WO2022116807A1 - 一种富含灵芝三萜的灵芝萃取油的制备方法 - Google Patents

一种富含灵芝三萜的灵芝萃取油的制备方法 Download PDF

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WO2022116807A1
WO2022116807A1 PCT/CN2021/130814 CN2021130814W WO2022116807A1 WO 2022116807 A1 WO2022116807 A1 WO 2022116807A1 CN 2021130814 W CN2021130814 W CN 2021130814W WO 2022116807 A1 WO2022116807 A1 WO 2022116807A1
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ganoderma lucidum
particles
extraction
spore powder
extract oil
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PCT/CN2021/130814
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English (en)
French (fr)
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冯鹏
周亚杰
钱一帆
沈建
胡浪
陈晓红
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中科健康产业集团股份有限公司
南京中科药业有限公司
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Publication of WO2022116807A1 publication Critical patent/WO2022116807A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B9/00—Essential oils; Perfumes
    • C11B9/02—Recovery or refining of essential oils from raw materials
    • C11B9/025—Recovery by solvent extraction
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4816—Wall or shell material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B9/00—Essential oils; Perfumes
    • C11B9/02—Recovery or refining of essential oils from raw materials
    • C11B9/027—Recovery of volatiles by distillation or stripping
    • 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
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

Definitions

  • the invention belongs to the technical field of pharmacy, and in particular relates to a preparation method of Ganoderma lucidum triterpenoid-rich Ganoderma lucidum extract oil.
  • Ganoderma lucidum also known as Swiss grass and fairy grass, has a flat nature and sweet taste. It is a precious traditional Chinese medicine in my country. Traditional Chinese medicine has long been regarded as a precious Chinese herbal medicine that nourishes and strengthens the body and strengthens the body.
  • Ganoderma lucidum is very rich in ingredients, including more than a dozen active ingredients such as Ganoderma lucidum polysaccharides, triterpenes and sterols, nucleosides, proteins and trace elements, which have good application value.
  • Ganoderma lucidum triterpenoids have anti-tumor, immune-enhancing, anti-aging, free radical scavenging, hypoglycemic and other effects, and have been the focus of research in recent years.
  • Triterpenes are one of the main active ingredients in Ganoderma lucidum fruiting bodies and Ganoderma lucidum spore powder, and have a wide range of pharmacological effects. Because of its low content in Ganoderma lucidum and difficult to separate, it brings great importance to the in-depth research and clinical application of triterpenes. limit.
  • Ganoderma lucidum extracts are mostly extracted with hot water or alcohol.
  • new technologies are needed, such as ultrasonic, microwave, supercritical CO2 extraction technology, etc.
  • Ganoderma lucidum spore powder is the extremely tiny oval germ cells ejected from the gills of Ganoderma lucidum during the growth and maturity of Ganoderma lucidum. With the development of Ganoderma lucidum cultivation technology, the yield of Ganoderma lucidum spore powder is getting higher and higher, even exceeding the yield of fruiting bodies. Because Ganoderma lucidum spores are rich in protein, amino acids, glycopeptides, vitamins, carotene, sterols, triterpenes, alkaloids, fatty acids, lactones and inorganic ions and other nutritionally active ingredients, Ganoderma lucidum spores are used as raw materials. The obtained products are well received by the market.
  • Ganoderma lucidum spore oil is a yellow transparent liquid prepared from Ganoderma lucidum spores as raw materials.
  • Ganoderma lucidum spore oil has the pharmacological functions of relieving pain, anti-inflammatory, detoxification, protecting liver, improving digestive organ function and lowering blood cholesterol and triglyceride.
  • Ganoderma lucidum spore oil can also reduce the occurrence of mental disorders, loss of appetite, vomiting, and hair loss caused by radiotherapy and chemotherapy.
  • supercritical CO 2 extraction technology has been widely used in many fields such as medicine, food, materials, chemical industry, waterless printing and dyeing and environmental protection.
  • the extraction pressure of supercritical CO 2 is mostly 300 bar and a few reach 500 bar. Under this pressure, the density of CO 2 is relatively low, and usually only fat-soluble components can be extracted.
  • a polar entrainer - ethanol is usually added to improve the polarity of supercritical CO 2 to achieve the ability to extract low-polar components.
  • Ganoderma lucidum fruiting body products on the market are mainly composed of Ganoderma lucidum fine powder or Ganoderma lucidum extract (the main formulation is capsules), and Ganoderma spore powder is mainly composed of broken wall Ganoderma lucidum spore powder or Ganoderma lucidum spore oil (the main formulations are capsules and Ganoderma lucidum spore oil). soft capsules).
  • the mixed components of Ganoderma lucidum fruiting body and Ganoderma lucidum spore powder are relatively few deep-processed products. Because the product composition is complex, there is no suitable formulation to encapsulate the product to prevent its oxidation and prolong its shelf life, so it is impossible to truly effectively utilize all the active ingredients of natural Ganoderma lucidum.
  • the object of the present invention is to overcome above-mentioned deficiency and provide a kind of preparation method of Ganoderma lucidum extract oil rich in Ganoderma lucidum triterpenes.
  • the Ganoderma lucidum fine powder and the Ganoderma lucidum spore powder are separately granulated to obtain Ganoderma lucidum fruiting body particles and Ganoderma lucidum spore powder particles, and then the obtained two kinds of particles are respectively dried, mixed and then subjected to carbon dioxide supercritical extraction.
  • the content of Ganoderma lucidum triterpenes in the prepared extract oil is higher than that in Ganoderma lucidum spore oil, and has significant antitumor pharmacological activity.
  • a preparation method of Ganoderma lucidum extract oil rich in Ganoderma lucidum triterpenes comprises the following steps:
  • Ganoderma lucidum fruiting body particles and Ganoderma lucidum spore powder particles soak in 95% ethanol for 1.5-2.5 hours, and carry out CO 2 supercritical extraction on the soaked mixture.
  • the pressure is 30-35MPa
  • the extraction time is 3-4h
  • the extract is collected;
  • the mixing ratio of Ganoderma lucidum fruiting body particles and Ganoderma lucidum spore powder particles ranges from 1 to 2:1 to 2.
  • the mixing ratio of Ganoderma lucidum fruiting body particles and Ganoderma lucidum spore powder particles is 1:1.
  • the CO 2 supercritical extraction conditions adopted in the present invention are based on a large number of pre-tests, and the factors such as extraction temperature, extraction pressure, extraction time, etc., which affect the supercritical extraction effect, adopt a single-factor investigation method, and the Ganoderma lucidum extraction oil yield is
  • the extraction process conditions were optimized, and the obtained extract was distilled under reduced pressure, and then the oil samples were collected and measured.
  • Ganoderma lucidum fruiting body particles and Ganoderma lucidum spore powder particles were mixed in a weight ratio of 1:1, soaked in 95% ethanol for 2 h, and the soaked mixture was subjected to CO 2 supercritical extraction. Under the condition of extraction time of 4h, 10 different gradient temperatures (32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C) were selected for investigation. Obtain the optimal extraction temperature for the index.
  • Ganoderma lucidum fruiting body particles and Ganoderma lucidum spore powder particles were mixed in a weight ratio of 1:1, soaked in 95% ethanol for 2 h, and the soaked mixture was subjected to CO 2 supercritical extraction.
  • Eight different gradient pressures (20MPa, 25MPa, 30MPa, 35MPa, 40MPa, 45MPa, 50MPa, 55MPa) were selected under the conditions of extraction temperature of 42°C and extraction time of 4h. pressure.
  • Ganoderma lucidum fruiting body particles and Ganoderma lucidum spore powder particles were mixed, soaked in 95% ethanol for 2 h, and the soaked mixture was subjected to CO 2 supercritical extraction. Under the conditions of extraction temperature of 42°C and extraction kettle pressure of 35MPa, 8 different gradient times (1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h) were selected for investigation, and the optimal extraction rate was obtained as an indicator. extraction time.
  • the single factor test shows that the optimal process conditions for supercritical CO 2 extraction of Ganoderma lucidum spore oil are: extraction kettle temperature 38 ⁇ 42°C, extraction kettle pressure 30 ⁇ 35MPa, extraction time 3 ⁇ 4h, the extraction rate under these conditions can reach 17%.
  • the material-to-liquid ratio is most preferably 1:1 during the above-mentioned 95% ethanol soaking.
  • the above-mentioned Ganoderma lucidum fruiting body particles are prepared by the following method: pulverize Ganoderma lucidum with a pulverizer, pass through a 40-50 mesh sieve to obtain Ganoderma lucidum fine powder; add water of 20-30% of the quality of Ganoderma lucidum fine powder to the Ganoderma lucidum fine powder, and granulate , 55-60 °C drying, Ganoderma lucidum fruiting body particles.
  • the above-mentioned Ganoderma lucidum spore powder particles are prepared by the following method: adding the Ganoderma lucidum spore powder with a wall-breaking rate of more than 70%, adding 20-30% water of the mass of the Ganoderma lucidum spore powder, granulating, and drying at 55-60°C.
  • the present invention combines Ganoderma lucidum particles and Ganoderma lucidum spore powder particles to carry out CO 2 supercritical extraction, on the one hand, the triterpenes and sterols in the ganoderma lucidum can be extracted, and on the other hand, the The oil substances in the spore powder combine with each other, coordinate with each other, interact with each other, and combine the advantages of the two to significantly improve the utilization rate of the composition and enhance the effect of inhibiting the transplanted tumor of animals.
  • the obtained extracted oil can be made into soft capsules, and the content of Ganoderma lucidum triterpenes is high, which can become an excellent new health food or medicine.
  • Ganoderma lucidum fine powder was added with 30% water by mass of Ganoderma lucidum fine powder, granulated in a granulator, and dried in a vacuum drying oven at 60° C. for 2 hours to obtain Ganoderma lucidum fruiting body particles.
  • Ganoderma lucidum particles and spore powder particles were mixed in equal weight ratios, put into supercritical extraction equipment, soaked in 95% ethanol with a material-to-liquid ratio of 1:1 for 2 hours, and the soaked mixture was subjected to CO 2 supercritical extraction.
  • the extraction conditions extraction kettle temperature 38 ⁇ 42°C, extraction kettle pressure 30 ⁇ 35MPa, extraction time 3 ⁇ 4h, collect the extract, obtain Ganoderma lucidum and spore powder extract, the extract is dark brown ethanol liquid containing oily substance, remove ethanol, A dark brown oily liquid was obtained and filtered to obtain a pure extracted oil.
  • Ganoderma lucidum fine powder was added with 30% water by mass of Ganoderma lucidum fine powder, granulated in a granulator, and dried in a vacuum drying oven at 60° C. for 2 hours to obtain Ganoderma lucidum fruiting body particles.
  • Ganoderma lucidum fruiting body particles and Ganoderma lucidum spore powder particles are mixed according to the weight ratio of 1:2, put into the supercritical extraction equipment, soaked in 95% ethanol with a material-to-liquid ratio of 1:1 for 2 hours, and the soaked mixture is subjected to CO2 extraction.
  • Supercritical extraction, extraction conditions extraction kettle temperature 38-42 °C, extraction kettle pressure 30-35 MPa, extraction time 3-4 h, collect the extract to obtain Ganoderma lucidum and spore powder extract, the extract is dark brown containing oily substances After removing the ethanol, a dark brown oily liquid is obtained, which is filtered to obtain a pure extracted oil.
  • the broken Ganoderma lucidum spore powder was added with 30% water, granulated, and dried in a vacuum drying box at 60° C. for 2 hours to obtain spore powder granules. Put the spore powder particles into the supercritical extraction equipment, and carry out CO 2 supercritical extraction.
  • the extraction conditions are as follows: the temperature of the extraction kettle is 38-42°C, the pressure of the extraction kettle is 30-35MPa, and the extraction time is 3-4h, and the extract is collected to obtain Ganoderma lucidum spore oil. .
  • the triterpene components of the extracted oil are subjected to ultraviolet detection, and the inspection method is as follows:
  • Tested drugs low, medium and high dose groups of the present invention, referred to as No. 1, No. 2, No. 3, Ganoderma lucidum extract group (all Ganoderma lucidum extracts, referred to as No. 4), Ganoderma spore oil group (all Ganoderma lucidum spore extracts, referred to as No. 5 for short) No).
  • Ganoderma lucidum fine powder Pulverize Ganoderma lucidum with a grinder to obtain Ganoderma lucidum fine powder, pass through a 40-50 mesh sieve. Ganoderma lucidum fine powder was added with 30% water, granulated in a granulator, and dried in a vacuum drying oven at 60°C for 2 hours to obtain Ganoderma lucidum granules.
  • Ganoderma lucidum particles were put into the supercritical extraction equipment, soaked with the same proportion of 95% ethanol for 2 hours, and the soaked mixture was subjected to CO 2 supercritical extraction. The extraction time is 3 to 4 hours, the extract is collected, and the ethanol is removed to obtain the Ganoderma lucidum extract.
  • the raw material Ganoderma lucidum spore powder is broken in the wall breaking machine, and the wall breaking rate reaches 70%.
  • the broken Ganoderma lucidum spore powder was added with 30% water, granulated, and dried in a vacuum drying oven at 60° C. for 2 hours to obtain spore powder granules.
  • the spore powder particles are put into the supercritical extraction equipment for CO 2 supercritical extraction.
  • the extraction conditions are as follows: the temperature of the extraction kettle is 38-42°C, the pressure of the extraction kettle is 30-35MPa, and the extraction time is 3-4h.
  • the extract is collected to obtain Ganoderma lucidum spore oil.
  • Blank control group normal saline
  • mice of the above specifications were inoculated with Heps solid type according to the transplanted tumor research method, the mice were weighed 24 hours after inoculation, and randomly divided into 7 groups, 10 mice in each group, half male and half male, blank control Group and CTX group were negative and positive control groups, respectively. 24 hours after inoculation, iv administration, once every other day, a total of 4 administrations, the mice were weighed on the 2nd day after drug withdrawal, the tumor-bearing mice were sacrificed and the tumor masses were separated and weighed.
  • Statistical processing t-test).
  • the results are shown in Table 1.
  • the results show that, compared with the blank control group, the iv administration of the No. 2 and No. 3 groups can significantly inhibit the tumor growth of Heps (P ⁇ 0.01, P ⁇ 0.01), and at the same time, the body weight of the experimental mice was significantly reduced. There is a reducing effect, but compared with the positive drug CTX group, the effect is small, and the effect is better than No. 1, No. 4 and No. 5.
  • Blank control group normal saline
  • mice of the above specifications were inoculated with S180 solid type according to the transplanted tumor research method, the mice were weighed 24 hours after inoculation, and randomly divided into 7 groups, 10 mice in each group, half male and half, blank control Group and CTX group were negative and positive control groups, respectively. 24 hours after inoculation, iv administration, once every other day, a total of 4 administrations, the mice were weighed on the 2nd day after drug withdrawal, the tumor-bearing mice were sacrificed and the tumor masses were separated and weighed.
  • Statistical processing t-test).

Abstract

本发明公开了一种富含灵芝三萜的灵芝萃取油的制备方法，该方法是先将灵芝细粉和破壁灵芝孢子粉与分别制粒，得到灵芝子实体颗粒和灵芝孢子粉颗粒，再将得到的两种颗粒依次分别干燥，混合后进行二氧化碳超临界萃取，最终使制得的萃取油灵芝三萜含量高于灵芝孢子油中灵芝三萜含量，并具有显著的抗肿瘤的药理活性。

Description

一种富含灵芝三萜的灵芝萃取油的制备方法 技术领域
本发明属于制药技术领域，具体涉及一种富含灵芝三萜的灵芝萃取油的制备方法。
背景技术
灵芝又称瑞草、仙草，性平味甘，为我国名贵的传统中药，中华传统医学长期以来一直视为滋补强身、固本扶正的珍贵中草药。灵芝的成分非常丰富，包括灵芝多糖、三萜及甾醇类化合物、核苷、蛋白质以及微量元素等十几种有效成分，具有很好的应用价值。灵芝三萜具有抗肿瘤、提高免疫力、抗衰老、清除自由基、降血糖等功效，近年来一直是人们研究的重点。三萜是灵芝子实体和灵芝孢子粉中的主要有效成分之一，具有广泛的药理作用，由于它在灵芝中的含量低且不易分离，给三萜的深入研究和临床应用带来很大的限制。
目前，灵芝提取物多采用热水浸提或酒精提取的方式，为了能够更加有效地提高溶剂的提取效率，增加灵芝三萜和甾醇的含量，需要新技术的辅助，如超声波、微波、超临界CO 2萃取技术等。
灵芝孢子粉是灵芝生长成熟期，从灵芝菌褶中弹射出来的极其微小的卵形生殖细胞。随着灵芝栽培技术的发展，灵芝孢子粉的产量越来越高，甚至超过子实体的产量。由于灵芝孢子中富含蛋白质、氨基酸类、糖肽类、维生素类、胡萝卜素、甾醇类、三萜类、生物碱类、脂肪酸类、内酯和无机离子等营养活性成分，以灵芝孢子为原料制得的产品深受市场欢迎。
灵芝孢子油是以灵芝孢子为原料制备得到的黄色透明液体，主要成分为三萜类灵芝酸、不饱和脂肪酸和灵芝多糖等。灵芝孢子油具有止痛、消炎、解毒、保肝、增进消化器官机能及降低血中胆固醇、甘油三酯的药理功能，还能抑制血小板凝结，消除血栓，降低血液黏度，提供血红蛋白，提高血液对心脑供氧能力和加速血液循环，帮助血液流通，软化血管，净化血液，消除细胞自由基，调整血管压力，增强机体新陈代谢和免疫调节能力。另外，灵芝孢子油还能减少因放、化疗引起的精神不佳、食欲减退、呕吐、 脱发等情况的发生。
超临界CO 2萃取技术，经过50多年来的快速发展，现已在医药、食品、材料、化学化工、无水印染和环境保护等许多领域获得广泛应用。目前，超临界CO 2萃取压力大都在300bar少数达到500bar，在这种压力下，CO 2的密度比较低，通常只能萃取脂溶性成分。为了提高萃取率，增加萃取低极性成分，通常采用加入极性夹带剂—乙醇，以提高超临界CO 2的极性达到萃取低极性成分的能力。
目前的天然灵芝虽具有较强的保健治疗作用，可有效提高人体的免疫力，但消费者无法有效利用其全部活性成分。市场上的灵芝子实体类产品主要以灵芝细粉或灵芝提取物为主成份(主要剂型是胶囊)，灵芝孢子粉主要以破壁灵芝孢子粉或灵芝孢子油为主成份(主要剂型是胶囊和软胶囊)。
灵芝子实体和灵芝孢子粉的混合组分深加工产品比较少，因为产品成分复杂，没有合适的剂型来包裹产品以防止其氧化，延长其保存期，所以无法真正高效利用天然灵芝的全部活性成分。
发明内容
本发明的目的是克服上述不足之处提供一种富含灵芝三萜的灵芝萃取油制备方法。该方法是先将灵芝细粉和破壁灵芝孢子粉与分别制粒，得到灵芝子实体颗粒和灵芝孢子粉颗粒，再将得到的两种颗粒依次分别干燥，混合后进行二氧化碳超临界萃取，最终使制得的萃取油灵芝三萜含量高于灵芝孢子油中灵芝三萜含量，并具有显著的抗肿瘤的药理活性。
本发明的目的具体是通过以下方式实现的：
一种富含灵芝三萜的灵芝萃取油制备方法，该方法包括以下步骤：
将灵芝子实体颗粒与灵芝孢子粉颗粒混合，用质量浓度为95％乙醇浸泡1.5-2.5h，将浸泡后的混合物进行CO 2超临界萃取，萃取条件：萃取釜温度38～42℃，萃取釜压力30～35MPa，萃取时间3～4h，收集萃取液；灵芝子实体颗粒与灵芝孢子粉颗粒混合比例范围1～2：1～2。优选灵芝子实体颗粒与灵芝孢子粉颗粒混合比例为1：1。
本发明采用的CO 2超临界萃取条件是在大量的预试验的基础上，对影响超临界萃取效果的萃取温度、萃取压力、萃取时间等因素采用单因素考察法，以灵芝萃取油得率为指标，进行萃取工艺条件优化，所得萃取液经减压蒸馏后，收集油样测得。
萃取油得率的计算：
Figure PCTCN2021130814-appb-000001
1、萃取温度的考察
将灵芝子实体颗粒与灵芝孢子粉颗粒按照1:1的重量比例混合，用质量浓度为95％乙醇浸泡2h，将浸泡后的混合物进行CO 2超临界萃取。在萃取时间4h条件下，选取10个不同梯度的温度(32℃、34℃、36℃、38℃、40℃、42℃、44℃46℃、48℃、50℃)进行考察，以萃取率为指标得出最优的萃取温度。
2、萃取压力的考察
将灵芝子实体颗粒与灵芝孢子粉颗粒按照1:1的重量比例混合，用质量浓度为95％乙醇浸泡2h，将浸泡后的混合物进行CO 2超临界萃取。在萃取温度42℃，萃取时间4h的条件下选取8个不同梯度的压力(20MPa、25MPa、30MPa、35MPa、40MPa、45MPa、50MPa、55MPa)进行考察，以萃取率为指标得出最优的萃取压力。
3、萃取时间的考察
将灵芝子实体颗粒与灵芝孢子粉颗粒混合，用质量浓度为95％乙醇浸泡2h，将浸泡后的混合物进行CO 2超临界萃取。在萃取温度42℃，萃取釜压力35MPa的条件下，选取8个不同梯度的时间(1h、2h、3h、4h、5h、6h、7h、8h)进行考察，以萃取率为指标得出最优的萃取时间。
4、结果
单因素试验表明，超临界CO 2萃取灵芝孢子油的最佳艺条件是：萃取釜温度38～42℃，萃取釜压力30～35MPa，萃取时间3～4h，在此条件下的萃取率可达17％。
上述95％乙醇浸泡时料液比最优选为1:1。
上述灵芝子实体颗粒是通过以下方法制备得到的：用粉碎机将灵芝粉碎，过40～50目筛，得灵芝细粉；灵芝细粉中加入灵芝细粉质量20-30％的水，制粒，55-60℃烘干，得灵芝子实体颗粒。
上述灵芝孢子粉颗粒是通过以下方法制备得到的：将破壁率达70％以上的灵芝孢子粉加入灵芝孢子粉质量20-30％水，制粒，55-60℃烘干。
与现有技术比较本发明的有益效果：本发明将灵芝颗粒和灵芝孢子粉颗粒组合在一起进行CO 2超临界萃取，一方面可以萃取出灵芝当中的三萜和甾醇类物质，一方面萃取 出孢子粉当中的油类物质，两者相互结合，相互协调，相互作用，结合二者优势，显著的提高组合物利用率，增强抑制动物移植性肿瘤的作用。所得萃取油可以做成软胶囊，其灵芝三萜的含量高，会成为一种优异的新型保健食品或药品。
具体实施方式
以下通过具体实施例对本发明进行解释说明：
实施例1
1)用粉碎机将灵芝粉碎，过40～50目筛，得灵芝细粉。
2)灵芝细粉加入灵芝细粉质量30％水，制粒机中制粒，真空干燥箱中60℃烘干2h，得灵芝子实体颗粒。
3)原料灵芝孢子粉，破壁机中破壁，破壁率达70％。
4)将破壁好的灵芝孢子粉加入30％水，制粒，真空干燥箱中60℃烘干2h，得孢子粉颗粒。
5)灵芝颗粒与孢子粉颗粒等重量比例混合，投入超临界萃取设备内，用料液比为1:1的95％乙醇进行浸泡2h，将浸泡后的混合物进行CO 2超临界萃取，萃取条件：萃取釜温度38～42℃，萃取釜压力30～35MPa，萃取时间3～4h，收集萃取液，得到灵芝及孢子粉萃取物，萃取物为含有油状物质的深棕色的乙醇液体，除去乙醇，得深棕色的油状液体，过滤，得纯净的萃取油。
实施例2
1)用粉碎机将灵芝粉碎，过40～50目筛，得灵芝细粉。
2)灵芝细粉加入灵芝细粉质量30％水，制粒机中制粒，真空干燥箱中60℃烘干2h，得灵芝子实体颗粒。
3)原料灵芝孢子粉，破壁机中破壁，破壁率达70％。
4)将破壁好的灵芝孢子粉加入30％水，制粒，真空干燥箱中60℃烘干2h，得孢子粉颗粒。
5)灵芝子实体颗粒与灵芝孢子粉颗粒按照重量比例为1：2混合，投入超临界萃取设备内，用料液比为1:1的95％乙醇进行浸泡2h，将浸泡后的混合物进行CO 2超临界萃取，萃取条件：萃取釜温度38～42℃，萃取釜压力30～35MPa，萃取时间3～4h，收集萃取液，得到灵芝及孢子粉萃取物，萃取物为含有油状物质的深棕色的乙醇液 体，除去乙醇，得深棕色的油状液体，过滤，得纯净的萃取油。
对比原料：灵芝孢子油
取灵芝孢子粉，破壁机中破壁，破壁率达70％。将破壁好的灵芝孢子粉加入30％水，制粒，真空干燥箱中60℃烘干2h，得孢子粉颗粒。将孢子粉颗粒投入超临界萃取设备内，进行CO 2超临界萃取，萃取条件：萃取釜温度38～42℃，萃取釜压力30～35MPa，萃取时间3～4h，收集萃取液，得到灵芝孢子油。
含量测定
对萃取油的三萜成分进行紫外检测，检验方法如下：
精密称取在105℃干燥至恒重的齐墩果酸对照品10mg，置于50ml容量瓶中，加氯仿溶解稀释至刻度，得齐墩果酸标准对照品溶液。
对照品溶液
精密吸取标准齐墩果酸对照品溶液0.1，0.2，0.4，0.6，0.8ml，分别置于10ml比色管中，加热挥去溶剂后，加入80g/L香草醛溶液0.5ml，硫酸溶液5.0ml，混匀后，于60℃水浴中保温30min，取出后冷水浴放置15min。在1cm比色皿中，以试剂空白液为参比调零，于波长500nm处测定吸光度，以吸光度对浓度进行回归，绘制标准曲线。
2.样品溶液
称取萃取油与灵芝孢子油样品120～150mg，加氯仿溶解于100ml容量瓶中。
精密吸取样品溶液0.2ml至10ml比色管中，按对照品溶液项下“加热挥去溶剂后”进行操作，测定吸光度，计算含量。
3.计算
Figure PCTCN2021130814-appb-000002
式中：
W-样品中总三萜的含量，％
C-从标准曲线中查得的齐墩果酸的含量，mg/ml
V-比色液体积，ml
F-稀释倍数
m-样品的质量，mg
4.结果：
Figure PCTCN2021130814-appb-000003
注：萃取油按照实施例1方法制备得到
试验例1
以下通过对动物移植性肿瘤的抑制作用的药效对本发明进行进一步说明：
1试验材料
1.1受试药物：本发明低中高剂量组，简称1号，2号，3号、灵芝萃取物组(全部灵芝萃取物，简称4号)、灵芝孢子油组(全部灵芝孢子萃取物，简称5号)。
灵芝萃取物组：
用粉碎机将灵芝粉碎，得灵芝细粉，过40～50目筛。灵芝细粉加入30％水，制粒机中制粒，真空干燥箱中60℃烘干2h，得灵芝颗粒。
灵芝颗粒投入超临界萃取设备内，用同比例的95％乙醇进行浸泡2h，将浸泡后的混合物进行CO 2超临界萃取，萃取条件：萃取釜温度38～42℃，萃取釜压力30～35MPa，萃取时间3～4h，收集萃取液，除去乙醇，得灵芝萃取物。
灵芝孢子油组：
原料灵芝孢子粉，破壁机中破壁，破壁率达70％。将破壁好的灵芝孢子粉加入30％水，制粒，真空干燥箱中60℃烘干2h，得孢子粉颗粒。
孢子粉颗粒投入超临界萃取设备内，进行CO 2超临界萃取，萃取条件：萃取釜温度38～42℃，萃取釜压力30～35MPa，萃取时间3～4h，收集萃取液，得到灵芝孢子油。
1.2动物：ICR种小白鼠，18-22g，雌雄各半，由中国药科大学动物实验室提供，饲料为颗粒饲料，饲养条件：空调房间，温度18-24℃，相对湿度70％。
1.3阳性药：环磷酰胺(CTX)，江苏盛迪医药有限公司。规格：0.2g/瓶。
2实验的主要内容
2.1 1号，2号，3号，4号、5号尾静脉注射对小鼠移植瘤Heps的抑制作用。
2.2 1号，2号，3号，4号、5号尾静脉注射对小鼠移植瘤S180的抑制作用。
3实验方法和步骤
3.1 1号，2号，3号，4号、5号尾静脉注射对小鼠移植瘤Heps的抑制作用。
3.1.1给药途径：1号，2号，3号，4号、5号尾静脉注射(iv)
3.1.2给药周期：按移植性肿瘤研究法接种Heps实体型，接种24小时后给药，iv给 药，隔天一次，共给药4次，停药后第2天处死解剖小鼠。
3.1.3剂量设置7组，分别为：
空白对照组(生理盐水)
1号：1mg/kg
2号：3mg/kg
3号：6mg/kg
4号：3mg/kg
5号：3mg/kg
CTX：30mg/kg
3.1.4给药体积：0.4ml/20g
3.1.5实验方法：取上述规格小鼠70只按移植性肿瘤研究方法接种Heps实体型，接种后24小时称鼠重，并随机分为7组，每组10只，雌雄各半，空白对照组与CTX组分别为阴、阳性对照组。接种24小时后给药，iv给药，隔天一次，共给药4次，于停药后第2天小鼠称重，处死荷瘤小鼠并分离瘤块，称瘤重，所得数据进行统计学处理(t检验)。
3.1.6实验结果
结果见表1，结果表明，与空白对照组相比，2号，3号组iv给药可显著地抑制Heps的肿瘤生长作用(P＜0.01，P＜0.01)，同时对实验小鼠的体重有减低作用，但与阳性药CTX组相比，影响很小，且效果优于1号、4号和5号。
表1 1号，2号，3号.4号，5号iv对小鼠移植瘤Heps的抑制作用(X±SD)(n＝10)
Figure PCTCN2021130814-appb-000004
注：与空白对照组比较，*P＜0.05**P＜0.01
3.2 1号，2号，3号，4号，5号尾静脉注射对小鼠移植瘤S180的抑制作用
3.2.1给药途径：1号，2号，3号，4号，5号尾静脉注射(iv)
3.2.2给药周期：按移植性肿瘤研究法接种S180实体型，接种24小时后给药，iv给药，隔天一次，共给药4次，停药后第2天处死解剖小鼠。
3.2.3剂量设置：共设7组，分别为：
空白对照组(生理盐水)
1号：1mg/kg
2号：3mg/kg
3号：6mg/kg
4号：3mg/kg
5号：3mg/kg
CTX：30mg/kg
3.2.4给药体积：0.4ml/20g
3.2.5实验方法：取上述规格小鼠70只按移植性肿瘤研究方法接种S180实体型，接种后24小时称鼠重，并随机分为7组，每组10只，雌雄各半，空白对照组与CTX组分别为阴、阳性对照组。接种24小时后给药，iv给药，隔天一次，共给药4次，于停药后第2天小鼠称重，处死荷瘤小鼠并分离瘤块，称瘤重，所得数据进行统计学处理(t检验)。
3.2.6结果见表4，结果表明，与空白对照组相比，2、3号组iv给药均可显著地抑制S180的肿瘤生长作用(P＜0.05，P＜0.01)，同时对实验小鼠的体重有减低作用，但与阳性药CTX组相比，影响很小，且效果明显优于1-2、4-5号。
表2 1号，2号，3号iv对小鼠移植瘤S180的抑制作用(X±SD)(n＝10)
Figure PCTCN2021130814-appb-000005
Figure PCTCN2021130814-appb-000006
注：与空白对照组比较，*P＜0.05**P＜0.01

Claims (5)

  1. 一种富含灵芝三萜的灵芝萃取油制备方法，其特征在于该方法包括以下步骤：
    将灵芝子实体颗粒与灵芝孢子粉颗粒混合，用质量浓度为95％乙醇浸泡1.5～2.5h，将浸泡后的混合物进行CO 2超临界萃取，萃取条件：萃取釜温度38～42℃，萃取釜压力30～35MPa，萃取时间3～4h，收集萃取液；灵芝子实体颗粒与灵芝孢子粉颗粒混合比例为1～2：1～2。
  2. 根据权利要求1所述的富含灵芝三萜的灵芝萃取油制备方法，其特征在于所述的灵芝子实体颗粒与灵芝孢子粉颗粒混合比例为1：1。
  3. 根据权利要求1所述的富含灵芝三萜的灵芝萃取油制备方法，其特征在于所述的95％乙醇浸泡时料液比为1:1。
  4. 根据权利要求1所述的富含灵芝三萜的灵芝萃取油制备方法，其特征在于所述的灵芝子实体颗粒是通过以下方法制备得到的：将灵芝粉碎，过40～50目筛，得灵芝细粉；灵芝细粉中加入灵芝细粉质量20～30％的水，制粒，55～60℃烘干，得灵芝子实体颗粒。
  5. 根据权利要求1所述的富含灵芝三萜的灵芝萃取油制备方法，其特征在于灵芝孢子粉颗粒是通过以下方法制备得到的：将破壁率达70％以上的灵芝孢子粉加入灵芝孢子粉质量20～30％水，制粒，55～60℃烘干。
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