WO2023207054A1 - 一种灵芝孢子油在制备延长肿瘤患者生存期药物的应用 - Google Patents

一种灵芝孢子油在制备延长肿瘤患者生存期药物的应用 Download PDF

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WO2023207054A1
WO2023207054A1 PCT/CN2022/133500 CN2022133500W WO2023207054A1 WO 2023207054 A1 WO2023207054 A1 WO 2023207054A1 CN 2022133500 W CN2022133500 W CN 2022133500W WO 2023207054 A1 WO2023207054 A1 WO 2023207054A1
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spore oil
ganoderma
ganoderma lucidum
tumor
lucidum spore
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PCT/CN2022/133500
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English (en)
French (fr)
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蒋兆健
蔡鸿飞
袁诚
刘菊妍
许文东
徐春草
韩亚明
李菁
毛禹康
张琴
张圳
杨阳
李康强
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广州白云山汉方现代药业有限公司
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Publication of WO2023207054A1 publication Critical patent/WO2023207054A1/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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • 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
    • C11B9/022—Refining
    • 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
    • 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

Definitions

  • the invention relates to the field of medical technology, and specifically relates to the application of Ganoderma lucidum spore oil in preparing drugs for prolonging the survival of tumor patients.
  • Ganoderma lucidum is a treasure in the treasure house of traditional Chinese medicine in the motherland. It is known as the "fairy grass” and can be used to treat restlessness, insomnia, palpitations, lung deficiency, cough and asthma, fatigue and shortness of breath, and inability to eat.
  • Ganoderma spores are extremely small spores ejected from the cap during the growth and maturity stage of Ganoderma lucidum. They are the reproductive cells of Ganoderma lucidum and contain all the genetically active substances of Ganoderma lucidum.
  • Ganoderma spore powder The chemical composition of Ganoderma spore powder is relatively complex, containing polysaccharides, triterpenes, nucleosides, vitamins, sterols, alkaloids, inorganic trace elements, etc.
  • Ganoderma spore oil is a fat-soluble active substance obtained from Ganoderma spore powder through wall breaking and supercritical carbon dioxide extraction. Its main components are triterpenes, sterols and triglycerides. Research shows that Ganoderma spore oil and Ganoderma spore powder have significant differences in the material basis for their medicinal effects.
  • Ganoderma spore polysaccharides, triterpenoids and Ganoderma spore proteins in Ganoderma spore powder exert good immunomodulatory activity.
  • the anti-tumor activity of Ganoderma spore powder is mainly composed of Polysaccharides and triterpenoids play a key role.
  • Polysaccharides are the main functional component of Ganoderma lucidum spore powder, and the triterpenoid content is low.
  • Ganoderma spore oil also has anti-tumor, immune-enhancing, and liver-protecting effects. It contains more triterpenes and a small amount of sterols, does not contain polysaccharides, and also contains a large amount of triglycerides.
  • the medical community proposes that the treatment of malignant tumors cannot simply pursue the killing rate of tumor cells, but must pay attention to the improvement of patients' quality of life and prolongation of survival time in order to more scientifically evaluate the actual efficacy of treating malignant tumors.
  • the object of the present invention is to provide a use of Ganoderma lucidum spore oil in preparing drugs for prolonging the survival of tumor patients. Based on the above reasons and needs, it was discovered that Ganoderma spore oil has new properties of prolonging the survival period of tumor patients, thereby providing a new use of Ganoderma spore oil as a drug, that is, its use as a drug to prolong the survival period of tumor patients.
  • the present invention adopts the following technical solutions:
  • the tumor includes one or more of liver cancer tumors, lung cancer tumors, gastric cancer tumors, intestinal cancer tumors, breast cancer tumors, and ascites cancer tumors.
  • the tumor patient has been treated with chemotherapy drugs.
  • the chemotherapeutic drug includes one or more of cyclophosphamide and paclitaxel.
  • the Ganoderma lucidum spore oil contains ergosterol, triterpenes, and triglycerides.
  • the Ganoderma lucidum spore oil has a triglyceride mass content of >70%, a triterpene mass content of 25% to 40%, an ergosterol mass content of 0.1% to 0.5%, and a moisture content of ⁇ 0.25%.
  • a method for preparing Ganoderma spore oil as described above including: taking broken Ganoderma spore powder and performing supercritical carbon dioxide extraction and refining to obtain the Ganoderma spore oil.
  • the wall-breaking rate of the broken Ganoderma lucidum spore powder is >95%.
  • the extraction and purification undergoes two-stage separation, and only the first-stage separation sample is retained to obtain the Ganoderma lucidum spore oil.
  • the conditions for the extraction essence include: extraction pressure is 25-35MPa, extraction temperature is 40-45°C, extraction time is 1-2h, CO2 flow rate is 40L/h, and first-stage separation pressure is 10-15MPa. , the separation temperature is 40 ⁇ 45°C, the second stage separation pressure is 5 ⁇ 8MPa, and the separation temperature is 40 ⁇ 45°C.
  • the present invention only retains the sample obtained by the first-level separation to obtain the Ganoderma lucidum spore oil. Through separation and refining, a large amount of impurities and moisture are removed, and the Ganoderma spore oil obtained is clear and translucent with stable component content. The experiment found that the sample after secondary separation contained many impurities, had a turbid appearance, high moisture content (about 10%), and was easy to deteriorate, so it was not retained.
  • Ganoderma spore oil is a fat-soluble active substance obtained from Ganoderma spores through wall breaking and supercritical carbon dioxide extraction. It contains sterols, triterpenes, triglycerides and other ingredients.
  • the present invention removes a large amount of impurities and moisture through separation and refining.
  • the Ganoderma spore oil obtained is clear and translucent, with few impurities and stable component content, ensuring the stability of the material basis of Ganoderma spore oil. Obviously the effect is also guaranteed.
  • the present invention conducts experiments and research on tumor-bearing mice using chemotherapy drugs and finds that Ganoderma lucidum spore oil has a significant effect on prolonging the survival period of tumor-bearing mice.
  • the Ganoderma lucidum spore oil of the present invention has the beneficial effect of being used to prepare drugs for prolonging the survival period of tumor patients.
  • the preparation method of Ganoderma lucidum spore oil of the present invention is as follows:
  • 500g of Ganoderma lucidum spore powder is taken for wall breaking, with a wall breaking rate of >95%, and then extracted with supercritical CO 2 and separated and refined to obtain the Ganoderma lucidum spore oil.
  • the extraction pressure is 25 ⁇ 35MPa
  • the extraction temperature is 40 ⁇ 45°C
  • the extraction time is 1 ⁇ 2h
  • the CO2 flow is 40L/h
  • the primary separation pressure is 10 ⁇ 15MPa
  • the separation temperature is 40 ⁇ 45°C
  • the secondary separation The pressure is 5 ⁇ 8MPa
  • the separation temperature is 40 ⁇ 45°C. Only the sample obtained by the primary separation is retained to obtain the Ganoderma lucidum spore oil.
  • the Ganoderma spore oil obtained is clear and translucent with stable component content.
  • ICR mouse male, weight 26 ⁇ 2g.
  • the animals were kept in SPF grade animal rooms with alternating light and dark (12h:12h) conditions, with free access to food and water.
  • the experiments began after the animals acclimated to the environment in the animal room for 7 days.
  • For Ganoderma spore oil use corn oil to make a solution of corresponding concentration before administration, and prepare it for immediate use.
  • Cyclophosphamide (CTX) prepare a solution of corresponding concentration with physiological saline before administration, and prepare it immediately.
  • EAC cell line (mouse Ehrlich ascites carcinoma cell line).
  • CTX group 16 tumor-bearing mice were administered an equal volume of corn oil by gavage; at the same time, 50 mg/Kg of cyclophosphamide was injected intraperitoneally, twice a week; the above administration was carried out until the animals died.
  • Ganoderma spore oil + CTX group 20 tumor-bearing mice, Ganoderma spore oil (5.1ml/Kg) was administered intragastrically, and cyclophosphamide 50mg/Kg was injected intraperitoneally, twice a week; the above administration was carried out until the animals died.
  • mice were continuously administered corn oil by gavage and intraperitoneally injected cyclophosphamide twice a week (CTX group), the average survival days of the mice was 31.5 ⁇ 8.8 days.
  • CTX group After continuous intragastric administration of Ganoderma lucidum spore oil (5.1ml/Kg) and intraperitoneal injection of cyclophosphamide twice a week, the average survival days of mice were 37.9 ⁇ 7.7d, which was statistically different from the CTX group, and their lives were prolonged. The rate is 20.2%.
  • the results showed that Ganoderma spore oil could significantly prolong the survival of tumor-bearing mice after being given chemotherapy drugs.
  • ICR mouse male, weight 26 ⁇ 2g.
  • the animals were kept in SPF grade animal rooms with alternating light and dark (12h:12h) conditions, with free access to food and water.
  • the experiments began after the animals acclimated to the environment in the animal room for 7 days.
  • For Ganoderma spore oil use corn oil to make a solution of corresponding concentration before administration, and prepare it for immediate use.
  • Cyclophosphamide (CTX) prepare a solution of corresponding concentration with physiological saline before administration, and prepare it immediately.
  • Cell line H22 liver cancer cell.
  • Solvent control group 25 tumor-bearing mice were administered an equal volume of corn oil by gavage; at the same time, 50 mg/Kg of cyclophosphamide was injected intraperitoneally, twice a week; until the animals died.
  • Ganoderma spore oil (2.72ml/Kg) + cyclophosphamide group 25 tumor-bearing mice, Ganoderma spore oil (2.72ml/Kg) was orally administered once a day for 6 consecutive days, then stopped for 1 day; at the same time Cyclophosphamide 50mg/Kg was injected intraperitoneally, twice a week; the above administration was carried out until the animal died.
  • Ganoderma spore oil (0.34ml/Kg) + cyclophosphamide group 25 tumor-bearing mice, Ganoderma spore oil (0.34ml/Kg) was orally administered once a day for 6 consecutive days, then stopped for 1 day; at the same time Cyclophosphamide 50mg/Kg was injected intraperitoneally, twice a week; the above administration was carried out until the animal died.
  • H22 tumor-bearing mice in the solvent control group were given corn oil by gavage continuously for 21 days, and cyclophosphamide (CTX) was intraperitoneally injected twice a week.
  • the average survival days of the mice was 41.12 ⁇ 2.77 days.
  • the mice were continuously given Ganoderma spore oil (2.72ml/Kg) by gavage and intraperitoneally injected with cyclophosphamide twice a week, the average survival days of the mice was 53.96 ⁇ 3.81d, which was significantly longer than that of the solvent control group (P ⁇ 0.05).
  • the life extension rate is 31.23%.
  • mice Male, body weight at the time of experiment was 22 ⁇ 4g (5-6 weeks old).
  • the animals were kept in SPF grade animal rooms with alternating light and dark (12h:12h) conditions, with free access to food and water.
  • the experiments began after the animals acclimated to the environment in the animal room for 7 days.
  • PTX Paclitaxel injection
  • Cell line Lewis lung cancer cells.
  • Paclitaxel alone group corn oil + PTX (20mg/Kg)
  • Ganoderma spore oil (high dose) combination group Ganoderma spore oil (3.4ml/Kg) + PTX (20mg/Kg)
  • Ganoderma spore oil (low dose) combination group Ganoderma spore oil (0.34ml/Kg) + PTX (20mg/kg)
  • mice in the paclitaxel alone treatment group were given corn oil by gavage continuously for 21 days, and paclitaxel (PTX) was intraperitoneally injected twice a week.
  • the average survival days of the mice was 17.68 ⁇ 1.42d.
  • Mice in the high-dose treatment group of Ganoderma spore oil combined with Paclitaxel were given Ganoderma spore oil (3.4ml/Kg) continuously by gavage and intraperitoneally injected Paclitaxel twice a week.
  • the average survival days of the mice was 22.05 ⁇ 1.49d, which was significantly higher.
  • the life extension rate was 22.89%.
  • mice in the low-dose treatment group of Ganoderma spore oil combined with Paclitaxel were given Ganoderma spore oil (0.34ml/Kg) continuously by gavage and intraperitoneally injected Paclitaxel twice a week.
  • the average survival days of the mice were 22.55 ⁇ 1.43d, which was significantly longer than In the solvent group (P ⁇ 0.05), the life extension rate was 27.55%.
  • the results suggest that Ganoderma lucidum spore oil can significantly prolong the survival time of tumor-bearing mice undergoing PTX chemotherapy.
  • Ganoderma lucidum spore oil of the present invention has a significant effect on prolonging the survival of tumor-bearing mice after administration of chemotherapy drugs.
  • Ganoderma lucidum spore oil of the present invention has the beneficial effect of being used to prepare drugs that extend the survival period of tumor-bearing mice after administration of chemotherapy drugs.

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Abstract

一种灵芝孢子油在制备延长肿瘤患者生存期药物的应用，灵芝孢子油具有延长荷瘤小鼠生存期的作用，用于制备延长肿瘤患者生存期的药物。

Description

一种灵芝孢子油在制备延长肿瘤患者生存期药物的应用 技术领域
本发明涉及医药技术领域，具体涉及一种灵芝孢子油在制备延长肿瘤患者生存期药物的应用。
背景技术
灵芝是祖国中医药宝库中的珍品，素有“仙草”之誉，可用于心神不宁，失眠心悸，肺虚咳喘，虚劳短气，不思饮食。灵芝孢子是灵芝生长成熟期从菌盖弹射出来极其细小的孢子，为灵芝的生殖细胞，具有灵芝的全部遗传活性物质。灵芝孢子粉的化学成分较为复杂，含有多糖类、三萜类、核苷类、维生素类、甾醇类、生物碱、无机微量元素等。灵芝孢子油是灵芝孢子粉经破壁、超临界二氧化碳萃取所得脂溶性活性物质，主要成分为三萜类、甾醇类和甘油三酯类。研究表明灵芝孢子油和灵芝孢子粉发挥药效的物质基础具有明显差异，灵芝孢子粉中灵芝孢子多糖、三萜化合物和灵芝孢子蛋白质发挥良好的免疫调节活性，灵芝孢子粉的抗肿瘤活性主要由多糖类和三萜类化合物起到关键性的作用，其中多糖是灵芝孢子粉的主要功效成分，三萜含量较低。而灵芝孢子油也具有抗肿瘤、增强免疫力、保肝护肝等功效，其含有较多的三萜和少量甾醇，不含多糖，此外含有大量的甘油三酯。
近年来癌症发病率呈现逐年攀升趋势，伴随着新型医疗模式的转变，医护工作人员在肿瘤治疗效果的基础上，更注重患者带瘤生存期的延长，我国的癌症患者生存期远低于国际水平，说明提高生存期是既我国癌症治疗需要迫切解决的问题，又是癌症患者最关心的问题。随着对肿瘤问题的认识不断深入，单纯以瘤体缩小来考察疗效的方式局限性越来越大。恶性肿瘤，尤其是晚期，患者即使肿瘤病灶缩小甚至达到完全缓解，但全身状况很差，生活质量很低。结果生存时间并无实质性延长，并未使患者真正受益。因此医学界提出恶性肿瘤的治疗不能单纯追求对肿瘤细胞杀伤率，而必须关注患者生活质量的改善和生存时间的延长，才能更科学地评估治疗恶性肿瘤的实际疗效。
如何更好地改善肿瘤患者、特别是肿瘤晚期患者的生活质量并有效地延长其生存时间，成为亟待解决的问题。
发明内容
本发明的目的在于，提供一种灵芝孢子油在制备延长肿瘤患者生存期药物方面的用途。基于上述的原因和需求，发现灵芝孢子油具有延长肿瘤患者生存期的新性能，进而提供一种灵芝孢子油作为药物的新用途，即在延长肿瘤患者生存期药物方面的用途。
为了解决上述技术问题，本发明采用如下技术方案：
一种灵芝孢子油在制药中的应用，用于延长肿瘤患者生存期。
在使用化疗药物的同时联用中医药是近年肿瘤治疗研究的热点。经过研究意外发现，将灵芝孢子粉经超临界二氧化碳萃取所得的灵芝孢子油，与化疗药物联用后有显著提高肿瘤患者生存期的影响，目前尚未见相关报道。
优选的，所述肿瘤包括肝癌肿瘤、肺癌肿瘤、胃癌肿瘤、肠癌肿瘤、乳腺癌肿瘤、腹水癌肿瘤中的一种或多种。
优选的，所述肿瘤患者经过化疗药物治疗。
优选的，所述化疗药物包括环磷酰胺、紫杉醇中的一种或多种。
优选的，所述灵芝孢子油含有麦角甾醇、三萜、甘油三酯。
优选的，所述灵芝孢子油中，甘油三酯质量含量＞70％，三萜质量含量为25％～40％，麦角甾醇质量含量为0.1％～0.5％，水分含量＜0.25％。
一种如上所述灵芝孢子油的制备方法，包括：取破壁灵芝孢子粉，经超临界二氧化碳萃取精制，获得所述灵芝孢子油。
优选的，所述破壁灵芝孢子粉的破壁率＞95％。
优选的，所述萃取精制经过二级分离，仅保留第一级分离样品，即获得所述灵芝孢子油。
优选的，所述萃取精的条件包括：萃取压力为25～35MPa，萃取温度为40～45℃，萃取时间为1～2h，CO 2流量为40L/h，第一级分离压力为10～15MPa，分离温度为40～45℃，第二级分离压力为5～8MPa，分离温度为40～45℃。
本发明仅保留一级分离得到的样品，即得所述灵芝孢子油。通过分离精制， 除去大量杂质和水分，获得的所述灵芝孢子油清澈透亮，组分含量稳定。实验发现二级分离后的样品含有较多杂质，外观浑浊，水分含量高(约含10％)，易变质，因此不予保留。
超临界二氧化碳萃取是特别适用于脂溶性、高沸点、热敏性物质提取的新型绿色提取分离技术。灵芝孢子油是灵芝孢子经破壁、超临界二氧化碳萃取所得的脂溶性活性物质，含甾醇、三萜、甘油三酯等成分。为获得质量稳定的目标灵芝孢子油，本发明通过分离精制，除去大量杂质和水分，获得的所述灵芝孢子油清澈透亮，杂质少、组分含量稳定，确保了灵芝孢子油物质基础的稳定，显然效果也得到了保障。
与现有技术相比较，实施本发明，具有如下有益效果：
本发明经过对使用化疗药物的荷瘤小鼠进行实验与研究，发现灵芝孢子油对荷瘤小鼠具有显著的延长生存期作用。本发明的灵芝孢子油具有可用于制备延长肿瘤患者生存期药物的有益效果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合具体实施例对本发明作进一步地详细描述，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1
灵芝孢子油的制备
本发明的灵芝孢子油制备方法如下：
取500g灵芝孢子粉进行破壁，破壁率＞95％，然后用超临界CO 2萃取，并通过分离精制，获得所述灵芝孢子油。萃取压力为25～35MPa，萃取温度为40～45℃，萃取时间为1～2h，CO 2流量为40L/h，一级分离压力为10～15MPa，分离温度为40～45℃，二级分离压力为5～8MPa，分离温度为40～45℃。仅保留一级分离得到的样品，即得所述灵芝孢子油。通过分离精制，除去大量杂质和水分，获得的所述灵芝孢子油清澈透亮，组分含量稳定。实验发现二级分离后 所得样品含有较多杂质，外观浑浊，水分含量高(约含10％)，易变质，因此不予保留。
实施例2
灵芝孢子油对接受环磷酰胺治疗的腹水癌细胞移植瘤小鼠生存期的影响
1.实验动物
ICR小鼠，雄性，体重26±2g。动物饲养于明暗交替(12h:12h)SPF级动物房中，自由进食饮水。动物在动物房中适应环境7天后开始进行实验。
2.实验材料
灵芝孢子油，给药前用玉米油配成相应浓度的溶液，现配现用。环磷酰胺(CTX)，给药前用生理盐水配成相应浓度的溶液，现配现用。
细胞株：EAC细胞株(小鼠艾氏腹水癌细胞株)。
3.实验方法
(1)荷瘤小鼠动物模型的建立：培养EAC细胞，取最佳生长状态的细胞，将细胞浓度调整至5×10 6/ml，在无菌条件下注入小鼠腹腔，每只小鼠注入0.2ml。腹腔传代保种10-12天，取腹部肿大、生长旺盛且无溃破的健康荷瘤小鼠，置超净工作台，腹部皮肤直接消毒后，用空针穿过腹部肌肉，抽取腹水，放入无菌容器内，置冰块上保存，用生理盐水洗涤、离心2次，生理盐水稀释后计数并调整细胞浓度约为5×10 7个/ml，在小鼠的右前肢腋下皮下注射上述瘤细胞悬液，每只小鼠0.2mL。在接种24h后，开始分组给药。
(2)实验分组
CTX组：荷瘤小鼠，16只，等容量玉米油灌胃；同时腹腔注射环磷酰胺50mg/Kg，每周2次；以上给药直至动物死亡。
灵芝孢子油+CTX组：荷瘤小鼠，20只，灵芝孢子油灌胃(5.1ml/Kg)，同 时腹腔注射环磷酰胺50mg/Kg，每周2次；以上给药直至动物死亡。
(3)灵芝孢子油对EAC实体瘤小鼠生存时间、生命延长率的影响
小鼠造模当天为第0天，从造模后第一天开始给予灵芝孢子油或玉米油灌胃，连续给药直至动物死亡。从造模后第一周开始给予腹腔注射环磷酰胺或生理盐水，每周两次，直至动物死亡。统计各组小鼠存活时间，计算各组平均生存时间，按下列公式计算生命延长率：生命延长率＝(药物组平均生存时间－模型组平均生存时间)/模型组平均生存时间×100％，并进行统计学处理。。
4.实验结果
EAC荷瘤小鼠在连续灌胃给予玉米油，同时每周2次腹腔注射环磷酰胺(CTX组)后，小鼠的平均生存天数为31.5±8.8d。在连续灌胃给予灵芝孢子油(5.1ml/Kg)，同时每周2次腹腔注射环磷酰胺后，小鼠平均生存天数为37.9±7.7d，与CTX组相比有统计学差异，生命延长率为20.2％。结果表明，灵芝孢子油可明显延长给予化疗药物后荷瘤小鼠的生存期。
实施例3
灵芝孢子油对接受环磷酰胺治疗的H22荷瘤小鼠生存期的影响
1.实验动物
ICR小鼠，雄性，体重26±2g。动物饲养于明暗交替(12h:12h)SPF级动物房中，自由进食饮水。动物在动物房中适应环境7天后开始进行实验。
2.实验材料
灵芝孢子油，给药前用玉米油配成相应浓度的溶液，现配现用。环磷酰胺(CTX)，给药前用生理盐水配成相应浓度的溶液，现配现用。
细胞株：H22肝癌细胞。
3.实验方法
(1)荷瘤小鼠动物模型的建立：培养H22细胞，取最佳生长状态的细胞，将细胞浓度调整至2.5×10 6/ml，在无菌条件下注入小鼠腹腔，每只小鼠注入0.2ml。腹腔传代保种10-12天，取腹部肿大、生长旺盛且无溃破的健康荷瘤小鼠，置超净工作台，腹部皮肤直接消毒后，用空针穿过腹部肌肉，抽取腹水，放入无菌容器内，置冰块上保存，用生理盐水洗涤、离心2次，生理盐水稀释后计数并调整细胞浓度约为2×10 7个/ml，在小鼠的右前肢腋下皮下注射上述瘤细胞悬液，每只小鼠0.2mL。接种后5天，选取在腋下可触及肿块的小鼠，开始分组给药。
(2)实验分组
溶剂对照组：荷瘤小鼠，25只，等容量玉米油灌胃；同时腹腔注射环磷酰胺50mg/Kg，每周2次；直至动物死亡。
灵芝孢子油(2.72ml/Kg)+环磷酰胺组：荷瘤小鼠，25只，灵芝孢子油灌胃(2.72ml/Kg)，每天一次，连续给药6天后，停药1天；同时腹腔注射环磷酰胺50mg/Kg，每周2次；以上给药直至动物死亡。
灵芝孢子油(0.34ml/Kg)+环磷酰胺组：荷瘤小鼠，25只，灵芝孢子油灌胃(0.34ml/Kg)，每天一次，连续给药6天后，停药1天；同时腹腔注射环磷酰胺50mg/Kg，每周2次；以上给药直至动物死亡。
(3)灵芝孢子油对H22实体瘤小鼠生存时间、生命延长率的影响
荷瘤小鼠随机分组后开始给予灵芝孢子油或玉米油灌胃，连续给药直至动物死亡。同时开始给予腹腔注射环磷酰胺(CTX)或生理盐水，每周两次，直至动物死亡。统计各组小鼠存活时间，计算各组平均生存时间，按下列公式计算生命延长率：生命延长率＝(药物组平均生存时间－模型组平均生存时间)/模型组平均生存时间×100％，并进行统计学处理。
4.实验结果
溶剂对照组H22荷瘤小鼠连续灌胃给予玉米油21天，同时每周2次腹腔注射环磷酰胺(CTX)，小鼠的平均生存天数为41.12±2.77d。小鼠连续灌胃给予灵芝孢子油(2.72ml/Kg)，同时每周2次腹腔注射环磷酰胺后，小鼠平均生存天数为53.96±3.81d，显著长于溶剂对照组(P<0.05)，生命延长率为31.23％。小鼠连续灌胃给予灵芝孢子油(0.34ml/Kg)，同时每周2次腹腔注射环磷酰胺后，小鼠平均生存天数为54.68±3.91d，显著长于溶剂组(P<0.05)，生命延长率为32.99％。结果提示，灵芝孢子油能够显著延长接受CTX化疗的H22荷瘤小鼠的生存期。
实施例4
灵芝孢子油对接受紫杉醇治疗的Lewis肺癌荷瘤小鼠生存期的影响
1.实验动物
C57BL/6小鼠，雄性，实验时体质量22±4g(5-6周龄)。动物饲养于明暗交替(12h:12h)SPF级动物房中，自由进食饮水。动物在动物房中适应环境7天后开始进行实验。
2.实验材料
灵芝孢子油，给药前用玉米油配成相应浓度的溶液，现配现用。紫杉醇注射液(PTX)。
细胞株：Lewis肺癌细胞。
3.实验方法
(1)荷瘤小鼠动物模型的建立：复苏、培养Lewis肺癌细胞，取最佳生长状态的细胞，将细胞浓度调整至7.5×10 6个/ml，在小鼠的右前肢腋下皮下注射上述瘤细胞悬液，每只小鼠0.2ml。在接种后第5天后，将成瘤的小鼠，按照体重大小进行随机分组。
(2)实验分组
紫杉醇单用组：玉米油+PTX(20mg/Kg)
灵芝孢子油(高剂量)联用组：灵芝孢子油(3.4ml/Kg)+PTX(20mg/Kg)
灵芝孢子油(低剂量)联用组：灵芝孢子油(0.34ml/Kg)+PTX(20mg/kg)
(3)灵芝孢子油对Lewis实体瘤小鼠生存时间、生命延长率的影响
小鼠造模当天为第0天，从造模后第一天开始给予灵芝孢子油或玉米油灌胃，连续给药直至动物死亡。从造模后第一周开始给予腹腔注射紫杉醇或生理盐水，每周两次，直至动物死亡。统计各组小鼠存活时间，计算各组平均生存时间，按下列公式计算生命延长率：生命延长率＝(药物组平均生存时间－模型组平均生存时间)/模型组平均生存时间×100％，并进行统计学处理。
4.实验结果
单用紫杉醇治疗组的Lewis荷瘤小鼠连续灌胃给予玉米油21天，同时每周2次腹腔注射紫杉醇(PTX)，小鼠的平均生存天数为17.68±1.42d。灵芝孢子油高剂量联用紫杉醇治疗组的小鼠连续灌胃给予灵芝孢子油(3.4ml/Kg)，同时每周2次腹腔注射紫杉醇后，小鼠平均生存天数为22.05±1.49d，显著高于溶剂组(P<0.05)，生命延长率为22.89％。灵芝孢子油低剂量联用紫杉醇治疗组的小鼠连续灌胃给予灵芝孢子油(0.34ml/Kg)，同时每周2次腹腔注射紫杉醇后，小鼠平均生存天数为22.55±1.43d，显著长于溶剂组(P<0.05)，生命延长率为27.55％。结果提示，灵芝孢子油能够显著延长荷瘤小鼠PTX化疗生存期。
综上，本发明的灵芝孢子油对荷瘤小鼠具有显著的延长给予化疗药物后荷瘤小鼠的生存期作用。本发明的灵芝孢子油具有可用于制备延长给予化疗药物后荷瘤小鼠的生存期药物的有益效果。
以上所揭露的仅为本发明的较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范 围。

Claims (10)

  1. 一种灵芝孢子油在制药中的应用，其特征在于，用于延长肿瘤患者生存期。
  2. 如权利要求1所述应用，其特征在于，所述肿瘤包括肝癌肿瘤、肺癌肿瘤、胃癌肿瘤、肠癌肿瘤、乳腺癌肿瘤、腹水癌肿瘤中的一种或多种。
  3. 如权利要求1所述应用，其特征在于，所述肿瘤患者经过化疗药物治疗。
  4. 如权利要求3所述应用，其特征在于，所述化疗药物包括环磷酰胺、紫杉醇中的一种或多种。
  5. 如权利要求1所述应用，其特征在于，所述灵芝孢子油含有麦角甾醇、三萜、甘油三酯。
  6. 如权利要求5所述应用，其特征在于，所述灵芝孢子油中，甘油三酯质量含量＞70％，三萜质量含量为25％～40％，麦角甾醇质量含量为0.1％～0.5％，水分含量＜0.25％。
  7. 一种如权利要求5所述灵芝孢子油的制备方法，其特征在于，包括：取破壁灵芝孢子粉，经超临界二氧化碳萃取精制，获得所述灵芝孢子油。
  8. 如权利要求7所述制备方法，其特征在于，所述破壁灵芝孢子粉的破壁率＞95％。
  9. 如权利要求7所述制备方法，其特征在于，所述萃取精制经过二级分离，仅保留第一级分离样品，即获得所述灵芝孢子油。
  10. 如权利要求9所述制备方法，其特征在于，所述萃取精制的条件包括：萃取压力为25～35MPa，萃取温度为40～45℃，萃取时间为1～2h，CO 2流量为40L/h，第一级分离压力为10～15MPa，分离温度为40～45℃，第二级分离压力为5～8MPa，分离温度为40～45℃。
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