provides a preparation method of tropolone-chitosan derivatives.
  • a kind of preparation method of tropolone-chitosan derivative comprises the following steps:
  • the mass ratio of the chitosan or chitosan derivative to tropolone or the substance containing tropolone structural unit is 5:(0.8-5); further preferably, the chitosan or chitosan
  • the mass ratio of the polysaccharide derivative to tropolone or a substance containing a tropolone structural unit is 5:(1-5). The less the amount of tropolone or the substance containing the tropolone structural unit is, the lower the preparation cost is.
  • the solvent contains water of organic acid, which helps the dissolving of chitosan.
  • the temperature of the heating reaction is 55-90°C; further preferably, the temperature of the heating reaction is 58-65°C.
  • the time for the heating reaction is more than 1 hour; more preferably, the time for the heating reaction is 1-3 hours.
  • the product is freeze-dried to obtain a powdery product.
  • the product is formulated into a solution with a mass fraction of 2-35%; further preferably, the product is formulated into a solution with a mass fraction of 3-30%.
  • the tropolone-chitosan derivative can be purified by washing with alcohol (such as ethanol).
  • Alcohol such as ethanol
  • Tropolone-chitosan derivatives are insoluble in ethanol.
  • Chitosan or chitosan derivatives, and tropolone or substances containing tropolone structural units are easily soluble in ethanol.
  • the third aspect of the present invention provides the application of a tropolone-chitosan derivative.
  • tropolone-chitosan derivatives in the preparation of antibacterial substances.
  • tropolone-chitosan derivative in the fields of medicine, cosmetics or food.
  • the present invention takes chitosan or chitosan derivatives, and tropolone or the material containing tropolone structural unit as reaction raw materials, and these two types of reactants react to obtain tropolone-shell by electrostatic interaction.
  • the polysaccharide derivatives, the tropolone-chitosan derivatives in the present invention have good antibacterial properties, and have good antibacterial effects on Gram-positive bacteria, Gram-negative bacteria, yeast and mold.
  • the tropolone-chitosan derivatives described in the present invention have good high temperature stability (such as 150°C, 180°C) and good solubility, which is very beneficial to the application of the tropolone-chitosan derivatives .
  • Fig. 1 is the proton nuclear magnetic resonance spectrogram of the tropolone-chitosan derivative that embodiment 1 makes.
  • the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels, or can be obtained by existing known methods.
  • Embodiment 1 the preparation of tropolone-chitosan derivative
  • R 1 represents -H
  • R 2 represents carboxymethyl
  • the value of x is 970
  • the value of y is 330.
  • the preparation method of above-mentioned tropolone-chitosan derivative comprises the following steps:
  • Fig. 1 is the proton nuclear magnetic resonance spectrogram of the tropolone-chitosan derivative that embodiment 1 makes. What " 1 " in Fig. 1 represents is the hydrogen nuclear magnetic resonance spectrum of beta-thujenin, and what "2" represents is the hydrogen nuclear magnetic resonance spectrum of the tropolone-chitosan derivative that embodiment 1 makes.
  • the upper left corner in Figure 1 is an enlargement of chemical shifts of 6.8-7.5 ppm.
  • 1 H-NMR analysis of the tropolone-chitosan derivative prepared in Example 1 confirmed the structure of the tropolone-chitosan derivative product prepared in Example 1.
  • the signals in the 1 H-NMR spectrum represent protons in different electronic environments and thus can be used to identify specific chemical groups, as well as changes in electron density.
  • the H NMR spectrum of ⁇ -thujenin (“1" in Figure 1) shows signals of different protons associated with the carbon atoms of this molecule.
  • the proton distribution is as follows (ppm): 1.17-1.18 (CH 3 , isopropyl group of ⁇ -thujenin), 2.9 (CH, isopropyl group of ⁇ -thujenin), 7.19-7.5 (CH, aromatic ring carbon 3 , 4, 5, 7).
  • the peak at 4.7 ppm is the solvent peak.
  • the H NMR spectrum of the tropolone-chitosan derivative ("2" in Fig. 1 ) prepared in Example 1 shows the proton signal combined with carboxymethyl chitosan and ⁇ -orientinin.
  • the protons (ppm) bound to carboxymethyl chitosan are: 1.98 (CH, non-deacetylated), 2.68 (CH, glucosamine ring carbon 2), 3.31-3.86 (CH, glucosamine ring carbon 3, 4 and 6), 3.91 (CH, carboxymethylated).
  • the number of protons combined with ⁇ -thujen is arranged in the following order (ppm): 1.14-1.1 (CH 3 , isopropyl group of ⁇ -thujen), 2.78 (CH, isopropyl group of ⁇ -thujen) .
  • Figure 1 confirms that both ⁇ -orientinin and carboxymethyl chitosan structures exist in tropolone-chitosan derivatives, and no covalent modification occurs.
  • Figure 1 also confirms that there is an interaction between ⁇ -thujen and carboxymethyl chitosan, because the protons of the aromatic ring of ⁇ -thujen (7.19-7.5ppm in its natural form) have moved to the front field (6.80-7.24ppm in the tropolone-chitosan derivative prepared in Example 1). This shift can be seen in the upper left corner of Figure 1 in the zoomed-in section for chemical shifts of 6.8-7.5 ppm.
  • Embodiment 2 the preparation of tropolone-chitosan derivative
  • a preparation method of tropolone-chitosan derivatives comprising the following steps:
  • carboxymethyl chitosan Take by weighing 10g carboxymethyl chitosan (the molecular weight of carboxymethyl chitosan is 300000Da, the deacetylation degree is 85%, the O substitution degree is 75%, the N substitution degree is 5%), dissolves in 1L water, and adds 1.5g ⁇ -Arborvitae, magnetically stirred at 150 rpm, and heated at 70°C for 1.5 hours, then freeze-dried, and the product was ground into powder, washed 5 times with ethanol, then freeze-dried, and ground into powder again, Tropolone-chitosan derivatives were prepared.
  • Embodiment 3 the preparation of tropolone-chitosan derivative
  • a preparation method of tropolone-chitosan derivatives comprising the following steps:
  • carboxybutyl chitosan Take by weighing 100g carboxybutyl chitosan (the molecular weight of carboxybutyl chitosan is 300000Da, the degree of deacetylation is 90%, the degree of O substitution is 75%, the degree of N substitution is 6%), dissolve in 1L of water, and add 40g D-mannitol was stirred magnetically at 150 rev/min, and heated at 75°C for 1.2 hours to produce a tropolone-chitosan derivative, and the prepared tropolone-chitosan derivative was reacted The mass concentration in the final solution was 14%.
  • Embodiment 4 the preparation of tropolone-chitosan derivative
  • a preparation method of tropolone-chitosan derivatives comprising the following steps:
  • carboxyethyl chitosan (the molecular weight of carboxyethyl chitosan is 350000Da, the degree of deacetylation is 80%, the degree of O substitution is 75%, the degree of N substitution is 5%), dissolve it in 1L of water, and add 5g of ⁇ -axacine, stirred magnetically at 150 rpm, and heated at 80°C for 1 hour, then freeze-dried, and the product was ground into powder, washed with ethanol for 5 times, then freeze-dried, and ground into powder again to prepare Tropolone-Chitosan Derivatives.
  • Embodiment 5 the preparation of tropolone-chitosan derivative
  • a preparation method of tropolone-chitosan derivatives comprising the following steps:
  • chitosan the molecular weight of chitosan is 250,000Da, the degree of deacetylation is 92%, the degree of O substitution is 75%, and the degree of N substitution is 5%
  • dissolve it in 1L of water add 10mL of acetic acid, and then add 8g of ⁇ -thugophenol, magnetically stirred at 150 rpm, and heated at 60°C for 1.5 hours, then freeze-dried, and the product was ground into powder, washed with ethanol for 5 times, then freeze-dried, and ground into powder again to prepare Tropolone-Chitosan Derivatives.
  • Embodiment 6 the preparation of tropolone-chitosan derivative
  • Example 6 Compared with Example 5, in Example 6, tropolone was used to replace ⁇ -Thukiol, and the rest of the preparation process was the same as in Example 5.
  • the cultured system is a commercially available nutrient broth (provided by Guangdong Huankai Microbial Technology Co., Ltd., model 022010), the pH of the cultured system is 6, and the culture conditions of the bacteria are as follows: at a temperature of 36 ° C for 7 days, The culture conditions of the yeast and the mold were cultured at 28° C. for 7 days, and the results are shown in Table 1.
  • Table 1 antibacterial effect (data in table 1 represent MIC, unit ppm)
  • the tropolone-chitosan derivatives prepared in Examples 1-6 of the present invention have better antibacterial effects than carboxymethyl chitosan. From Table 1, it can be seen that ⁇ -abrevitin also has good antibacterial properties, but compared with the antibacterial properties of the tropolone-chitosan derivatives obtained in Examples 1-6 of the present invention, ⁇ -orientinin The antibacterial performance of the antibacterial property is still relatively weak (except Pseudomonas aeruginosa), and the commercially available price of ⁇ -thujen is more than 3 times of the price of the tropolone-chitosan derivative that the embodiment of the present invention 1-6 makes , so the tropolone-chitosan derivatives prepared in Examples 1-6 of the present invention are cost-effective.
  • Get the tropolone-chitosan derivative that embodiment 1 makes test it through different temperature conditions treatment (different temperature conditions are specifically divided into: normal temperature 25 °C process 1 hour, high pressure wet heat treatment (101KPa, 121 °C, 15 minutes ), the antibacterial effect after 150° C. of normal pressure oil bath for 1 hour, and 180° C. of normal pressure oil bath for 1 hour), and then the stability of the tropolone-chitosan derivatives obtained in Example 1 can be known.
  • the antibacterial results are shown in Table 2.
  • the cultured system is a commercially available nutrient broth (provided by Guangdong Huankai Microbial Technology Co., Ltd., model 022010), the pH of the cultured system is 6, and the culture conditions of the bacteria are as follows: at a temperature of 36 ° C for 7 days, The culture conditions of the yeast and mold were cultured at 28° C. for 7 days.
  • the tropolone-chitosan derivatives prepared in the remaining examples have similar high-temperature stability as the tropolone-chitosan derivatives prepared in the above-mentioned Example 1.
  • Solubility (g/100g water) Solubility Chitosan ⁇ 0.01 insoluble/hardly soluble ⁇ -Arborvitin ⁇ 0.01 insoluble/hardly soluble Example 1 10 Soluble Example 2 10 Soluble Example 3 >10 soluble Example 4 10 Soluble Example 5 10 Soluble
  • the tropolone-chitosan derivative that the embodiment of the present invention 1-6 makes has better water solubility relative to chitosan and ⁇ -thujen.
  • the application and effect of chitosan derivatives have a very important influence.
  • Test strains Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa; fungi: Aspergillus niger, Candida albicans.
  • TSB solid medium tryptone soy medium
  • SDB solid medium Sabouraud dextrose medium
  • lecithin Tween 80 nutrient agar medium tiger red medium.
  • Inoculation of bacterial strains each bacterial strain was activated and subcultured for 3 generations to make a certain concentration of bacterial suspension.
  • the amount of bacteria contained in the sample is 1 ⁇ 10 6 cfu/mL or 1 ⁇ 10 6 cfu/g, and the amount of fungi contained is 1 ⁇ 10 4 cfu/mL or 1 ⁇ 10 4 cfu/g;
  • Hydrophobic samples Weigh 10g of the sample for testing, put it in a sterilized mortar, add 10mL of sterilized liquid paraffin, and grind it into a viscous Thick, then add 10mL sterilized Tween 80, grind until dissolved, add 70mL sterilized normal saline, mix thoroughly in a 45°C water bath to make a 1:10 suspension, and then make a 10-fold dilution series concentration The gradient test solution is ready for use;
  • the sample contains bacteria at 10 2 cfu/g-10 3 cfu/g (or cfu/mL), and the sample can pass the challenge test conditionally, that is, when the product contains protein or other animal and plant materials
  • the composition is not particularly high, and the hygienic environment of the production meets the requirements, and the packaging is not prone to secondary pollution, the anti-corrosion system can be used, otherwise it cannot;
  • the sample contains bacteria at 10cfu/g-100cfu/g (or cfu/mL), indicating that the antiseptic system of the sample has a strong inhibitory effect on microorganisms.
  • the product is in production, Not susceptible to microbial contamination during storage and use;
  • the ingredients of the test sample cream are shown in Table 4.
  • composition of the sample essence for inspection is shown in Table 5.
  • Table 5 Component list of the sample essence for inspection
  • Table 9 shows the antiseptic challenge results of the blank control essence (that is, without the tropolone-chitosan derivative prepared in Example 1).
  • tropolone-chitosan derivatives of the present invention have better inhibitory and killing effects, broad spectrum and high efficiency, and have broad application prospects.
  • Oral daily products mainly include toothpaste, mouthwash, tooth powder, etc.
  • the application of tropolone-chitosan derivatives of the present invention in such oral daily products can not only solve the above-mentioned antiseptic problems, but also act on oral cavity Anaerobic bacteria, inhibit the reproduction of oral anaerobic bacteria in the oral cavity.
  • oral anaerobic infection is bad breath after getting up in the morning and gradually aggravating, followed by symptoms such as dry mouth, pulpitis, periodontitis, gingivitis, nasal congestion or gingival swelling and pain.
  • the anaerobic bacteria that cause this series of problems include Fusobacterium nucleatum subsp.
  • the embodiment of the present invention 1-6 produces The obtained tropolone-chitosan derivatives all have strong inhibitory effects on the above oral anaerobic bacteria, and the antibacterial results are shown in Table 10 below.
  • Table 10 Minimum inhibitory concentration (MIC, unit ppm) of tropolone-chitosan derivatives to oral anaerobic bacteria
  • tropolone-chitosan derivatives prepared by the present invention have a strong inhibitory effect on oral anaerobic bacteria, thereby effectively reducing the symptoms of oral anaerobic bacteria infection.

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Abstract

本发明属于抗菌物质技术领域，公开了一种托酚酮-壳聚糖衍生物及其制备方法和应用。该托酚酮-壳聚糖衍生物的结构式如式(1)所示。其中，R1表示-H、含羟基的烷基或含羧基的烷基中的至少一种；R2表示-H、含羟基的烷基、含羧基的烷基或含羰基的烷基中的至少一种；R3、R4、R5、R6、R7分别独立表示-H、羟基、羧基、烯基、取代的烷基或未取代的烷基中的至少一种；所述x和y分别独立表示正整数。该托酚酮-壳聚糖衍生物具有良好的抗菌性，对革兰氏阳性菌、革兰氏阴性菌、酵母和霉菌具有很好的抗菌效果，同时具备良好的耐高温稳定性，溶解性良好，十分有利于托酚酮-壳聚糖衍生物在医药、化妆品或食品领域中的应用。

Description

一种托酚酮-壳聚糖衍生物及其制备方法和应用 技术领域
本发明属于抗菌物质技术领域，特别涉及一种托酚酮-壳聚糖衍生物及其制备方法和应用。
背景技术
甲壳素是一种天然高分子化合物，它是由N-乙酰-D-葡萄糖胺和D-葡萄糖胺通过β-(1-4)糖苷键连接而成。可以对其部分或完全脱乙酰化，得到我们常用的壳聚糖。市面上大多数壳聚糖是由虾、蟹壳等经一系列处理(酸洗或碱洗)而得到，无毒、无味，已广泛应用于食品、医药、化妆品等行业中，具有优越的保湿性、免疫调节活性、抗菌性等优点。然而壳聚糖也存在一些局限性，一是壳聚糖不溶于水，只能在酸性条件下溶解；二是作为一种抗菌剂，需要添加至较高浓度，导致性价比不高；三是其携带的正电荷易与体系中的阴离子原料发生絮凝，导致体系的稳定性较差。
壳聚糖含有氨基和羟基，在较温和的反应条件下可进行化学改性，在医药、化妆品领域中，水溶性壳聚糖衍生物(例如羧甲基壳聚糖)较为常用。但现有的相关技术中，经过改性后的壳聚糖衍生物的抗菌性能往往不佳，导致要达到较好的抗菌效果，通常需要加入较高浓度的壳聚糖衍生物，这不利于壳聚糖衍生物的应用。另外，现有的壳聚糖衍生物对高温(例如120℃)的稳定性较差，经过高温处理过的壳聚糖衍生物的抗菌性能下降明显。
因此，亟需提供一种新的壳聚糖衍生物，该壳聚糖衍生物的抗菌性较之前有显著提升，稳定性良好，应用成本较低，这十分有助于壳聚糖衍生物的应用。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出一种托酚酮-壳聚糖衍生物及其制备方法和应用，所述托酚酮-壳聚糖衍生物具有良好的抗菌性能，特别是经过高温(例如大于140℃，具体的有150℃、180℃)处理后的托酚酮-壳聚糖衍生物，仍然具有良好的抗菌性能，可见本发明所述托酚酮-壳聚糖衍生 物对温度的稳定性良好。
本发明的发明构思：本发明以壳聚糖或壳聚糖衍生物，以及托酚酮或含托酚酮结构单元的物质为反应原料，这两类反应物通过静电交互作用，反应得到一种化合物(也可称为一种盐)，本发明所述化合物具有良好的抗菌性，特别是具备良好的耐高温稳定性(例如150℃、180℃)，溶解性良好，制备成本较低，十分有利于所述化合物的应用。
本发明的第一方面提供一种托酚酮-壳聚糖衍生物。
具体的，一种托酚酮-壳聚糖衍生物，所述托酚酮-壳聚糖衍生物的结构式如式(1)所示：
Figure PCTCN2021117058-appb-000001
其中，R 1表示-H、含羟基的烷基或含羧基的烷基中的至少一种；
R 2表示-H、含羟基的烷基、含羧基的烷基或含羰基的烷基中的至少一种；
R 3、R 4、R 5、R 6、R 7分别独立表示-H、羟基、羧基、烯基、取代的烷基或未取代的烷基中的至少一种；
所述x和y分别独立表示正整数。
优选的，所述R 3、R 4、R 5、R 6、R 7分别独立表示的取代的烷基包括烷羰基、烷氧基、烷酯基、烷羟基，例如-OCH 3、-COCH 3。
优选的，所述R 1选自-H、-CH 2COOH、-CH 2CH 2OH或-CH 2CH 2(OH)CH 3中的至少一种。
优选的，所述R 2选自-H、-COCH 3、CH 2COOH或CH 2CH 2(OH)CH 3中的至少一 种。
优选的，所述R 3选自-H、-OH或-CH(CH 3) 2中的至少一种。
优选的，所述R 4选自-H、-CH(CH 3) 2、-CH＝CH 2CH 3、-COCH 3或-COOH中的至少一种。
优选的，所述R 5选自-H、-CH(CH 3) 2、-CH 2CH＝C(CH3) 2或-CH 2CH 2C(OH)(CH 3) 2中的至少一种。
优选的，所述R 6选自-H或-OH中的至少一种。
优选的，所述R 7选自-H或-OCH 3中的至少一种。
优选的，所述x的取值范围为1-50000；进一步优选的，所述x的取值范围为1-46300。
优选的，所述y的取值范围为1-50000；进一步优选的，所述y的取值范围为1-46300。
优选的，制备所述托酚酮-壳聚糖衍生物的原料组分包括壳聚糖或壳聚糖衍生物，以及托酚酮或含托酚酮结构单元的物质。
优选的，所述壳聚糖衍生物的脱乙酰度为60％-100％，优选80％-99％之间，壳聚糖衍生物的分子量可为100-10000000Da之间。
优选的，所述壳聚糖衍生物，氨基取代度越低越好，优选的低于10％。因为氨基取代度越低可释放出更多的氨基，使壳聚糖衍生物与托酚酮或含托酚酮结构单元的物质反应，增强了最终产物的抗菌性。
优选的，所述壳聚糖衍生物的第3、6位的羟基取代度可为1-100％，优选60-100％。这样有助于提高壳聚糖衍生物的水溶性。
优选的，所述壳聚糖衍生物选自羧甲基壳聚糖、羧乙基壳聚糖、羧乙基壳聚糖、壳聚糖的钾盐或壳聚糖的钠盐中的至少一种。
优选的，所述含托酚酮结构单元的物质选自α-侧柏素、β-侧柏素、γ-侧柏素、侧柏素酚、4-乙酰基-托酚酮、前提素、3,6-二羟基-5-氧代-1,3,6-环庚三烯-1-羧酸、β-斧松素、α-罗汉柏酚、异矮柏醚或甘露醇(或D-甘露糖醇)中的至少一种。
本发明的第二方面提供一种托酚酮-壳聚糖衍生物的制备方法。
具体的，一种托酚酮-壳聚糖衍生物的制备方法，包括以下步骤：
将壳聚糖或壳聚糖衍生物溶解于溶剂中，然后加入托酚酮或含托酚酮结构单元的物质，加热反应，制得所述托酚酮-壳聚糖衍生物。
优选的，所述壳聚糖或壳聚糖衍生物与托酚酮或含托酚酮结构单元的物质的质量比为5:(0.8-5)；进一步优选的，所述壳聚糖或壳聚糖衍生物与托酚酮或含托酚酮结构单元的物质的质量比为5:(1-5)。托酚酮或含托酚酮结构单元的物质的用量越少，制备成本越低。
优选的，所述溶剂中含有有机酸的水，有助于壳聚糖的溶解。
优选的，所述加热反应的温度为55-90℃；进一步优选的，所述加热反应的温度为58-65℃。
优选的，所述加热反应的时间为1小时以上；进一步优选的，所述加热反应的时间为1-3小时。
优选的，所述加热反应结束后，对产物进行冻干，获得粉末状的产物。
优选的，所述加热反应结束后，将产物配制成质量分数为2-35％的溶液；进一步优选的，将产物配制成质量分数为3-30％的溶液。
优选的，所述加热反应结束后，可使用醇(例如乙醇)洗涤，可提纯托酚酮-壳聚糖衍生物。托酚酮-壳聚糖衍生物不溶于乙醇。壳聚糖或壳聚糖衍，以及托酚酮或含托酚酮结构单元的物质易溶于乙醇。
本发明的第三方面提供一种托酚酮-壳聚糖衍生物的应用。
上述托酚酮-壳聚糖衍生物在制备抗菌物质中的应用。
所述的托酚酮-壳聚糖衍生物在医药、化妆品或食品领域中的应用。
相对于现有技术，本发明的有益效果如下：
(1)本发明以壳聚糖或壳聚糖衍生物，以及托酚酮或含托酚酮结构单元的物质为反应原料，这两类反应物通过静电交互作用，反应得到托酚酮-壳聚糖衍生物，本发明所述托酚酮-壳聚糖衍生物具有良好的抗菌性，对革兰氏阳性菌、革兰氏阴性菌、酵母和霉菌具有很好的抗菌效果。
(2)本发明所述托酚酮-壳聚糖衍生物具备良好的耐高温稳定性(例如150℃、 180℃)，溶解性良好，十分有利于托酚酮-壳聚糖衍生物的应用。
(3)本发明所述托酚酮-壳聚糖衍生物的制备过程以及纯化过程简单，制备成本低，可广泛应用在医药、化妆品或食品领域。
附图说明
图1为实施例1制得的托酚酮-壳聚糖衍生物的核磁共振氢谱图。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案，现列举以下实施例进行说明。需要指出的是，以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明，均可从常规商业途径得到，或者可以通过现有已知方法得到。
实施例1：托酚酮-壳聚糖衍生物的制备
本实施例1制得的托酚酮-壳聚糖衍生物的结构式如式(2)所示：
Figure PCTCN2021117058-appb-000002
其中R 1表示-H，R 2表示羧甲基，x取值为970，y的取值为330。
上述托酚酮-壳聚糖衍生物的制备方法，包括以下步骤：
称取10g羧甲基壳聚糖钠(羧甲基壳聚糖钠的分子量为280000Da，脱乙酰度为85％，O取代度为75％，N取代度为5％)，溶于1L水中，并加入2gβ-侧柏素，于150转/分钟磁力搅拌，并在60℃加热反应1小时，随后冷冻干燥，并将产物研磨成粉末，用乙醇清洗5次，然后冷冻干燥，再次研磨成粉末，制得托酚酮-壳聚糖衍生物。
图1为实施例1制得的托酚酮-壳聚糖衍生物的核磁共振氢谱图。图1中的“1”表示的是β-侧柏素的核磁共振氢谱，“2”表示的是实施例1制得的托酚酮-壳聚糖衍生物的核磁共振氢谱。图1中的左上角是对化学位移为6.8-7.5ppm的放大。
对实施例1制得的托酚酮-壳聚糖衍生物进行 1H-NMR分析证实了实施例1制得的托酚酮-壳聚糖衍生物产物的结构。 1H-NMR谱中的信号代表不同电子环境中的质子，因此可以用来识别特定的化学基团，以及电子密度的变化。
β-侧柏素的核磁共振氢谱(图1中的“1”)显示了与这个分子的碳原子有关的不同质子的信号。质子分配如下(ppm)：1.17-1.18(CH 3，β-侧柏素的异丙基)，2.9(CH，β-侧柏素的异丙基)，7.19-7.5(CH，芳香环碳3、4、5、7)。
4.7ppm处的峰为溶剂峰。实施例1制得的托酚酮-壳聚糖衍生物(图1中的“2”)的核磁共振氢谱显示了与羧甲基壳聚糖和β-侧柏素相结合的质子信号。与羧甲基壳聚糖结合的质子(ppm)分别为：1.98(CH，非脱乙酰基)、2.68(CH，葡萄糖胺环碳2)、3.31-3.86(CH，葡萄糖胺环碳3、4和6)、3.91(CH，羧甲基化基)。与β-侧柏素结合的质子数按以下顺序排列(ppm)：1.14-1.1(CH 3，β-侧柏素的异丙基)，2.78(CH，β-侧柏素的异丙基)。
图1证实了β-侧柏素和羧甲基壳聚糖结构均存在于托酚酮-壳聚糖衍生物中，且未发生共价修饰。图1还证实了β-侧柏素和羧甲基壳聚糖之间存在相互作用，因为β-侧柏素的芳香环的质子(在其自然形式下为7.19-7.5ppm)已经移动到了前场(在实施例1制得的托酚酮-壳聚糖衍生物中的6.80-7.24ppm)。此转移在图1中的左上角对化学位移为6.8-7.5ppm的放大部分可以看出。
核磁共振氢谱图的变化与电子密度的变化有关。图1显示β-侧柏素的芳香环的电子密度增加。β-侧柏素结构形成了一个阴离子，通过其共振结构使负电荷离域且稳定。
实施例2：托酚酮-壳聚糖衍生物的制备
一种托酚酮-壳聚糖衍生物的制备方法，包括以下步骤：
称取10g羧甲基壳聚糖(羧甲基壳聚糖的分子量为300000Da，脱乙酰度为85％，O取代度为75％，N取代度为5％)，溶于1L水中，并加入1.5gγ-侧柏素，于150 转/分钟磁力搅拌，并在70℃加热反应1.5小时，随后冷冻干燥，并将产物研磨成粉末，用乙醇清洗5次，然后冷冻干燥，再次研磨成粉末，制得托酚酮-壳聚糖衍生物。
实施例3：托酚酮-壳聚糖衍生物的制备
一种托酚酮-壳聚糖衍生物的制备方法，包括以下步骤：
称取100g羧丁基壳聚糖(羧丁基壳聚糖的分子量为300000Da，脱乙酰度为90％，O取代度为75％，N取代度为6％)，溶于1L水中，并加入40g D-甘露糖醇，于150转/分钟磁力搅拌，并在75℃加热反应1.2小时，制得托酚酮-壳聚糖衍生物，制得的托酚酮-壳聚糖衍生物在反应后的溶液中的质量浓度为14％。
实施例4：托酚酮-壳聚糖衍生物的制备
一种托酚酮-壳聚糖衍生物的制备方法，包括以下步骤：
称取10g羧乙基壳聚糖(羧乙基壳聚糖的分子量为350000Da，脱乙酰度为80％，O取代度为75％，N取代度为5％)，溶于1L水中，并加入5gβ-斧松素，于150转/分钟磁力搅拌，并在80℃加热反应1小时，随后冷冻干燥，并将产物研磨成粉末，用乙醇清洗5次，然后冷冻干燥，再次研磨成粉末，制得托酚酮-壳聚糖衍生物。
实施例5：托酚酮-壳聚糖衍生物的制备
一种托酚酮-壳聚糖衍生物的制备方法，包括以下步骤：
称取10g壳聚糖(壳聚糖的分子量为250000Da，脱乙酰度为92％，O取代度为75％，N取代度为5％)，溶于1L水中，并加入10mL的乙酸，再加入8gα-罗汉柏酚，于150转/分钟磁力搅拌，并在60℃加热反应1.5小时，随后冷冻干燥，并将产物研磨成粉末，用乙醇清洗5次，然后冷冻干燥，再次研磨成粉末，制得托酚酮-壳聚糖衍生物。
实施例6：托酚酮-壳聚糖衍生物的制备
与实施例5相比，实施例6中用托酚酮代替α-罗汉柏酚，其余制备过程与实施例5相同。
产品效果测试
1.抗菌效果测试
取实施例1-6制得的托酚酮-壳聚糖衍生物，以及羧甲基壳聚糖、β-侧柏素(羧 甲基壳聚糖、β-侧柏素作为对照)，测试其对细菌(包括革兰氏阳性菌和革兰氏阴性菌，其中，革兰氏阳性菌包括金黄色葡萄球菌、蜡样芽孢杆菌、枯草芽孢杆菌、植物乳酸杆菌，革兰氏阴性菌包括大肠杆菌、绿脓杆菌)、酵母(白色念珠菌)、霉菌(黑曲霉)的最低抑菌浓度(MIC)(样品的浓度梯度为10000ppm、5000ppm、4000ppm、3000ppm、2000ppm、1000ppm、500ppm、400ppm、375ppm、300ppm)。
培养的体系为市售的营养肉汤(由广东环凯微生物科技有限公司提供，型号为022010)，培养的体系的pH为6，细菌的培养条件为，在温度为36℃下培养7天，酵母和霉菌的培养条件为，在温度为28℃下培养7天，结果如表1所示。
表1：抗菌效果(表1中数据表示MIC，单位ppm)
Figure PCTCN2021117058-appb-000003
Figure PCTCN2021117058-appb-000004
备注：表1中“/”表示没有抑菌效果。
从表1可以看出，本发明实施例制1-6得的托酚酮-壳聚糖衍生物相对于羧甲基壳聚糖，具有更好的抗菌效果。从表1中可知β-侧柏素也有较好的抑菌性能，但相较于本发明实施例1-6制得的托酚酮-壳聚糖衍生物的抗菌性能，β-侧柏素的抗菌性能还是相对较弱(除了绿脓杆菌)，且β-侧柏素市售的价格是本发明实施例1-6制得的托酚酮-壳聚糖衍生物的价格的3倍以上，所以本发明实施例制1-6所制得的托酚酮-壳聚糖衍生物性价比高。
2.高温稳定性测试
取实施例1制得的托酚酮-壳聚糖衍生物，测试其经不同温度条件处理(不同温度条件具体分为：常温25℃处理1小时、高压湿热处理(101KPa，121℃，15分钟)、常压油浴150℃处理1小时、常压油浴180℃处理1小时)后的抗菌效果，进而可得知实施例1制得的托酚酮-壳聚糖衍生物的稳定性。抗菌结果如表2所示。
培养的体系为市售的营养肉汤(由广东环凯微生物科技有限公司提供，型号为022010)，培养的体系的pH为6，细菌的培养条件为，在温度为36℃下培养7天，酵母和霉菌的培养条件为，在温度为28℃下培养7天。
表2：抗菌效果(表2中数据表示最小抑菌浓度MIC，单位为ppm)
Figure PCTCN2021117058-appb-000005
Figure PCTCN2021117058-appb-000006
从表2可以看出，经过高压湿热处理1小时、常压油浴150℃处理1小时、常压油浴180℃处理1小时等条件处理后，实施例1制得的托酚酮-壳聚糖衍生物对革兰氏阳性菌、革兰氏阴性菌、酵母和酵母的抗菌效果与常温25℃保持一致，表明本发明实施例1制得的托酚酮-壳聚糖衍生物具有良好的耐高温稳定性。
其余实施例制得的托酚酮-壳聚糖衍生物具有类似上述实施例1制得的托酚酮-壳聚糖衍生物类似的耐高温稳定性。
3.溶解性能测试
取实施例1-6制得的托酚酮-壳聚糖衍生物，以及壳聚糖、β-侧柏素，测试在20℃的温度下在水中的溶解度你，结果如表3所示。
表3：溶解度测试结果
  溶解度(g/100g水) 溶解性
壳聚糖 ＜0.01 不溶/难溶
β-侧柏素 ＜0.01 不溶/难溶
实施例1 10 可溶
实施例2 10 可溶
实施例3 ＞10 易溶
实施例4 10 可溶
实施例5 10 可溶
实施例6 10 可溶
从表3可以看出，本发明实施例1-6制得的托酚酮-壳聚糖衍生物相对壳聚糖、β-侧柏素，具有更好的水溶性，这对托酚酮-壳聚糖衍生物的应用及效果的发挥有着非常重要的影响。
4.托酚酮-壳聚糖衍生物在化妆品中的防腐效果测试
试验菌株：金黄色葡萄球菌、大肠杆菌、铜绿假单胞杆菌；真菌：黑曲霉、白色念珠菌。
培养基：TSB固体培养基(胰酪大豆胨培养基)；SDB固体培养基(沙氏葡萄糖培养基)；卵磷脂吐温80营养琼脂培养基、虎红培养基。
防腐挑战实验具体步骤如下：
(1)菌株的接种：各菌株活化传代培养3代后制成一定浓度的菌悬液，供试化妆品样品制成后分装成30mL或30g的包装形式，分别加入菌悬液后，使化妆品样品含细菌的量为1×10 6cfu/mL或1×10 6cfu/g、含真菌的量为1×10 4cfu/mL或1×10 4cfu/g；
(2)供检样品的制备：称取10g供检样品，加到装有玻璃珠及90mL灭菌生理盐水的三角瓶中，充分振荡混匀，静置15min，取其上清液作为1:10的检液，然后依次制成10倍稀释系列浓度梯度检液待用，疏水性样品：称取10g供检样品，放到灭菌的研钵中，加入10mL灭菌液体石蜡，研磨成粘稠状，再加入10mL灭菌吐温80，研磨待溶解后，加入70mL灭菌生理盐水，在45℃水浴中充分混合，制成1：10的悬液，后依次制成10倍稀释系列浓度梯度检液待用；
(3)供试样品的检测：分别在加入菌液后的第0d、4h、1d、7d、14d、21d、28d(“d”表示“天”)按上述取样方法进行取样检测，取上述制备的10倍稀释系列检液1mL加入灭菌平板中，再倒入冷却至45℃的卵磷脂吐温80营养琼脂培养基或虎红培养基适量，逆时针摇动混合均匀，每个稀释梯度做2个平行的倒平板，待平板凝固后，细菌置于培养箱中37℃培养48小时后计数，真菌置于培养箱中28℃培养72小时后计数；
(4)防腐体系的效能评价标准：
(a)第28天时，样品中含细菌或霉菌>10 3cfu/g(或cfu/mL)，该样品不能通过微生物攻击的挑战性实验，表明样品的防腐体系不能有效地起到抑制微生物的作用，产品在生产、贮藏和使用中很容易受到微生物的污染；
(b)第28天时，样品中含细菌在10 2cfu/g-10 3cfu/g(或cfu/mL)，该样品有条件地通过挑战性实验，即当产品中蛋白质或其它动植物材料成分不是特别高，同时生产的卫生环境符合要求，包装物不易发生二次污染时，该防腐体系可以使用，否则不能；
(c)第28天，样品中含细菌在10cfu/g-100cfu/g(或cfu/mL)，表明该样品的防腐体系对微生物有较强的抑杀效果，通过挑战试验，产品在生产、贮藏和使用时不容易受到微生物污染；
(d)从第7天起，样品中的细菌<10cfu/g(或cfu/mL)，说明该样品的防腐体系对微生物有特强的抑杀作用，通过挑战试验，产品在生产、贮藏和使用时很不容易被微生物污染。
(5)供检样品的制备：
供检样品乳霜的成分如表4所示。
表4：供检样品乳霜成分
Figure PCTCN2021117058-appb-000007
Figure PCTCN2021117058-appb-000008
供检样品精华液的成分如表5所示。
表5:供检样品精华液成分表
Figure PCTCN2021117058-appb-000009
实施例1制得的托酚酮-壳聚糖衍生物应用于乳霜的防腐挑战结果如表6所示。表6：含实施例1制得的托酚酮-壳聚糖衍生物的乳霜的防腐挑战结果
Figure PCTCN2021117058-appb-000010
Figure PCTCN2021117058-appb-000011
空白对照乳霜(即不含实施例1制得的托酚酮-壳聚糖衍生物)的防腐挑战结果如表7所示。
表7：空白对照乳霜的防腐挑战结果
Figure PCTCN2021117058-appb-000012
含实施例1制得的托酚酮-壳聚糖衍生物的精华液的防腐挑战结果如表8所示。
表8：含实施例1制得的托酚酮-壳聚糖衍生物的精华液的防腐挑战结果
Figure PCTCN2021117058-appb-000013
Figure PCTCN2021117058-appb-000014
空白对照精华液(即不含实施例1制得的托酚酮-壳聚糖衍生物)的防腐挑战结果如表9所示。
表9：空白对照精华液的防腐挑战结果
Figure PCTCN2021117058-appb-000015
由表6-9可知，在乳霜和精华液对金黄色葡萄球菌、大肠杆菌、铜绿假单胞杆菌、黑曲霉、白色念球菌的防腐试验中，从第7天起，含实施例1制得的托酚酮-壳聚糖衍生物的样品中的细菌及真菌均<10cfu/g(或cfu/mL)，说明含实施例1制得的托酚酮-壳聚糖衍生物的样品的防腐体系对微生物有特强的抑杀作用，说明添加有本发明的托酚酮-壳聚糖衍生物的产品在生产、贮藏和使用时很不容易被微生物污染。将实施例1制得的托酚酮-壳聚糖衍生物替换成实施例2-6制得的托酚酮-壳聚糖衍生物，也具有类似的防腐效果。
由此可证，本发明的托酚酮-壳聚糖衍生物具有较好的抑制和杀死作用，广谱高 效，具有广阔的应用前景。
5.在口腔日用产品中的应用效果
口腔日用产品主要包括牙膏、漱口水、牙粉等，本发明的托酚酮-壳聚糖衍生物在这类口腔日用产品中的应用，除了能解决上述防腐的问题，也能作用于口腔厌氧菌，抑制口腔厌氧菌在口腔内的繁殖。众所周知，口腔厌氧菌感染最明显的症状就是晨起后口臭并逐渐加重，随之出现口干，牙髓炎，牙周炎，牙龈炎鼻塞或者牙龈肿痛等症状。而引起这一系列问题的厌氧菌包括具核梭杆菌聚核亚种、粘性放线菌、伴放线放线杆菌、远缘链球菌、变异链球菌等，本发明实施例1-6制得的托酚酮-壳聚糖衍生物对以上口腔厌氧菌均有较强的抑制效果，其抑菌结果如下表10所示。
表10：托酚酮-壳聚糖衍生物对口腔厌氧菌的最小抑菌浓度(MIC，单位ppm)
Figure PCTCN2021117058-appb-000016
由表10可知，本发明制得的托酚酮-壳聚糖衍生物对口腔厌氧菌有很强的抑制作用，从而能有效减轻口腔厌氧菌感染的症状。

Claims (13)

  1. 一种托酚酮-壳聚糖衍生物，其特征在于，所述托酚酮-壳聚糖衍生物的结构式如式(1)所示：
    Figure PCTCN2021117058-appb-100001
    其中，R 1表示-H、含羟基的烷基或含羧基的烷基中的至少一种；
    R 2表示-H、含羟基的烷基、含羧基的烷基或含羰基的烷基中的至少一种；
    R 3、R 4、R 5、R 6、R 7分别独立表示-H、羟基、羧基、烯基、取代的烷基或未取代的烷基中的至少一种；
    所述x和y分别独立表示正整数。
  2. 根据权利要求1所述的托酚酮-壳聚糖衍生物，其特征在于，所述R 3、R 4、R 5、R 6、R 7分别独立表示的取代的烷基包括烷羰基、烷氧基、烷酯基、烷羟基。
  3. 根据权利要求1所述的托酚酮-壳聚糖衍生物，其特征在于，所述R 1选自-H、-CH 2COOH、-CH 2CH 2OH或-CH 2CH 2(OH)CH 3中的至少一种。
  4. 根据权利要求1所述的托酚酮-壳聚糖衍生物，其特征在于，所述R 2选自-H、-COCH 3、CH 2COOH或CH 2CH 2(OH)CH 3中的至少一种。
  5. 根据权利要求1所述的托酚酮-壳聚糖衍生物，其特征在于，所述R 3选自-H、-OH或-CH(CH 3) 2中的至少一种。
  6. 根据权利要求1或2所述的托酚酮-壳聚糖衍生物，其特征在于，所述R 4选自-H、-CH(CH 3) 2、-CH＝CH 2CH 3、-COCH 3或-COOH中的至少一种。
  7. 根据权利要求1所述的托酚酮-壳聚糖衍生物，其特征在于，所述R 5选自-H、-CH(CH 3) 2、 -CH 2CH＝C(CH3) 2或-CH 2CH 2C(OH)(CH 3) 2中的至少一种。
  8. 根据权利要求1所述的托酚酮-壳聚糖衍生物，其特征在于，所述R 6选自-H或-OH中的至少一种。
  9. 根据权利要求1-4中任一项所述的托酚酮-壳聚糖衍生物，其特征在于，制备所述托酚酮-壳聚糖衍生物的原料组分包括壳聚糖或壳聚糖衍生物，以及托酚酮或含托酚酮结构单元的物质。
  10. 根据权利要求9所述的托酚酮-壳聚糖衍生物，其特征在于，所述含托酚酮结构单元的物质选自α-侧柏素、β-侧柏素、γ-侧柏素、侧柏素酚、4-乙酰基-托酚酮、前提素、3，6-二羟基-5-氧代-1，3，6-环庚三烯-1-羧酸、β-斧松素、α-罗汉柏酚、异矮柏醚或甘露醇中的至少一种。
  11. 权利要求1-10中任一项所述的托酚酮-壳聚糖衍生物的制备方法，其特征在于，包括以下步骤：
    将所述壳聚糖或壳聚糖衍生物溶解于溶剂中，然后加入所述托酚酮或含托酚酮结构单元的物质，加热反应，制得所述托酚酮-壳聚糖衍生物。
  12. 权利要求1-10中任一项所述的托酚酮-壳聚糖衍生物在制备抗菌物质中的应用。
  13. 权利要求1-10中任一项所述的托酚酮-壳聚糖衍生物在医药、化妆品或食品领域中的应用。
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