WO2013155885A1 - 灵芝人参药性菌质在制备治疗肺癌的药物中的应用 - Google Patents

灵芝人参药性菌质在制备治疗肺癌的药物中的应用 Download PDF

Info

Publication number
WO2013155885A1
WO2013155885A1 PCT/CN2013/070663 CN2013070663W WO2013155885A1 WO 2013155885 A1 WO2013155885 A1 WO 2013155885A1 CN 2013070663 W CN2013070663 W CN 2013070663W WO 2013155885 A1 WO2013155885 A1 WO 2013155885A1
Authority
WO
WIPO (PCT)
Prior art keywords
ginseng
group
ganoderma lucidum
lung cancer
ganoderma
Prior art date
Application number
PCT/CN2013/070663
Other languages
English (en)
French (fr)
Inventor
邱智东
Original Assignee
Qiu Zhidong
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qiu Zhidong filed Critical Qiu Zhidong
Publication of WO2013155885A1 publication Critical patent/WO2013155885A1/zh

Links

Classifications

    • 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
    • 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)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of bioengineering technology, in particular to an application of a ganoderma lucidum medicinal fungus in preparing a medicament for treating lung cancer.
  • Lung cancer is the most common primary malignant tumor of the lung. Most lung cancer originates from the bronchial mucosa epithelium, so it is also called bronchial lung cancer. In the past 50 years, the incidence and mortality of lung cancer have risen rapidly in countries all over the world, especially in developed industrial countries. Among male patients who died of cancer, lung cancer has ranked first. Therefore, effective treatment of lung cancer is one of the current research hotspots.
  • cisplatin discovered by B. Rosenborg et al., which is a common chemical drug for cancer treatment, and has many drugs such as lung cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, and the like. Solid tumors can show efficacy and have a high effect on lung cancer.
  • cisplatin is a heavy metal complex with a combination of two chlorine atoms and two ammonia molecules in the center. It can cause bone marrow suppression, gastrointestinal reactions, renal toxicity, neurotoxicity, allergic reactions, electrolytes. There are many limitations in the usage and dosage of adverse reactions such as disorders.
  • Shenyi Capsule is a capsule prepared by extracting ginseng saponin Rg3 from red ginseng by biological enzyme extraction and transformation technology. It has the effect of cultivating solids and replenishing qi and blood.
  • the patient's symptoms of qi deficiency help to improve the efficacy of primary lung cancer and liver cancer.
  • the toxic side effect of the ginseng mites is small, it needs to be prepared by chemical hydrolysis, enzymatic hydrolysis, microbial fermentation, etc. The process is cumbersome, the reaction conditions and product structure are difficult to control, and the environmental pollution is large.
  • the Chinese patent No. 201010176053.0 discloses a Ganoderma lucidum ginseng fungus which is obtained by using ginseng as a substrate and Ganoderma lucidum as a strain through a relatively simple silent solid fermentation.
  • the ginseng medicinal fungus contains not only the active ingredients of traditional ginseng. And contains the active ingredients of Ganoderma lucidum, not only poison Very low in nature, and it helps to improve sleep and promote growth and development.
  • the technical problem to be solved by the present invention is to provide a Ganoderma lucidum medicinal fungus for use in the preparation of a medicament for treating lung cancer, which has high curative effect and less toxic side effects.
  • the invention provides an application of a ganoderma ginseng medicinal fungus in preparing a medicament for treating lung cancer, wherein the ginseng ginseng medicinal fungus is obtained by using ginseng as a substrate and a ganoderma lucidum as a strain, and is obtained by two-way solid fermentation, and the specific process is as follows: :
  • the ginseng is pulverized, moistened with water, and sterilized to obtain a fermentation substrate.
  • the weight of the added water is 100% to 150% of the weight of the ginseng, and the wetting time is 61! ⁇ 12h;
  • the culture is expanded to obtain a fermentation strain
  • the fermenting species is inoculated into a fermentation substrate, and subjected to bidirectional solid fermentation at 24 ° C to 28 ° C in the dark for 10 days to 20 days.
  • the ganoderma ginseng medicinal fungus prepared by the above method contains various ginsenosides such as ginsenoside Rgl, Rb Re, Rg3, and Rlil, and further contains ginseng polysaccharide, ganoderma lucidum polysaccharide and the like, and the prior art discloses that it is non-toxic and helpful. For improving sleep.
  • the inventors have found that the Ganoderma lucidum ginseng medicinal fungus can also be used for the preparation of a medicament for treating lung cancer, and the effect is comparable to that of cisplatin and ginseng glucoside, and is superior to Ganoderma lucidum, ginseng and humans in participating in Ganoderma lucidum.
  • the ganoderma ginseng medicinal fungus and the preparation method thereof are described in detail in the Chinese patent document No. 201010176053.0, and the present invention will not be described again.
  • the present invention preferably pretreats the Ganoderma lucidum ginseng fungus according to the following method:
  • Ganoderma ginseng medicinal fungus is added with water or ethanol, and water or ethanol is recovered, concentrated, dried, and pulverized to obtain Ganoderma lucidum ginseng bactericidal powder.
  • the active ingredient of Ganoderma lucidum ginseng fungus is preferably extracted by using water or ethanol as a solvent, and the extraction process is not particularly limited in the present invention, and the extraction method well known to those skilled in the art may be used.
  • the weight of the water is preferably 6 to 8 times, more preferably 6.5 to 7.5 times the weight of the Ganoderma lucidum medicinal fungus;
  • the weight of the ethanol is preferably the weight of the Ganoderma lucidum ginseng 10% to 90%, more preferably 20% to 80%.
  • the Ganoderma lucidum ginseng fungus is preferably extracted multiple times, more preferably three times or more, and after the extraction is completed, the filtrate obtained by filtration is combined, according to the technical field.
  • the method known to the skilled person recovers the solvent, concentrates, dries, and pulverizes, and obtains Ganoderma lucidum ginseng bactericidal powder.
  • the Lewis lung cancer was studied as an in vivo therapeutic drug, and the mice were intragastrically administered at a dose of 0.5 g/kg, 1.0 g/kg, 2.0 g/kg, respectively.
  • the blank group, the model group, the cisplatin group, the Shenyi capsule group, the ginseng group, the Ganoderma lucidum group and the human group participated in the Ganoderma lucidum group as a control, and the results showed that the therapeutic effect of the Ganoderma lucidum ginseng drug on lung cancer was compared with that of cisplatin and ginseng.
  • the drugs such as sputum are quite good, and it is better than Ganoderma lucidum, ginseng and people to participate in Ganoderma
  • a culture medium containing 2 wt/ 0 glucose, 0.5% peptone, 0.1 wt% yeast powder, 0.15 wt% KH 2 PO 4 and 0.1 wt% M:gSO 4 was prepared.
  • the lOOmL strain culture solution was placed in a 250 mL flask, O.lMPa, and sterilized at 121 °C for 30 min.
  • four 0.5 cm x 0.5 cm mycelium were cut into the culture medium of the flask, and shake culture was carried out for 5 days at 28 ° C and 150 r / min to obtain a seed liquid containing hyphae;
  • mice Female C57BL/6 mice, purchased from the Shrek Animal Center of the Shanghai Branch of the Chinese Academy of Sciences, were randomly divided into sputum groups according to their body weight:
  • A is a blank group
  • B is a model group
  • C is a positive drug cisplatin group
  • D is a Shenyi capsule group
  • E is a Ganoderma lucidum ginseng low-dose group (0.5g/kg)
  • F is a Ganoderma lucidum ginseng drug-like fungus.
  • G is Ganoderma lucidum ginseng high-dose group (2g / kg)
  • H is ginseng group (2g kg)
  • I is Ganoderma lucidum group (2g/kg)
  • J is a person who participates in the Ganoderma lucidum group (2g/kg);
  • Each group consisted of '12, each with 2 cages and 6 cages per cage.
  • mice were intragastrically administered 400 L/20 g body weight once daily.
  • Example 2 5 g of the Ganoderma lucidum ginseng bactericidal powder prepared in Example 1 was dissolved in 100 mL of sterile water, diluted to ⁇ 5 g / mL, and the mice were intragastrically administered 40 ( ⁇ L / 20 g body weight, that is, 0, 5 g / kg;
  • Example 2 5 g of the Ganoderma lucidum ginseng powder prepared in Example 1 was dissolved in 100 mL of sterile water, which was 0.05 g/mL, and the mice were intragastrically administered 400 ⁇ /2 (body weight, ie, 1.0 g/kg;
  • mice were intragastrically administered 40 ( ⁇ L / 20g body weight, 2g/kg once in the afternoon and afternoon;
  • mice Take 2.5g of Ganoderma lucidum powder and 2,5g of ginseng powder and mix it in lOOmL of sterile water. 0.05g/mL, mice were intragastrically administered 40 ( ⁇ L/20g body weight, 2g/kg once in the afternoon and afternoon. 2.1.3 Experimental equipment and materials
  • Oral tube provided by the Experimental Animal Center of Xiamen University;
  • Sterile disposable syringe purchased from the warehouse of Xiamen University Medical College;
  • Sterile scalpel provided by the Experimental Animal Center of Xiamen University Medical College;
  • Flow cytometry sorting flow cytometry, provided by the Central Laboratory of Xiamen University Medical College; Elispot kit: Shenzhen Dakco is pre-packaged for the company;
  • CD4 antibody purchased from eBiokgend, FITC label
  • CD8 antibody purchased from eBioscience, PE label.
  • Lewis lung cancer-bearing mice 14 days after passage, were sacrificed by cervical dislocation, fixed on the rat plate, routinely disinfected, and the tumor tissue was removed from the ankle subcutaneously in the ultra-clean workbench, and the fibrous capsule and necrotic tissue were removed.
  • the cells were cut in a sterile dish, gently ground with 4 ° C physiological saline in a tissue grinder, and filtered to prepare a tumor fine I bag suspension, and the cell concentration was adjusted to 1 X 10 7 /mL.
  • mice were subcutaneously injected with 0.2 mL of the above tumor cell suspension in the right ankle to establish a Lewis lung cancer animal model.
  • the inoculated tumor cells were continued to be administered with the corresponding drugs on the next day.
  • Group C was intraperitoneally administered once a day for 14 days.
  • the diet and tumor growth of the mice were observed every day. Twenty-four hours after the last intragastric administration, the mice were weighed and sacrificed, and the antitumor activities of the drugs in each group were evaluated.
  • Tumor growth inhibition rate (%) model group tumor weight Organs 3 ⁇ 4 quantity (mg) ⁇ ⁇
  • the ratio of CD4 and CD8 cells in the spleen was measured to preliminarily determine the effect on immune cells; the production of CTL (number of killer T cells) in the spleen was examined.
  • Group A and Group B continued to receive distilled water 400:L/20g
  • group C was intraperitoneally injected with 0.1mg/mL, 200 ⁇ /20g
  • group D was intragastrically administered 0.4mg/mL, 400 ⁇ /20g
  • G The group was intragastrically administered with 0.05g/mL, 400 ⁇ / 20g in the afternoon and the afternoon
  • the group F was administered with 0.05g/mL and 400 ⁇ /20
  • the sputum group was administered with 0.025g/mL, 40 ( ⁇ L/20g
  • group H, group I, J The group was intragastrically administered with 0.05 g/mL and 400 ⁇ 2 (3 ⁇ 4) in the afternoon and afternoon.
  • mice were sacrificed, tumors, thymus, spleen, and liver were taken and weighed separately. The ratio of CD4 CD8 cells in the spleen was measured, and the production of CTL in the spleen was detected.
  • mice were weighed and sacrificed.
  • the tumors, liver, spleen and thymus of each group were weighed and weighed separately.
  • the tumor suppression growth of each group of mice was calculated according to the formula described above. Rate and organ index, see Table ⁇ / br> 1.
  • Table 1 shows the index of mouse organ and tumor growth inhibition rate.
  • the number of CD4 and CD8 positive cells in the spleen of each group was significantly higher than that in the model group, and the highest dose of Ganoderma lucidum ginseng medicinal high dose group was the highest.
  • Ganoderma lucidum ginseng medicinal low dose group and Shenyi capsule group The increase in the amount of ginseng group, Ganoderma lucidum group and people participating in the Ganoderma lucidum group is equivalent.
  • the spleen CTL ie the production of killer T cells, was detected by ELISPOT. See Table 2 for the scar, and Table 3 shows the results of spleen CTL assay.
  • Ganoderma lucidum ginseng has the anti-tumor effect in mice, can regulate the production of CD4, CD8, CTL cells, and does not produce liver, parotid and spleen toxicity, and its effect and cisplatin
  • the effects of Shenyi Capsule and other drugs are quite good, and it is better than Ganoderma lucidum, ginseng and people to participate in Ganoderma lucidum.

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Medical Informatics (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种灵芝人参药性菌质在制备治疗肺癌的药物中的应用，所述灵芝人参药性菌质为以人参为基质、以灵芝为菌种，经过双向固体发酵后得到，其中含有人参皂苷Rg1、Rb1、Re、Rg3、Rh1等多种人参皂苷，还含有人参多糖、灵芝多糖等成分，可用于制备治疗肺癌的药物。

Description

灵芝人参药性菌质在制备治疗肺癌的药物中的应用
本申请要求于 2012年 4月 18日提交中国专利局、申请号为 20Ί 2 10115166.9、 发明名称为 "灵芝人参药性菌盾在制备治疗肺癌的药物中的应用"的中国专利 申请的优先权， 其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物工程技术领域，尤其涉及一种灵芝人参药性菌质在制备治 疗肺癌的药物中的应用。
背景技术
肺癌是最常见的肺原发性恶性肿瘤， 绝大多数肺癌起源于支气管粘膜上 皮， 故亦称支气管肺癌。 近 50多年来， 世界各国特别是工业发发达国家， 肺 癌的发病率和病死率均迅速上升，死于癌症的男性病人中肺癌已居首位。因此， 对肺癌进行有效治疗是目前的研究热点之一。
目前， 现有技术公开了多种用于治疗肺癌的药物， 如 B. Rosenborg等发现 的顺铂， 顺铂是癌症治疗的常用化学药物， 对肺癌、 鼻咽癌、 卵巢癌、 前列腺 癌等多种实体肿瘤均能显示疗效， 对肺癌具有较高的疗效。 但是， 顺铂是中心 以二价铂通两个氯原子和两个氨分子结合的重金属络合物，使用时会出现骨髓 抑制、 胃肠道反应、 腎脏毒性、神经毒性、过敏反应、 电解质紊乱等不良反应， 其用法及用量都存在诸多限制。
与顺铂等化学合成类药物相比， 中药具有毒性小、安全性高等优点而广泛 用于癌症的治疗中， 如黄芪、 白花蛇舌草、 茯苓、 白术、 川芎、 甘草、 红参、 女贞子、 三七、 当归、 大黄、 桃仁等均可用于治疗癌症。 其中， 参一胶囊是采 用生物酶提取转化技术提取红参中的人参皂苷 Rg3 制备的胶囊剂， 其具有培 元固本、 补益气血的功效， 可配合化疗用药， 提高基体免疫能力， 改善肿瘤患 者的气虚症状， 有助于提高原发性肺癌、 肝癌的功效。 虽然参一胶嚢的毒副作 用较小， 但是其需要采用化学水解、 酶水解、 微生物发酵等方法制备， 工艺过 程较为繁瑣， 对反应条件及产物结构不易控制， 且对环境污染较大。
申请号为 201010176053.0 的中国专利公开了一种以人参作为基质， 以灵 芝作为菌种经过较为简单的默向固体发酵得到的灵芝人参药性菌质，该灵芝人 参药性菌质不仅含有传统人参的有效成分， 而且含有灵芝的有效成分， 不仅毒 性极低， 而且有助于改善睡眠、 促进生长发育。 但是， 目前并没有任何文献公 开灵芝人参药性菌质在治疗肿瘤方面的应用， 尤其是在治疗肺癌方面的应用。 发明内客
有鉴于此，本发明所要解决的技术问题在于提供一种灵芝人参药性菌质在 制备治疗肺癌的药物中的应用， 其疗效较高且毒副作用较小。
本发明提供了一种灵芝人参药性菌质在制备治疗肺癌的药物中的应用，所 述灵芝人参药性菌质为以人参为基质、 以灵芝为菌种， 经过双向固体发酵后得 到， 具体过程如下：
将人参粉碎后加水润湿， 灭菌后得到发酵基质， 加水的重量为人参千重的 100%〜 150%， 所述润湿的时间为 61！〜 12h；
将灵芝菌种活化后进行扩大培养， 得到发酵菌种；
将所述发酵菌种接种于发酵基质中， 在 24°C〜28°C、 避光的条件下进行双 向固体发酵 10天〜 20天。
釆用上述方法制备得到的灵芝人参药性菌质中含有人参皂苷 Rgl、 Rb Re、 Rg3、 Rlil等多种人参皂苷， 还含有人参多糖、 灵芝多糖等成分， 现有技 术公开其无毒性， 有助于改善睡眠。 发明人研究发现， 所述灵芝人参药性菌质 还可以用于制备治疗肺癌的药物， 效果与顺铂、 参一胶嚢等药物相当， 优于灵 芝、 人参和人参加灵芝。 灵芝人参药性菌质及其制备方法在申请号为 201010176053.0的中国专利文献中有详细阐述， 本发明不再赘述。
为了提高灵芝人参药性菌质对肺癌的治疔效果，本发明优选对所述灵芝人 参药性菌质按照以下方法进行预处理：
向灵芝人参药性菌质加入水或乙醇提取，回收水或乙醇后进行浓缩、千燥、 粉碎， 得到灵芝人参药性菌质粉。
本发明优选以水或乙醇为溶剂提取灵芝人参药性菌质的有效成分，本发明 对所述提取过程没有特殊限制， 本领域技术人员熟知的提取方法即可。在进行 提取时， 所述水的重量优选为所述灵芝人参药性菌质重量的 6〜8倍， 更优选为 6.5~7.5倍； 所述乙醇的重量优选为所述灵芝人参药性菌质重量的 10%〜90%, 更优选为 20%〜80%。 本发明优选对所述灵芝人参药性菌质进行多次提取， 更 优选进行 3次以上的提取，提取完毕后将过滤得到的滤液合并，按照本领域技 术人员熟知的方法回收溶剂、 浓缩、 千燥、 粉碎， 得到灵芝人参药性菌质粉。 得到灵芝人参药性菌质粉后， 以其作为体内治疗药物对小鼠 Lewis肺癌进 行研究， 分别以 0.5g/kg、 1.0g/kg, 2.0g/kg的剂量对小鼠进行灌胃， 同时以空 白組、 模型组、 顺铂组、 参一胶囊组、 人参組、 灵芝组和人参加灵芝组作为对 照， 结果表明， 所述灵芝人参药性菌质对于肺癌的治疗效果与顺铂和参一胶嚢 等药物相当， 优于灵芝、 人参和人参加灵芝。
具体实施方式
实施例 1
将长春中医药大学药用真菌研究所保存的灵芝菌株接种在 PDA斜面培养 基上、 27°C±1 °C时进行培养， 培养 7天得到菌丝体备用；
制作含 2wt°/0葡萄糖、 0.5 %蛋白胨、 0.1wt%酵母粉、 0.15wt%KH2PO4 和 0.1wt%M:gSO4的菌种培养液。取 lOOmL菌种培养液装入 250mL三角瓶中， O.lMPa, 121 °C灭菌 30min。 在无菌室内切取 4块 0.5cmx0.5cm的菌丝体接入 三角瓶的菌种培养液中， 28°C、 150r/min的条件下振荡培养 5天后， 得到含有 菌丝球的种子液；
取 50g无虫蛀、 无霉变的生晒人参粉碎过 60目筛，加 50g水润湿 12h后， 将得到的人参装入 500mL三角瓶中， O.lMPa灭菌 60mm后， 接入 50mL种子 液， 28Ό时黑暗培养 10天， 得到灵芝人参药性菌质；
向所述灵芝人参药性菌质中加入 8倍重量的水提取 3次，合并滤液， 回收 水， 浓缩、 千燥、 粉碎后得到灵芝人参药性菌质粉。
实施例 2
灵芝人参药性菌质体内对小鼠 Lewis肺癌的抑制作用
2.1实验材料
2.1.1实验动物及分组
雌性 C57BL/6 小鼠， 购于中国科学院上海分院史莱克动物中心， 按体重 随机分为 Ί组：
A为空白组、 B为.模型组、 C为阳性药顺铂组、 D为参一胶囊组、 E为灵 芝人参药性菌质低剂量组（ 0.5g/kg ), F为灵芝人参药性菌质中剂量组（ lg/kg ) G为灵芝人参药性菌质高剂量组（2g/kg ), H为人参组（2g kg )， I为灵芝组 ( 2g/kg ), J为人参加灵芝组（2g/kg );
每组' 12只， 每组分为 2笼， 每笼 6只。
2,1 ,2实验试剂
2,1 ,2.1参一胶囊
吉林亚泰制药股份有限公司， 国药准字 Z20030044。 每粒含人参皂苷 Rg3
10mg, 按体表面积折算为小鼠剂量为 8mg/kg， 以蒸馏水配为 0.4mg/mL， 给药 体积 20mL/kg。 小鼠灌胃 400 L/20g体重， 每日一次。
2,1 ,2.2顺铂：
按体表面积一次 20mg/m2， 每只鼠 0.2mg/20g体重， 腹腔注射， 连续 14 天。
取顺铂 16mg/mL， 以无菌水稀释到 lmg/mL , 腹腔注射 0.2mlL 即为 0.2mg/20g体重。
2.1.2.3灵芝人参药性菌盾低剂量组
取 5g实施例 1制备的灵芝人参药性菌质粉溶于 l OOmL无菌水，倍比稀释 到瞧 5g/mL， 小鼠灌胃 40(^L /20g体重， 即为 0,5g/kg;
2.1.2.4灵芝人参药性菌盾中剂量组
取 5g 实施例 1 制备的灵芝人参药性菌质粉溶于 lOOmL 无菌水， 即为 0.05g/mL, 小鼠灌胃 400μ /2( 体重， 即为 l .Og/kg;
2.1.2.5灵芝人参药性菌质高剂量组
取 5g 实施例 1 制备的灵芝人参药性菌质粉溶于 lOOmL 无菌水， 即为
0.05g/mL, 小鼠灌胃 40(^L /20g体重， 上下午各灌胃一次即为 2g/kg;
2丄 2.6人参组
取 5g人参细粉溶于 lOOmL无菌水， 即为 0.05g/mL， 小鼠灌胃 400μ /2(¾ 体重， 上下午各灌胃一次即为 2g/kg;
2丄 2,7灵芝组
取 5g灵芝细粉溶于 lOOmL无菌水， 即为 0,05g/mL， 小鼠灌胃 400μϋ2(% 体重， 上下午各灌胃一次即为 2g/kg;
2丄 2,8人参加灵芝组
取 2.5g 灵芝细粉和 2,5g 人参细粉混合后溶于 lOOmL 无菌水， 即为 0.05g/mL, 小鼠灌胃 40(^L/20g体重， 上下午各灌胃一次即为 2g/kg。 2.1 .3实验设备及材料
电子天平： 德国赛昂纳斯；
灌胃管： 厦门大学实验动物中心提供；
无菌一次性注射器： 购自厦门大学医学院仓库；
普通饲料： 由厦门大学医学院动物中心提供；
无菌手术刀： 厦门大学医学院实验动物中心提供；
流式细胞仪： 分选型流式细胞仪， 厦门大学医学院中心实验室提供； Elispot试剂盒： 深圳达科为公司预包被板；
CD4抗体： 购自 eBiokgend公司， FITC标记；
CD8抗体： 购自 eBioscience公司， PE标记。
2.2实验方案
2.2.1荷瘤动物模型的建立
取传代后 14天的 Lewis肺癌瘤源小鼠， 脱颈处死， 固定于鼠板上， 常规 消毒，于超净工作台中，从腋部皮下剥取肿瘤组织，剔除纤维包膜和坏死组织， 在无菌平皿中剪碎， 置组织研磨器中加 4°C生理盐水轻轻研磨， 过滤， 制成瘤 细 I包悬液， 调整细胞浓度为 1 X 107/mL。
将上述动物分组并给予相应药物 5天后， 除 A组外， 其他各组小鼠均于 右腋部皮下注射上述瘤细胞悬液 0.2mL以建立荷 Lewis肺癌动物模型。接种瘤 细胞次日开始继续灌胃给予相应药物， C组腹腔注射给药， 每天一次， 连续 14天， 每天观察小鼠饮食及肿瘤生长情况。 末次灌胃给药后 24h， 小鼠称重， 处死， 评价各组药物的抗肿瘤活性。
2.2.2观察指标
观察胂瘤重量变化，评价灵芝人参药性菌质体内对胂瘤生长的影响，确定 其体内的抗肿瘤活性， 同时通过观察动物一般表现、体重变化及用药后动物的 胸腺和脾脏 肝脏重量的改变，初步评价药物的潜在的一般毒性及其对动物免 疫功能的影响， 肿瘤生长抑制率和脏器指数分别按照以下公式计算：
模型组瘤重 给药组瘤重 ι ηΛ
^ X 100
肿瘤生长抑制率 （％) = 模型组瘤重 脏器¾量（mg) ι η
τττ : ^x lO
脏器指数 （mg/lOg体重） = 体重（g)
检测脾脏的 CD4、 CD8细胞的比值， 以初步判断对免疫细胞的影响； 检测脾脏 CTL (杀伤性 T细胞的数量） 的产生情况。
2.2.3实验步骤：
第 1天至第 5天： A组与 B组灌胃蒸馏水 400 L/20g; D组灌胃 0.4mg/mL、
40(^L/20g; G组灌胃 0.05g/mL， 上 T午各 40(^L/20g; F组灌胃 0.05g/mL、 400μΙ/2(¾; E组灌胃 0.025g/mL、 400 L/20g， H:组、 ί组、 J组灌胃 0.05g/mL， 上下午各 40(^L/20g。
第 6天： 给除 A组以外的小鼠接种肿瘤。
第 Ί天至第 25天： A组与 B组继续灌以蒸馏水 400 :L/20g， C组腹腔注 射 0.1mg/mL， 200μ /20g; D组灌胃 0.4mg/mL, 400μ /20g; G组灌胃 0.05g/mL, 上下午各 400μ /20g; F组灌胃 0.05g/mL、 400μΕ/20 ; Ε组灌胃 0.025g/mL、 40(^L/20g; H组 I组、 J组灌胃 0.05g/mL、 上下午各 400μϋ2(¾。
第 26天： 处死小鼠， 取肿瘤、 胸腺、 脾脏、 肝脏， 并分别称重， 检测脾 脏 CD4 CD8细胞的比值， 检测脾脏 CTL的产生情况。
2.3实验结果
2.3.1肿瘤抑制生长率结杲
末次灌胃给药后 24h， 小鼠称重， 处死， 取各组小鼠的肿瘤、 肝脏、 脾脏、 胸腺并分别称重，并按照上文所述的公式计算各组小鼠的肿瘤抑制生长率和脏 器指数， 结杲参见表' 1.， 表 1为小鼠脏器指数及肿瘤抑制生长率结杲。
01
^ ^ ^m m %QD D ζττ
° ,两 f寺首 ^
YWH¥ Y 11 一 f 古陣 萆 Y ¥
¾ ' ι γ ¥Ψ首 ' m^^m- ^ ^fs^〖 年
Z£'LP ■ 6Γ0干 6S'066'£ 干 9S'0£ 9'1^干££'00£ 9ΐ'0干 Γ I9SXI¾l7- 0^0"0Tr 00'lT 8-9I9 IL'££ ZZQ^ZLQ WZZ ZL'69 S£'09干 98'£9980御 Γ 199Ό mi'66【 ΙΟ'Ο干 frr086'0干 80·£ΐ I 96ΊΡ 8Γ0干 ^9'08 ££干88'£968'99干 S 6£9 ΟΐΌ干 Γ l½'8[.干 IS'SO O'O干 £Γ0ΠΊ干 εε ΐΗ £VIL VY^Zi'mi'LZ VYS9 干 9 9 Π-'O IWK干 ^'ΐΟ^ΟΌ干 £Γ0Ι.ΓΙ干
0S'L£ 0£Ό干 0£'0S0O£干 t Z88t'6S干 8Γ0 [ ΠΌ干 9Γ 1.Ι.Γ|7[.干 86'86[J.0'0干 SrOI.6'0干 fr8'£l J
Figure imgf000008_0001
I1'8S干 6 ί ΠΌ干 IC 'O 干 ϋ9'68[.Μ)Ό干 W( 8'0干 1.0'£ΐ3
Zi'LP v. 'Ο干 6 086 Ι干 98'0£ £ £p 6VZ0L εΐ'Ο干 Γ I【9'6干69'£0 ΙΟ'Ο干 9Γ088Ό干 99·£ΐα li'ZL Γ0干 ί£'0 干 8i'6Sv0l'6S干 »'6Ι?990'0干 l「 1.06'£ 干 86'6£[.S0'0干 ίΓΟΟΓΙ干 '£ΐ3 OO'O 0干 Π.·1 £LTS^££' 66V£ 19'L£L Μ.Ό干 8Γ 干 18'80^0Ό干 干 6f 9ΐ 8 00Ό干讓 ^' ΐτΐ8'£9ΐ6 Ιΐτ85"9ε .½Ότ6Γ 干 τθ Ο'Ο干 9£'0S0'I干 吉
Figure imgf000008_0002
fi 善罩 善幕翁 i 幕 ¾屮
Figure imgf000008_0003
C990.0/CT0ZN3/X3d S88SSl/CT0Z OAV 表 2脾脏 CD4、 CD8细胞数量及比值结果
组别 CD4细包比例 （％) CD8细包比例 （％) CD4/ CD8
A 20.60±2,07 13.36±1.61 1.54±0.06
B 11.79土 0.65s 6.28土 0.53 1.88±0.0958
C 21.40±1.75Δ 13.87±0,40Δ 1.54士 0.132Δ
D 19.59±0,54Δ 10.97±0.93κΔ L79±0.179s
E 19.04土 0.52Δ 10.64±0.80κΔ 1.79±0.083s
F 17.13±0.46¾Δ 11.64±0.77*Δ 1.48±0.084Δ
G 22.57±0.89¾Δ 15.22±0.87¾Δ 1.48±0.056Δ
H 16.98±0.35¾Δ 11.03±0.69*Δ 1.54±0.057Δ
I 17.02±0.43¾Δ 10.77±0.72¾Δ 1.58±0.077Δ
J 17.56±0.54¾Δ 11.55±0.80*Δ 1.52±0.062Δ 表 2中， *表示与空白组比较 Ρ < 0,05， Δ表示与模型组比较 Ρ < 0.05。
由表 2可知， 给药各组小鼠脾脏的 CD4和 CD8阳性细胞数比模型组明显 增多， 灵芝人参药性菌质高剂量组增加量最多， 灵芝人参药性菌质低剂量组与 参一胶囊组、 人参组、 灵芝组及人参加灵芝组增加量相当。
2.3.3脾脏 CTL结果
用 ELISPOT检测脾脏 CTL， 即杀伤性 T细胞的产生情况， 结杲参见表 2， 表 3为脾脏 CTL检测结果。
表 3 脾脏 CTL检测结杲
组别 CTL细胞数量
A 1
B 3.75土 0.5
C 24,5±5.92*
D 8.0土 2.0s
E 9±0.82a
F 15.5±3.32sA
G 23.5±3.87'Δ
H 10.8±3.2ΓΔ
I 11.6±2.58¾Δ
J 12.3±3.03'Δ
表 3中， *表示与空白组比较 Ρ < 0,05， Δ表示与模型组比较 Ρ < 0.05。 由表 3可知， 给药各组小鼠脾脏的 CTL细胞产生增多， 其中灵芝人参药 性菌质高剂量组 CTL细胞产生增量与顺铂组相当， 高于其他组； 灵芝人参药 性菌质低剂量組与参一胶嚢组、 人参组、 灵芝组及人参加灵芝组增加量相当。
由上述实施例可知， 灵芝人参药性菌质在体内有抗小鼠成瘤的作用， 可调 节 CD4、 CD8、 CTL 细胞产生， 且不会产生肝脏、 驹腺、 脾脏毒性， 其效杲 与顺铂、 参一胶囊等药物效果相当， 优于灵芝、 人参和人参加灵芝。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指 出， 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 可以对本发明进行若千改进和修饰 ,这些改进和修饰也落入本发明权利要求的 保护范围内。

Claims

权 利 要 l、 灵芝人参药性菌质在制备治疗肺癌的药物中的应用。
2、 根据权利要求 1所述的应用， 其特征在于， 所述灵芝人参药性菌质按 照以下方法进行预处理：
向灵芝人参药性菌质加入水或乙醇提取，回收水或乙醇后进行浓缩、千燥、 粉碎， 得到灵芝人参药性菌质粉。
3、 根据权利要求 2所述的应用， 其特征在于， 所述水的重量为所述灵芝 人参药性菌质重量的 6〜8倍。
4、 根据权利要求 2所述的应用， 其特征在于， 所述乙醇的重量为所述灵 芝人参药性菌质重量的 10%〜90%。
PCT/CN2013/070663 2012-04-18 2013-01-18 灵芝人参药性菌质在制备治疗肺癌的药物中的应用 WO2013155885A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210115166.9 2012-04-18
CN2012101151669A CN102614238A (zh) 2012-04-18 2012-04-18 灵芝人参药性菌质在制备治疗肺癌的药物中的应用

Publications (1)

Publication Number Publication Date
WO2013155885A1 true WO2013155885A1 (zh) 2013-10-24

Family

ID=46554687

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/070663 WO2013155885A1 (zh) 2012-04-18 2013-01-18 灵芝人参药性菌质在制备治疗肺癌的药物中的应用

Country Status (2)

Country Link
CN (1) CN102614238A (zh)
WO (1) WO2013155885A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102614238A (zh) * 2012-04-18 2012-08-01 邱智东 灵芝人参药性菌质在制备治疗肺癌的药物中的应用
CN105925655A (zh) * 2016-06-27 2016-09-07 中国农业科学院特产研究所 一种利用松杉灵芝液体发酵得到稀有人参皂苷的方法
CN112106981A (zh) * 2020-09-21 2020-12-22 绿之韵生物工程集团有限公司 一种抗疲劳的灵芝制剂及其制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1105594A (zh) * 1994-11-16 1995-07-26 潘自航 降癌灵丹
WO2010076922A1 (ko) * 2008-12-31 2010-07-08 주식회사한국신약 버섯 발효인삼 분말을 함유하는 항암용 건강식품
CN101816689A (zh) * 2010-05-19 2010-09-01 陈心智 人参的处理方法
CN101829172A (zh) * 2010-05-19 2010-09-15 陈心智 灵芝人参药性菌质、药物制剂、药物组合物和食品组合物
CN102406669A (zh) * 2011-11-11 2012-04-11 吉林天强制药有限公司 一种中药组合物及其制备方法和制剂
CN102614238A (zh) * 2012-04-18 2012-08-01 邱智东 灵芝人参药性菌质在制备治疗肺癌的药物中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1907308A (zh) * 2005-08-02 2007-02-07 孙毅 一种治疗肿瘤的中药组合物

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1105594A (zh) * 1994-11-16 1995-07-26 潘自航 降癌灵丹
WO2010076922A1 (ko) * 2008-12-31 2010-07-08 주식회사한국신약 버섯 발효인삼 분말을 함유하는 항암용 건강식품
CN101816689A (zh) * 2010-05-19 2010-09-01 陈心智 人参的处理方法
CN101829172A (zh) * 2010-05-19 2010-09-15 陈心智 灵芝人参药性菌质、药物制剂、药物组合物和食品组合物
CN102406669A (zh) * 2011-11-11 2012-04-11 吉林天强制药有限公司 一种中药组合物及其制备方法和制剂
CN102614238A (zh) * 2012-04-18 2012-08-01 邱智东 灵芝人参药性菌质在制备治疗肺癌的药物中的应用

Also Published As

Publication number Publication date
CN102614238A (zh) 2012-08-01

Similar Documents

Publication Publication Date Title
Cao et al. Antitumor activity of polysaccharide extracted from Pleurotus ostreatus mycelia against gastric cancer in vitro and in vivo
CN102008528B (zh) 复方海参制剂及其制备方法
CN101418325B (zh) 桦褐孔菌胞内外混合多糖及其制备方法和药物用途
JP6389958B2 (ja) 瓦草５環性トリテルペンサポニン類化合物抗腫瘍の薬物用途
TW201618801A (zh) 用於輔助化療藥物之醫藥組合物及其用途
Wei et al. The immunomodulatory effects of active ingredients from Nigella sativa in RAW264. 7 cells through NF-κB/MAPK signaling pathways
WO2013155885A1 (zh) 灵芝人参药性菌质在制备治疗肺癌的药物中的应用
WO2018166151A1 (zh) 一种用于治疗肿瘤的中药组合物及其制备方法和用途
CN101380346A (zh) 一种治疗肿瘤的中药组合物及其制作方法
CN101537036A (zh) 一种皂荚皂苷提取物及其制备方法与应用
CN102406669B (zh) 一种中药组合物及其制备方法和制剂
CN100336552C (zh) 一种治疗肺癌的药物
TWI601535B (zh) 牛樟段木栽培牛樟芝子實體及固態培養菌絲體水及乙醇萃取物之組合物應用於抗癌藥輔助劑
CN102178701B (zh) 一种具有抗肿瘤活性多糖组合物的制备方法
CN115300624A (zh) 人参皂苷联合pd-1阻断剂在制备抗头颈鳞癌药物中的应用
CN107893035A (zh) 一种猴头菌的培养基、黄精生物转化菌丝体、黄精生物转化菌丝体的提取物及其用途
US20220000952A1 (en) Method preparing antrodia cinnamomea extract and antrodia cinnamomea composition, and pharmaceutical composition
RU2642256C2 (ru) Применение композиции в получении медицинской продукции или лекарственного средства для профилактики и лечения вызванной лучевой и химиотерапией лейкопении
TW200920387A (en) Composition of mushroom beta-glucan for anti-cancer and preparation method thereof
CN102440994A (zh) 灵芝酸g在肿瘤治疗中作为免疫增效剂和超抗原依赖的治疗药物的应用
CN101732340A (zh) 一种花蕊石提取物的用途及制备方法
CN104382926A (zh) 一种中药复方多糖组合物及其提取方法和新用途
CN102078351B (zh) 一种治疗癌症的组合物
CN102309493B (zh) 一种抗癌药物组合物
Li et al. Inhibition of Yuyihe Powder on tumor growth in mice models of Sarcoma 180

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13778381

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13778381

Country of ref document: EP

Kind code of ref document: A1