WO2006128378A1 - Use of ganoderic acid in treating tumour - Google Patents

Use of ganoderic acid in treating tumour Download PDF

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Publication number
WO2006128378A1
WO2006128378A1 PCT/CN2006/001171 CN2006001171W WO2006128378A1 WO 2006128378 A1 WO2006128378 A1 WO 2006128378A1 CN 2006001171 W CN2006001171 W CN 2006001171W WO 2006128378 A1 WO2006128378 A1 WO 2006128378A1
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Prior art keywords
cancer
tumor
cells
ganoderic acid
ganoderma lucidum
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PCT/CN2006/001171
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French (fr)
Chinese (zh)
Inventor
Jianjiang Zhong
Jianwen Liu
Wen Tang
Yingbo Hong
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East China University Of Science And Technology
Shanghai Ying-Xing Biotechnology Co., Ltd.
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Priority claimed from CNB200510026378XA external-priority patent/CN100408042C/en
Priority claimed from CN 200510112304 external-priority patent/CN1823788A/en
Application filed by East China University Of Science And Technology, Shanghai Ying-Xing Biotechnology Co., Ltd. filed Critical East China University Of Science And Technology
Publication of WO2006128378A1 publication Critical patent/WO2006128378A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Definitions

  • the present invention relates to new uses of ganoderic acid, in particular to the new use of Me (Ganoderic acid Me) and Ganoic acid T (Ganoderic acid T). Background technique
  • Ganoderma lucidum is a precious medicinal fungus that has been used for thousands of years. Ancient medical scientists have fully recognized the medicinal value of Ganoderma lucidum. They believe that Ganoderma lucidum has the function of "supporting the body and strengthening the body” and can be used to treat a variety of diseases. D pharmacological research proves that Ganoderma lucidum has a wide range of pharmacological effects, such as anti-tumor, anti-HIV. Virus, immune regulation, anti-myocardial ischemia, regulation of blood lipids, blood sugar lowering, sedative effect, liver protection, anti-radiation and anti-chemotherapy, anti-hypoxia and anti-aging effects.
  • Triterpenoids are one of the main chemical constituents of Ganoderma lucidum, and many triterpenoids have important physiological activities. Since the first separation of such compounds by T Kubota (Helv. Chim. Acta, 65 (2), 611-619, 1982) in 1982, more than 100 similar compounds have been isolated to date.
  • Ganoderma lucidum Me or Ganoderma lucidum T (GA- ⁇ ) are extracted from the fermentation of Ganoderma lucidum mycelium, respectively (Chem. Pharm. Bull., 34 (5), 2282-2285, 1986; Agric Biol. Chem., 51 (2), 619-622, 1987)
  • the technique of the cockroach is prepared for extraction with a purity greater than 99%, which was first isolated and identified in 1983 (Tetrahedron Lett., 24 (10), 1081 -1084, 1983) and 1987 (Agric. Biol. Chem., 51 (2), 619-622, 1987).
  • MDR reversal agent should have the following conditions: 1 safe, less toxic to normal tissues; 2 can achieve effective concentration in vitro and in tumor cells; 3 itself has certain anti-tumor activity; 4 stable, long half-life in vivo; Metabolites are also effective.
  • the urgent problem to be solved in the field of tumor chemotherapy is to find new anticancer drugs that are low in toxicity, high in efficiency, and difficult to produce multidrug resistance.
  • Ganoderma lucidum Me or Ganoderma lucidum T can inhibit tumor proliferation and growth, and a small dose produces an effect of inhibiting the growth of various tumor cells in a dose-dependent manner, which is different for different human cancer cells and human normal cells. Cytotoxicity, especially IC 5D for cancer cells is much lower than normal cells 1 (: 5 () , and the difference between the two is significant (p ⁇ 0.05). Moreover, low doses of Ganoderma lucidum Me or Ganoderma lucidum T It can increase the sensitivity of multidrug-resistant tumors to chemotherapeutic drugs that have developed resistance.
  • High-dose use has the effect of inhibiting the growth of multiple multidrug-resistant tumor cells and is dose-dependent, so they can also be used as Drugs and sensitizing synergists for the treatment of multidrug resistant tumors are used in the chemotherapy of tumors.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier.
  • the carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as a diluent, an excipient such as water, a filler such as starch, sucrose, etc., a binder such as a cellulose derivative gelatin, polyvinylpyrrolidone, etc., a lubricant , such as talcum powder. 9% ⁇ The 9%, the content of the ginseng acid is 0. 5-99. 9%.
  • the pharmaceutical composition consists essentially of Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be used not only to inhibit tumor proliferation and growth (especially proliferation and growth of multidrug resistant tumor cells), but also to increase multidrug resistant tumor-resistant chemotherapy drugs. Sensitivity.
  • the composition further comprises the chemotherapeutic agent, such as doxorubicin, vinblastine, vincristine, verapamil, and the like.
  • the invention further relates to a method of treating a tumor comprising administering a therapeutically effective amount of the above composition of the invention to a subject in need of such treatment.
  • the present invention also provides the use of Ganoderma lucidum Me and/or Ganoderma lucidum T in the preparation of a medicament for the treatment of tumors.
  • Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier may be administered to a patient in need of treatment in the form of a composition, generally at a dose of 1-100 mg / (kg body weight per day), depending on the patient Age, condition, etc. change.
  • composition of the ganoderic acid Me or ganoderic acid T of the present invention and the above carrier can be applied to a patient in need of such treatment by oral, nasal, intravenous or subcutaneous injection.
  • it can be prepared into a conventional tablet, powder or oral solution; when it is used for injection, it can be prepared into an injection solution by a method conventional in the art; the meic acid Me or the ganoderic acid T of the present invention
  • Various preparations, including powders, pills, tablets, capsules and the like, solid preparations, ointments, patches, injections and the like can be prepared by a conventional method in the pharmaceutical field.
  • the composition or preparation provided by the present invention has the effect of enhancing the sensitivity of multidrug resistant tumor cells to chemotherapeutic drugs at a low concentration (the low concentration means less than l-10 mg / (kg body weight per day) Dose), at At high concentrations, it has the effect of inhibiting the proliferation of multidrug resistant tumor cells (the high concentration refers to a dose higher than 10-100 mg / (kg body weight ⁇ day)), indicating that it not only has tumor cell killing
  • the role of the tumor cells can also inhibit the infinite proliferation of tumor cells, and thus can play a greater and greater degree of treatment of tumors.
  • the anticancer agent and the sensitizing synergist in the multidrug resistant tumor chemotherapy provided by the invention have the same cytotoxicity to the multidrug resistant cancer cells and the non-multidrug resistant cancer cells, and the difference therebetween Not significant (p ⁇ 0.05).
  • the Ganoderma lucidum Me (GA-Me) or Ganoderma lucidum T (GA- ⁇ ) mentioned herein is inhibited in the prior art and extracted from the fermentation of Ganoderma lucidum mycelium, and can be used in the literature (Chem. Pharm. Bull., 34 (5), 2282-2285, 1986 ; Agric. Biol. Chem., 51 (2), 619-622, 1987)
  • the tumor of the present invention includes various types of tumors medically belonging, including benign tumors and malignant tumors.
  • the malignant tumors include: malignant melanoma, malignant lymphoma, tumors of the digestive organs (gastric cancer, colon cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer), lung cancer, breast cancer, testicular cancer, ovarian cancer, uterine cancer , prostate cancer, upper jaw cancer, tongue cancer, oral cancer, throat cancer, thyroid cancer, brain tumor, various sarcoma, osteosarcoma, leukemia, nervous system tumor, bladder tumor, skin cancer, skin accessory organ cancer and skin metastases .
  • the tumor is a multi-drug resistant tumor.
  • the animal test proves that the anticancer agent and the sensitizing synergist of the present invention are low in toxicity and are suitable for oral or parenteral administration of human and mammalian animals.
  • the anticancer agent and the sensitizing synergist in the multidrug resistant tumor chemotherapy provided by the invention can usually be combined with the auxiliary material which does not affect the pharmacological action to prepare a preparation for oral or parenteral administration.
  • ganoderic acid inhibits the growth of cancer cells by inducing apoptosis.
  • the present invention also investigates the related apoptotic pathways in which cancer cells are induced after GAs treatment.
  • DNA degradation was observed, the membrane potential of mitochondria decreased, and cytochrome C was released from mitochondria.
  • caspase-8 there was no significant change in the activity of caspase-8, and the activity of caspase-3 was rapidly increased.
  • An increase in the expression of p53 and an increase in the expression of Bax were observed, while the expression of Bel-2 did not change.
  • Drug resistance of chemotherapy drugs is a common cause of ineffective cancer treatment. Finding new drug leaders that can simultaneously anti-tumor and enhance sensitization has become an urgent problem in tumor chemotherapy.
  • the present invention also investigates the possibility that ganoderic acid increases the sensitivity of multidrug resistance KB-A-1 cells to doxorubicin and its possible mechanism of action, and the results indicate that ganoderic acid is relatively high in concentration.
  • Doxorubicin-sensitive and insensitive cancer cell lines are highly cytotoxic, and the multidrug resistance induced by doxorubicin does not affect the inhibitory effect of ganoderic acid on cancer cells. At lower concentrations, the multidrug resistance induced by doxorubicin can be reversed The sensitivity of the hormone increases.
  • ganoderic acid acts as a triterpene molecule to inhibit tumor proliferation by inducing apoptosis, and apoptosis of cancer cells is induced by the mitochondrial pathway. Further studies have found that inhibiting the activity of Topoisomerase l is the cause of apoptosis. At the same time, ganoderic acid has certain sensitization and synergistic effects on multidrug resistant cells. Combined with the application history of Ganoderma lucidum for thousands of years, it can be speculated that they may be an effective tumor suppressor or lead compound.
  • the present invention provides two natural compounds having the functions of simultaneous anti-tumor and sensitization and synergy, overcoming the drawback of traditionally using Ganoderma lucidum mixed extract as a medicine, and using modern biotechnology as a means of invention, clearly
  • These two compounds have significant simultaneous anti-tumor and sensitizing effects (in vitro and in vivo experiments), and have less cytotoxicity to human normal cells, and have good application prospects.
  • Figure 1 shows the inhibitory effects of different concentrations of GA-Me and GA-T on multidrug resistance KB-A-1 cells.
  • Figure 2 shows that different concentrations of GA-Me and GA-T increase the sensitivity of KB-A-1 cells to doxorubicin.
  • Figure 3 shows that different concentrations of GA-Me and GA-T increase the content of doxorubicin in KB-A-1 cells.
  • Figure 4 shows that different concentrations of GA-Me and GA-T increase the content of Rhodanminl23 in KB-A-1 cells.
  • Figure 5 shows the sensitivity of GA-Me and GA-T to the anticancer drug doxorubicin in nude mice.
  • Figure 6 shows the inhibition of proliferation of multidrug resistant solid tumors in nude mice by GA-Me and GA-T.
  • Figure 7 shows the inhibitory effects of different concentrations of GA-Me and GA-T on HeLa cells.
  • Figure 8 shows the inhibitory effects of different concentrations of GA-Me and GA-T on HeLa cells.
  • Figure 9 shows the inhibitory effects of different concentrations of GA-Me and GA-T on 95-D cells.
  • Figure 10 is a comparison of IC 5D of several tumor cells and normal cells L02 by GA-Me and GA-T.
  • Figure 11 shows the comparison of different drugs and concentrations to inhibit tumor growth in nude mice. detailed description
  • 95-D is a human high metastatic lung cancer cell
  • Hela is a human cervical cancer cell
  • SMMC-7721 is a human liver cancer cell
  • L02 is a human liver cell
  • Chinese medical name is an oral epidermoid carcinoma cell line, English medical name for cell line of epidermal carcinoma of the mouth 0 which is a derived from a human oral epithelial cancer cell lines, the cell lines showed inducible multidrug resistance properties.
  • Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM medium.
  • Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with RPMI-1640 medium.
  • the toxic effects of GA-Me and GA-T on 95-D cells were determined by thiazole blue (MTT) rapid colorimetry.
  • Logarithmic growth phase cells Inoculated in a 96-well culture plate, 0.2 ml per well, in the presence of 5 g / ml of ganoderic acid, a certain concentration of doxorubicin was added, each concentration was parallel 4 wells, and the control group was added with an equal volume of the culture solution.
  • the intracellular doxorubicin content was measured by high performance liquid chromatography (HPLC). Sensitive logarithmic growth phase cells were seeded in 24-well plates at a concentration of 10 5 cells/ml. Cells were treated with a combination of doxorubicin and ganoderic acid for 4 hours. The control was treated with doxorubicin alone. After the treated cells were washed three times with cold PBS, the cells were suspended, the concentration of each group was adjusted, and 0.5 ml of water was added thereto, and the cells were repeatedly thawed and centrifuged for 30 minutes at 12,000 rpm, and the supernatant was used for doxorubicin detection.
  • HPLC high performance liquid chromatography
  • the analytical conditions are:
  • the detector is a fluorescence monitor with excitation and emission wavelengths of 495 and 560 dishes.
  • the flow rate is 0. 8 ml/min.
  • the flow rate is 0. 8 ml/min.
  • the flow rate is 0. 8 ml/min.
  • the results are shown in Figure 3.
  • Rhodamine 123 The intracellular accumulation of Rhodamine 123 was detected by a fluorescence spectrophotometer. Sensitive logarithmic growth phase cells were seeded in 96-well plates at a concentration of 10 5 cells/well. The cells were first adherently cultured for 4 hours, and then the cells were treated with ganoderic acid for 4 hours. The control was doxorubicin alone (0. 75 ⁇ ) Treated and treated with ganoderic acid alone. Then add Rodin name 123, the final concentration is 20 ⁇ , and continue to culture for 60 minutes.
  • the fluorescence spectrophotometer has excitation and emission wavelengths of 485 nm and 530 nm, respectively. The result is shown in Figure 4.
  • KB-A-1 cells were inoculated into 6 nude mice. The cell concentration was 10 5 / 0.2 ml, and growth was carried out for 15 days under SPF conditions. The nude mice were sacrificed, and the solid tumors were removed. The cells were cut to a size of 0.5 mm under aseptic conditions, and the nude mice were used to enter the experimental nude mice, one on each side. Five rats in each group were administered with a control drug and a positive drug three days later. Both the control drug and the positive drug were administered by intraperitoneal injection. Five hours after the administration of the control drug, the positive drug was administered. After four days, the positive drug was administered again, and the drug was administered twice. The control drug was administered continuously for 12 days. After stopping the drug for 6 days, the nude mice were sacrificed and the solid tumor was removed. Weigh and analyze the inhibition rate of solid tumors.
  • Tumor inhibition rate (%) [ (the average weight of the solid tumor in the control group - the average weight of the solid tumor in the administration group)
  • KB-A-1 cells in logarithmic growth phase were prepared under aseptic conditions, and about 2.5 X lOVmL cell suspension was prepared.
  • 0.2 mL/mouse was inoculated subcutaneously in the lower limbs of two nude mice. After 15 days of growth, the animals were sacrificed. The solid tumor of the mouse was dissected under the conditions of the bacteria, cut open, the dead cells at the center of the tumor were removed, and the living tissue was cut to a size of 0.5 mm, and washed with PBS.
  • the nude mice were inoculated subcutaneously in the lower limbs of the experimental nude mice.
  • Tumor inhibition rate (%) [(Average weight of control solid tumors - Average weight of solid tumors in the administration group) I Control entity Average tumor weight] X 100
  • the culture medium was placed in an incubator at 37 ° C, 5% ⁇ 2 and saturated humidity for 1-4 days, and ⁇ ⁇ was added to each well 4 hours before the end of the experiment, and 0. 04 ⁇ was added to each well after the end of the culture.
  • Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM medium.
  • the toxic effects of GA - Me and GA-T on HeLa cells were determined by MTT rapid colorimetry.
  • the logarithmic growth phase cells (106 cells. ml-1) were inoculated into 96-well culture plates at 0.2 ml per well, and treated with a certain concentration of GA-Me and GA-T, respectively, each concentration was parallel 4 wells, the control group was added, etc.
  • the volume of the culture solution was incubated at 37 ° C, 5% C02 and saturated humidity in an incubator for 1-4 days. 5 mg.
  • ml-1 MTT ⁇ was added to each well 4 hours before the end of the experiment, and 0 was added to each well after the completion of the culture.
  • the absorbance, the inhibition rate of the tumor cells and the inhibition rate of the drug to the cells were calculated, and the half-inhibitory concentration (IC50) of the cells was determined by plotting the different concentrations of the drug and the inhibition rate of the cells.
  • the experimental results are shown in Figure 8 below.
  • Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with RPMI-1640 medium.
  • the toxic effects of GA-Me and GA-T on 95-D cells were determined by MTT rapid colorimetry.
  • the logarithmic growth phase cells (106 cells. ml-1) were inoculated into a 96-well culture plate at 0. 2 ml per well, and a certain concentration of GA-Me and GA-T were added respectively. Each concentration was parallelized with 4 wells, and the control group was added.
  • the volume of the culture medium was cultured in an incubator at 37 ° C, 5% CO 2 and saturated humidity for 1-4 days. 5 mg.
  • Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM or RPMI-1640 medium.
  • the toxic effects of GA-Me and GA-T on several cells were determined by MTT rapid colorimetry.
  • the logarithmic growth phase cells (106cell.ml-l) were inoculated into 96-well culture plates at 0.2 ml per well, and treated with a certain concentration of GA-Me and GA-T, each concentration was parallel 4 wells, the control group was added, etc.
  • the volume of the culture medium was cultured in an incubator at 37 ° C, 5% CO 2 and saturated humidity for 1-4 days. 5 mg.
  • ral-1 MTT ⁇ was added to each well 4 hours before the end of the experiment, and 0 was added to each well after the completion of the culture.
  • Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, formulated in PBS and diluted to the desired concentration.
  • Two male nude mice, forefoot subcutaneous injection 95-D fine Cells (1 x 106 cells/ml) were sacrificed after four weeks of feeding. Solid tumors of nude mice were stripped, cut and incubated with PBS (7.4), and filtered through an 80 mesh screen. Formulated as a cell suspension for use. Male nude mice were randomly divided into groups according to their body weight. The 95-D cell suspension (1x106 cells/ml) was injected into the forelimbs for one week.
  • the cells were stimulated with Ganoderma lucidum T at 25, 50 g/mL, washed twice with PBS, trypsinized, serum stopped, and centrifuged to collect cells (1500 rpm, 10 min) with Binding solution (see 3.3).
  • the cell concentration was 2x10 5 / mL.
  • Aiuiexin V-FITC solution was added to a final concentration of 10 ng/mL, incubated for 15 minutes at room temperature in the dark, and then stained with PI to a final concentration of 5 g/mL. Flow cytometry was used for analysis.
  • Ganoderma acid inhibits the proliferation of highly metastatic lung cancer cells by inducing apoptosis.
  • apoptosis The most important feature of apoptosis is the fragmentation of DNA. As the apoptosis increases, the DNA fragment in increments of 180 bp gradually increases. The lipogel gel appears as a ladder-like electrophoresis band. Cancer cells treated with ganoderic acid also showed significant DNA ladders.
  • ganoderic acid can induce apoptosis and limit cell cycle in cancer cells, and is concentration dependent. It can be speculated that ganoderic acid mainly expresses its cytotoxicity by inducing apoptosis of cancer cells and limiting cell cycle.
  • cytochrome c There are two main pathways for apoptosis, and the mitochondrial pathway is generally accompanied by the leakage of cytochrome c. Normally, it exists in the lumen between the mitochondrial inner membrane and the outer membrane. The apoptotic signal stimulates it to release from the mitochondria to the cytosol, and initiates the caspase cascade after binding with apaf-1 (apoptotic protease activating factor-1). : The cytochrome C/Apaf-1 complex activates caspase_9, which in turn activates caspase-3 and other downstream caspase. The increase in cytochrome C content was observed in the cytoplasm of cancer cells treated with ganoderic acid, as shown in Figure 4.2.
  • cytochrome C After 4 hours of treatment, the cytochrome C content was increased by a factor of 2 compared to the control, and after 8 hours of treatment, the content was increased by a factor of 3.
  • cytochrome C plays an important role in apoptosis.
  • Ganoderma acid treatment significantly stimulated the leakage of cytochrome c. Therefore, it can be speculated that ganoderic acid has a direct or indirect effect on mitochondria.
  • the amount of intracellular reactive oxygen species (R0S) in cancer cells treated with ganoderic acid varies with treatment time The extension has increased. After 8 hours of treatment, it increased by 40% compared to the control.
  • ganoderic acid affects the membrane potential of mitochondria, free radicals inside the mitochondria leak into the cytoplasm, causing an increase in intracellular free radicals. It is the result of apoptosis.
  • ganoderic acid may interfere with the redox balance in the cell, leading to the dysregulation of the free radical scavenging mechanism, thereby accumulating free radicals and further causing apoptosis.
  • the increase in free radicals in this case is the cause of apoptosis.
  • Ganoderma acid affects the activity of Caspase-3, but has no effect on the activity of Caspase-8
  • Ganoderma acid treatment of cancer cells causes leakage of cytochrome C, which in turn activates caspase-3 and other downstream caspa Se .
  • the process of apoptosis is allowed to proceed.
  • caspase-3 activity was also significantly increased as the treatment time increased. After 8 hours of treatment, the activity increased nearly threefold.
  • Activation of Caspase-3 occurs in both pathways of apoptosis. That is, the caspase-8 of the FAS pathway can be linked to the mitochondrial pathway via the BID protein. Therefore, we tested the activity of caspase-8 to determine if the addition of the FAS pathway resulted in the activation of caspase-3. The results are shown in the figure.
  • caspase-8 After treatment with ganoderic acid, the activity of caspase-8 did not change within 8 hours. Thus, the influence of the FAS pathway can be ruled out, and it can be inferred that the activation of caspase-3 is caused by the complex of cytochrome C.
  • ganoderic acid may activate downstream mitochondrial-related apoptotic events through cells, including activation of casp aS e3, and induction of apoptosis in cancer cells by activating the mitochondrial pathway of CaS pa S e3.
  • Ganoderma acid T affects the expression of apoptosis-related proteins
  • the Bel-2 family which includes the apoptosis-promoting factors Bax, Bak, Bid, and the apoptosis inhibitors Bel-2 and Bel-x. These promoting factors and inhibitors maintain an equilibrium state, and once the balance is broken, it will cause the occurrence and suppression of apoptosis.
  • Another important protein that affects apoptosis is P53. When DNA damage occurs, more P53 is released for maintenance of genomic DNA stability. It can also guide P53 extranuclear output by mediating P53 monoubiquitination. Mitochondria, which directly activate Bax, initiate mitochondrial non-transcription-dependent apoptosis. When DNA damage has been repaired, active P53 is rapidly downregulated to restore normal cell homeostasis.

Abstract

A pharmaceutical composition comprising ganoderic acid T and/or ganoderic acid Me, as well as its application for inhibiting tumor growth or proliferation.Ganoderic acid T and/or ganoderic acid Me have obvious action for inhibiting human tumor cell growth or proliferation, at the same time its toxicity for normal cells is low.

Description

灵芝酸在癌症治疗中的应用 技术领域  Application of Ganoderma Acid in Cancer Therapy
本发明涉及灵芝酸的新用途, 尤其涉及灵芝酸 Me (Ganoderic acid Me) 和灵 芝酸 T (Ganoderic acid T) 的新用途。 背景技术  The present invention relates to new uses of ganoderic acid, in particular to the new use of Me (Ganoderic acid Me) and Ganoic acid T (Ganoderic acid T). Background technique
灵芝是一种珍贵的药用真菌, 已有几千年的应用历史。 古代医药学家对灵芝 的药用价值已有充分认识,认为灵芝具有 "扶正固本"作用,可用于治疗多种疾病 D 药理研究证明, 灵芝有广泛的药理作用, 如抗肿瀹, 抗 HIV病毒, 免疫调节, 抗 心肌缺血, 调节血脂, 降血糖, 镇静作用, 保肝作用, 抗放射和抗化疗, 抗缺氧和 抗衰老作用等。 Ganoderma lucidum is a precious medicinal fungus that has been used for thousands of years. Ancient medical scientists have fully recognized the medicinal value of Ganoderma lucidum. They believe that Ganoderma lucidum has the function of "supporting the body and strengthening the body" and can be used to treat a variety of diseases. D pharmacological research proves that Ganoderma lucidum has a wide range of pharmacological effects, such as anti-tumor, anti-HIV. Virus, immune regulation, anti-myocardial ischemia, regulation of blood lipids, blood sugar lowering, sedative effect, liver protection, anti-radiation and anti-chemotherapy, anti-hypoxia and anti-aging effects.
三萜类化合物是灵芝类的主要化学成分之一, 很多三萜类化合物具有重要的 生理活性。 自 1982年 T Kubota (Helv. Chim. Acta, 65 (2), 611-619, 1982) 等 人首次分离得到该类化合物以后, 迄今为止已分离得到 100多种同类化合物。  Triterpenoids are one of the main chemical constituents of Ganoderma lucidum, and many triterpenoids have important physiological activities. Since the first separation of such compounds by T Kubota (Helv. Chim. Acta, 65 (2), 611-619, 1982) in 1982, more than 100 similar compounds have been isolated to date.
灵芝酸 Me (GA-Me) 或灵芝酸 T (GA- Τ)分别由灵芝菌丝体发酵中提取, 可采 用文献 (Chem. Pharm. Bull. , 34 (5) , 2282-2285, 1986 ; Agric. Biol. Chem., 51 (2) , 619-622, 1987) 公幵的技术进行制备提取, 纯度大于 99%, 其最先被分离 鉴定于 1983年(Tetrahedron Lett., 24 (10) , 1081-1084, 1983)和 1987年(Agric. Biol. Chem. , 51 (2), 619-622, 1987)。 现有技术中已经有了关于发酵生产灵芝酸、 灵芝抗肿瘤和灵芝酸分离的专利和文献 (例如参见日本专利公开特许公报 特开平 4-304890 s 中国专利出版物 CN1264743A、 CN1483423、 CN 1480208、 CN 1286119 和 CN 1181271等)。 然而, 现有技术中并未见有关灵芝酸单体及其应用的报道 肿瘤化疗是目前治疗肿瘤的重要手段, 影响治疗效果的主要问题是肿瘤能形 成多药耐药性。已有的增敏增效药物都存在使用上的缺陷,寻找新的能同时抗肿瘤 和增敏增效的药物资源已成为急需解决的问题。 理想的 MDR逆转剂应具备以下条 件: ①安全, 对正常组织毒性小; ②在体内及肿瘤细胞能达到体外有效浓度; ③本 身具有一定的抗肿瘤活性; ④稳定、 体内半衰期较长; ⑤其代谢物也有效。  Ganoderma lucidum Me (GA-Me) or Ganoderma lucidum T (GA- Τ) are extracted from the fermentation of Ganoderma lucidum mycelium, respectively (Chem. Pharm. Bull., 34 (5), 2282-2285, 1986; Agric Biol. Chem., 51 (2), 619-622, 1987) The technique of the cockroach is prepared for extraction with a purity greater than 99%, which was first isolated and identified in 1983 (Tetrahedron Lett., 24 (10), 1081 -1084, 1983) and 1987 (Agric. Biol. Chem., 51 (2), 619-622, 1987). There are already patents and literatures on the production of ganoderic acid, ganoderma lucidum anti-tumor and ganoderic acid in the prior art (see, for example, Japanese Patent Laid-Open Publication No. Hei-4-304890 s Chinese Patent Publication No. CN1264743A, CN1483423, CN 1480208, CN 1286119 and CN 1181271, etc.). However, there is no report on the application of ganoderic acid monomer and its application in the prior art. Tumor chemotherapy is an important means for treating tumors at present, and the main problem affecting the therapeutic effect is that tumors can form multidrug resistance. Existing sensitizing and potentiating drugs have drawbacks in use, and finding new drug resources capable of simultaneously anti-tumor and sensitizing and increasing efficiency has become an urgent problem to be solved. The ideal MDR reversal agent should have the following conditions: 1 safe, less toxic to normal tissues; 2 can achieve effective concentration in vitro and in tumor cells; 3 itself has certain anti-tumor activity; 4 stable, long half-life in vivo; Metabolites are also effective.
因此, 肿瘤化疗治疗领域急需解决的问题是寻找出新的低毒、高效和不易产生 多药抗药性的抗癌药物。  Therefore, the urgent problem to be solved in the field of tumor chemotherapy is to find new anticancer drugs that are low in toxicity, high in efficiency, and difficult to produce multidrug resistance.
1 1
确 认 本 发明内容 Confirmation Summary of the invention
发明人发现, 灵芝酸 Me或灵芝酸 T能够抑制肿瘤的增殖和生长, 小剂量即产 生抑制多种肿瘤细胞生长的作用且具有剂量依赖性,其对不同的人类癌细胞和人类 正常细胞有不同的细胞毒性, 尤其是对癌细胞的 IC5D要比正常细胞的 1(:5()低很多, 而且两者差异显著(p〈0. 05)。而且, 小剂量灵芝酸 Me或灵芝酸 T能增加多药耐药 性肿瘤对已产生耐药性的化疗药物的敏感性,大剂量使用有抑制多种多药耐药性肿 瘤细胞生长的作用且具有剂量依赖性,因此还可将它们作为治疗多药耐药性肿瘤的 药物和增敏增效剂, 应用于肿瘤的化学治疗中。 The inventors have found that Ganoderma lucidum Me or Ganoderma lucidum T can inhibit tumor proliferation and growth, and a small dose produces an effect of inhibiting the growth of various tumor cells in a dose-dependent manner, which is different for different human cancer cells and human normal cells. Cytotoxicity, especially IC 5D for cancer cells is much lower than normal cells 1 (: 5 () , and the difference between the two is significant (p < 0.05). Moreover, low doses of Ganoderma lucidum Me or Ganoderma lucidum T It can increase the sensitivity of multidrug-resistant tumors to chemotherapeutic drugs that have developed resistance. High-dose use has the effect of inhibiting the growth of multiple multidrug-resistant tumor cells and is dose-dependent, so they can also be used as Drugs and sensitizing synergists for the treatment of multidrug resistant tumors are used in the chemotherapy of tumors.
具体而言, 本发明涉及一种药物组合物, 包括治疗有效量的灵芝酸 Me或灵芝 酸 T和医药学上可接受的载体。所说的载体是指药学领域常规的药物载体,如稀释 剂、 赋形剂如水等, 填充剂, 如淀粉、 蔗糖等, 粘合剂, 如纤维素衍生物明胶、 聚 乙烯吡咯烷酮等, 润滑剂, 如滑石粉等。 在该组合物中, 灵芝酸 Me或灵芝酸 T的 重量含量为 0. 1-99. 9%, 优选的含量为 0. 5-99. 9%。 在一个实施方案中, 所述药物 组合物基本上由灵芝酸 Me或灵芝酸 T和医药学上可接受的载体组成。 所述药物组 合物不仅能够用于抑制肿瘤的增殖和生长 (尤其是多药耐药性肿瘤细胞的增殖和生 长), 而且还能增加多药耐药性肿瘤对己产生耐药性的化疗药物的敏感性。 因此, 在一个较佳方案中, 所述组合物还包含所述化疗药物, 如阿霉素、 长春碱、 长春新 碱、 维拉帕米等。  In particular, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier. The carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as a diluent, an excipient such as water, a filler such as starch, sucrose, etc., a binder such as a cellulose derivative gelatin, polyvinylpyrrolidone, etc., a lubricant , such as talcum powder. 9%。 The 9%, the content of the ginseng acid is 0. 5-99. 9%. In one embodiment, the pharmaceutical composition consists essentially of Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used not only to inhibit tumor proliferation and growth (especially proliferation and growth of multidrug resistant tumor cells), but also to increase multidrug resistant tumor-resistant chemotherapy drugs. Sensitivity. Accordingly, in a preferred embodiment, the composition further comprises the chemotherapeutic agent, such as doxorubicin, vinblastine, vincristine, verapamil, and the like.
本发明还涉及一种肿瘤治疗方法, 该方法包括将治疗有效量的本发明上述组 合物给予需要该治疗的对象。 本发明还提供了灵芝酸 Me和 /或灵芝酸 T在制备肿 瘤治疗药物中的应用。  The invention further relates to a method of treating a tumor comprising administering a therapeutically effective amount of the above composition of the invention to a subject in need of such treatment. The present invention also provides the use of Ganoderma lucidum Me and/or Ganoderma lucidum T in the preparation of a medicament for the treatment of tumors.
可以将灵芝酸 Me或灵芝酸 T与医药学上可接受的载体以组合物的形式, 施加 于需要治疗的患者, 一般的剂量为 l-100mg/ (公斤体重 ·天), 具体可根据患者的年 龄、 病情等进行变化。  Ganoderma lucidum Me or Ganoderma lucidum T and a pharmaceutically acceptable carrier may be administered to a patient in need of treatment in the form of a composition, generally at a dose of 1-100 mg / (kg body weight per day), depending on the patient Age, condition, etc. change.
本发明的灵芝酸 Me或灵芝酸 T与上述载体构成的组合物, 可以通过口服、 鼻 吸、静脉注射或皮下注射的形式施加于需要这种治疗的患者。用于口服时, 可以将 其制备成为常规的片剂、粉剂或口服液; 用于注射时, 可以将其采用本领域常规的 方法, 制备成为注射液; 本发明的芝酸 Me或灵芝酸 T的各种制剂, 包括散剂, 丸 剂, 片剂, 胶囊剂等固型制剂, 软膏, 贴敷剂, 注射剂等可以釆用药学领域常规的 方法进行制备。  The composition of the ganoderic acid Me or ganoderic acid T of the present invention and the above carrier can be applied to a patient in need of such treatment by oral, nasal, intravenous or subcutaneous injection. When used orally, it can be prepared into a conventional tablet, powder or oral solution; when it is used for injection, it can be prepared into an injection solution by a method conventional in the art; the meic acid Me or the ganoderic acid T of the present invention Various preparations, including powders, pills, tablets, capsules and the like, solid preparations, ointments, patches, injections and the like can be prepared by a conventional method in the pharmaceutical field.
本发明提供的组合物或制剂在低浓度的情况下具有增强多药耐药性肿瘤细胞 对化疗药物敏感性的作用(所说的低浓度指低于 l-10mg/ (公斤体重 ·天)的剂量),在 高浓度的情况下具有抑制多药耐药性肿瘤细胞增殖的作用 (所说的高浓度指的是高 于 10-100mg/ (公斤体重 ·天)的剂量), 说明其不仅具有杀灭肿瘤细胞的作用而且可 以遏制肿瘤细胞的无限增殖能力, 因而可以发挥更进一步更大程度治疗肿瘤的效 果。 The composition or preparation provided by the present invention has the effect of enhancing the sensitivity of multidrug resistant tumor cells to chemotherapeutic drugs at a low concentration (the low concentration means less than l-10 mg / (kg body weight per day) Dose), at At high concentrations, it has the effect of inhibiting the proliferation of multidrug resistant tumor cells (the high concentration refers to a dose higher than 10-100 mg / (kg body weight · day)), indicating that it not only has tumor cell killing The role of the tumor cells can also inhibit the infinite proliferation of tumor cells, and thus can play a greater and greater degree of treatment of tumors.
本发明提供的多药耐药性肿瘤化疗中的抗癌剂和增敏增效剂,对多药耐药性 癌细胞和非多药耐药性癌细胞有相同的细胞毒性,而且两者差异不显著(p〈0. 05)。  The anticancer agent and the sensitizing synergist in the multidrug resistant tumor chemotherapy provided by the invention have the same cytotoxicity to the multidrug resistant cancer cells and the non-multidrug resistant cancer cells, and the difference therebetween Not significant (p<0.05).
本文所说的灵芝酸 Me (GA-Me) 或灵芝酸 T (GA- Τ) 是现有技术中抑制的, 分 别由灵芝菌丝体发酵中提取,可采用文献(Chem. Pharm. Bull. , 34 (5), 2282-2285, 1986 ; Agric. Biol. Chem. , 51 (2) , 619-622, 1987) 公开的技术进行制备提取, 纯度大于 99% , 其最先被分离鉴定于 1983 年 (Tetrahedron Lett. , 24 (10), 1081-1084, 1983) 和 1987年 (Agric. Biol. Chem. , 51 (2) , 619-622, 1987)。  The Ganoderma lucidum Me (GA-Me) or Ganoderma lucidum T (GA- Τ) mentioned herein is inhibited in the prior art and extracted from the fermentation of Ganoderma lucidum mycelium, and can be used in the literature (Chem. Pharm. Bull., 34 (5), 2282-2285, 1986 ; Agric. Biol. Chem., 51 (2), 619-622, 1987) The disclosed technique for preparative extraction with a purity greater than 99%, which was first isolated and identified in 1983 (Tetrahedron Lett., 24 (10), 1081-1084, 1983) and 1987 (Agric. Biol. Chem., 51 (2), 619-622, 1987).
本发明所述的肿瘤包括为医学上所属的各类肿瘤, 包括良性肿瘤和恶性肿瘤。 而所说的恶性肿瘤包括: 恶性黑色素瘤, 恶性淋巴瘤, 消化器官的肿瘤(胃癌, 肠 癌, 肝癌, 胆囊癌, 胆道癌, 胰腺癌), 肺癌, 乳癌, 睾丸癌, 卵巢癌, 子宫癌, 前列腺癌, 上颚癌, 舌癌, 口腔癌, 咽喉癌, 甲状腺癌, 脑部肿瘤, 各类肉瘤, 骨 肉瘤, 白血病, 神经系统肿瘤,膀胱肿瘤, 皮肤癌, 皮肤附属器官癌和皮肤转移癌。 在较佳的实施方案中, 所述肿瘤是多药耐药性肿瘤。  The tumor of the present invention includes various types of tumors medically belonging, including benign tumors and malignant tumors. The malignant tumors include: malignant melanoma, malignant lymphoma, tumors of the digestive organs (gastric cancer, colon cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer), lung cancer, breast cancer, testicular cancer, ovarian cancer, uterine cancer , prostate cancer, upper jaw cancer, tongue cancer, oral cancer, throat cancer, thyroid cancer, brain tumor, various sarcoma, osteosarcoma, leukemia, nervous system tumor, bladder tumor, skin cancer, skin accessory organ cancer and skin metastases . In a preferred embodiment, the tumor is a multi-drug resistant tumor.
动物试验证明, 本发明的抗癌剂和增敏增效剂, 低毒, 适合于人和哺乳类动 物经口或非经口给药。本发明提供的多药耐药性肿瘤化疗中的抗癌剂和增敏增效剂, 通常能与不影响药理学作用的辅助料配合制成经口或非经口给药的制剂。  The animal test proves that the anticancer agent and the sensitizing synergist of the present invention are low in toxicity and are suitable for oral or parenteral administration of human and mammalian animals. The anticancer agent and the sensitizing synergist in the multidrug resistant tumor chemotherapy provided by the invention can usually be combined with the auxiliary material which does not affect the pharmacological action to prepare a preparation for oral or parenteral administration.
进一步的研究表明, 灵芝酸通过诱导凋亡来抑制癌细胞的生长的。 本发明还 研究了 GAs处理后,癌细胞被诱导的相关凋亡途径。在实验中,观察到了 DNA降解, 线粒体的膜电位下降, 细胞色素 C从线粒体中释放。 此外, caspase- 8的活性没有 发生显著的变化, 而 caspase- 3 的活性快速增加。 观察到了 p53 表达量的增加, Bax表达量的上调, 而 Bel- 2的表达量没有发生变化。 综合以上结果表明, GAs抑 制高转移肺癌生长主要是通过细胞内的与 P53相关的线粒体途径来实现的。  Further studies have shown that ganoderic acid inhibits the growth of cancer cells by inducing apoptosis. The present invention also investigates the related apoptotic pathways in which cancer cells are induced after GAs treatment. In the experiment, DNA degradation was observed, the membrane potential of mitochondria decreased, and cytochrome C was released from mitochondria. In addition, there was no significant change in the activity of caspase-8, and the activity of caspase-3 was rapidly increased. An increase in the expression of p53 and an increase in the expression of Bax were observed, while the expression of Bel-2 did not change. Taken together, the above results indicate that GAs inhibit high-metastasis lung growth mainly through intracellular P53-associated mitochondrial pathway.
化疗药物的耐药性是导致癌症治疗无效的常见原因。 寻找新的能同时抗肿瘤 和增敏增效的药物先导物已成为肿瘤化疗中急需解决的问题。本发明还研究了灵芝 酸增加多药耐药性 KB- A- 1细胞对阿霉素的敏感性的可能性及其可能的作用机理, 结果表明,灵芝酸在相对较高的浓度条件下对阿霉素敏感和不敏感的癌细胞株都有 很强的细胞毒性,阿霉素诱导出的多药耐药性并未影响到灵芝酸对癌细胞的抑制作 用。在较低的浓度条件下, 可以逆转阿霉素诱导出的多药耐药性, 使癌细胞对阿霉 素的敏感性增加。进一步研究表明, 灵芝酸处理后的细胞, 能使多药耐药性对阿霉 素的吸收量增加。 通过对 Rhodanminl23的吸收试验表明, 灵芝酸处理可能影响到 Pgp蛋白的活性。动物实验表明: 灵芝酸抑制裸鼠体内多药耐药性实体瘤的增殖并 增加对阿霉素的敏感性, 阿霉素和灵芝酸的混合使用并没有引起动物死亡,表明灵 芝酸并没有导致毒副作用的增加。 Drug resistance of chemotherapy drugs is a common cause of ineffective cancer treatment. Finding new drug leaders that can simultaneously anti-tumor and enhance sensitization has become an urgent problem in tumor chemotherapy. The present invention also investigates the possibility that ganoderic acid increases the sensitivity of multidrug resistance KB-A-1 cells to doxorubicin and its possible mechanism of action, and the results indicate that ganoderic acid is relatively high in concentration. Doxorubicin-sensitive and insensitive cancer cell lines are highly cytotoxic, and the multidrug resistance induced by doxorubicin does not affect the inhibitory effect of ganoderic acid on cancer cells. At lower concentrations, the multidrug resistance induced by doxorubicin can be reversed The sensitivity of the hormone increases. Further studies have shown that cells treated with ganoderic acid can increase the absorption of doxorubicin by multidrug resistance. The absorption test of Rhodanminl23 showed that ganoderic acid treatment may affect the activity of Pgp protein. Animal experiments showed that: Ganoderma lucidum inhibited the proliferation of multidrug-resistant solid tumors in nude mice and increased the sensitivity to doxorubicin. The combination of doxorubicin and ganoderic acid did not cause animal death, indicating that ganoderic acid did not cause Increased toxic side effects.
综合以上结果, 可以发现灵芝酸做为一种三萜分子具有通过诱导凋亡来抑制 肿瘤增殖的活性,诱导癌细胞的凋亡是通过线粒体途径实现的。进一步研究发现抑 制 Topoisomerase l的活性是诱导凋亡的原因。 同时, 灵芝酸对多药耐药性细胞有 一定的增敏增效作用。结合灵芝几千年的应用历史,可以推测它们可能是一种有效 的肿瘤抑制剂或先导化合物。 综上所述, 本发明提供的具有同时抗肿瘤和增敏增效的作用的两种天然化合 物,克服了传统上以灵芝混合提取物作为药物的缺陷,用现代生物技术作为发明的 手段, 明确了有效成分, 运用纯化合物作为抗肿瘤药物和增敏增效剂, 减少了未知 成分引起负作用的可能性。这两种化合物具有显著的同时抗肿瘤和增敏增效的作用 (体外和体内实验), 同时对人类正常细胞的细胞毒性较小,具有良好的应用前景。 附图说明  Based on the above results, it can be found that ganoderic acid acts as a triterpene molecule to inhibit tumor proliferation by inducing apoptosis, and apoptosis of cancer cells is induced by the mitochondrial pathway. Further studies have found that inhibiting the activity of Topoisomerase l is the cause of apoptosis. At the same time, ganoderic acid has certain sensitization and synergistic effects on multidrug resistant cells. Combined with the application history of Ganoderma lucidum for thousands of years, it can be speculated that they may be an effective tumor suppressor or lead compound. In summary, the present invention provides two natural compounds having the functions of simultaneous anti-tumor and sensitization and synergy, overcoming the drawback of traditionally using Ganoderma lucidum mixed extract as a medicine, and using modern biotechnology as a means of invention, clearly The active ingredient, using pure compounds as anti-tumor drugs and sensitizing synergists, reduces the possibility of negative effects caused by unknown components. These two compounds have significant simultaneous anti-tumor and sensitizing effects (in vitro and in vivo experiments), and have less cytotoxicity to human normal cells, and have good application prospects. DRAWINGS
图 1为不同浓度的 GA- Me 和 GA-T对多药耐药性 KB- A-1细胞的抑制作用。 ' 图 2为不同浓度的 GA-Me和 GA-T增加 KB- A- 1细胞对阿霉素的敏感性。  Figure 1 shows the inhibitory effects of different concentrations of GA-Me and GA-T on multidrug resistance KB-A-1 cells. Figure 2 shows that different concentrations of GA-Me and GA-T increase the sensitivity of KB-A-1 cells to doxorubicin.
图 3为不同浓度的 GA-Me和 GA-T增加 KB- A- 1细胞内阿霉素的含量。  Figure 3 shows that different concentrations of GA-Me and GA-T increase the content of doxorubicin in KB-A-1 cells.
图 4为不同浓度的 GA- Me和 GA-T增加 KB- A- 1细胞内 Rhodanminl23的含量。 图 5为 GA- Me和 GA- T增加荷瘤裸鼠对抗癌药阿霉素的敏感性。  Figure 4 shows that different concentrations of GA-Me and GA-T increase the content of Rhodanminl23 in KB-A-1 cells. Figure 5 shows the sensitivity of GA-Me and GA-T to the anticancer drug doxorubicin in nude mice.
图 6为 GA- Me和 GA- T抑制裸鼠体内多药耐药性实体瘤的增殖。  Figure 6 shows the inhibition of proliferation of multidrug resistant solid tumors in nude mice by GA-Me and GA-T.
图 7为不同浓度的 GA- Me 和 GA-T对 HeLa细胞的抑制作用。  Figure 7 shows the inhibitory effects of different concentrations of GA-Me and GA-T on HeLa cells.
图 8为不同浓度的 GA- Me和 GA- T对 HeLa细胞的抑制作用。  Figure 8 shows the inhibitory effects of different concentrations of GA-Me and GA-T on HeLa cells.
图 9为不同浓度的 GA- Me和 GA-T对 95-D细胞的抑制作用。  Figure 9 shows the inhibitory effects of different concentrations of GA-Me and GA-T on 95-D cells.
图 10为 GA-Me和 GA- T对几种肿瘤细胞和正常细胞 L02的 IC5D比较。 Figure 10 is a comparison of IC 5D of several tumor cells and normal cells L02 by GA-Me and GA-T.
图 11为不同药物及浓度抑制裸鼠体内肿瘤生长的比较。 具体实施方式  Figure 11 shows the comparison of different drugs and concentrations to inhibit tumor growth in nude mice. detailed description
下面通过实施例对本发明内容作进一步说明。 所举之例并不限制本发明的保 护范围。 The contents of the present invention will be further described below by way of examples. The examples given do not limit the protection of the present invention. Range of protection.
实施例中的 95-D, Hela, SMMC-7721 , L02和 KB- A- 1细胞株的供应商是中国 科学院上海细胞库。 其中 95- D 是人高转移肺癌细胞; Hela 是人宫颈癌细胞; SMMC- 7721是人肝癌细胞; L02是人肝细胞; KB- A-1细胞的中文医学名称为口腔表 皮样癌细胞株, 英文医学名称为 cell line of epidermal carcinoma of the mouth 0 它是一种来源于人口腔上皮的癌细胞株, 该细胞株表现出可诱导的多药耐药性特 性。 The supplier of 95-D, Hela, SMMC-7721, L02 and KB-A-1 cell lines in the examples is the Shanghai Cell Bank of the Chinese Academy of Sciences. Among them, 95-D is a human high metastatic lung cancer cell; Hela is a human cervical cancer cell; SMMC-7721 is a human liver cancer cell; L02 is a human liver cell; KB-A-1 cell Chinese medical name is an oral epidermoid carcinoma cell line, English medical name for cell line of epidermal carcinoma of the mouth 0 which is a derived from a human oral epithelial cancer cell lines, the cell lines showed inducible multidrug resistance properties.
实验用 BALB/c裸鼠由中国科学院实验动物中心提供。 等级: SPF。 质量合格 证号码: SCXK (沪) 2003-0003。 动物实验过程遵循中华人民共和国相关的动物实验 和伦理学法规。 实施例 1 GA-Me和 GA-T对 KB-A- 1细胞的毒性作用  Experimental BALB/c nude mice were provided by the Experimental Animal Center of the Chinese Academy of Sciences. Rating: SPF. Quality certificate number: SCXK (Shanghai) 2003-0003. The animal experiment process follows the relevant animal experiments and ethics regulations of the People's Republic of China. Example 1 Toxicity of GA-Me and GA-T to KB-A-1 cells
GA-Me和 GA-T均为从发酵生产的灵芝菌丝体中分离得到的纯天然产物, 纯度 大于 99%, 用 DMEM培养液稀释成所需浓度。  Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM medium.
采用噻唑蓝 (MTT) 快速比色法测定 GA- Me和 GA- T对 HeLa细胞的毒性作用。 将对数生长期细胞 (
Figure imgf000007_0001
接种于 96孔培养板, 每孔 0. 2ml, 分别加入一 定浓度的 GA- Me和 GA-T处理, 每浓度平行 4孔, 对照组加等量体积的培养液, 置 37°C , 5% C02及饱和湿度的培养箱培养 1-4天,实验终止前 4小时每孔加入 Smg'mr1 MTT ΙΟμΙ , 培养结束后每孔加入 0. 04 Ν二甲基亚砜 (DMS0), 每孔 150 μ 1, 振荡 10 min, 待 MTT还原产物完全溶解, 用 BioRad 550型酶标仪, 以 550 nm为实验波 长, 655 nra 为参照波长测定其吸收度, 计算药物对细胞的抑制率, 并以药物的不 同浓度和细胞的抑制率作图, 确定细胞的半数抑制浓度(IC5Q)。 实验结果如下图 1 所示。
The toxic effects of GA-Me and GA-T on HeLa cells were determined by MTT rapid colorimetry. Logarithmic growth phase cells (
Figure imgf000007_0001
Inoculate in a 96-well culture plate, 0.2 ml per well, and add a certain concentration of GA-Me and GA-T respectively. Each concentration is paralleled with 4 wells, and the control group is added with an equal volume of the culture solution at 37 ° C, 5%. Incubate C0 2 and saturated humidity incubator for 1-4 days, add Smg'mr 1 MTT ΙΟμΙ to each well 4 hours before the end of the experiment, and add 0.04 Ν dimethyl sulfoxide (DMS0) per well after the end of the culture. After 150 μ 1 , shaking for 10 min, the MTT reduction product was completely dissolved. The absorbance was measured with a BioRad 550 microplate reader at 550 nm and the reference wavelength of 655 nra, and the inhibition rate of the drug was calculated. The concentration of the drug and the inhibition rate of the cells were plotted to determine the half-inhibitory concentration of the cells (IC 5Q ). The experimental results are shown in Figure 1 below.
以上实施例说明, GA- Me和 GA- T均具有良好的杀灭 KB- A- 1肿瘤细胞的作用, 而且这种抑制效果还呈现出剂量依赖性。 实施例 2 GA-Me和 GA- T增加 KB- A- 1细胞对抗癌药阿霉素的敏感作用  The above examples demonstrate that both GA-Me and GA-T have a good effect on killing KB-A-1 tumor cells, and this inhibitory effect is also dose-dependent. Example 2 GA-Me and GA-T increase the sensitivity of KB-A-1 cells to the anticancer drug doxorubicin
GA-Me和 GA-T均为从发酵生产的灵芝菌丝体中分离得到的纯天然产物, 纯度 大于 99%, 用 RPMI- 1640培养液稀释成所需浓度。 采用噻唑蓝 (MTT) 快速比色法 测定 GA- Me和 GA- T对 95- D细胞的毒性作用。 将对数生长期细胞 (
Figure imgf000007_0002
) 接种于 96孔培养板, 每孔 0. 2ml, 在 5 g/ml灵芝酸的存在下, 分别加入一定浓度 的阿霉素处理, 每浓度平行 4孔, 对照组加等量体积的培养液, 置 37°C, 5% C02 N2006/001171 及饱和湿度的培养箱培养 3天, 实验终止前 4小时每孔加入 δι^'ηύ^ΜΤΤ ΙΟμΙ, 培 养结束后每孔加入 0. 04 N DMS0, 每孔 150μ1, 振荡 10 min, 待 MTT还原产物完全 溶解, 用 BioRad 550型酶标仪, 以 550 rnn为实验波长, 655 nm 为参照波长测定 其吸收度,计算阿霉素对癌细胞的抑制率,并以药物的不同浓度和细胞的抑制率作 图, 确定 IC5。。 实验结果见图 2。
Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with RPMI-1640 medium. The toxic effects of GA-Me and GA-T on 95-D cells were determined by thiazole blue (MTT) rapid colorimetry. Logarithmic growth phase cells (
Figure imgf000007_0002
Inoculated in a 96-well culture plate, 0.2 ml per well, in the presence of 5 g / ml of ganoderic acid, a certain concentration of doxorubicin was added, each concentration was parallel 4 wells, and the control group was added with an equal volume of the culture solution. , set 37 ° C, 5% C0 2 N2006/001171 and the incubator of saturated humidity were cultured for 3 days, and δι^'ηύ^ΜΤΤ ΙΟμΙ was added to each well 4 hours before the end of the experiment. After the completion of the culture, 0. 04 N DMS0 was added to each well, and 150 μl per well was shaken for 10 min. The MTT reduction product was completely dissolved. The absorbance was measured with a BioRad 550 microplate reader at 550 rnn as the experimental wavelength and 655 nm as the reference wavelength. The inhibition rate of doxorubicin on cancer cells was calculated, and the drug concentration and cells were determined. The inhibition rate is plotted to determine IC 5 . . The experimental results are shown in Figure 2.
以上实施例说明, GA-Me和 GA- T均具有良好的增加 KB-A- 1细胞对阿霉素的敏 感性作用。 实施例 3 GA-Me和 GA-T增加 KB- A-1细胞内阿霉素的含量  The above examples demonstrate that both GA-Me and GA-T have a good increase in the sensitivity of KB-A-1 cells to doxorubicin. Example 3 GA-Me and GA-T increase the content of doxorubicin in KB-A-1 cells
细胞内的阿霉素含量用高效液相色谱 (HPLC) 检测。 敏感性对数生长期细胞 接种在 24孔板中, 浓度为 105个 /ml, 细胞在阿霉素和灵芝酸联合作用下处理 4小 时, 对照为单独阿霉素处理的细胞。 处理后的细胞以冷 PBS洗三次后, 悬浮细胞, 统一各组细胞浓度, 加入 0. 5m水, 反复冻融三次, 12000rpm离心 30分钟, 上清 用于阿霉素检测。 分析条件为: 检测器为荧光监测器, 激发和发射波长为 495 和 560皿。 流动相为 5mM的磷酸 /甲醇 /乙腈 /异丙醇: 8: 7: 2: 3 (体积比), 使用前 用 0. 45μπι滤膜过滤, 流速为 0. 8 ml/min。 结果见图 3。 The intracellular doxorubicin content was measured by high performance liquid chromatography (HPLC). Sensitive logarithmic growth phase cells were seeded in 24-well plates at a concentration of 10 5 cells/ml. Cells were treated with a combination of doxorubicin and ganoderic acid for 4 hours. The control was treated with doxorubicin alone. After the treated cells were washed three times with cold PBS, the cells were suspended, the concentration of each group was adjusted, and 0.5 ml of water was added thereto, and the cells were repeatedly thawed and centrifuged for 30 minutes at 12,000 rpm, and the supernatant was used for doxorubicin detection. The analytical conditions are: The detector is a fluorescence monitor with excitation and emission wavelengths of 495 and 560 dishes. The flow rate is 0. 8 ml/min. The flow rate is 0. 8 ml/min. The flow rate is 0. 8 ml/min. The results are shown in Figure 3.
以上实施例说明, GA- Me和 GA- T使癌细胞内的抗癌药物浓度增加, 增加了多 药耐药性癌细胞对阿霉素的敏感性。 实施例 4 GA-Me和 GA- T增加 KB- A-1细胞内 Rhodanminl23的含量  The above examples demonstrate that GA-Me and GA-T increase the concentration of anticancer drugs in cancer cells and increase the sensitivity of multidrug resistant cancer cells to doxorubicin. Example 4 GA-Me and GA-T increase the content of Rhodanminl23 in KB-A-1 cells
细胞内累集的 Rhodamine 123用荧光分光光度计检测。 敏感性对数生长期细 胞接种在 96孔板中, 浓度为 105个 /孔, 细胞首先贴壁培养 4小时, 然后细胞在灵 芝酸作用下处理 4小时, 对照为单独阿霉素 (0. 75 μΜ) 处理和单独灵芝酸处理的 细胞。 然后加入罗丹名 123, 终浓度为 20μΜ,继续培养 60分钟。 处理后的细胞以 冷 PBS洗三次后, 悬浮细胞, 统一各组细胞浓度, 加入 0. 5ml水, 反复冻融三次, 12000rpm离心 30分钟, 上清用于 Rhodamine 123检测。 荧光分光光度计, 激发和 发射波长分别为 485 nm和 530nm。 结果如图 4所示。 The intracellular accumulation of Rhodamine 123 was detected by a fluorescence spectrophotometer. Sensitive logarithmic growth phase cells were seeded in 96-well plates at a concentration of 10 5 cells/well. The cells were first adherently cultured for 4 hours, and then the cells were treated with ganoderic acid for 4 hours. The control was doxorubicin alone (0. 75 μΜ) Treated and treated with ganoderic acid alone. Then add Rodin name 123, the final concentration is 20 μΜ, and continue to culture for 60 minutes. After the treated cells were washed three times with cold PBS, the cells were suspended, the concentration of each group was adjusted, and 0.5 ml of water was added thereto, and the cells were repeatedly thawed and centrifuged for 30 minutes at 12,000 rpm, and the supernatant was used for Rhodamine 123 detection. The fluorescence spectrophotometer has excitation and emission wavelengths of 485 nm and 530 nm, respectively. The result is shown in Figure 4.
以上实施例说明, GA- Me和 GA-T能使癌细胞内的抗癌药物浓度增加, 增加了 多药耐药性癌细胞对抗癌药物的敏感性。 实施例 5 GA-Me和 GA- T增加荷瘤裸鼠对阿霉素的敏感性实验  The above examples demonstrate that GA-Me and GA-T increase the concentration of anticancer drugs in cancer cells and increase the sensitivity of multidrug resistant cancer cells to anticancer drugs. Example 5 GA-Me and GA-T increase the sensitivity of tumor-bearing nude mice to doxorubicin
采用裸鼠进行实验。 敏感性的对数生长期 KB-A-1细胞接种在 6只裸鼠腋下, 细胞浓度为 105个 /0.2ml, SPF条件下生长 15天。 处死裸鼠, 剥取实体瘤, 无菌条 件下剪碎至 0.5mm大小, 用大号接种针打入实验裸鼠腋下, 左右各一。 每组 5只, 三天后进行对照药物和阳性药物给药。 对照药物和阳性药物均以腹腔注射方式给 药。 对照药物给药完 5小时后, 阳性药物给药,隔四天后阳性药物再次给药, 共给 药 2次, 对照药物连续给药 12天, 停药 6天后处死裸鼠, 剥取实体瘤, 称重并分 析实体瘤的抑制率。 Experiments were performed using nude mice. Sensitive logarithmic growth phase KB-A-1 cells were inoculated into 6 nude mice. The cell concentration was 10 5 / 0.2 ml, and growth was carried out for 15 days under SPF conditions. The nude mice were sacrificed, and the solid tumors were removed. The cells were cut to a size of 0.5 mm under aseptic conditions, and the nude mice were used to enter the experimental nude mice, one on each side. Five rats in each group were administered with a control drug and a positive drug three days later. Both the control drug and the positive drug were administered by intraperitoneal injection. Five hours after the administration of the control drug, the positive drug was administered. After four days, the positive drug was administered again, and the drug was administered twice. The control drug was administered continuously for 12 days. After stopping the drug for 6 days, the nude mice were sacrificed and the solid tumor was removed. Weigh and analyze the inhibition rate of solid tumors.
肿瘤抑制率(%) =[ (对照组实体瘤平均重量数一给药组实体瘤平均重量数) Tumor inhibition rate (%) = [ (the average weight of the solid tumor in the control group - the average weight of the solid tumor in the administration group)
I对照组实体瘤平均重量数 ] X 100 I control group of solid tumors average weight] X 100
结果见附图图 5,在图 5中:  The results are shown in Figure 5 of the accompanying drawings, in Figure 5:
阳性药物阿霉素 (lmg/kg) 组;  Positive drug doxorubicin (1mg/kg) group;
阳性药物阿霉素 ( lrag/kg) +灵芝酸 T (25 mg/kg) 组;  The positive drug doxorubicin (lrag/kg) + ganoderic acid T (25 mg/kg) group;
阳性药物阿霉素 (lmg/kg) +灵芝酸 Me (25 rag/kg) 组。  The positive drug doxorubicin (1 mg/kg) + ganoderic acid Me (25 rag/kg) group.
以上实施例说明, GA-Me和 GA-T能增加荷瘤裸鼠对阿霉素的敏感性, 具有 增敏增效作用。 实施例 6 GA-Me和 GA- T抑制裸鼠体内多药耐药性实体瘤的实验  The above examples demonstrate that GA-Me and GA-T can increase the sensitivity of tumor-bearing nude mice to doxorubicin, and have a sensitizing and synergistic effect. Example 6 Inhibition of multidrug resistance solid tumors in nude mice by GA-Me and GA-T
采用裸鼠进行实验。 无菌条件下取对数生长期的 KB-A-1细胞, 制备成约 2.5 X lOVmL细胞悬液, 于两只裸鼠上肢下侧皮下接种 0.2mL/鼠, 生长 15天后, 处死 动物, 无菌条件下剖取小鼠的实体瘤, 剪开, 除去瘤中心的死细胞, 剪碎外表活组 织至 0.5mm大小, 用 PBS清洗。 于实验裸鼠上肢下侧皮下接种, 生长 3天后, 待 长出米粒大小, 可触摸到的硬实体瘤后, 按实验设计方案给药, 5周后处死各组动 物, 剖取各组小鼠的实体瘤, 测每鼠实体瘤的重量, 按下列公式计算肿瘤抑制率: 肿瘤抑制率(%) =[ (对照组实体瘤平均重量数一给药组实体瘤平均重量数) I对照组实体瘤平均重量数 ] X 100  Experiments were performed using nude mice. KB-A-1 cells in logarithmic growth phase were prepared under aseptic conditions, and about 2.5 X lOVmL cell suspension was prepared. 0.2 mL/mouse was inoculated subcutaneously in the lower limbs of two nude mice. After 15 days of growth, the animals were sacrificed. The solid tumor of the mouse was dissected under the conditions of the bacteria, cut open, the dead cells at the center of the tumor were removed, and the living tissue was cut to a size of 0.5 mm, and washed with PBS. The nude mice were inoculated subcutaneously in the lower limbs of the experimental nude mice. After 3 days of growth, the size of the rice grains was allowed to grow, and the hard solid tumors that could be touched were administered according to the experimental design. After 5 weeks, the animals in each group were sacrificed, and the mice in each group were dissected. For solid tumors, the weight of each solid tumor was measured. The tumor inhibition rate was calculated according to the following formula: Tumor inhibition rate (%) = [(Average weight of control solid tumors - Average weight of solid tumors in the administration group) I Control entity Average tumor weight] X 100
结果见附图图 6, 在图 6中:  The results are shown in Figure 6, in Figure 6:
灵芝酸 T (25mg/kg) 组;  Ganoderma acid T (25mg/kg) group;
灵芝酸 Me (25mg/kg) 组。  Ganoderma lucidum Me (25 mg/kg) group.
以上实施例说明, GA-Me和 GA-T能抑制裸鼠体内多药耐药性实体瘤的生长, 具有抑制多药耐药性肿瘤增殖的活性。 实施例 7  The above examples demonstrate that GA-Me and GA-T inhibit the growth of multidrug resistant solid tumors in nude mice and have the activity of inhibiting the proliferation of multidrug resistant tumors. Example 7
将 1重量份的 GA-Me或 GA- T与 199份蒸馏水混合均匀,分装,即获得口服液。 P T/CN2006/001171 实施例 8 1 part by weight of GA-Me or GA-T was uniformly mixed with 199 parts of distilled water, and the mixture was dispensed to obtain an oral solution. PT/CN2006/001171 Example 8
将 1重量份的 GA- Me或 GA- T与 199重量份的注射用生理盐水混合均勾, 即获 得注射液。 实施例 9  One part by weight of GA-Me or GA-T was mixed with 199 parts by weight of physiological saline for injection to obtain an injection. Example 9
采用噻唑蓝 (MTT) 快速比色法测定 GA- Me和 GA-T对 KB, HeLa和 95-D肿瘤 细胞以及 HLF人正常细胞的毒性作用。 将对数生长期的各种细胞 (
Figure imgf000010_0001
The toxic effects of GA-Me and GA-T on KB, HeLa and 95-D tumor cells and HLF human normal cells were determined by MTT rapid colorimetry. Will be a variety of cells in the logarithmic growth phase (
Figure imgf000010_0001
接种于 96孔培养板,每孔 0. 2ml,分别加入 50, 100, 150和 200 μ§ ml—1浓度的 GA-Me 和 GA- T处理, 每浓度平行 4孔, 对照组加等量体积的培养液, 置 37°C, 5%∞2及 饱和湿度的培养箱培养 1-4天, 实验终止前 4小时每孔加入 δπ^πιΓ^ΜΤΤ ΙΟμΙ , 培 养结束后每孔加入 0. 04 Ν二甲基亚砜 (DMS0), 每孔 150μ1, 振荡 10 min, 待 MTT 还原产物完全溶解, 用 BioRad 550型酶标仪, 以 550 nm为实验波长, 655 nm 为 参照波长测定其吸收度, 计算药物对细胞的抑制率, 并以细胞的半数抑制浓度 (IC50) 作图。 统计实验数据的结果如图 Ί所示。 Inoculate in a 96-well culture plate, 0.2 ml per well, and add 50, 100, 150, and 200 μ § ml- 1 concentrations of GA-Me and GA-T, respectively. Each concentration is parallel to 4 wells, and the control group is added with an equal volume. The culture medium was placed in an incubator at 37 ° C, 5% ∞ 2 and saturated humidity for 1-4 days, and δπ^πιΓ^ΜΤΤ ΙΟμΙ was added to each well 4 hours before the end of the experiment, and 0. 04 每 was added to each well after the end of the culture. Dimethyl sulfoxide (DMS0), 150 μl per well, shaking for 10 min, until the MTT reduction product was completely dissolved, and the absorbance was measured using a BioRad 550 microplate reader with 550 nm as the experimental wavelength and 655 nm as the reference wavelength. Drug inhibition rate of the cell, and a cell half maximal inhibitory concentration (IC 50) plotted. The results of statistical experimental data are shown in Figure 。.
以上实施例说明, GA-Me和 GA-T均具有良好杀灭 KB-A-1, HeLa和 95-D肿 瘤细胞株的作用, 而对人体正常细胞株的毒性较小。 实施例 10 GA-Me和 GA-T对 HeLa细胞的毒性作用  The above examples demonstrate that both GA-Me and GA-T have a good killing effect on KB-A-1, HeLa and 95-D tumor cell lines, but less on normal human cell lines. Example 10 Toxicity of GA-Me and GA-T to HeLa cells
GA-Me和 GA-T均为从发酵生产的灵芝菌丝体中分离得到的纯天然产物, 纯度 大于 99%,用 DMEM培养液稀释成所需浓度。采用噻唑蓝 (MTT)快速比色法测定 GA - Me 和 GA-T对 HeLa细胞的毒性作用。 将对数生长期细胞 (106cell. ml- 1 ) 接种于 96 孔培养板, 每孔 0. 2ml, 分别加入一定浓度的 GA- Me和 GA- T处理, 每浓度平行 4 孔, 对照组加等量体积的培养液, 置 37°C, 5% C02及饱和湿度的培养箱培养 1 - 4 天, 实验终止前 4小时每孔加入 5mg. ml-1 MTT ΙΟμΙ, 培养结束后每孔加入 0. 04 Ν 二甲基亚砜 (DMS0), 每孔 150 μ 1, 振荡 10 min, 待 MTT还原产物完全溶解, 用 BioRad 550型酶标仪, 以 550 nm为实验波长, 655 nm为参照波长测定其吸收度, 计算肿瘤细胞的抑制率和药物对细胞的抑制率,并以药物的不同浓度和细胞的抑制 率作图, 确定细胞的半数抑制浓度 (IC50)。 实验结果如下图 8所示。  Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM medium. The toxic effects of GA - Me and GA-T on HeLa cells were determined by MTT rapid colorimetry. The logarithmic growth phase cells (106 cells. ml-1) were inoculated into 96-well culture plates at 0.2 ml per well, and treated with a certain concentration of GA-Me and GA-T, respectively, each concentration was parallel 4 wells, the control group was added, etc. The volume of the culture solution was incubated at 37 ° C, 5% C02 and saturated humidity in an incubator for 1-4 days. 5 mg. ml-1 MTT ΙΟμΙ was added to each well 4 hours before the end of the experiment, and 0 was added to each well after the completion of the culture. 04 Ν Dimethyl sulfoxide (DMS0), 150 μl per well, shaking for 10 min, until the MTT reduction product is completely dissolved, using BioRad 550 microplate reader, 550 nm as the experimental wavelength, 655 nm as the reference wavelength The absorbance, the inhibition rate of the tumor cells and the inhibition rate of the drug to the cells were calculated, and the half-inhibitory concentration (IC50) of the cells was determined by plotting the different concentrations of the drug and the inhibition rate of the cells. The experimental results are shown in Figure 8 below.
以上实施例说明, GA- Me和 GA- T均具有良好的杀灭 HeLa肿瘤细胞的作用, 而 且这种抑制效果还具有剂量依赖性。 实施例 11 GA-Me和 GA- T对 95-D细胞的毒性作用 The above examples show that both GA-Me and GA-T have a good effect of killing HeLa tumor cells, and this inhibitory effect is also dose-dependent. Example 11 Toxicity of GA-Me and GA-T to 95-D cells
GA-Me和 GA- T均为从发酵生产的灵芝菌丝体中分离得到的纯天然产物, 纯度 大于 99%, 用 RPMI-1640培养液稀释成所需浓度。 采用噻唑蓝 (MTT) 快速比色法 测定 GA- Me和 GA- T对 95-D细胞的毒性作用。 将对数生长期细胞 (106cell. ml-1 ) 接种于 96孔培养板, 每孔 0. 2ml, 分别加入一定浓度的 GA- Me和 GA-T处理, 每浓 度平行 4孔, 对照组加等量体积的培养液, 置 37°C, 5% C02及饱和湿度的培养箱 培养 1-4天, 实验终止前 4小时每孔加入 5mg. ml-1 MTT ΙΟμΙ, 培养结束后每孔加 入 0. 04 N DMS0, 每孔 150μ1, 振荡 10 min, 待 MTT还原产物完全溶解, 用 BioRad 550型酶标仪, 以 550 nm为实验波长, 655 nm为参照波长测定其吸收度, 计算肿 瘤细胞的抑制率和药物对细胞的抑制率, 并以药物的不同浓度和细胞的抑制率作 图, 确定 IC50。 实验结果见图 9。  Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with RPMI-1640 medium. The toxic effects of GA-Me and GA-T on 95-D cells were determined by MTT rapid colorimetry. The logarithmic growth phase cells (106 cells. ml-1) were inoculated into a 96-well culture plate at 0. 2 ml per well, and a certain concentration of GA-Me and GA-T were added respectively. Each concentration was parallelized with 4 wells, and the control group was added. The volume of the culture medium was cultured in an incubator at 37 ° C, 5% CO 2 and saturated humidity for 1-4 days. 5 mg. ml-1 MTT ΙΟμΙ was added to each well 4 hours before the end of the experiment, and 0 was added to each well after the completion of the culture. 04 N DMS0, 150μ1 per well, shaking for 10 min, until the MTT reduction product is completely dissolved, the absorbance is determined by the BioRad 550 microplate reader with 550 nm as the experimental wavelength and 655 nm as the reference wavelength, and the inhibition rate of the tumor cells is calculated. And the inhibition rate of the drug to the cells, and the IC50 is determined by plotting the different concentrations of the drug and the inhibition rate of the cells. The experimental results are shown in Figure 9.
以上实施例说明, GA-Me和 GA-T均具有良好的杀灭 95- D肿瘤细胞的作用, 而 且这种抑制效果具有剂量依赖性。 实施例 12 GA-Me和 GA- T对几种肿瘤细胞和正常细胞 L02的细胞毒性比较 The above examples show that both GA-Me and GA-T have a good effect of killing 95-D tumor cells, and this inhibitory effect is dose-dependent. Example 12 Comparison of cytotoxicity of GA-Me and GA-T against several tumor cells and normal cells L02
GA-Me和 GA-T均为从发酵生产的灵芝菌丝体中分离得到的纯天然产物, 纯度 大于 99%, 用 DMEM或 RPMI- 1640培养液稀释成所需浓度。 采用噻唑蓝 (MTT) 快速 比色法测定 GA-Me 和 GA-T 对几种细胞的毒性作用。 将对数生长期细胞 ( 106cell. ml-l ) 接种于 96孔培养板, 每孔 0. 2ml, 分别加入一定浓度的 GA - Me 和 GA- T处理, 每浓度平行 4孔, 对照组加等量体积的培养液, 置 37°C, 5% C02 及饱和湿度的培养箱培养 1-4天,实验终止前 4小时每孔加入 5mg. ral-1 MTT ΙΟμΙ , 培养结束后每孔加入 0. 04 Ν DMSO (二甲基亚砜), 每孔 150 y l, 振荡 10 rain, 待 MTT还原产物完全溶解, 用 BioRad 550型酶标仪, 以 550 nm为实验波长, 655 nm 为参照波长测定其吸收度, 计算肿瘤细胞的抑制率和药物对细胞的抑制率, 确定 IC50并作图, 结果见图 10。 Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, and diluted to the desired concentration with DMEM or RPMI-1640 medium. The toxic effects of GA-Me and GA-T on several cells were determined by MTT rapid colorimetry. The logarithmic growth phase cells (106cell.ml-l) were inoculated into 96-well culture plates at 0.2 ml per well, and treated with a certain concentration of GA-Me and GA-T, each concentration was parallel 4 wells, the control group was added, etc. The volume of the culture medium was cultured in an incubator at 37 ° C, 5% CO 2 and saturated humidity for 1-4 days. 5 mg. ral-1 MTT ΙΟμΙ was added to each well 4 hours before the end of the experiment, and 0 was added to each well after the completion of the culture. 04 DMSO DMSO (dimethyl sulfoxide), 150 yl per well, shaking for 10 min, until the MTT reduction product is completely dissolved, using BioRad 550 microplate reader, 550 nm as the experimental wavelength, 655 nm as the reference wavelength to determine its absorption Degree, calculate the inhibition rate of tumor cells and the inhibition rate of the drug on the cells, determine the IC50 and map, the results are shown in Figure 10.
以上实施例说明, GA- Me和 GA- T对人类肿瘤细胞的细胞毒性较大, 对正常细 胞的细胞毒性较弱, 即 GA-Me和 GA- T对某些人类肿瘤细胞具有一定的靶向抑制作 用。 实施例 13 GA-Me和 GA- T抑制裸鼠体内肿瘤生长活性的比较  The above examples demonstrate that GA-Me and GA-T are more cytotoxic to human tumor cells and less cytotoxic to normal cells, ie, GA-Me and GA-T have certain targeting to certain human tumor cells. Inhibition. Example 13 Comparison of GA-Me and GA-T inhibition of tumor growth activity in nude mice
GA-Me和 GA-T均为从发酵生产的灵芝菌丝体中分离得到的纯天然产物, 纯度 大于 99%, 用 PBS配制并稀释成所需浓度。 雄性裸鼠两只, 前肢腋下注射 95-D细 胞(1x106个 /ml ), 喂养四周后处死, 剥离裸鼠的实体瘤, 剪碎并用 PBS (7. 4)研 磨, 80 目筛网过滤。 配成细胞悬浮液备用。 雄性裸鼠按体重随机分组, 前肢腋下 注射 95- D细胞悬浮液 (1x106个 /ml ), 喂养一周, 待实体瘤长出后, 不同的药物 腹腔隔日注射, 阴性对照为生理盐水 (PBS)。 以抗癌药物为阳性对照, 注射 2周后 停药, 再伺养 10天, 处死, 剥离裸鼠的实体瘤称重。 与对照比较计算抑制率。 药 物的不同浓度对肿瘤的抑制率的影响的实验结果如图 11所示。 Both GA-Me and GA-T are pure natural products isolated from the fermented mycelium of Ganoderma lucidum, with a purity greater than 99%, formulated in PBS and diluted to the desired concentration. Two male nude mice, forefoot subcutaneous injection 95-D fine Cells (1 x 106 cells/ml) were sacrificed after four weeks of feeding. Solid tumors of nude mice were stripped, cut and incubated with PBS (7.4), and filtered through an 80 mesh screen. Formulated as a cell suspension for use. Male nude mice were randomly divided into groups according to their body weight. The 95-D cell suspension (1x106 cells/ml) was injected into the forelimbs for one week. After the solid tumors were grown, different drugs were injected intraperitoneally every other day. The negative control was normal saline (PBS). . The anticancer drug was used as a positive control. After 2 weeks of injection, the drug was stopped, and then it was sacrificed for 10 days. The patient was sacrificed and the solid tumor of the nude mice was weighed. The inhibition rate was calculated in comparison with the control. The experimental results of the effects of different concentrations of the drug on the inhibition rate of the tumor are shown in FIG.
以上实施例说明, GA- Me和 GA-T在体内也能抑制肿瘤的生长, 同时也具有剂 量依赖性。 实施例 14灵芝酸诱导 95- D肿瘤细胞凋亡的分子途径研究  The above examples demonstrate that GA-Me and GA-T also inhibit tumor growth in vivo and are also dose dependent. Example 14 Molecular Pathway of Ganoderma Acid-induced Apoptosis of 95-D Tumor Cells
材料和方法  Materials and Method
细胞周期的检测  Cell cycle detection
对数生长期细胞经 25, 5(^g/mL两种浓度的药物处理特定的时间后, 各种处 理过的细胞经胰酶消化, 离心收集细胞(1500 rpmxlO min) , PBS 洗两遍, 以 PBS 调整细胞浓度在 106个 /mL,加入 RNASE,终浓度为 100U/mL, 37Gc保温 30min, 加 入 PI, 终浓度为 50 g/mL,在暗中常温下反应 30min, 然后 300目滤膜过滤,进行流 式细胞仪分析凋亡和细胞周期, 每次记录 10000个细胞。 After the logarithmic growth phase cells were treated with 25, 5 (^g/mL two concentrations of the drug for a specific period of time, the various treated cells were trypsinized, centrifuged to collect the cells (1500 rpm x 10 min), and washed twice with PBS. The cell concentration was adjusted to 10 6 /mL with PBS, RNASE was added, the final concentration was 100 U/mL, 37 G c was kept for 30 min, PI was added, the final concentration was 50 g/mL, and the reaction was carried out in the dark at room temperature for 30 min, then 300 mesh filtration. Membrane filtration, flow cytometry analysis of apoptosis and cell cycle, recording 10,000 cells each time.
凋亡的检测  Detection of apoptosis
细胞经过 25, 50 g/mL两种浓度的灵芝酸 T刺激后, PBS洗涤两遍, 加胰酶 消化, 血清中止, 离心收集细胞 (1500 rpm, 10 min) ,以 Binding液 (见 3.3 ) 制 成单细胞悬浮液, 细胞浓度在 2xl05个 /mL。 加入 AiuiexinV-FITC液, 终浓度为 10 ng/mL,室温暗中孵育 15分钟, 然后以 PI染色, 终浓度为 5 g/mL.用流式细胞仪分 析。 The cells were stimulated with Ganoderma lucidum T at 25, 50 g/mL, washed twice with PBS, trypsinized, serum stopped, and centrifuged to collect cells (1500 rpm, 10 min) with Binding solution (see 3.3). Into a single cell suspension, the cell concentration was 2x10 5 / mL. Aiuiexin V-FITC solution was added to a final concentration of 10 ng/mL, incubated for 15 minutes at room temperature in the dark, and then stained with PI to a final concentration of 5 g/mL. Flow cytometry was used for analysis.
结果和分析  Results and analysis
1)灵芝酸抑制高转移肺癌细胞的增殖是通过诱导凋亡来实现的  1) Ganoderma acid inhibits the proliferation of highly metastatic lung cancer cells by inducing apoptosis.
MTT法和克隆形成实验。 发现灵芝酸 T对高转移肺癌细胞有较强的细胞毒性。 MTT 检测方法虽然可以看出药物对细胞增殖的影响, 但它不能提供更进一步的解 释。即无法确定影响细胞增殖是通过抑制细胞数量的增加还是促进细胞的死亡来实 现的。 为了解释抑制增殖的原因, 进行了细胞凋亡检测。 结果发现, 灵芝酸 T处理 的癌细胞出现了凋亡现象, 而且凋亡程度随着灵芝酸浓度的增加而增加。 在 50 μ§ ΐ的浓度下作用 8小时, 导致了超过 40%的细胞凋亡。 细胞凋亡最主要的特 征是 DNA的片断化, 随着凋亡的增加, 以 180bp为单位的 DNA片断逐渐增加, 在琼 脂糖凝胶上表现为梯状电泳条带。 经过灵芝酸处理的癌细胞, 也表现出明显的 DNA 梯状条带。 MTT assay and colony formation experiments. It was found that Ganoderma lucidum T has strong cytotoxicity against high metastatic lung cancer cells. Although the MTT assay can show the effect of drugs on cell proliferation, it does not provide further explanation. That is, it is impossible to determine whether affecting cell proliferation is achieved by inhibiting an increase in the number of cells or promoting cell death. To explain the reason for inhibiting proliferation, apoptosis detection was performed. It was found that the cancer cells treated with Ganoderma lucidum T showed apoptosis, and the degree of apoptosis increased with the increase of the concentration of ganoderic acid. Acting for 8 hours at a concentration of 50 μ § , resulted in more than 40% apoptosis. The most important feature of apoptosis is the fragmentation of DNA. As the apoptosis increases, the DNA fragment in increments of 180 bp gradually increases. The lipogel gel appears as a ladder-like electrophoresis band. Cancer cells treated with ganoderic acid also showed significant DNA ladders.
表 1 灵芝酸限制 95- D癌细胞的细胞周期  Table 1 Ganoderma acid limit cell cycle of 95-D cancer cells
G2-M G 2 -M
细胞系 浓度 ^g/mL) GQ-GJ (%) S (%)  Cell line concentration ^g/mL) GQ-GJ (%) S (%)
(%)  (%)
0 26. 59 25. 53 47. 88 0 26. 59 25. 53 47. 88
95-D 25 45 21. 51 33. 48 95-D 25 45 21. 51 33. 48
50 60. 67 16. 99 22. 35 体外癌细胞经过灵芝酸 T处理后, 引起凋亡的同时, 还限制了细胞生长周期在 G1期, 在同一时间内, 灵芝酸浓度越高, 累积的 G1期细胞越多。  50 60. 67 16. 99 22. 35 In vitro cancer cells treated with ganoderic acid T cause apoptosis and also limit the cell growth cycle in G1 phase. At the same time, the higher the concentration of ganoderic acid, the cumulative G1 The more cells there are.
以上结果表明, 灵芝酸能诱导癌细胞的凋亡和限制细胞周期, 而且具有浓度依 赖性。由此可以推测灵芝酸主要是通过诱导癌细胞的凋亡和限制细胞周期来表现它 的细胞毒性的。  These results indicate that ganoderic acid can induce apoptosis and limit cell cycle in cancer cells, and is concentration dependent. It can be speculated that ganoderic acid mainly expresses its cytotoxicity by inducing apoptosis of cancer cells and limiting cell cycle.
2) 灵芝酸 Τ影响癌细胞线粒体的功能  2) Ganoderma lucidum Τ affects the function of cancer cell mitochondria
凋亡的发生主要有两条途径, 线粒体途径一般伴随着细胞色素 C的泄漏。正常 情况下,它存在于线粒体内膜和外膜之间的腔中,凋亡信号刺激使其从线粒体释放 至细胞液, 结合 Apaf-1 ( apoptotic protease activating factor-1 ) 后启动 caspase 级联反应: 细胞色素 C/Apaf- 1 复合物激活 caspase_9, 后者再激活 caspase-3和其它下游 caspase。 经过灵芝酸处理的癌细胞细胞质中发现细胞色素 C含量的增加, 如图 4. 2所示。 经过 4小时处理后, 细胞色素 C的含量比对照增加 了 2倍, 而 8小时处理后, 含量增加了 3倍。 细胞色素 C作为一种信号物质, 在 细胞凋亡中发挥着重要的作用。灵芝酸处理很显著的刺激了细胞色素 C的泄漏。因 而可以推测, 灵芝酸是对线粒体产生了直接或间接的影响。  There are two main pathways for apoptosis, and the mitochondrial pathway is generally accompanied by the leakage of cytochrome c. Normally, it exists in the lumen between the mitochondrial inner membrane and the outer membrane. The apoptotic signal stimulates it to release from the mitochondria to the cytosol, and initiates the caspase cascade after binding with apaf-1 (apoptotic protease activating factor-1). : The cytochrome C/Apaf-1 complex activates caspase_9, which in turn activates caspase-3 and other downstream caspase. The increase in cytochrome C content was observed in the cytoplasm of cancer cells treated with ganoderic acid, as shown in Figure 4.2. After 4 hours of treatment, the cytochrome C content was increased by a factor of 2 compared to the control, and after 8 hours of treatment, the content was increased by a factor of 3. As a signal substance, cytochrome C plays an important role in apoptosis. Ganoderma acid treatment significantly stimulated the leakage of cytochrome c. Therefore, it can be speculated that ganoderic acid has a direct or indirect effect on mitochondria.
诱导肿瘤细胞凋亡与线粒体的功能状态有关。如改变线粒体膜完整性。利用流 式细胞仪检测灵芝酸对癌细胞的线粒体内膜转膜电位影响,结果如图 4. 3所示。线 粒体内的荧光强度下降随着处理时间的增加而降低, 8小时的处理后, 荧光强度大 约降低 20 %。 结果提示灵芝酸影响了癌细胞的转膜电位, 使转膜电位下降, 从而 导致细胞色素 C的泄漏。  Induction of tumor cell apoptosis is associated with functional status of mitochondria. Such as changing the integrity of mitochondrial membranes. Flow cytometry was used to detect the effect of ganoderic acid on the mitochondrial inner membrane transmembrane potential of cancer cells. The results are shown in Figure 4.3. The decrease in fluorescence intensity in the mitochondria decreased with increasing processing time, and after 8 hours of treatment, the fluorescence intensity decreased by approximately 20%. The results suggest that ganoderic acid affects the transmembrane potential of cancer cells, causing the membrane potential to decrease, leading to leakage of cytochrome c.
3) 灵芝酸 T刺激活性氧的产生  3) Ganoderma acid T stimulates the production of reactive oxygen species
经过灵芝酸处理的癌细胞, 细胞内活性氧自由基 (R0S ) 的含量随着处理时间 的延长而有所增加。 经过 8小时的处理, 比对照增加了 40%。 自由基的增加有多 种可能的方式, 首先, 由于灵芝酸影响了线粒体的膜电位, 导致线粒体内部的自由 基泄漏到细胞质中, 引起胞内自由基的增加,这种情况的自由基的增加是由于凋亡 所引起的结果。其次, 灵芝酸可能干扰了细胞内的氧化还原平衡, 导致自由基清除 机制的失调, 从而使自由基积累, 进一步引起凋亡。这种情况的自由基的增加是产 生凋亡的原因。 The amount of intracellular reactive oxygen species (R0S) in cancer cells treated with ganoderic acid varies with treatment time The extension has increased. After 8 hours of treatment, it increased by 40% compared to the control. There are many possible ways to increase free radicals. First, because ganoderic acid affects the membrane potential of mitochondria, free radicals inside the mitochondria leak into the cytoplasm, causing an increase in intracellular free radicals. It is the result of apoptosis. Secondly, ganoderic acid may interfere with the redox balance in the cell, leading to the dysregulation of the free radical scavenging mechanism, thereby accumulating free radicals and further causing apoptosis. The increase in free radicals in this case is the cause of apoptosis.
4) 灵芝酸影响 Caspase-3的活性, 但对 Caspase- 8的活性没有影响  4) Ganoderma acid affects the activity of Caspase-3, but has no effect on the activity of Caspase-8
灵芝酸处理癌细胞导致细胞色素 C的泄漏, 后者再激活 caspase- 3和其它下 游 caspaSe。 使凋亡进程得以进行下去。 通过检测处理细胞的 Caspase- 3发现, 随 着处理时间的增加, caspase- 3活性也显著增加。 8小时处理后, 活性增加近三倍。 Caspase-3的激活在凋亡的两条途径中都有发生。即 FAS途径的 caspase- 8可以经 过 BID 蛋白与线粒体途径发生联系。 因此, 我们检测了 caspase- 8的活性, 以确 定是否有 FAS途径的加入导致 caspase- 3的激活。结果如图所示,经过灵芝酸处理 后, 在 8小时内 caspase- 8的活性没有发生变化。 由此可以排除 FAS途径的影响, 可以推断 caspase- 3的激活是由于细胞色素 C的复合物所导致的。 Ganoderma acid treatment of cancer cells causes leakage of cytochrome C, which in turn activates caspase-3 and other downstream caspa Se . The process of apoptosis is allowed to proceed. By detecting the Caspase-3 of the treated cells, caspase-3 activity was also significantly increased as the treatment time increased. After 8 hours of treatment, the activity increased nearly threefold. Activation of Caspase-3 occurs in both pathways of apoptosis. That is, the caspase-8 of the FAS pathway can be linked to the mitochondrial pathway via the BID protein. Therefore, we tested the activity of caspase-8 to determine if the addition of the FAS pathway resulted in the activation of caspase-3. The results are shown in the figure. After treatment with ganoderic acid, the activity of caspase-8 did not change within 8 hours. Thus, the influence of the FAS pathway can be ruled out, and it can be inferred that the activation of caspase-3 is caused by the complex of cytochrome C.
为了进一步验证癌细胞内灵芝酸在诱导凋亡过程中对 caspase酶活性的影响, 使用了专一性的 caspase抑制剂处理。结果表明, Caspase 3抑制剂或 pan- caspase 抑制剂能明显减少由灵芝酸引起的细胞凋亡, 而 caspase 8 抑制剂不能抑制药物 诱导的细胞凋亡。这些数据进一步验证了灵芝酸是通过激活 caspase 3的活性而不 是激活 caspase 8活性的方式诱导癌细胞凋亡。 因为 caspase8是外部途径的关键 酶, 这表明外部途径并没有参与到灵芝酸诱导的凋亡途径中。 因此, 灵芝酸可能通 过细胞激活了与线粒体相关的下游凋亡事件, 包括激活 caspaSe3的活性, 并通过 激活 CaSpaSe3的线粒体途径诱导癌细胞凋亡。 In order to further verify the effect of ganoderic acid on the activity of caspase in the induction of apoptosis in cancer cells, a specific caspase inhibitor treatment was used. The results showed that Caspase 3 inhibitors or pan- caspase inhibitors significantly reduced apoptosis induced by ganoderic acid, whereas caspase 8 inhibitors did not inhibit drug-induced apoptosis. These data further demonstrate that ganoderic acid induces apoptosis in cancer cells by activating caspase 3 activity rather than activating caspase 8 activity. Because caspase8 is a key enzyme in the external pathway, this suggests that the external pathway is not involved in the ganic acid-induced apoptosis pathway. Therefore, ganoderic acid may activate downstream mitochondrial-related apoptotic events through cells, including activation of casp aS e3, and induction of apoptosis in cancer cells by activating the mitochondrial pathway of CaS pa S e3.
5) 灵芝酸 T影响凋亡相关蛋白质的表达  5) Ganoderma acid T affects the expression of apoptosis-related proteins
细胞内凋亡的发生伴随着凋亡相关蛋白的表达。 调控细胞凋亡的关键因子为 The occurrence of intracellular apoptosis is accompanied by the expression of apoptosis-related proteins. The key factor regulating apoptosis is
Bel— 2家族, 该家族包括凋亡促进因子 Bax、 Bak、 Bid以及凋亡抑制因子 Bel— 2 和 Bel— x。 这些促进因子和抑制因子维护着平衡状态, 一旦平衡被打破将引起凋 亡的发生和抑制。 另外一个凋亡的重要影响蛋白是 P53. DNA损伤时, 释放出更多 的 P53来进行基因组 DNA稳定性的维护工作; 亦可通过介导 P53单泛素化而引导 P53核外输出, 定位于线粒体, 直接激活 Bax, 启动线粒体非转录依赖性细胞凋亡。 当 DNA损伤已修复, 活性 P53迅速下调以恢复细胞正常稳态时。 The Bel-2 family, which includes the apoptosis-promoting factors Bax, Bak, Bid, and the apoptosis inhibitors Bel-2 and Bel-x. These promoting factors and inhibitors maintain an equilibrium state, and once the balance is broken, it will cause the occurrence and suppression of apoptosis. Another important protein that affects apoptosis is P53. When DNA damage occurs, more P53 is released for maintenance of genomic DNA stability. It can also guide P53 extranuclear output by mediating P53 monoubiquitination. Mitochondria, which directly activate Bax, initiate mitochondrial non-transcription-dependent apoptosis. When DNA damage has been repaired, active P53 is rapidly downregulated to restore normal cell homeostasis.
灵芝酸对相关蛋白表达的影响结果见图 4. 8所示。 首先灵芝酸处理后, 细胞内 P53的表达量增加。在我们的试验中, 已经发现灵芝酸可以诱导 DNA的片断化, P53 的表达增加可能主要是由 DNA的损伤引起的。 在 Bel— 2家族的蛋白中, BCL- 2的 表达量未见明显的变化,而 BAX的表达量却有所增加。因而 BCL-2/BAX的比值下降。 与在大部分的抗癌研究的结果一致,但是有关灵芝酸单体对这一比值的影响还是第 一次发现。 尽管本发明描述了具体的例子, 但是有一点对于本领域技术人员来说是明显 的,即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。因此, 所附权利要求覆盖了所有这些在本发明范围内的变动。本文引用的所有出版物、专 利和专利申请均纳入本文作参考。 The results of the effect of ganoderic acid on the expression of related proteins are shown in Figure 4. 8. First, after treatment with ganoderic acid, intracellular The expression level of P53 is increased. In our experiments, it has been found that ganoderic acid can induce DNA fragmentation, and the increased expression of P53 may be mainly caused by DNA damage. In the Bel-2 family of proteins, there was no significant change in the expression of BCL-2, but the expression of BAX increased. Thus the ratio of BCL-2/BAX decreases. Consistent with the results of most anti-cancer studies, but the effect of ganoderic acid monomer on this ratio was first discovered. While the invention has been described with respect to the specific embodiments, it will be apparent to those skilled in the art Therefore, the appended claims are intended to cover all such modifications within the scope of the invention. All publications, patents and patent applications cited herein are hereby incorporated by reference.

Claims

权利要求 Rights request
1. 一种药物组合物, 它包括治疗有效量的灵芝酸 Me和 /或灵芝酸 T和医药学 上可接受的载体。 A pharmaceutical composition comprising a therapeutically effective amount of Ganoderma lucidum Me and/or Ganoderma lucidum T and a pharmaceutically acceptable carrier.
2. 如权利要求 1所述的药物组合物, 其中它还包含化疗药。  2. The pharmaceutical composition according to claim 1, wherein it further comprises a chemotherapeutic agent.
3. 如权利要求 1所述的药物组合物, 其中以组合物的重量计, 灵芝酸 Me或 灵芝酸 T的重量含量为 0. 1-99. 9%。  9%。 9%. 9%. 9%. 9%. 9%.
4. 灵芝酸 Me在制备肿瘤生长和增殖的抑制剂、 多药耐药性癌症化疗增敏增 效剂和抑制剂中的应用。  4. Ganoderma lucidum Me is used in the preparation of inhibitors of tumor growth and proliferation, multidrug resistant cancer chemotherapy sensitizers and inhibitors.
5. 灵芝酸 T在制备肿瘤生长和增殖的抑制剂、 多药耐药性癌症化疗增敏增效 剂和抑制剂中的应用。  5. The application of ganoderic acid T in the preparation of inhibitors of tumor growth and proliferation, multidrug resistant cancer chemotherapy sensitizers and inhibitors.
6. 如权利要求 4或 5所述的应用, 其中所述肿瘤选自恶性黑色素瘤, 恶性淋 巴瘤, 消化器官的肿瘤 (胃癌, 肠癌, 肝癌, 胆囊癌, 胆道癌, 胰腺癌) , 肺癌, 乳癌, 睾丸癌, 卵巢癌, 子宫癌, 前列腺癌, 上颚癌, 舌癌, 口腔癌, 咽喉癌, 甲 状腺癌, 脑部肿瘤, 各类肉瘤, 骨肉瘤, 白血病, 神经系统肿瘤, 膀胱肿瘤, 皮肤 癌, 皮肤附属器官癌和皮肤转移癌。  6. The use according to claim 4 or 5, wherein the tumor is selected from the group consisting of malignant melanoma, malignant lymphoma, tumor of the digestive organ (gastric cancer, intestinal cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer), lung cancer , breast cancer, testicular cancer, ovarian cancer, uterine cancer, prostate cancer, upper jaw cancer, tongue cancer, oral cancer, throat cancer, thyroid cancer, brain tumor, various sarcoma, osteosarcoma, leukemia, nervous system tumor, bladder tumor, Skin cancer, skin accessory organ cancer and skin metastases.
7. 一种抑制对象体内肿瘤增殖或生长的方法, 该方法包括将治疗有效量的权 利要求 1所述的组合物给予需要该治疗的对象。  A method of inhibiting tumor proliferation or growth in a subject, the method comprising administering a therapeutically effective amount of the composition of claim 1 to a subject in need of such treatment.
8. 如权利要求 7所述的方法,其中所述肿瘤选自恶性黑色素瘤,恶性淋巴瘤, 消化器官的肿瘤 (胃癌, 肠癌, 肝癌, 胆囊癌, 胆道癌, 胰腺癌) , 肺癌, 乳癌, 睾丸癌, 卵巢癌, 子宫癌, 前列腺癌, 上颚癌, 舌癌, 口腔癌, 咽喉癌, 甲状腺癌, 脑部肿瘤, 各类肉瘤, 骨肉瘤, 白血病, 神经系统肿瘤, 膀胱肿瘤, 皮肤癌, 皮肤 附属器官癌和皮肤转移癌。  8. The method according to claim 7, wherein the tumor is selected from the group consisting of malignant melanoma, malignant lymphoma, tumor of the digestive organ (gastric cancer, intestinal cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer), lung cancer, breast cancer , testicular cancer, ovarian cancer, uterine cancer, prostate cancer, upper jaw cancer, tongue cancer, oral cancer, throat cancer, thyroid cancer, brain tumor, various sarcoma, osteosarcoma, leukemia, nervous system tumor, bladder tumor, skin cancer , skin accessory organ cancer and skin metastases.
9. 如权利要求 7所述的方法, 其中将治疗有效量的权利要求 1所述的组合 物联合化疗药一同给予需要该治疗的对象。  9. The method of claim 7, wherein a therapeutically effective amount of the composition of claim 1 in combination with a chemotherapeutic agent is administered to a subject in need of such treatment.
10. —种增强对象体内肿瘤对已产生耐药性的化疗药物的敏感性的方法, 该 方法包括将有效量的权利要求 1所述的组合物给予需要所述对象。  10. A method of enhancing the sensitivity of a tumor in a subject to a chemotherapeutic drug that has developed resistance, the method comprising administering an effective amount of the composition of claim 1 to the subject in need thereof.
PCT/CN2006/001171 2005-06-01 2006-05-31 Use of ganoderic acid in treating tumour WO2006128378A1 (en)

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