WO2023001161A1 - 一种灵芝孢子油及其在制备抗癌因性疲乏的药物中的应用 - Google Patents

一种灵芝孢子油及其在制备抗癌因性疲乏的药物中的应用 Download PDF

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WO2023001161A1
WO2023001161A1 PCT/CN2022/106599 CN2022106599W WO2023001161A1 WO 2023001161 A1 WO2023001161 A1 WO 2023001161A1 CN 2022106599 W CN2022106599 W CN 2022106599W WO 2023001161 A1 WO2023001161 A1 WO 2023001161A1
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spore oil
ganoderma lucidum
ganoderma
oil
preparation
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PCT/CN2022/106599
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English (en)
French (fr)
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蔡鸿飞
许文东
袁诚
刘菊妍
李菁
韩亚明
曹霖
张琴
蒋兆健
毛禹康
徐春草
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广州白云山汉方现代药业有限公司
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Priority to KR1020247002129A priority Critical patent/KR20240022647A/ko
Priority to AU2022314013A priority patent/AU2022314013A1/en
Priority to CA3224500A priority patent/CA3224500A1/en
Priority to EP22845326.2A priority patent/EP4344704A1/en
Publication of WO2023001161A1 publication Critical patent/WO2023001161A1/zh
Priority to US18/415,519 priority patent/US20240197799A1/en

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    • 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
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • 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
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • 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
    • A61K9/08—Solutions
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • 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
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • 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
    • A61K2236/55—Liquid-liquid separation; Phase separation
    • 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
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    • 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 relates to the technical field of medicine, in particular to ganoderma lucidum spore oil and its application in the preparation of anti-cancer-related fatigue medicines.
  • Cancer-related fatigue is one of the most common symptoms of cancer patients and the most common side effect of cancer treatment.
  • NCCN National Comprehensive Cancer Network
  • the International Classification of Diseases (ICD) describes the symptoms of cancer-related fatigue as non-specific weakness, weakness, general decline, lethargy, and fatigue. According to statistics, more than 75% of cancer patients can see the symptoms of cancer-related fatigue. However, there is no definite conclusion on the mechanism of cancer-related fatigue, and there is no drug for the treatment of cancer-related fatigue.
  • cancer-related fatigue is mostly caused by emotional disorders, cancer toxin invasion, diet fatigue, etc., which cause damage to qi, blood and body fluids, and damage the qi of the viscera, which will not be resolved after a long time. Therefore, the treatment of cancer-related fatigue is mainly based on the tumor itself, based on syndrome differentiation and treatment according to the deficiency and excess of qi, blood, yin and yang in the viscera, with strengthening the body and eliminating pathogenic factors as the main treatment principle, mainly tonifying, supplemented by eliminating pathogenic factors, and eliminating fatigue as much as possible In addition to the cause of formation, attention should also be paid to overall regulation.
  • Ganoderma lucidum is a treasure in the treasure house of traditional Chinese medicine in the motherland. It is known as the "fairy herb”.
  • Ganoderma lucidum spores are extremely small spores ejected from the cap during the mature growth period of Ganoderma lucidum. They are the germ cells of Ganoderma lucidum and have all the genetically active substances of Ganoderma lucidum. Modern pharmacological studies have shown that Ganoderma lucidum spores have the functions of anti-tumor, enhancing immune regulation, lowering blood sugar, lowering blood fat, anti-inflammation, anti-hypoxia, and scavenging free radicals.
  • Ganoderma lucidum spore oil is a fat-soluble active substance obtained by breaking the wall of Ganoderma lucidum spores and extracting it with supercritical carbon dioxide. Modern research has shown that it has the effects of anti-tumor, enhancing immunity, and protecting the liver.
  • the object of the present invention is to provide a ganoderma lucidum spore oil and its application in the preparation of anti-cancer-related fatigue medicine. Based on the above reasons and needs, it is found that Ganoderma lucidum spore oil has new properties of anti-cancer-related fatigue, and then provides a new application of Ganoderma lucidum spore oil as a medicine, that is, the application in the preparation of drugs for treating or preventing cancer-related fatigue.
  • the present invention adopts the following technical solutions:
  • a ganoderma spore oil in which no ganoderma triterpenoids are detected.
  • the content of triglycerides in the Ganoderma lucidum spore oil is >90%.
  • the triglyceride content in the ganoderma lucidum spore oil of the present invention is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • Ganoderma lucidum spore oil has the effect of enhancing immunity and anti-tumor because it is rich in Ganoderma lucidum triterpenoids. According to the existing research results, it should be considered that with the decrease of the content of triterpenoids, various effects of spore oil will be weakened or even disappeared.
  • the present invention unexpectedly found through research that in the face of pathological fatigue, especially cancer-related fatigue, making the content of triglycerides in Ganoderma lucidum spore oil >90% has a good anti-cancer-related fatigue effect.
  • the spore oil containing Ganoderma lucidum triterpenoids it has a better anti-cancer fatigue effect, and significantly improves the relief effect on mental fatigue and physical fatigue caused by it. It was unimaginable before.
  • a kind of preparation method of Ganoderma lucidum spore oil as above, comprises the steps:
  • Ganoderma lucidum spores are subjected to wall breaking, granulation, drying and supercritical CO2 extraction to obtain crude spore oil;
  • the mass ratio of the crude spore oil to the silica gel is 1: (10-40).
  • the volume ratio of petroleum ether to ethyl acetate is (8.5-9.5):(0.5-1.5).
  • the volume ratio of petroleum ether to ethyl acetate is 9.2:0.8.
  • the ratio of the diameter of the elution column to the height of the packed column is 1: (10-20).
  • the medicine is tablet, capsule, buccal preparation, granule, granule, pill, powder, ointment, pill, suspension, solution, injection, suppository, cream, spray, drop or one of the patches.
  • the present invention finds that the spore oil with triglyceride>90% and ergosterol 0%-0.5% has significant anti-fatigue effect on tumor-bearing mice. And it is surprisingly found that compared with the spore oil containing Ganoderma lucidum triterpenoids, the Ganoderma lucidum spore oil of the present invention significantly improves the relieving effect on the resulting mental fatigue and physical fatigue, which is expected to be significant Improving the quality of daily life of cancer patients.
  • the Ganoderma lucidum spore oil of the present invention has the beneficial effect of being used to prepare medicines for preventing and treating cancer-related fatigue, and at the same time enriching and separating triterpenoids is also expected to develop other new products with higher triterpenoid content.
  • Fig. 1 is the statistical diagram of the average body weight of human gastric cancer transplanted tumor mouse model in effect example 4 as the experiment time changes;
  • Fig. 2 is a statistical diagram of the average daily food intake of the human gastric cancer transplanted tumor mouse model in Effect Example 4 as a function of the experimental time.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • a ganoderma spore oil is obtained by extracting the ganoderma spores through the following steps: the ganoderma spores are obtained by breaking the wall, granulating, drying, and extracting with supercritical CO 2 , and then adsorbing and separating the ganoderma through a silica gel column.
  • the triglyceride content in the ganoderma lucidum spore oil is more than 90%, the ergosterol content is 0%-0.5%, and the ganoderma lucidum triterpenoids are not detected.
  • Nude mice, SPF grade, half male and half female, 32 in total, feeding environment feeding in nude mice in SPF grade animal room, free to eat, 12h light/12h dark, animal room ambient temperature 16-26°C, relative humidity 40% ⁇ 70%.
  • Test product 1 commercially available ganoderma lucidum spore oil containing ganoderma triterpenoids
  • test product 2 ganoderma spore oil obtained by the process of the present application, triglyceride content > 90%, ergosterol content of 0% to 0.5% , Ganoderma lucidum triterpenoids were not detected
  • corn oil was used to make a solution of corresponding concentration before administration, and it was prepared and used now.
  • Cell line human lung cancer NCI-H460 cell line.
  • nude mice were randomly divided into 4 groups: blank control group (Control), tumor model group (Model), test product 1 group (Sample 1), test product 2 group (Sample 2). 8 in each group, half male and half male.
  • Route of administration intragastric administration of test products 1 and 2 and corn oil.
  • test article 1, 2 and corn oil are given once a day.
  • test article 1, 2 and corn oil were administered continuously for 4 weeks.
  • Dosing volume 0.1mL/10g body weight, that is, 10mL/kg.
  • the tail suspension test of tumor-bearing mice was carried out on the 28th day after administration. Stick the tail of the mouse (2cm away from the tip of the tail) on the tester with adhesive tape, so that the mouse is in an upside-down state. The mouse struggles continuously to overcome the abnormal position. "Moving”, record the cumulative immobility time of the mouse within 6 minutes.
  • test product 1 and test product 2 could significantly shorten the tail-suspension time of tumor-bearing mice, and test product 2 could further significantly shorten the time of tumor-bearing mice compared with test product 1.
  • the tail suspension time of the mice even reached the level of normal tumor-free mice (blank control group).
  • the ganoderma lucidum spore oil without triterpenoids prepared by the present invention has a more significant anti-fatigue effect on tumor-bearing mice than the spore oil containing ganoderma triterpenoids, and can even reach the level of a cancer-free state , Significantly improves the relief effect on the mental fatigue and physical fatigue caused by it.
  • Kunming mice SPF grade, female, 40, feeding environment: fed in SPF grade animal room mice, free to eat, 12h light/12h dark, animal room ambient temperature 16-26°C, relative humidity 40%-70% .
  • test product ganoderma lucidum spore oil obtained by the process of the present application
  • corn oil was used to make a solution of corresponding concentration before administration, and it was prepared for immediate use.
  • Chemotherapy drug cyclophosphamide
  • batch number: 8D231A 200 mg/bottle, produced by Baxter Oncology GmbH
  • a solution of corresponding concentration is prepared with normal saline before administration, and is prepared and used immediately.
  • Cell line mouse H22 liver cancer cell line.
  • mice xenograft tumor model after H22 cells were cultured to the required number, digested and counted, 0.1ml per mouse was inoculated near the right upper limb of the mouse to build a model. Rats were randomly divided into 4 groups: model control group (Control), cyclophosphamide group (CTX), Ganoderma lucidum spore oil (GLSO), cyclophosphamide + Ganoderma lucidum spore oil group (CTX+GLSO). 10 in each group.
  • CTX model control group
  • GLSO Ganoderma lucidum spore oil
  • CTX+GLSO Ganoderma lucidum spore oil
  • Administration route ganoderma lucidum spore oil administration; cyclophosphamide tail vein administration.
  • Weekly dosing frequency 1 dose per day, 5 consecutive days of administration followed by 2 days of rest.
  • Dosing cycle continuous administration for 3 weeks.
  • Dosing volume 0.1mL/10g body weight, that is, 10mL/kg.
  • the exhaustive swimming test of tumor-bearing mice was performed on the 13th day and the 20th day after administration, respectively.
  • the tumor-bearing mice were put into swimming tanks for swimming, the water depth was not less than 30cm, and the water temperature was 25 ⁇ 1°C.
  • the time from swimming to exhaustion of tumor-bearing mice was recorded.
  • the limbs of each tumor-bearing mouse were kept moving. If the limbs of the tumor-bearing mice were floating on the water surface, they could be stirred with a wooden stick, so that the tumor-bearing mice could not float independently after sinking under the water surface for 10 seconds. Judgment is exhaustion. When the average swimming time exceeds 360s, it will be counted as 360s.
  • Nude mice, SPF grade, female, 40, feeding environment feeding among nude mice in SPF grade animal room, free to eat, 12h light/12h dark, animal room ambient temperature 16-26°C, relative humidity 40%-70% .
  • test product ganoderma lucidum spore oil obtained by the process of the present application
  • corn oil was used to make a solution of corresponding concentration before administration, and it was prepared for immediate use.
  • Chemotherapy drug oxaliplatin
  • batch number: 190911AM 50mg/bottle, produced by Jiangsu Hengrui Pharmaceutical Co., Ltd., made into a solution of corresponding concentration with normal saline before administration, and prepared for immediate use.
  • Cell line human gastric cancer MNK45 cell line.
  • mice were randomly divided into 4 groups: model control group (Control), chemotherapy drug group (Oxaliplatin), chemotherapy+test product group (Oxaliplatin+Sample), chemotherapy+corn oil group (Oxaliplatin+Oil). 10 in each group.
  • model control group Control
  • chemotherapy drug group Oxaliplatin
  • chemotherapy+test product group Oxaliplatin+Sample
  • chemotherapy+corn oil group Oxaliplatin+Oil
  • Administration route intragastric administration of the test product and corn oil; tail vein administration of oxaliplatin.
  • Dosing cycle chemotherapeutic drugs are administered for 6 weeks; after 6 weeks of administration of the test product and corn oil, the administration is continued until the end of the test.
  • test product 10mL/kg; oxaliplatin 2mg/kg.
  • Dosing volume 0.1mL/10g body weight, that is, 10mL/kg.
  • the exhaustive swimming test of tumor-bearing mice was performed on the 37th and 49th day after administration, respectively.
  • the tumor-bearing mice were put into swimming tanks for swimming, the water depth was not less than 30cm, and the water temperature was 25 ⁇ 1°C.
  • the time from swimming to exhaustion of tumor-bearing mice was recorded. Keep the limbs of each tumor-bearing mouse moving during the whole experiment. If the limbs of the tumor-bearing mice float on the water surface and do not move, you can use a wooden stick to stir it nearby, so that the tumor-bearing mice cannot float independently after sinking under the water surface for 10 seconds. Judgment is exhaustion. When the average swimming time exceeds 360s, it will be counted as 360s.
  • Ganoderma lucidum spore oil does not affect the body weight of tumor-bearing mice
  • the Ganoderma lucidum spore oil of the present invention has a significant anti-fatigue effect on tumor-bearing mice. Moreover, the animals in the Ganoderma lucidum spore oil combined with chemotherapy drugs group did not die or lose weight significantly, and did not affect the food intake of tumor-bearing mice, suggesting that the Ganoderma lucidum spore oil has good safety in animals.
  • the ganoderma lucidum spore oil of the invention has the beneficial effect of being used for preparing medicines for preventing and treating cancer-related fatigue.

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Abstract

一种灵芝孢子油及其在制备抗癌因性疲乏的药物中的应用。所述灵芝孢子油中甘油三酯类成分含量＞90%，未检出灵芝三萜类化合物。所述灵芝孢子油采用超临界CO2萃取，经硅胶柱，石油醚-乙酸乙酯混合液洗脱，减压浓缩，真空干燥获得。所述灵芝孢子油具有显著的改善因癌症引起的疲乏作用，且具有良好的安全性，可用于制备治疗癌因性疲乏的药物。

Description

一种灵芝孢子油及其在制备抗癌因性疲乏的药物中的应用 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及其在制备抗癌因性疲乏的药物中的应用。
背景技术
癌因性疲乏(cancer-related fatigue，CRF)是肿瘤患者最常见的症状之一，也是肿瘤治疗中最常见的副作用。美国国家综合癌症网络(NCCN)定义为“一种痛苦的、持续的、主观的、有关躯体、情感或认知方面的疲乏感或疲惫感，与近期的活动量不符，与癌症或癌症的治疗有关，并且妨碍日常生活。”国际疾病分类标准(ICD)描述癌因性疲乏的症状为非特异的无力、虚弱、全身衰退、嗜睡、疲劳。据统计，超过75％肿瘤患者可见癌因性疲乏症状。但目前对于癌因性疲乏的机制研究还没有确切定论，也没有针对癌因性疲乏的治疗药物。
古代中医文献中并无出现“癌因性疲乏”相关病名的记载，多描述为“虚劳”。《素问·通评虚实论》云：“精气夺则虚”，可视为虚劳最早的定义。《金匮要略·虚劳篇》认为凡是由劳伤致脏腑经络、阴阳气血诸虚，有一系列劳损症状皆为虚劳。现代中医经过不断发展，普遍认为癌因性疲乏多由情志失调、癌毒侵犯、饮食劳倦等造成气血津液受损，伤及脏腑之气，日久不复所致。因此，癌因性疲乏的治疗主要立足于肿瘤疾病本身，根据脏腑气血阴阳的虚实的不同进行辨证论治，以扶正祛邪为主要治则，补益为主，辅以祛邪，在尽可能消除疲劳成因之外，还应重视整体调节。
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。现代药理研究表明，灵芝孢子具有抗肿瘤、增强免疫调节、降血糖、降血脂、抗炎、抗缺氧能力、清除自由基等作用。灵芝孢子油是灵芝孢子经破壁、超临界二氧化碳萃取所得脂溶性活性物质，现代研究表明具有抗肿瘤、增强免疫力、 保肝护肝等功效。
现有研究一般认为灵芝的主要有效成分为灵芝三萜类化合物，对灵芝的各种功效研究也都更集中在灵芝三萜上。也因此现有孢子油一般追求更高的灵芝三萜类化合物占比，含量可达到10～30％。而对于孢子油中的其他组分，例如占比最大的甘油三酯类化合物的成分研究和功效分析，目前尚未见相关报道。另一方面，现有研究对灵芝缓解疲劳效果也都集中在缓解生理性疲劳，对于病理性疲劳、特别是癌因性疲乏方面的作用效果也鲜见报道。
发明内容
本发明的目的在于，提供一种灵芝孢子油及其在制备抗癌因性疲乏的药物中的应用。基于上述的原因和需求，发现灵芝孢子油具有抗癌因性疲乏的新性能，进而提供一种灵芝孢子油作为药物的新应用，即在制备治疗或预防癌因性疲乏药物中的应用。
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，所述灵芝孢子油中，灵芝三萜类化合物未检出。
优选的，所述灵芝孢子油中，甘油三酯类成分含量＞90％。
本发明的灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。目前，大部分研究认为灵芝孢子油具有增强免疫力及抗肿瘤作用是由于其富含灵芝三萜类化合物，因此，对灵芝孢子油的成分研究主要偏重于灵芝三萜类化合物的富集。根据现有研究结果，应认为随着三萜类化合物含量的下降，孢子油各种功效均会随之减弱甚至消失。
但是，本发明经过研究意外地发现，在面对病理性疲劳、特别是癌因性疲乏时，使灵芝孢子油甘油三酯类成分含量＞90％，具有良好的抗癌因性疲乏效果。特别是，相较于含有灵芝三萜类化合物的孢子油，能具有更好的抗癌因性疲乏效果，显著提高了对因此而造成的精神上的疲乏和体力上的疲乏的缓解作用，这在此前是无法想象的。
一种如上所述灵芝孢子油的制备方法，包括如下步骤：
A、灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，获得孢子油粗品；
B、取硅胶适量，用石油醚-乙酸乙酯混合溶液溶解，湿法装柱，获得洗脱柱；
C、取所述孢子油粗品往所述洗脱柱上样，用所述石油醚-乙酸乙酯混合溶液常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到所述灵芝孢子油。
经检测，该灵芝孢子油中，甘油三酯＞90％，麦角甾醇0％～0.5％，三萜未检出。根据该工艺可以得到几乎未含有三萜类化合物的孢子油，另一方面，其实也同时对三萜类化合物进行了富集，有希望获得更高三萜类含量的其它新产品。
优选的，所述孢子油粗品质量与所述硅胶质量比为1：(10～40)。
优选的，所述石油醚-乙酸乙酯混合溶液中，石油醚与乙酸乙酯的体积比为(8.5～9.5)：(0.5～1.5)。
优选的，所述石油醚-乙酸乙酯混合溶液中，石油醚与乙酸乙酯的体积比为9.2：0.8。
优选的，所述洗脱柱的柱直径与填充柱高之比为1：(10～20)。
一种如上所述灵芝孢子油在制备抗癌因性疲乏药物中的应用。
一种含有如上所述灵芝孢子油的药物，所述药物还含有药学上可接受的载体。
优选的，所述药物为片剂、胶囊剂、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、溶液剂、注射剂、栓剂、霜剂、喷雾剂、滴剂或贴剂中的一种。
与现有技术相比较，实施本发明，具有如下有益效果：
本发明经过对三萜类化合物以外的灵芝成分进行实验与研究，发现甘油三酯＞90％，麦角甾醇0％～0.5％的孢子油对荷瘤小鼠具有显著的抗疲乏作用。并且令人意外地发现，相较于含有灵芝三萜类化合物的孢子油，本发明的灵芝孢子油显著提高了对因此而造成的精神上的疲乏和体力上的疲乏的缓解作用，有希望显著提高癌症患者的日常生活质量。而且本发明灵芝孢子油联合化疗药物组的动物没有死亡或体重明显下降、没有影响荷瘤小鼠的摄食量，提示灵芝孢子油在动物体内具有良好的安全性。本发明的灵芝孢子油具有可用于制备癌因性疲乏的防治药物的有益效果，而同时富集分离的三萜类化合物也有望开发出三萜类含量更高的其它新产品。
附图说明
图1为效果例4中人胃癌移植瘤小鼠模型体重平均值随实验时间变化统计图；
图2为效果例4中人胃癌移植瘤小鼠模型每日摄食量平均值随实验时间变化统计图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
实施例1
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝9.2:0.8(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:10，取孢子油上样，孢子油质量与硅胶质量比为1:28，用石油醚：乙酸乙酯＝9.2:0.8(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。
实施例2
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝9.2:0.8(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:13，取孢子油上样，孢子油质量与硅胶质量比为1:28，用石油醚：乙酸乙酯＝9.2:0.8(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。
实施例3
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝9.2:0.8(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:15，取孢子油上样，孢子油质量与硅胶质量比为1:28，用石油醚：乙酸乙酯＝9.2:0.8(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。
实施例4
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝9.2:0.8(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:15，取孢子油上样，孢子油质量与硅胶质量比为1:30，用石油醚：乙酸乙酯＝9.2:0.8(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三 萜类化合物未检出。
实施例5
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝9.3:0.7(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:15，取孢子油上样，孢子油质量与硅胶质量比为1:35，用石油醚：乙酸乙酯＝9.3:0.7(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。
实施例6
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝9.2:0.8(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:15，取孢子油上样，孢子油质量与硅胶质量比为1:33，用石油醚：乙酸乙酯＝9.2:0.8(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。
实施例7
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝9.0:1.0(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:17，取孢子油上样，孢子油质量与硅胶质量比为1:35，用石油醚：乙酸乙酯＝9.0:1.0(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。
实施例8
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝9.5:0.5(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:10，取孢子油上样，孢子油质量与硅胶质量比为1:10，用石油醚：乙酸乙酯＝9.5:0.5(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。
实施例9
一种灵芝孢子油，通过以下步骤进行提取得到：灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，再经硅胶柱吸附分离得到。分离工艺如下：取硅胶适量，用石油醚：乙酸乙酯＝8.5:1.5(体积比)溶解，湿法装柱，柱直径与填充柱高之比为1:20，取孢子油上样，孢子油质量与硅胶质量比为1:40，用石油醚：乙酸乙酯＝8.5:1.5(体积比)常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到孢子油样品。
该灵芝孢子油中甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出。
效果例1
灵芝孢子油对人肺癌移植瘤小鼠模型的抗疲乏作用
1.实验动物
裸小鼠，SPF级，雌雄各半，共32只，饲养环境：饲养于SPF级动物房裸鼠间，自由摄食，12h光照/12h黑暗，动物房环境温度16～26℃，相对湿度40％～70％。
2.实验材料
供试品1(市售含有灵芝三萜类成分的灵芝孢子油)，供试品2(本申请工艺获得的灵芝孢子油，甘油三酯含量＞90％，麦角甾醇含量为0％～0.5％，灵芝三萜类化合物未检出)，给药前用玉米油配成相应浓度的溶液，现配现用。
细胞株：人源肺癌NCI-H460细胞株。
3.实验方法
(1)裸鼠移植瘤模型的构建：NCI-H460细胞培养至所需数量后，消化计数后按0.15ml/只，接种至裸鼠右上肢附近造模(空白对照组不接种)，待肿瘤体积长至100mm 3左右，将裸鼠随机分成4组：空白对照组(Control)、肿瘤模型组(Model)、供试品1组(Sample 1)、供试品2组(Sample 2)。每组8只，雌雄各半。
(2)给药方法
给药途径：供试品1、2和玉米油灌胃给药。
每周给药频率：供试品1、2和玉米油每天给药1次。
给药周期:供试品1、2和玉米油连续给药4周。
给药剂量：供试品10mL/kg。
给药体积：按0.1mL/10g体重给药，即10mL/kg。
(3)悬尾试验
于给药后第28天进行荷瘤小鼠悬尾试验。将小鼠尾(距尾尖2cm处)用胶布粘在测试仪上，使小鼠呈倒挂状态，小鼠为克服不正常体位而不断挣扎， 以小鼠完全停止挣扎，呈静止的状态记为“不动”，记录小鼠在6min内的累计不动时间。
4.实验结果
结果如表3所示，给药第28天，肿瘤模型组荷瘤小鼠的平均悬尾不动时间显著高于空白对照组，且两组比较具有显著性差异(P<0.05)；供试品1组和供试品2组荷瘤小鼠的平均悬尾不动时间显著低于肿瘤模型组，且两组与肿瘤模型组比较均具有显著性差异(P<0.05)；供试品2组荷瘤小鼠的平均悬尾不动时间显著低于供试品1组，且两组比较依然具有显著性差异(P<0.05)。
表3灵芝孢子油对雌性荷瘤小鼠行为学的影响
Figure PCTCN2022106599-appb-000001
注：与肿瘤模型组比较，*P＜0.05；与供试品1组比较， #P＜0.05
悬尾试验中，由于小鼠处于不正常体位中，只要精神和体力上允许，就会不断地维持挣扎，企图克服不正常体位。即小鼠如果全程没有疲乏感，则理论悬尾不动时间为0。相反地，如果小鼠在挣扎过程中感受到精神上或者体力上的疲乏，就会停止挣扎开始休息，这段时间就会被记录。因此平均悬尾不动时间越长，小鼠在这个过程中的精神疲乏和体力疲乏就越严重。
悬尾试验结果表明，给药供试品1和供试品2可明显缩短荷瘤小鼠悬尾不动时间，且供试品2与供试品1相比也可进一步明显缩短荷瘤小鼠悬尾不动时间，甚至达到正常无肿瘤小鼠(空白对照组)的水平。这表明本发明制备获得的不含三萜的灵芝孢子油，相较于含有灵芝三萜类化合物的孢子油，对荷瘤小鼠具有更显著的抗疲乏作用，甚至可以达到无癌状态的水平，显著提高了对因此而造成的精神上的疲乏和体力上的疲乏的缓解作用。
基于此，仅以本发明制备获得的不含三萜的灵芝孢子油做进一步的药物组合效果实验和安全性实验。
效果例2
灵芝孢子油对肝癌移植瘤小鼠模型的抗疲乏作用
1.实验动物
昆明小鼠，SPF级，雌性，40只，饲养环境：饲养于SPF级动物房小鼠间，自由摄食，12h光照/12h黑暗，动物房环境温度16～26℃，相对湿度40％～70％。
2.实验材料
供试品(本申请工艺获得的灵芝孢子油)，给药前用玉米油配成相应浓度的溶液，现配现用。化疗药物(环磷酰胺)，批号：8D231A，200mg/瓶，Baxter Oncology GmbH生产，给药前用生理盐水配成相应浓度的溶液，现配现用。
细胞株：小鼠H 22肝癌细胞株。
3.实验方法
(1)小鼠移植瘤模型的构建：H 22细胞培养至所需数量后，消化计数后按0.1ml/只，接种至小鼠右上肢附近造模，待肿瘤体积长至100mm 3左右，小鼠随机分成4组：模型对照组(Control)、环磷酰胺组(CTX)、灵芝孢子油(GLSO)、环磷酰胺+灵芝孢子油组(CTX+GLSO)。每组10只。
(2)给药方法
给药途径：灵芝孢子油灌胃给药；环磷酰胺尾静脉给药。
每周给药频率：每天给药1次，连续给药5天后休息2天。
给药周期:连续给药3周。
给药剂量：灵芝孢子油10mL/kg；环磷酰胺30mg/kg。
给药体积：按0.1mL/10g体重给药，即10mL/kg。
(3)力竭游泳试验
分别于给药后第13天和第20天进行荷瘤小鼠力竭游泳试验。将荷瘤小鼠置入游泳箱中游泳，水深不少于30cm，水温25±1℃。记录荷瘤小鼠从游泳开始至力竭的时间。在整个实验过程中使每只荷瘤小鼠四肢保持运动，如果荷瘤小鼠漂浮在水面四肢不动，可用木棒在其附近搅动，以荷瘤小鼠沉于水面下10s后不能自主浮出判断为力竭。当平均游泳时间超过360s时，以360s进行统计。
4.实验结果
结果如表1所示，给药第13天和第20天，单独给予灵芝孢子油后，荷瘤小鼠平均游泳时间显著高于模型组，且两组比较具有显著性差异(P<0.05)；给药第13天和第20天，联合给予环磷酰胺和灵芝孢子油后，荷瘤小鼠的平均游泳时间显著高于模型组，且两组比较具有显著性差异(P<0.05)。
表1灵芝孢子油对雌性荷瘤小鼠行为学的影响
Figure PCTCN2022106599-appb-000002
注：与模型对照组比较，*P＜0.05
结果表明，灵芝孢子油可明显延长荷瘤小鼠力竭游泳时间，提示本发明所述灵芝孢子油对肝癌荷瘤小鼠具有显著的抗疲乏作用。
效果例3
灵芝孢子油对人胃癌移植瘤小鼠模型的抗疲乏作用
1.实验动物
裸小鼠，SPF级，雌性，40只，饲养环境：饲养于SPF级动物房裸鼠间，自由摄食，12h光照/12h黑暗，动物房环境温度16～26℃，相对湿度40％～70％。
2.实验材料
供试品(本申请工艺获得的灵芝孢子油)，给药前用玉米油配成相应浓度的溶液，现配现用。化疗药物(奥沙利铂)，批号：190911AM，50mg/瓶，江苏恒瑞医药股份有限公司生产，给药前用生理盐水配成相应浓度的溶液，现配现用。
细胞株：人源胃癌MNK45细胞株。
3.实验方法
(1)裸鼠移植瘤模型的构建：MNK45细胞培养至所需数量后，消化计数后按0.1ml/只，接种至裸鼠右上肢附近造模，待肿瘤体积长至100mm 3左右，将裸鼠随机分成4组：模型对照组(Control)、化疗药物组(Oxaliplatin)、化疗+供试品组(Oxaliplatin+Sample)、化疗+玉米油组(Oxaliplatin+Oil)。每组10只。
(2)给药方法
给药途径：供试品和玉米油灌胃给药；奥沙利铂尾静脉给药。
每周给药频率：供试品和玉米油每天给药1次，连续给药5天后休息2天。奥沙利铂，每周给药2次，D1天和D4天给药。
给药周期:化疗药物给药6周；供试品和玉米油给药6周后继续给药至试验结束。
给药剂量：供试品10mL/kg；奥沙利铂2mg/kg。
给药体积：按0.1mL/10g体重给药，即10mL/kg。
(3)力竭游泳试验
分别于给药后第37天和第49天进行荷瘤小鼠力竭游泳试验。将荷瘤小鼠置入游泳箱中游泳，水深不少于30cm，水温25±1℃。记录荷瘤小鼠从游泳开始至力竭的时间。在整个实验过程中使每只荷瘤小鼠四肢保持运动，如果荷瘤小鼠漂浮在水面四肢不动，可用木棒在其附近搅动，以荷瘤小鼠沉于水面下10s 后不能自主浮出判断为力竭。当平均游泳时间超过360s时，以360s进行统计。
4.实验结果
结果如表2所示，给药第37天，给予化疗药物奥沙利铂同时给予灵芝孢子油后，荷瘤小鼠的平均游泳时间显著高于模型组，且两组比较具有显著性差异(P<0.05)；停止使用化疗药物1周后即给药第49天，荷瘤小鼠的平均游泳时间依然显著高于模型组且有显著性差异(P<0.05)。
表2灵芝孢子油对雌性荷瘤小鼠行为学的影响
Figure PCTCN2022106599-appb-000003
注：与模型对照组比较，*P＜0.05
结果表明，给药供试品可明显延长荷瘤小鼠力竭游泳时间，提示本发明所述灵芝孢子油对荷瘤小鼠具有显著的抗疲乏作用。
效果例4
灵芝孢子油体内安全性的研究
1.灵芝孢子油不影响荷瘤小鼠的体重
结果如图1所示，灵芝孢子油联合奥沙利铂后并未引起荷瘤小鼠体重显著降低。
2.灵芝孢子油不影响荷瘤小鼠的摄食量
结果如图2所示，灵芝孢子油联合奥沙利铂后并未引起荷瘤小鼠摄食量显著减少。
综上，本发明的灵芝孢子油对荷瘤小鼠具有显著的抗疲乏作用。而且本发明灵芝孢子油联合化疗药物组的动物没有死亡或体重明显下降、没有影响荷瘤 小鼠的摄食量，提示灵芝孢子油在动物体内具有良好的安全性。本发明的灵芝孢子油具有可用于制备癌因性疲乏的防治药物的有益效果。
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，其特征在于，所述灵芝孢子油中，灵芝三萜类化合物未检出。
  2. 如权利要求1所述灵芝孢子油，其特征在于，所述灵芝孢子油中，甘油三酯类成分含量＞90％。
  3. 一种如权利要求1所述灵芝孢子油的制备方法，其特征在于，包括如下步骤：
    A、灵芝孢子经破壁、制粒、干燥和超临界CO 2萃取，获得孢子油粗品；
    B、取硅胶适量，用石油醚-乙酸乙酯混合溶液溶解，湿法装柱，获得洗脱柱；
    C、取所述孢子油粗品往所述洗脱柱上样，用所述石油醚-乙酸乙酯混合溶液常压洗脱，收集洗脱液，合并，减压浓缩除去溶剂，真空干燥，得到所述灵芝孢子油。
  4. 如权利要求1所述灵芝孢子油的制备方法，其特征在于，所述孢子油粗品质量与所述硅胶质量比为1：10～40。
  5. 如权利要求1所述灵芝孢子油的制备方法，其特征在于，所述石油醚-乙酸乙酯混合溶液中，石油醚与乙酸乙酯的体积比为8.5～9.5：0.5～1.5。
  6. 如权利要求5所述灵芝孢子油的制备方法，其特征在于，所述石油醚-乙酸乙酯混合溶液中，石油醚与乙酸乙酯的体积比为9.2：0.8。
  7. 如权利要求1所述灵芝孢子油的制备方法，其特征在于，所述洗脱柱的柱直径与填充柱高之比为1：10～20。
  8. 一种如权利要求1所述灵芝孢子油在制备抗癌因性疲乏药物中的应用。
  9. 一种含有如权利要求1所述灵芝孢子油的药物，其特征在于，所述药物还含有药学上可接受的载体。
  10. 如权利要求1所述灵芝孢子油的药物，其特征在于，所述药物为片剂、胶囊剂、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、溶液剂、注射剂、栓剂、霜剂、喷雾剂、滴剂或贴剂中的一种。
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