WO2021082889A1 - 一种含抗炎活性成分的组合物的环保制备方法和应用 - Google Patents

一种含抗炎活性成分的组合物的环保制备方法和应用 Download PDF

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WO2021082889A1
WO2021082889A1 PCT/CN2020/120294 CN2020120294W WO2021082889A1 WO 2021082889 A1 WO2021082889 A1 WO 2021082889A1 CN 2020120294 W CN2020120294 W CN 2020120294W WO 2021082889 A1 WO2021082889 A1 WO 2021082889A1
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extraction
composition containing
active ingredients
root
inflammatory active
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PCT/CN2020/120294
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English (en)
French (fr)
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王新亮
卢伊娜
田军
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上海珈凯生物科技有限公司
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Priority to US17/280,069 priority Critical patent/US11458180B2/en
Publication of WO2021082889A1 publication Critical patent/WO2021082889A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539—Scutellaria (skullcap)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/489—Sophora, e.g. necklacepod or mamani
    • 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
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the present invention relates to the technical field of biomedicine and cosmetics, in particular to an environmentally friendly preparation method and application of a composition containing anti-inflammatory active ingredients.
  • Scutellaria baicalensis has the effects of clearing heat, removing dampness, purging fire and detoxification, and is used for fever, pneumonia, carbuncle and boils.
  • Sophora flavescens has the functions of clearing away heat and dampness, killing insects, and diuresis, and is used for eczema, eczema, skin itching and so on.
  • Licorice has the effects of clearing away heat and toxins, eliminating phlegm and relieving cough, relieving emergency and relieving pain, and reconciling various drugs.
  • glycyrrhizic acid has an antagonistic effect on many links such as inflammation, development and response.
  • different extraction methods may have different compositions of active ingredients, resulting in different effects.
  • the traditional three-medicine co-decocting method needs to be decocted immediately, and the operation is troublesome, which is not conducive to long-term storage and inconvenient to use.
  • the patent CN2016109373139 discloses a method for ethanol extraction of "licorice, sophorae, scutellaria, angelica, chuanxiong, nepeta, parsnip and white willow bark” ", but not according to the nature of the active substance of each medicinal material in a certain order of the aqueous solution extraction method.
  • Literature 2 Zhao Yang, Zhang Tao, Jia Hongmei, et al. Optimization of the extraction process of Sophora flavescens-Glycyrrhizae and analysis of its chemical components[J].Chinese Journal of Experimental Formulas,2017(03):26-32.) A method for co-extracting licorice and matrine with a ratio of 1:1 and refluxing 60% ethanol to increase the total alkalis and glycyrrhizic acid of matrine.
  • none of the above methods provide a method for co-extracting the three medicinal materials of Sophora flavescens, Scutellaria baicalensis Georgi and licorice in aqueous solution, so as to shorten the process, reduce the steps, reduce the risk of contamination, and save energy and environmental protection.
  • the above method does not deliberately propose specific solutions for the stability of solvents, equipment, solution-type products that require attention in scale-up production, and changes in efficiency caused by process changes.
  • the purpose of the present invention is to provide an environmentally friendly preparation method of a composition containing anti-inflammatory active ingredients, specifically the three medicinal materials of Sophora flavescens root, Scutellaria baicalensis root and Glycyrrhiza inflata root
  • the preparation method of co-extracting biologically active ingredients by feeding materials in order to achieve:
  • the present invention provides an environmentally friendly preparation method of a composition containing anti-inflammatory active ingredients, which includes the following steps:
  • step S2 adjust the pH of the solution extracted in step S1, then add the root of Glycyrrhiza inflata, the temperature is raised to boiling water for extraction;
  • step S3 adding Scutellaria baicalensis root to the boiling aqueous solution extracted in step S2 for extraction, filtering, and adjusting the pH;
  • step S4 adding the filtrate obtained in step S3 to activated carbon for heat preservation and filtering;
  • step S5 Add the filtrate obtained in step S4 to a clarifying agent, stand still, and filter;
  • step S6 Perform membrane separation on the filtrate obtained in step S6 to obtain the composition containing the anti-inflammatory active ingredient.
  • the ingredient ratio of the root of Scutellaria baicalensis Georgi, Sophora flavescens root and Glycyrrhiza inflata root is 21-40:21-40:21-40.
  • the composition containing the anti-inflammatory active ingredient can be prepared.
  • the present invention specifically selects the root of Glycyrrhiza inflata, which can achieve better anti-inflammatory effect and better stability of the final product after compound extraction with the root of Scutellaria baicalensis Georgi and the root of Sophora flavescens.
  • the anti-inflammatory effect of the compound extracted with scutellaria root and flavescens root is not as good as that of the present invention, and the ingredients contained in other licorice roots will affect the color of the final product.
  • the appearance and product stability bring negative effects.
  • the amount of water used is 5-10 times the total amount of medicinal materials; the pH value is 3.0-7.0; the temperature of the high-temperature insulation extraction is 60-80°C, and the extraction time is 30 min- 2h.
  • step S2 1-10% NaOH is used to adjust the pH to 8-10, and the boiling water extraction time is 30min-2h.
  • the alkaline condition of matrine is conducive to the dissolution of glycyrrhizic acid, and the surface activity of glycyrrhizic acid is conducive to the dissolution of matrine.
  • the pH is adjusted to a slightly alkaline condition, while ensuring that matrine is not destroyed, glycyrrhizic acid is in an ionic state, which is more conducive to the dissolution of glycyrrhizic acid and other active ingredients.
  • step S3 the extraction time is 30 min-2 h; the pH is adjusted to 5.5-7.5.
  • the root of Scutellaria baicalensis contains special baicalinase, which is easy to hydrolyze baicalin, causing the decrease of baicalin; therefore, it is necessary to use boiling water to kill baicalinase and retain the components of baicalin to increase the efficacy.
  • baicalin is a flavonoid compound with multiple phenolic hydroxyl groups. It is easier to rupture plant cell walls under alkaline conditions of pH 8-10 and increase the dissolution of flavonoids in the roots of Scutellaria baicalensis Georgi.
  • matrine forms hydrogen bonds with baicalin under alkaline conditions to enhance the solubility of baicalin.
  • glycyrrhizic acid can increase the surface tension of the solution, which is more conducive to the extraction efficiency of matrine and baicalin.
  • the present invention found through preliminary tests:
  • the extraction rate of matrine can be kept unchanged, but during the extraction process, under acidic environment heating conditions, it will promote licorice.
  • the acid hydrolysis of the acid causes the glycyrrhizic acid to be hydrolyzed into glycyrrhetinic acid, thereby reducing the actual content of glycyrrhizic acid in the product by 18.7%.
  • baicalin has very poor water solubility, it must be under strong alkaline conditions to achieve a high dissolution rate. Furthermore, according to the aforementioned document 1, the solubilizing effect of glycyrrhizic acid on baicalin cannot be exerted. Compared with the patent process, the extraction rate of baicalin is reduced by 21.4%.
  • the added amount of activated carbon is 0.2-1% of the total filtrate; the holding temperature is 85-95° C., and the holding time is 0.5-1 hour.
  • the color of the obtained solution is darker, and activated carbon is used to remove part of the pigment.
  • the clarifying agent is one or a combination of chitosan, ZTC clarifying agent I-IV, and 101 juice clarifying agent, and the added amount of clarifying agent is 1-5% of the total filtrate;
  • the standing time is 0.5-1h.
  • the step of membrane separation includes first separating with a microfiltration membrane, then separating with an ultrafiltration membrane, and finally using a reverse osmosis membrane for desalination.
  • the method further includes a step of sterilizing and/or preserving the composition prepared in step S6;
  • the sterilization method adopts one of high temperature sterilization and moist heat sterilization, and is suitable for applications in the field of biomedicine through different carriers.
  • the preservative used in the preservative treatment is one or a combination of two or more of benzoic acid and salt-potassium sorbate, sodium dehydroacetate, and sodium diacetate, or an alcohol component with preservative effect, suitable for food and beverages In the use of health care products; the added amount of the preservative is 0.01-50% of the filtrate.
  • the alcohol component with antiseptic effect includes one or a combination of two or more of butanediol, 1,2-hexanediol, and glycerin, which is suitable for cosmetic applications.
  • the invention also provides an application of the composition prepared according to the aforementioned method in food, medicine, and cosmetics.
  • composition prepared by the present invention can be used in cosmetics to enhance inhibition of inflammation, so as to relieve skin sensitivity; in addition, it can also repair cell damage to achieve the effect of improving skin cell vitality; in addition, it can also promote cell gene expression, thereby repairing skin Barrier to reduce skin diseases caused by external stimuli.
  • the present invention has the following beneficial effects:
  • the present invention adopts a method in which three medicinal materials of Sophora flavescens root, Glycyrrhiza inflated root, and Scutellaria baicalensis root are successively fed and co-extracted, so that the active ingredients of the three medicinal materials promote each other's extraction efficiency during the whole extraction process, and can promote mutual stability.
  • the process steps are shortened, energy consumption is reduced, cost is lowered, and operation is simple and convenient, and it is suitable for continuous and large-scale production.
  • the method of the present invention is a green and environmentally friendly extraction technology for biologically active ingredients with broad prospects, and is suitable for industrialized production.
  • the present invention adopts a method of co-extraction of three medicinal materials such as Sophora flavescens root, Glycyrrhiza inflated root and Scutellaria root, which improves the extraction efficiency of active ingredients while shortening the process, ensuring that the process is completed in a short time, and reducing water-soluble samples In the production, due to the risk of long-term production of bacteria, it is conducive to the use and preservation of the product.
  • three medicinal materials such as Sophora flavescens root, Glycyrrhiza inflated root and Scutellaria root
  • the present invention does not use organic solvents in the extraction, separation and purification process, which reduces the solvent residues of medicines, health products, foods and cosmetics, which is beneficial to the wide use of products; at the same time, it reduces the recovery, storage and discharge of organic solvents, which is beneficial to the use of factory production equipment and is more beneficial energy saving.
  • An environmentally friendly preparation method of a composition containing anti-inflammatory active ingredients including the following steps:
  • step (2) Add 10% NaOH to the solution in step (1), adjust the pH to 10, add 40 g of Glycyrrhiza inflata root, heat to boiling water, and extract for 30 min;
  • step (3) Add 40 g of scutellaria root to the solution of step (2), extract for 2 hours, filter to obtain the filtrate, and adjust the pH to 7.5;
  • step (3) Add 1% activated carbon to the filtrate obtained in step (3), raise the temperature to 95°C, keep the reaction for 1 hour, and filter to obtain the filtrate;
  • step (6) Separate the filtrate obtained in step (5) with a microfiltration membrane to remove suspended solids, bacteria, etc.; then use an ultrafiltration membrane to remove macromolecular organics, proteins, etc. to separate and concentrate, and finally use a reverse osmosis membrane for desalination to obtain a filtrate;
  • the extraction rate of matrine was 95%
  • the extraction rate of glycyrrhizic acid in Glycyrrhiza inflata was 94%
  • the extraction rate of baicalin was 91%.
  • An environmentally friendly preparation method of a composition containing anti-inflammatory active ingredients including the following steps:
  • step (3) Add 30 g of Scutellaria baicalensis root to the solution of step (2), extract for 1 hour, filter to obtain the filtrate, and adjust the pH to 6.5;
  • step (3) The filtrate obtained in step (3) is added to 0.5% activated carbon, the temperature is raised to 90°C, the reaction is incubated for 45 minutes, and filtered to obtain the filtrate;
  • step (6) Separate the filtrate obtained in step (5) with a microfiltration membrane to remove suspended solids, bacteria, etc.; then use an ultrafiltration membrane to remove macromolecular organics, proteins, etc. to separate and concentrate, and finally use a reverse osmosis membrane for desalination to obtain a filtrate;
  • the extraction rate of matrine is 96%
  • the extraction rate of glycyrrhizic acid in Glycyrrhiza inflata is 96%
  • the extraction rate of baicalin is 93%.
  • An environmentally friendly preparation method of a composition containing anti-inflammatory active ingredients including the following steps:
  • step (3) Add 21 g of scutellaria root to the solution of step (2), extract for 30 min, filter to obtain the filtrate, and adjust the pH to 5.5;
  • step (3) The filtrate obtained in step (3) is added to 0.2% activated carbon, the temperature is raised to 85°C, the reaction is incubated for 0.5 hours, and filtered to obtain the filtrate;
  • step (6) Separate the filtrate obtained in step (5) with a microfiltration membrane to remove suspended solids, bacteria, etc.; then use an ultrafiltration membrane to remove macromolecular organics, proteins, etc., to separate and concentrate, and finally use a reverse osmosis membrane for desalination to obtain a filtrate;
  • the extraction rate of matrine is 96%
  • the extraction rate of glycyrrhizic acid in Glycyrrhiza inflata is 97%
  • the extraction rate of baicalin is 94%.
  • Example 1 The composition solutions obtained in Example 1 and Comparative Examples 1, 2, and 3 were tested for efficacy on skin-related cells, and the specific results are as follows.
  • Cytotoxicity refers to a harmful effect on cell structure and/or the basic processes of cell survival, proliferation and function. Toxicity is a result of non-specific changes in "basic cell functions" (for example: mitochondria, lysosomes, plasma membrane integrity, etc.), which may eventually lead to changes in the body's organ-specific functions or death.
  • basic cell functions for example: mitochondria, lysosomes, plasma membrane integrity, etc.
  • the mouse macrophage cell line Raw 264.7 cells have a strong ability to adhere and swallow antigens. They are commonly used cell lines for the study of microbiology and immunology, and are also one of the most classic cell lines for studying anti-inflammatory effects.
  • Neutral red (NR) is a weakly cationic in vitro reactive dye that easily diffuses through the plasma membrane and accumulates in the lysosome, and combines with the anionic lysosome matrix to form an electronically stable state. Toxic substances change the cell surface or lysosomal membrane, causing lysosomal fragility and other harmful changes, which are gradually irreversible. These harmful changes can cause cell death and/or inhibit cell growth, leading to a decrease in the amount of NR stored in the cell. Therefore, the NR uptake experiment can reflect the cytotoxic effect.
  • test substances are the different concentrations of the compositions prepared in Examples 1-3 and Comparative Examples 1-3 after being diluted (starting from 5% concentration, set according to 4-fold dilution, the final concentration points include 5%, 1.25 %, 0.3125%, 0.078%) composition.
  • the compositions prepared in each example and comparative example were diluted to form a 1.25% concentration of each composition.
  • the cell viability of the remaining groups decreased to 60%.
  • the high-concentration composition has a certain toxic effect on cells.
  • the cell viability of each example was significantly higher than that of the three comparative examples, indicating that the cytotoxicity of the examples was relatively lower.
  • Comparative example 1 1.25 104.92 0.3125 100.73 Comparative example 2 1.25 21.30 0.3125 32.48 Comparative example 3 1.25 20.78 0.3125 31.10 Example 1 1.25 13.69 0.3125 116.20 Example 2 1.25 32.59 0.3175 108.44 Example 3 1.25 55.34 0.3175 106.31
  • Inflammation refers to a basic pathological process mainly defensive response that occurs in living tissues with vascular system to the stimulation of various injury factors.
  • the clinical manifestations of inflammation include a series of phenomena such as redness, swelling, heat, itching, and pain.
  • LPS Bacterial lipopolysaccharides
  • PGE-2 prostaglandin E2
  • NO nitric oxide
  • test substance is a composition of different concentrations (0.3125%, 0.078%) formed after dilution of the compositions prepared in Example 1 and Comparative Examples 1-3;
  • Dex is dexamethasone (10 ⁇ M).
  • the positive control Dex can inhibit the expression of PGE-2 in a dose-dependent manner, and the inhibition rate can reach 75.27% at 10 ⁇ M (Dex-10 group shown in Figure 1).
  • the liquid plant extract composition prepared in Example 1 can reduce the expression of PGE-2 in a dose-dependent manner in the range of 0.078-0.3125%. Compared with the three comparative examples, the difference is statistically significant (**P ⁇ 0.01) . It can achieve 72.00% inhibition at a concentration of 0.3125%, and an inhibition rate of 41.17% at a concentration of 0.078%, which is equivalent to the anti-inflammatory effect of the positive control Dex.
  • Example 1 the composition prepared in Example 1 is not only less toxic to mouse macrophages Raw264.7 compared with Comparative Examples 1-3, but also has a greater effect on the expression of the inflammatory factor PGE-2 induced by LPS. Strong suppression effect.
  • compositions prepared in Examples 2 and 3 are also tested to have lower toxicity on the mouse macrophage Raw 264.7, and have a strong inhibitory effect on the expression of the inflammatory factor PGE-2 induced by LPS.

Abstract

本发明公开了一种含抗炎成分的组合物的环保制备方法及其应用，所述方法包括：S1、称取一定体积水，调pH，加入苦参根，进行高温保温提取；S2、对步骤S1提取后的溶液调pH，然后加入胀果甘草根，升温至沸水进行提取；S3、于步骤S2提取后的沸水溶液中加入黄芩根进行提取，过滤，调pH；S4、将步骤S3所得滤液加入活性炭保温，过滤；S5、将步骤S4所得滤液加入澄清剂，静置，过滤；S6、对步骤S6所得滤液进行膜分离，即得。本发明工艺步骤少，无有机溶剂、无废水，节能环保，适合于连续化、规模化生产。本发明制备的生物活性成分水溶液能够运用于生物医药、食品保健品、化妆品领域，尤其在皮肤上的功效能够得到提高和扩增。

Description

一种含抗炎活性成分的组合物的环保制备方法和应用 技术领域
本发明涉及生物医药和化妆品技术领域，具体地，涉及一种含抗炎活性成分的组合物的环保制备方法和应用。
背景技术
自古以来，黄芩具有清热燥湿、泻火解毒作用，用于温热病、肺炎、痈肿疖疮等症。苦参具有清热燥湿，杀虫，利尿之功，用于湿疹，湿疮，皮肤瘙痒等。而甘草具有清热解毒，祛痰止咳，缓急止痛，调和诸药之功效，其中甘草酸对炎症发生、发展和反应等许多环节具有对抗作用。然而，不同的提取方法活性成分组成可能不同，造成的功效也不同。采用传统的三药共煎方式需现煎现服，且操作麻烦，不利于长期保存，使用不便。
关于苦参、黄芩和甘草这三种单味药的主要活性成分提取纯化工艺专利和文献中已有大量报道，主要包括以下几种方法：水提法、醇提法等。然而，对于大规模生产而言，乙醇属于甲类危险品，储存库需按甲类消防建筑设计，且不宜量大，故生产工艺在考虑环保、经济情况下，需考虑少用或不用乙醇。而对于水提取法，水提取效率较低，且在高温环境下容易变质、长菌，大规模生产时需考虑缩短工艺、提高效率、节能环保。
关于苦参、黄芩和甘草这三种药材两两提取活性成分专利中无详细报道，文献中有少量报道。
如专利CN2016109373139“一种具有抗皮肤刺激功效的中药提取物组合物及其制备方法”公开了将“甘草、苦参、黄芩、当归、川芎、荆芥、防风和白柳皮的进行乙醇提取的方法”，但未根据各药材活性物的性质按一定顺序先后投料的水溶液提取方法。
如文献1((鲁冰,杨璇,王宝华,et al.甘草酸对黄芩苷增溶作用的研究[J].北京中医药大学学报,2014,37(9):620-624.)提出了甘草与黄芩水加热回流共提取，提高黄芩苷收率的方法，得到甘草酸0.22g/L，黄芩苷164mg/38ml。
文献2(赵洋,张涛,贾红梅,et al.苦参-甘草药对提取工艺的优化及其化学成分分析[J].中国实验方剂学杂志,2017(03):26-32.)提出了甘草与苦参以1:1比例，60％乙醇回流共提取，提高苦参总碱和甘草酸的方法。
文献3(刘斌,石任兵,朱丽君,et al.苦参汤黄酮类成分HPLC指纹图谱及其与组方药味黄芩和苦参的相关性研究[J].中国中药杂志,2007,32(16):1631-1634.)提出了苦参-黄芩-生地黄(3：2：8)，70％乙醇回流共提取，研究其指纹图谱的方法，但未有实验证明其提取的生物活性成分与药效之间的关系。
然而上述方法均未提供一种苦参、黄芩和甘草三种药材水溶液共提取的方法，以达到缩短工艺、减少步骤，减少染菌风险，节能环保的目的。另外，上述方法中也未特意针对放大生产需注意的溶剂、设备、溶液型产品稳定性，以及工艺改变所带来的功效改变提出具体解决方案。
发明内容
针对现有技术的缺陷，本发明的目的在于提供一种含抗炎活性成分的组合物的环保制备方法，具体为在生产中将苦参根、黄芩根和胀果甘草根三种药材按一定顺序先后投料共提取生物活性成分的制备方法，以求达到：
1)缩短工艺，不使用有机溶剂，减少废水，减少染菌风险、节能环保的作用。
2)同时，通过此发明涉及工艺的改进，拓展产品在生物医药、食品保健品、化妆品中的应用，尤其是皮肤上的功效作用得以增强和扩增。
本发明的目的是通过以下技术方案实现的：
本发明提供了一种含抗炎活性成分的组合物的环保制备方法，包括以下步骤：
S1、称取一定体积水，调pH，加入苦参根，进行高温保温提取；
S2、对步骤S1提取后的溶液调pH，然后加入胀果甘草根，升温至沸水进行提取；
S3、于步骤S2提取后的沸水溶液中加入黄芩根进行提取，过滤，调pH；
S4、将步骤S3所得滤液加入活性炭保温，过滤；
S5、将步骤S4所得滤液加入澄清剂，静置，过滤；
S6、对步骤S6所得滤液进行膜分离，得所述含抗炎活性成分的组合物。
优选地，所述黄芩根、苦参根、胀果甘草根的配料比为21-40:21-40:21-40。在该配料比范围内，均可制备得到所述含抗炎活性成分的组合物。
本发明特定选择胀果甘草根，其与黄芩根、苦参根复合提取后，可达到更好的抗炎效果、最终产品的稳定性也更好。而若采用其他如乌拉尔甘草根、光果甘草根等，则其与黄芩根、苦参根复合提取后的组合物抗炎效果不如本发明，而且其它甘草中含有的成分会对最终产品的颜色外观和产品稳定性带来负面效果。
优选地，步骤S1中，所述水的用量为药材总量的5-10倍量；所述pH值为3.0-7.0；所述高温保温提取的温度为60-80℃，提取时间为30min-2h。
优选地，步骤S2中，采用1-10％NaOH调所述pH为8-10，所述沸水提取时间为30min-2h。苦参碱的偏碱性条件有利于甘草酸的溶出，甘草酸的表面活性性能又有利于苦参碱的溶出。同时，pH调成偏碱性条件，在保证苦参碱不被破坏的同时，甘草酸呈离子状态，更加有利于甘草酸等有效成分的溶出。
优选地，步骤S3中，所述提取时间为30min-2h；所述调pH至5.5-7.5。
在此步骤中，黄芩根中含有特殊的黄芩苷酶，易水解黄芩苷，造成黄芩苷的降低；故需用沸水灭黄芩苷酶，保留黄芩苷成分，增加药效。另外，黄芩苷属于黄酮类化合物，具有多个酚羟基，在pH为8-10碱性条件下更易使植物细胞壁破裂，增大黄芩根中黄酮类成分的溶出。同时，在碱性条件苦参碱又与黄芩苷形成氢键增强黄芩苷的溶解性。另外，甘草酸能提高溶液的表面张力，更有利于苦参碱和黄芩苷的提取效率。
本发明通过前期试验发现：
①按传统的三药共煎方式，即取黄芩根，苦参根、胀果甘草根同时投料，水提取，所得滤液调pH至5.5-7.5后，按步骤S4-S6处理。实验结果表明该工艺过滤困难，造成生产工艺困难。
②若三种原料的添加顺序改变，例如：
A.取水，先用10％NaOH调成pH＝8-10,先加胀果甘草根提取,再加苦参根、最后黄芩根,过滤，得到滤液，pH调至5.5-7.5后，按步骤S4-S6处理。因为在偏碱性条件下，苦参碱呈游离状态，水溶性不够，本发明从环保角度考虑采用水提，故水提取溶液中有效成分苦参碱含量降低。与本专利工艺相比，苦参碱的提取率降低32.3％。
而若首先提取胀果甘草根之后,如果将ph调到酸性，再提取苦参根，虽可做到苦参碱提取率不变，但是在提取过程中，酸性环境加热条件下，会促进甘草酸的酸水解，使甘草酸水解成甘草次酸，从而降低产品中甘草酸的实际含量，降低幅度18.7％。
B.取水，先提黄芩根，然后再提苦参根，最后将溶液用10％NaOH调成pH＝8-10,加胀果甘草根提取，过滤，得到滤液，pH调至5.5-7.5后，按步骤S4-S6处理。此方案则也将导致黄芩苷活性物含量降低。因为黄芩苷水溶性极差，必须在较强的碱性条件下，溶出率才高。而且根据前述文献1，无法发挥甘草酸对黄芩苷的助溶作用。与本专利工艺相比，黄芩苷的提取率降低21.4％。
C.取水，先提苦参根，得苦参根提取液，然后加入10％NaOH调pH＝9，加入胀果甘草 根，黄芩根，升温至沸水，提取，过滤，得到滤液，调pH至5.5-7.5后，按步骤S4-S6处理。此方案则也将导致黄芩苷活性物含量降低36.1％。表面上甘草酸、黄芩苷都需要碱性条件溶出率才高，看似合理，实际上长时间在沸水中，在碱性环境下，会促进黄芩苷C环结构的开环变异，从而大幅降低黄芩苷的实际含量。
由此，若改变三种原料的添加顺序，将会显著降低药材中主要有效物质的提取得率，最终导致制备得到的组合物中关键活性物质的含量降低，进而影响组合物的功效；也会提高非有效物质的含量进而导致副作用的增加。
优选地，步骤S4中，所述活性炭加入量为滤液总量的0.2-1％；所述保温温度为85-95℃、保温时间为0.5-1小时。
在上述步骤中，所得溶液颜色较深，采用活性炭去除部分色素。
优选地，步骤S5中，所述澄清剂为壳聚糖、ZTC澄清剂I-IV、101果汁澄清剂中一种或两种以上组合，澄清剂加入量为滤液总量的1-5％；所述静置时间为0.5-1h。
在上述步骤中，因为采用水作为提取溶剂，水提取除了提取小分子生物活性成分，植物中大量的水溶性杂质如糖、蛋白质等物质也易提取出来，导致生物活性分子含量不高，并且糖类、蛋白质等容易长菌，长期放置会导致组合物变质。本发明中采用澄清剂去除易长菌的杂质成分，使组合物减少长菌风险，并且使得生物活性成分含量增高，活性增强。
所述膜分离的步骤包括先用微滤膜分离，然后用超滤膜分离，最后采用反渗透膜进行脱盐处理。
优选地，所述方法还包括将步骤S6制得的组合物进行灭菌和/或防腐处理的步骤；
所述灭菌方法采用高温灭菌和湿热灭菌中的一种，经不同载体，适用生物医药领域运用中。
所述防腐处理采用的防腐剂为苯甲酸及盐-山梨酸钾、脱氢乙酸钠，双乙酸钠中的一种或两种以上组合，或采用具有防腐作用的醇类成分，适用食品、饮料、保健品运用中；所述防腐剂添加量为滤液的0.01-50％。
更优选地，所述具有防腐作用的醇类成分包括丁二醇、1，2-己二醇、甘油中的一种或两种以上组合，适用化妆品运用。
本发明还提供了一种根据前述方法制备的组合物在食品、药品、化妆品中的应用。
本发明制备的组合物运用于化妆品中能增强抑制炎症反应，从而达到舒缓皮肤敏感作用；另外，还能修复细胞损伤从而达到提升皮肤细胞活力作用；另外，还能促进细胞基因表达，从而修复皮肤屏障，减少外来刺激引起的皮肤疾病。
与现有技术相比，本发明具有如下的有益效果：
1)本发明采用苦参根、胀果甘草根、黄芩根三种药材先后投料共提取的方法，使得三种药材活性成分在提取的全过程中互相促进提取效率，又能互相促进稳定性，比单味药材单独提取和两两药材提取缩短了工艺步骤，减少了耗能，降低了成本低，操作简便，适合于连续化、规模化生产等特点。本发明的方法是一种绿色环保、前景广阔的生物活性成分提取技术，适宜于工业化生产。
2)本发明采用苦参根、胀果甘草根、黄芩根三种药材先后投料共提取的方法，在提高活性成分提取效率的同时缩短工艺，保证工艺在短时间内完成，减少了水溶性样品在生产中由于长时间生产染菌的风险，有利于产品的运用和保存。
3)本发明提取分离纯化过程中不使用有机溶剂，减少医药、保健品、食品和化妆品溶剂残留，有利产品广泛使用；同时减少有机溶剂的回收、储存、排放，有利工厂生产设备使用，更加有利环保节能。
4)该制备工艺除必要的清洗废水产生，工艺中所用到的溶剂都作为产品的溶剂，无其他废水产生，节能环保。
[根据细则91更正 05.01.2021]　
5)通过上述的本发明采用苦参根、胀果甘草根、黄芩根三种药材先后投入共提取的方法，生物活性成分含量增高，且在溶液中由于共分子作用更加稳定，使得最终产品的功效得以提高和扩增。

附图说明
图1为各受试物组浓度为0.3125%时对PGE-2的抑制率；
图2为各受试物组浓度为0.078%时对PGE-2的抑制率。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
实施例1
一种含抗炎活性成分的组合物的环保制备方法，包括以下步骤：
(1)称取水1000g，加入提取罐中，用稀HCl调pH＝4.5，加热至80℃；然后称取苦参根40g加入提取罐中，在80℃下提取2小时；
(2)于步骤(1)的溶液加入10％NaOH，调pH＝10，加入胀果甘草根40g，升温至沸水，提取30min；
(3)于步骤(2)的溶液加入黄芩根40g，提取2h，过滤，得到滤液，调pH至7.5；
(4)将步骤(3)所得滤液加入1％活性炭，升温至95℃，保温反应1小时，过滤，得到滤液；
(5)将步骤(4)所得滤液加入5％ZTC澄清剂II，静置1h，过滤；
(6)将步骤(5)所得滤液用微滤膜去除悬浮固体、细菌等进行分离；然后用超滤膜去除大分子有机物、蛋白等分离浓缩，最后用反渗透膜进行脱盐，得滤液；
(7)向步骤(6)所得滤液中加入丁二醇，使丁二醇含量为35％，得一浅黄色透明液体，即所述组合物。
所得组合物中，苦参碱的提取率为95％、胀果甘草中甘草酸的提取率为94％、黄芩苷的提取率为91％。
实施例2
一种含抗炎活性成分的组合物的环保制备方法，包括以下步骤：
(1)称取水1000g，加入提取罐中，用稀HCl调pH＝4，加热至70℃；然后称取苦参根30g加入提取罐中，在70℃下提取1小时；
(2)于步骤(1)的溶液加入10％NaOH，调pH＝9，加入胀果甘草根40g，升温至沸水，提取60min；
(3)于步骤(2)的溶液加入黄芩根30g，提取1h，过滤，得到滤液，调pH至6.5；
(4)将步骤(3)所得滤液加入0.5％活性炭，升温至90℃，保温反应45min，过滤，得到滤液；
(5)将步骤(4)所得滤液加入0.5％壳聚糖澄清剂，静置45min，过滤；
(6)将步骤(5)所得滤液用微滤膜去除悬浮固体、细菌等进行分离；然后用超滤膜去除大分子有机物、蛋白等分离浓缩，最后用反渗透膜进行脱盐，得滤液；
(7)向步骤(6)所得滤液中加入丁二醇，使丁二醇含量为35％，得一浅黄色透明液体，即所述组合物。
所得组合物中，苦参碱的提取率为96％、胀果甘草中甘草酸的提取率为96％、黄芩苷的提取率为93％。
实施例3
一种含抗炎活性成分的组合物的环保制备方法，包括以下步骤：
(2)称取水1000g，加入提取罐中，用稀HCl调pH＝3，加热至60℃；然后称取苦参根21g加入提取罐中，在60℃下提取30min；
(2)于步骤(1)的溶液加入1％NaOH，调pH＝8，加入胀果甘草根21g，升温至沸水，提取120min；
(3)于步骤(2)的溶液加入黄芩根21g，提取30min，过滤，得到滤液，调pH至5.5；
(4)将步骤(3)所得滤液加入0.2％活性炭，升温至85℃，保温反应0.5小时，过滤，得到滤液；
(5)将步骤(4)所得滤液加入1％ZTC澄清剂II，静置0.5h，过滤；
(6)将步骤(5)所得滤液用微滤膜去除悬浮固体、细菌等进行分离；然后用超滤膜去除大分子有机物、蛋白等分离浓缩，最后用反渗透膜进行脱盐，得滤液；
(7)向步骤(6)所得滤液中加入丁二醇，使丁二醇含量为35％，得一浅黄色透明液体，即所述组合物。
所得组合物中，苦参碱的提取率为96％、胀果甘草中甘草酸的提取率为97％、黄芩苷的提取率为94％。
对比例1
取苦参根30g，胀果甘草根40g，同时投料，80℃水提取2h，得滤液1；取黄芩根30g，单独采用80℃提取2h，得滤液2。将滤液1和滤液2混合，进行实施例1的步骤(4)-(7)的处理，得组合物。
对比例2
取黄芩根30g，胀果甘草根40g同时投料，80℃水提取2h，得滤液1；取苦参根30g，单独采用80℃提取2h，得滤液2。将滤液1和滤液2混合，进行实施例1的步骤(4)-(7)的处理，得组合物。
对比例3
取黄芩根30g，苦参根30g同时投料，80℃水提取2h，得滤液1；取胀果甘草根30g， 单独采用80℃提取2h，得滤液2。将滤液1和滤液2混合，进行实施例1的步骤(4)-(7)的处理，得组合物。
效果验证：
将实施例1和对比例1，2，3获得的组合物溶液在皮肤相关的细胞上进行功效验证，具体结果如下。
1.细胞毒性效应
细胞毒性是指一种对细胞结构和/或细胞生存、增殖和功能的基本过程造成的有害效应。毒性是“基本细胞功能”(例如：线粒体、溶酶体、质膜完整性等)非特异性改变的一种结果，最后可能会导致机体的器官特异性功能改变或死亡。
小鼠巨噬细胞系Raw 264.7细胞具有很强的黏附和吞噬抗原的能力，是研究微生物学、免疫学的常用细胞株，也是研究抗炎效果最经典的细胞株之一。中性红(NR)是一种弱阳离子的体外活性染料，极易通过质膜扩散并在溶酶体内聚集，并与阴离子溶酶体基质结合形成电子稳定的状态。毒性物质改变细胞表面或者溶酶体膜，引起溶酶体脆性和其他有害改变并逐渐不可逆。这些有害改变可以引起细胞死亡和/或抑制细胞生长，导致细胞中储留NR含量下降。因此，NR摄取实验可反应细胞毒性效应。
本实验先研究各实施例和对比例所获得组合物作用于Raw 264.7细胞后，对细胞造成的毒性，来评判各组合物的安全性。实验所用主要试剂及仪器如表1所示，选用Raw264.7细胞进行NR摄取实验。
表1.实验所用主要试剂及仪器
仪器试剂名称 品牌 型号
Raw264.7细胞 ATCC TIB-71
DMEM培养基 Gibco 10569-010
胎牛血清FBS Corning 35-076-CV
青链霉素PS Corning 15140-122
0.25％胰酶 Gibco 25200-056
磷酸缓冲液PBS Sigma P4417-50TAB
NR染料 Sigma N7005-1G
倒置显微镜 江南永兴 XD202
细胞计数仪 Thermo Countess II
96孔板 Corning 3599
二氧化碳培养箱 Thermo 371
微孔板震荡仪 其林贝尔 QB9001
酶标仪 Thermo MultiSkan FC
取对数期生长的Raw264.7细胞，弃去培养液，用PBS清洗2次，加入0.25％胰酶，37℃放置5min，用含10％FBS﹑1％PS的DMEM完全培养液终止消化后，收集细胞1000rpm离心3min，弃去上清液，用完全培养液重悬细胞，进行计数，调节细胞浓度为5×10 5个/ml，每孔100μl加入96孔板中。24h后，细胞贴壁，每孔加入100μl不同浓度的受试物样品。混匀，于培养箱中继续孵育24±1h。
实验分组如下表2所示：
表2
分组名称 样品处理
空白对照组 完全培养液
阴性对照组 完全培养液+Raw264.7细胞
受试物组 完全培养液+Raw264.7细胞+受试物
备注：受试物为实施例1-3和对比例1-3制备的组合物经稀释后形成的不同浓度(从5％浓度开始，按照4倍稀释来设置，最终浓度点包括5％、1.25％、0.3125％、0.078％)组合物。
处理结束后，弃去培养液，每孔加入200μl中性红染液(33μg/ml)，培养箱中放置3±0.1h。弃上清(不留残留)，每孔加入150μl中性红洗脱液(1％冰醋酸+50％无水乙醇+49％超纯水)，室温避光震荡20-45min。于酶标仪540nm处测定吸光值(A)，计算细胞活性。细胞活性％＝A 受试物组/A 阴性对照×100％
如表3所示，将各实施例和对比例制备的组合物稀释后形成1.25％浓度的各组合物处理Raw 264.7细胞24h后，除对比例1外，其余组的细胞活性均下降至60％以下，说明高浓度的组合物对细胞有一定的毒性作用。0.3125％浓度的各组合物处理细胞后，各实施例的细胞活性明显高于3个对比例，说明实施例的细胞毒性相对来说更小。
表3.不同浓度组合物对细胞活性的影响
样品处理 浓度1，％ 细胞活性1，％ 浓度2，％ 细胞活性2，％
阴性对照组 - 100.00 - 100.00
对比例1 1.25 104.92 0.3125 100.73
对比例2 1.25 21.30 0.3125 32.48
对比例3 1.25 20.78 0.3125 31.10
实施例1 1.25 13.69 0.3125 116.20
实施例2 1.25 32.59 0.3175 108.44
实施例3 1.25 55.34 0.3175 106.31
2.抗炎效果
炎症(inflammation)是指具有血管系统的活体组织对各种损伤因子的刺激所发生的一种以防御反应为主的基本病理过程。炎症的临床表征包括红、肿﹑热﹑痒﹑疼等一系列现象。
细菌脂多糖(Lipopolysaccharides，LPS)是革兰氏阴性杆菌细胞壁的主要组分之一，是诱导产生炎症反应的主要物质之一。在炎性反应过程中，巨噬细胞通过产生多种不同的细胞因子或者释放溶酶体酶，间接或直接参与各种炎症性疾病的反应过程，并且能够对细胞外LPS的刺激产生应答，启动细胞内一系列由相应信号蛋白的激活引起的级联反应，产生各种炎症介质，如前列腺素E2(PGE-2)﹑一氧化氮(NO)等。
本实验通过LPS刺激Raw 264.7细胞，收集细胞培养液，用酶联免疫方法(ELISA)测定炎症因子PGE-2的表达量，比较各实施例和对比例所获得组合物对抗炎效果的影响。实验所用主要试剂及仪器如表4所示，选用Raw264.7细胞和LPS进行实验。
表4.实验所用主要试剂及仪器
仪器试剂名称 品牌 型号
Raw264.7细胞 ATCC TIB-71
DMEM培养基 Gibco 10569-010
胎牛血清FBS Corning 35-076-CV
青链霉素PS Corning 15140-122
0.25％胰酶 Gibco 25200-056
磷酸缓冲液PBS Sigma P4417-50TAB
LPS Sigma L3012-10MG
地塞米松Dex Sigma D4902-25MG
PGE-2 ELISA试剂盒 Cayman 514010
倒置显微镜 江南永兴 XD202
细胞计数仪 Thermo Countess II
96孔板 Corning 3599
二氧化碳培养箱 Thermo 371
微孔板震荡仪 其林贝尔 QB9001
酶标仪 Thermo MultiSkan FC
取对数期生长的Raw264.7细胞，弃去培养液，用PBS清洗2次，加入0.25％胰酶，37℃放置5min，用含10％FBS﹑1％PS的DMEM完全培养液终止消化后，收集细胞1000rpm离心3min，弃去上清液，用完全培养液重悬细胞，进行计数，调节细胞浓度为5×10 5个/ml，每孔100μl加入96孔板中。24h后，细胞贴壁，每孔先加入100μl不同浓度的受试物样品及阳性对照﹑阴性对照和1μg/ml的LPS。混匀，于培养箱中继续孵育24h±1h。根据细胞毒性的结果，选择对细胞活性影响较小的浓度点(0.3125％以下)进行此抗炎实验的验证。实验分组如下表5所示：
表5
分组名称 样品处理
空白对照组 完全培养液
阴性对照组 完全培养液+Raw264.7细胞
LPS组(模型组) 完全培养液+Raw264.7细胞+LPS
阳性对照组 完全培养液+Raw264.7细胞+LPS+Dex
受试物组 完全培养液+Raw264.7细胞+LPS+受试物
备注：受试物为实施例1和对比例1-3制备的组合物经稀释后形成的不同浓度(0.3125％、0.078％)组合物；Dex为地塞米松(10μM)。
处理结束后，将细胞培养液收集至新的96孔板，按照试剂盒说明书对PGE-2的表达量进行ELISA检测，根据标准曲线计算PGE-2的含量(E)，并与LPS组相比，计算各样品的相对抑制率(抑制率％＝(1-E 受试物组/E LPS组)×100％)。
如图1和图2所示，在Raw 264.7细胞上，LPS显著诱导PGE-2的表达。阳性对照Dex可剂量依赖性地抑制PGE-2的表达，10μM时(图1所示的Dex-10组)抑制率可达到75.27％。实施例1制得的液体植物提取物组合物在0.078-0.3125％范围可剂量依赖性地降低PGE-2的表达，与3个对比例相比，差异有统计学意义(**P<0.01)。其在0.3125％ 的浓度即可达到72.00％的抑制效果，在0.078％的浓度抑制率也达到41.17％，与阳性对照Dex的抗炎效果相当。
综合来看，实施例1所制得的组合物，与对比例1-3相比，不仅对小鼠巨噬细胞Raw264.7的毒性较低，对LPS诱导的炎症因子PGE-2表达也有较强的抑制效果。
实施例2和3所制得的组合物经测试也同样对小鼠巨噬细胞Raw 264.7的毒性较低、对LPS诱导的炎症因子PGE-2表达有较强的抑制效果。
本发明具体应用途径很多，以上所述仅是本发明的优选实施方式。应当指出，以上实施例仅用于说明本发明，而并不用于限制本发明的保护范围。对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。

Claims (10)

  1. 一种含抗炎活性成分的组合物的环保制备方法，其特征在于，包括以下步骤：
    S1、称取一定体积水，调pH，加入苦参根，进行高温保温提取；
    S2、对步骤S1提取后的溶液调pH，然后加入胀果甘草根，升温至沸水进行提取；
    S3、于步骤S2提取后的沸水溶液中加入黄芩根进行提取，过滤，调pH；
    S4、将步骤S3所得滤液加入活性炭保温，过滤；
    S5、将步骤S4所得滤液加入澄清剂，静置，过滤；
    S6、对步骤S6所得滤液进行膜分离，得所述含抗炎活性成分的组合物。
  2. 根据权利要求1所述的含抗炎活性成分的组合物的环保制备方法，其特征在于，所述黄芩根、苦参根、胀果甘草根的配料比为21-40:21-40:21-40。
  3. 根据权利要求1所述的含抗炎活性成分的组合物的环保制备方法，其特征在于，步骤S1中，所述水的用量为药材总量的5-10倍量；所述pH值为3.0-7.0；所述高温保温提取的温度为60-80℃，提取时间为30min-2h。
  4. 根据权利要求1所述的含抗炎活性成分的组合物的环保制备方法，其特征在于，步骤S2中，采用1-10％NaOH调所述pH为8-10，所述沸水提取时间为30min-2h。
  5. 根据权利要求1所述的含抗炎活性成分的组合物的环保制备方法，其特征在于，步骤S3中，所述提取时间为30min-2h；所述调pH至5.5-7.5。
  6. 根据权利要求1所述的含抗炎活性成分的组合物的环保制备方法，其特征在于，步骤S4中，所述活性炭加入量为滤液总量的0.2-1％；所述保温温度为85-95℃、保温时间为0.5-1小时。
  7. 根据权利要求1所述的含抗炎活性成分的组合物的环保制备方法，其特征在于，步骤S5中，所述澄清剂为壳聚糖、ZTC澄清剂I-IV、101果汁澄清剂中一种或两种及以上组合，澄清剂加入量为滤液总量的1-5％；所述静置时间为0.5-1h；
  8. 根据权利要求1所述的含抗炎活性成分的组合物的环保制备方法，其特征在于，步骤S6中，所述膜分离的步骤包括先用微滤膜分离，然后用超滤膜分离浓缩，最后用反渗透膜进行脱盐处理。
  9. 根据权利要求1所述的含抗炎活性成分的组合物的环保制备方法，其特征在于，所述方法还包括将步骤S6制得的组合物进行灭菌和/或防腐处理的步骤；
    所述灭菌方法采用高温灭菌和湿热灭菌中的一种；
    所述防腐处理采用的防腐剂为苯甲酸及盐-山梨酸钾、脱氢乙酸钠，双乙酸钠中的一种或两种以上组合；或采用具有防腐作用的醇类成分，所述防腐剂添加量为滤液的0.01-50％。
  10. 一种根据权利要求1所述方法制备的含抗炎活性成分的组合物在保健食品、药品、化妆品中的应用。
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Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08208465A (ja) * 1994-11-11 1996-08-13 Kanebo Ltd 感冒治療薬
CN1312077A (zh) * 2001-01-04 2001-09-12 贺平 甘草和苦参有效提取物组成的复方组份及其应用
CN103976905A (zh) * 2014-05-29 2014-08-13 上海清轩生物科技有限公司 一种具有消炎抗过敏作用的植物提取液混合物
CN107308049A (zh) * 2017-07-04 2017-11-03 甘肃青黛中草药美容研究有限责任公司 一种以中药组分为活性成分的祛痘清印护肤品
CN109394634A (zh) * 2018-12-27 2019-03-01 上海伽誉生物科技有限公司 抗炎修复组合物及其制备方法和其应用
CN110623999A (zh) * 2019-10-30 2019-12-31 上海珈凯生物科技有限公司 一种含抗炎活性成分的组合物的环保制备方法和应用
CN110638861A (zh) * 2019-10-30 2020-01-03 上海珈凯生物科技有限公司 一种组合物在皮肤屏障基因修复中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7482029B2 (en) * 2005-04-01 2009-01-27 Bionovo, Inc. Composition for treatment of menopause
CN107375086A (zh) * 2017-08-10 2017-11-24 广州市金原生物科技有限公司 一种美白防晒护肤品及其制备方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08208465A (ja) * 1994-11-11 1996-08-13 Kanebo Ltd 感冒治療薬
CN1312077A (zh) * 2001-01-04 2001-09-12 贺平 甘草和苦参有效提取物组成的复方组份及其应用
CN103976905A (zh) * 2014-05-29 2014-08-13 上海清轩生物科技有限公司 一种具有消炎抗过敏作用的植物提取液混合物
CN107308049A (zh) * 2017-07-04 2017-11-03 甘肃青黛中草药美容研究有限责任公司 一种以中药组分为活性成分的祛痘清印护肤品
CN109394634A (zh) * 2018-12-27 2019-03-01 上海伽誉生物科技有限公司 抗炎修复组合物及其制备方法和其应用
CN110623999A (zh) * 2019-10-30 2019-12-31 上海珈凯生物科技有限公司 一种含抗炎活性成分的组合物的环保制备方法和应用
CN110638861A (zh) * 2019-10-30 2020-01-03 上海珈凯生物科技有限公司 一种组合物在皮肤屏障基因修复中的应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEN, BIN: "Extraction and Isolation of Baicalin from Scutellaria Baicalensis Georgi)", TECHNIQUES FOR EXTRACTION AND ISOLATION OF NATURAL DRUGS, 31 July 2007 (2007-07-31), pages 87 - 88, XP009527879, ISBN: 978-7-5349-3701-9 *
LI, GANGRONG: "Licorice", COMPENDIUM OF MATERIA MEDICA" A QUICK CHECK BOOK OF CHINESE MEDICINE HEALTH, 30 September 2016 (2016-09-30), pages 338, XP009527878, ISBN: 978-7-5502-8587-3 *
LIU, BIN ET AL.: "Design and Implementation of QoS Control Frames Based on RSVP for Internet", CHINESE TRADITIONAL PATENT MEDICINE, vol. 30, no. 4, 30 April 2008 (2008-04-30), pages 574 - 576, XP055809112, ISSN: 1001-1528 *
LU, BING ET AL.: "Solubilizing Effect of Glycyrrhizic Acid on Baicalin", JOURNAL OF BEIJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE, vol. 37, no. 9, 30 September 2014 (2014-09-30), pages 620 - 624, XP055809114, ISSN: 1006-2157 *
ZI, FULIN: "Introduction to medical chemistry experiment technology", 31 March 1998, CHENGDU UNIVERSITY OF SCIENCE AND TECHNOLOGY PRESS, CN, ISBN: 7-5616-3636-9, article ZI, FULIN: "Types of Solvents", XP009527900 *

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