WO2013170637A1 - Application of paeoniflorin compound in preparation of antitumor drug - Google Patents

Application of paeoniflorin compound in preparation of antitumor drug Download PDF

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WO2013170637A1
WO2013170637A1 PCT/CN2013/000683 CN2013000683W WO2013170637A1 WO 2013170637 A1 WO2013170637 A1 WO 2013170637A1 CN 2013000683 W CN2013000683 W CN 2013000683W WO 2013170637 A1 WO2013170637 A1 WO 2013170637A1
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cancer
tumor
paeoniflorin
cell
cells
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PCT/CN2013/000683
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Chinese (zh)
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李梢
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北京京朋汇药业研究发展有限公司
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Publication of WO2013170637A1 publication Critical patent/WO2013170637A1/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
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • A61P11/04Drugs for disorders of the respiratory system for throat disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Definitions

  • the invention belongs to the field of anti-tumor medicines, and relates to the use of paeoniflorin compounds in preparing anti-tumor medicines, and particularly relates to the use of paeoniflorin and paeoniflorin in the preparation of anti-tumor drugs. Background technique
  • Tumors are the body's normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in the clonal dysplasia of new organisms, which appear as a mass. Tumor cells differ from normal cells in terms of external morphology, metabolism, and function, and exhibit persistent proliferation. According to effective statistics, tumors are the first of three major factors that threaten the health of human beings around the world. Therefore, the development of anti-tumor drugs has become a hot topic in recent years.
  • Cytotoxic drugs including drugs that destroy DNA structure and function (such as cyclophosphamide, irinotecan), Drugs that affect the biosynthesis of nucleic acids (such as 5-fluorouracil, cytarabine, methotrexate);
  • drugs that affect hormone balance including anti-estrogen drugs (such as toremifene), anti-androgens Drugs (such as bicalutamide) and aromatase inhibitors (such as letrozole);
  • other and auxiliary drugs including immune function regulators (such as interleukins, interferons), biological response modifiers (such as Ero Tini, gefitinib), cell differentiation inducers (such as retinoic acid, arsenic acid), antifolate preparations (Libitai), monoclonal antibodies (such as Avastin) and adjuvant analges
  • cytotoxic drugs generally cause moderate to severe digestive system reactions (such as malignant vomiting, stomatitis), myelosuppression (such as leukopenia, thrombocytopenia) and organ toxicity (such as Neurotoxicity, liver and kidney toxicity), hormone balance interference drugs can cause mild to moderate gastrointestinal reactions, reproductive system damage and even depressive symptoms in patients, most regulators and inducers can also cause different degrees of skin reactions and Liver and kidney dysfunction.
  • Irinotecan Hydrochloride is commonly used in the treatment of adult metastatic colorectal cancer, but the effect is significant, but after administration, 20% of patients have gastrointestinal adverse reactions - severe diarrhea, 78.7% of patients have been neutral In neutropenia, 9% of patients develop transient acute cholinergic syndrome (Bret Wallace et al. Science, 330, 2010.; Sun Wei et al. Shuo Pharmacy, 18(35), 2007.; Wu Yuhong et al. Anhui Medicine, 14(10), 2010.
  • Pemetrexed disodium is the first anti-pleural mesothelioma drug that causes bone marrow suppression in patients, including neutropenia, thrombocytopenia, anemia or each Hematopoietic reduction, liver and kidney dysfunction are hanged (Zheng Hang et al. Cancer Research, 34(4), 2007.; Wang Jianying et al. Chinese and Foreign Health Digest, 12, 201 1. ) ; Bevaciz b Avastin is a monoclonal antibody to vascular endothelial growth factor VEGF that binds to VEGF and prevents its binding to receptors on the surface of endothelial cells.
  • anti-tumor Chinese medicines have less damage to the body.
  • the mechanism of anti-tumor Chinese medicine mainly involves cytotoxicity, improving immunity, inducing tumor cell apoptosis and differentiation, and inhibiting tumor angiogenesis.
  • anti-tumor Chinese medicine can be divided into the following categories: 1. Detoxification, such as Astragalus and Daqingye; 2. Blood-activating and stasis-like, such as rhubarb and Salvia; 3.
  • Fuzheng Peiben Such as ginseng, astragalus, asparagus; four, phlegm and stagnation, such as Pinellia, melon, Tiannanxing; 5, Lishui and dampness, such as scorpion, psyllium, buckwheat; 6, topical drugs, such as Realgar, horse money.
  • Some of the active ingredients in the above anti-tumor Chinese medicines, such as taxol in Taxus br ev ifolic, are often used in anti-tumor research and have achieved good results (Tang Zhaohui et al. Chinese Clinical Rehabilitation, 27 , 2006.; Jiang Zhiwu et al. Heilongjiang Science and Technology Information, 7, 2010; Pluznilc, DH et al. Cancer Research, 54 (15), 1994). Therefore, the in-depth exploration of the active ingredients of anti-tumor Chinese medicine has important theoretical significance and clinical significance. Guiding significance.
  • paclitaxel extracted from yew is a specific stabilizer for microtubules, which promotes the assembly of microtubules and maintains the stability of microtubules.
  • the accumulation of these microtubules interferes with various functions of cells, especially the cell division stops at mitosis. Period, which blocks the normal division of cells and exerts an anti-cancer effect.
  • paclitaxel has a significant side effect on patients.
  • White peony is the root of Paeonia a piano ora Pa!
  • Known pharmacological effects include analgesia, sedation, anticonvulsant, anti-inflammatory, anti-pathogenic microorganisms and liver protection, and also on the immune system and smooth muscle.
  • Radix Paeoniae Alba is a paeoniflorin compound, represented by paeoniflorin (English name: Albiflorin; English name Paeonilactone C; AL) and paeoniflorin (English name Paeoniflorin). It provides a new treatment for paeoniflorin compounds and provides new options for the prevention and treatment of clinical tumors. Summary of the invention
  • the object of the present invention is to provide a novel antitumor drug, paeoniflorin, which is derived from a traditional Chinese medicine and can be used for the prevention and/or treatment of various tumors in the clinic.
  • a paeoniflorin compound or a pharmaceutically acceptable salt, solvate, polymorph, enantiomer or racemic mixture thereof for the manufacture of a medicament for the prevention and/or treatment of a tumor.
  • the paeoniflorin compound is selected from the group consisting of paeoniflorin of structural formula 1 and/or paeoniflorin of structural formula 2 -
  • the tumor is a solid tumor.
  • the tumor is selected from the group consisting of colon cancer, lung cancer, liver cancer, neuroblastoma, brain tumor, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal carcinoma, pancreatic cancer, laryngeal cancer, prostate cancer, renal cancer, adrenal cortex
  • colon cancer lung cancer, liver cancer, neuroblastoma, brain tumor, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal carcinoma, pancreatic cancer, laryngeal cancer, prostate cancer, renal cancer, adrenal cortex
  • tumor skin cancer, melanoma, ovarian cancer, bladder cancer, lymphoma, and gastric cancer.
  • the tumor is selected from the group consisting of a colon cancer, a lung cancer, a neuroblastoma, a breast cancer, a cervical cancer, a rhabdomyosarcoma, a nasopharyngeal cancer, a pancreatic cancer, a laryngeal cancer, a prostate cancer, a renal cancer, and an adrenocortical tumor.
  • a colon cancer a lung cancer
  • a neuroblastoma a breast cancer
  • a cervical cancer a rhabdomyosarcoma
  • a nasopharyngeal cancer a pancreatic cancer
  • laryngeal cancer a prostate cancer
  • a renal cancer and an adrenocortical tumor.
  • an adrenocortical tumor kind or more.
  • the paeoniflorin compound is a paeoniflorin
  • the tumor is selected from the group consisting of intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, and prostate cancer.
  • the tumor is selected from the group consisting of intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, and prostate cancer.
  • the paeoniflorin compound is paeoniflorin
  • the tumor is selected from one or more of laryngeal cancer, cervical cancer, nasopharyngeal cancer, and pancreatic cancer.
  • the intestinal cancer is selected from one or more of colon cancer, rectal cancer, and colorectal cancer.
  • the prevention of tumor refers to the treatment of precancerous lesions.
  • the preventing tumor refers to treating precancerous lesions of intestinal cancer and preventing the conversion of severe enteritis into intestinal cancer.
  • the medicament is in any of the clinically acceptable dosage forms.
  • the dosage form comprises a preparation for parenteral administration and a preparation for parenteral administration. More preferably, the preparation for parenteral administration is selected from the group consisting of a powder, a tablet, a granule, a capsule, a dropping tablet, an emulsion or a suspension; the parenteral preparation is selected as an injection, a spray Agent, suppository, infusion, patch or ointment.
  • paeoniflorin and paeoniflorin can be used as the prophylactic and/or therapeutic agent for tumors, respectively.
  • the sole active ingredient may also be used together as an active ingredient of the preventive and/or therapeutic tumor drug; the preventive and/or therapeutic tumor drug may also be prepared separately or together with other substances.
  • paeoniflorin compounds of the present invention can significantly inhibit intestinal cancer, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal cancer, pancreatic cancer, laryngeal cancer, prostate cancer, lung cancer, neuroblastoma, renal cancer and A variety of tumors such as adrenal cortex have broad-spectrum anti-tumor function; Paeoniflorin can also treat precancerous lesions, effectively block the conversion of severe enteritis to intestinal cancer, treat precancerous lesions of intestinal cancer, and reduce intestinal cancer. The incidence rate is of great significance. Moreover, there are some differences in the dominant anti-tumor spectrum of paeoniflorin and paeoniflorin.
  • Paeoniflorin can significantly inhibit intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer and prostate cancer. Paeoniflorin can significantly inhibit laryngeal Cancer, cervical cancer, nasopharyngeal cancer and pancreatic cancer. Specifically,
  • intestinal cancer cell line HT29 intestinal cancer cell line CT26, rhabdomyosarcoma cell line A204, nasopharyngeal carcinoma cell CNE-2Z, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1, prostate cancer cell DU145, cervical cancer
  • pancreatic cancer cell PANC1, prostate cancer cell DU145 cervical cancer
  • intestinal cancer cells HT29, pancreatic cancer cells PANC1, laryngeal cancer cells Hep2, cervical cancer cells Hela, breast cancer cells MDA231 and prostate cancer cells DU145 are particularly sensitive to paeoniflorin.
  • nasopharyngeal carcinoma cells CNE-2Z nasopharyngeal carcinoma cells CNE-2Z
  • laryngeal carcinoma cells Hep2 pancreatic cancer cells PANC1
  • cervical cancer cells Hela rhabdomyosarcoma cells A204.
  • nasopharyngeal carcinoma CNE-2Z cells, laryngeal carcinoma Hep2 cells, pancreatic cancer PANC1 cells, and cervical cancer Hela cells are particularly sensitive to paeoniflorin.
  • tumor cell migration assay showed that paeoniflorin on intestinal cancer cell CT26, cervical cancer cell line Hela, neuroblastoma cell line SY5Y, breast cancer cell MDA231, breast cancer cell T47D, breast cancer cell MCF7, rhabdomyosarcoma cell A204, Renal cancer Wilms cell G401, nasopharyngeal carcinoma cell CNE2Z, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, lung cancer cell A549, lung cancer cell LLC, pancreatic cancer cell PANC1 and adrenocortical tumor cell SW13 have significant inhibition.
  • paeoniflorin inhibited the migration of cervical cancer cell line Hela, intestinal cancer cell line CT26, breast cancer cell line MDA231, nasopharyngeal carcinoma cell line CNE2Z and pancreatic cancer cell line PANC1.
  • the results of tumor cell migration assay showed that paeoniflorin on rhabdomyosarcoma cell A204, nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1 and adrenocortical tumor cell SW13, etc.
  • the migration has a significant inhibitory effect.
  • paeoniflorin the inhibitory effect on intestinal cancer cell CT26, nasopharyngeal carcinoma cell CNE2Z, laryngeal carcinoma cell Hep2 and adrenal cortical tumor cell SW13 was particularly significant.
  • paeoniflorin can significantly inhibit the length and metastasis of CT26 intestinal cancer, LLC lung cancer, MDA231 breast cancer and Hela cervical cancer.
  • the inhibition rate of paeoniflorin on CT26 intestinal cancer can reach 54.41%.
  • the inhibition rate of Hela cervical cancer can reach 52.3%.
  • Figure 1 Effect of paeoniflorin on tumor cell migration (Transwell assay).
  • FIG. 1 Effect of paeoniflorin on tumor cell migration (Scratch Analysis).
  • FIG. 3 Effect of paeoniflorin on tumor cell migration (Transwell assay).
  • 4B tumor growth curve of LLC lung cancer nude mice
  • 4C tumor growth curve of MDA231 breast cancer nude mice
  • 4D Tumor growth curve of Hela cervical cancer nude mice.
  • Figure 6 Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwell assay).
  • Figure 7 Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwell assay). detailed description
  • the paeoniflorin and paeoniflorin used in the following examples were purchased from Shanghai Dingrui Chemical Co., Ltd. (purity was 98% or more), and the total glucoside capsule (Pavlin) was purchased from Ningbo Lihua Pharmaceutical Co., Ltd.
  • Triazolium blue (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment paeoniflorin treatment of rhabdomyosarcoma A204, Nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, intestinal cancer HT29, neuroblastoma cell SY5Y, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC 1, cervical cancer cell Hela, breast cancer cell MDA231, breast cancer cell T47D, breast cancer cell MCF7, skin squamous cell carcinoma A431, renal cancer Wilms Cell G401 and prostate cancer cell DU145 were for about 24 hours; after MTT staining for 4 hours, dimethyl sulfoxide (DMSO) was dissolved, and the absorbance value was measured at 570 nm.
  • DMSO dimethyl sulfoxide
  • Paeoniflorin was found to be pancreatic cancer cell PANC1, intestinal cancer cell line CT26, intestinal cancer cell line HT29, laryngeal cancer cell Hep2, cervical cancer cell line Hela, breast cancer cell MDA231, prostate cancer cell DU145, nasopharyngeal carcinoma cell CNE2Z,
  • the proliferation of rhabdomyosarcoma cell A204, breast cancer cell T47D and breast cancer cell MCF7 cells all had significant inhibitory effects.
  • paeoniflorin is most sensitive to the effects of intestinal cancer cells HT29, pancreatic cancer cells PANC1, laryngeal carcinoma cells Hep2, cervical cancer cells Hela, breast cancer cells MDA231 and prostate cancer cells DU145.
  • the specific results are shown in Table 1.
  • A204 (rhabdomyosarcoma cells) 45 ⁇ / ⁇ 1
  • HT29 intestinal cancer cells 33 g/ml
  • CT26 intestinal cancer cells
  • Hep2 (laryngeal cancer cell) 36 g/ml
  • PANC 1 pancreatic cancer cells
  • MDA231 (breast cancer cells) 40 ⁇
  • T47D breast cancer cells 56 g/nil
  • MCF7 breast cancer cells 61 g/ml
  • A431 skin squamous cell carcinoma 92 g/ml
  • A204 cells rhizomatous muscle cells
  • CNE-2Z cells (nasopharynx cancer cells) 50 ⁇
  • CT26 cells (intestinal cancer cells) 82 ⁇
  • Hep2 cells (laryngeal cancer cells) 44
  • PANC1 cells pancreatic cancer cells 53 g/ml
  • Hela cells (cervical cancer cells) 49 ⁇
  • HT29 cells (intestinal cancer cells) 96 g/ml
  • MDA231 cells (breast cancer cells) ⁇ 15 ⁇ ]
  • G401 cells (kidney cancer Wilms cells) 89 ⁇ ⁇
  • Paeoniflorin can significantly inhibit the migration of rhabdomyosarcoma cell ⁇ 204, nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1 and adrenal cortical tumor cell SW13. It was shown that the number of cells in the administration group was significantly reduced relative to the control group. Among them, paeoniflorin inhibited intestinal cancer cell line CT26, nasopharyngeal carcinoma cell line CNE2Z, laryngeal carcinoma cell line Hep2 and adrenocortical tumor cell line SW13. See Figure 3 for details.
  • mice of each solid tumor transplantation model were randomly divided into 3 groups: control group (inoculated with tumor mass without administration), pre-dosing group of paeoniflorin (referring to the day of inoculation of tumor block) and paeoniflorin
  • control group inoculated with tumor mass without administration
  • pre-dosing group of paeoniflorin referring to the day of inoculation of tumor block
  • paeoniflorin The drug-administered group (administered when the tumor-forming volume reached 70-100 mm 3 or more), 5-6 nude mice per group.
  • the pre-administration group of paeoniflorin was intraperitoneally administered on the day of inoculation of the tumor block at a dose of 200 m g/kg, the frequency of administration was once every 2 days for 3 weeks, and the mice were sacrificed the day after the end of the administration. .
  • the paeoniflorin administration group when the tumor volume reached 70-100 mm 3 or more, the paeoniflorin was administered intraperitoneally at a dose of 300 mg/kg, and the administration frequency was once every 2 days for 2 weeks. Mice were sacrificed the day after the end. Tumor volume and weight were measured and the tumor inhibition rate was calculated.
  • the growth rate of A. LLC tumor and CT26 tumor is equivalent, and the volume reaches 100 mm 3 and is about 12 days;
  • MDA231 cells are human. Compared with LLC and CT26 tumors, the tumor formation time in nude mice is slower, and the tumor growth rate is relatively slow. The volume reaches 70 mm 3 and is about 18 days.
  • Hela cells are human. Compared with LLC and CT26 tumors, the tumor formation time in nude mice is slower, and the tumor growth rate is relatively slow. The volume reaches 70 mm 3 and is about 20 days.
  • mice The dominant tumors of paeoniflorin in vitro, pancreatic cancer PANC1 cells and cervical cancer Hela cells, were selected to establish a subcutaneous tumor-bearing model in nude mice. After the tumor volume reached 70 mm 3 , paeoniflorin was intraperitoneally injected at a dose of 300 mg/kg, and the frequency of administration was once every 2 days for 2 weeks. The model group was used as a control. Mice were sacrificed the day after the end of dosing. Tumor volume and weight were measured and the tumor inhibition rate was calculated.
  • Control group Paeoniflorin administration group dose (mg/kg) 300 mg/g
  • mice were divided into 3 groups: normal group, model group and paeoniflorin intervention group, 5 in each group.
  • the model group and the intervention group were intraperitoneally injected with the gene mutation agent oxidized azomethane (AOM, 20 mg/g).
  • AOM gene mutation agent oxidized azomethane
  • the paeoniflorin dry group was administered intraperitoneally (100 mg/Kg) on the 2nd day of AOM injection, once every two days for 3 times.
  • the model group did not have any drug treatment.
  • One week after AOM administration 3% sodium dextran sulfate (DSS) was used to replace normal drinking water. The mice were continuously consumed for 7 days; after that, they were returned to normal drinking water, and the mice were continuously consumed for 14 days.
  • DSS sodium dextran sulfate
  • mice in the model group had obvious colonic mucosal congestion, uneven intestinal thickness, and several tumors had been formed.
  • the mice in the paeoniflorin intervention group had a uniform colon thickness and no obvious bleeding points. See tumor formation, see Figure 5A for details.
  • the HE staining results showed that the glandular structure of normal mouse gland was intact and distributed, no obvious inflammatory cell infiltration, no submucosal hemorrhage and ulceration.
  • mucosal ulcers appeared in the intestinal tissues of mice, multiple glandular deletions accompanied by inflammatory cell infiltration, and cancer cells infiltrated into the intestinal mucosa and submucosa, presenting typical features of intestinal cancer in situ.
  • the paeoniflorin T pre-group mouse intestinal tissue showed focal inflammatory cell infiltration, individual gland loss, but no obvious cancer cell infiltration, showing the typical characteristics of enteritis, see Figure 5B.
  • thiazolyl blue (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, paeoniflorin and paeoniflorin treatment A204, CNE-2Z, CT26, Hep2 and PANC l tumor cells were used for 24 hours, then MTT staining for 4 hours, dimethyl sulfoxide (DMSO) was dissolved, and the absorbance value was measured at 570 nm.
  • DMSO dimethyl sulfoxide
  • A204 rhabdomyosarcoma cells 45 g/ml 61 g/ml
  • Hep2 (laryngeal cancer cell) 36 g/ml 39 ⁇ ⁇
  • PANC1 pancreatic cancer cells
  • HT29 has an inhibitory effect, but the IC 5 o (half inhibition rate) of paeoniflorin on nasopharyngeal carcinoma cell line CNE2Z, cervical cancer cell line Hela and intestinal cancer cell line HT29 is lower than total glucosides of paeony, indicating that paeoniflorin The inhibition of tumor cell proliferation is stronger than that of total glucosides of paeony.
  • Table 6 The specific results are shown in Table 6.
  • Tumor cell Hela (cervical cancer cell) 49 ⁇ 55 ⁇ / ⁇ 1
  • paeoniflorin can effectively inhibit intestinal cancer, lung cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, prostate cancer, nasopharyngeal carcinoma, rhabdomyosarcoma, lung cancer, sputum mesothelioma, kidney cancer and adrenal cortex
  • Tumor growth and metastasis can effectively inhibit the growth and metastasis of colorectal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer and prostate cancer
  • paeoniflorin can effectively inhibit laryngeal cancer, cervical cancer, nasopharyngeal cancer, pancreatic cancer and
  • the growth and metastasis of rhabdomyosarcoma is particularly effective in inhibiting the growth and metastasis of laryngeal, cervical, nasopharyngeal and pancreatic cancers.
  • Paeoniflorin can also effectively treat precancerous lesions of colorectal cancer and significantly block the conversion of severe ulcerative enteritis to intestinal cancer. Therefore, the results of in vitro and in vivo experiments show that paeoniflorin can effectively inhibit the growth and metastasis of a variety of tumors, and has a broad-spectrum anti-tumor effect; paeoniflorin compounds can also treat precancerous lesions of intestinal cancer, blocking severe The conversion of enteritis to intestinal cancer.

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Abstract

An application of a paeoniflorin compound, or a pharmaceutically acceptable salt, solvate, polymorph, enantiomer, or racemic mixture thereof in preparation of drugs for preventing and/or treating tumors. Preferably, the paeoniflorin compound is selected from albiflorin and/or paeoniflorin. The tumors are solid tumors.

Description

芍药苷类化合物在制备抗肿瘤药物中的用途 相关申请的交叉引用  Use of paeoniflorin in the preparation of antitumor drugs CROSS REFERENCE TO RELATED APPLICATIONS
本申请要求 2012年 5月 15日递交的中国专利申请号为 201210151425.3的专利申请的权 益, 在此将其全部内容引入作为参考。 技术领域  The present application claims the benefit of the Japanese Patent Application Serial No. 2012. Technical field
本发明属于抗肿瘤药物领域, 涉及芍药苷类化合物在制备抗肿瘤药物中的用途, 具体涉 及芍药内酯苷和芍药苷在制备抗肿瘤药物中的用途。 背景技术  The invention belongs to the field of anti-tumor medicines, and relates to the use of paeoniflorin compounds in preparing anti-tumor medicines, and particularly relates to the use of paeoniflorin and paeoniflorin in the preparation of anti-tumor drugs. Background technique
肿瘤是机体在各种致癌因素作用下, 细胞在基因水平上失去对其生长的正常调控, 导致 其克隆性异常增生而形成的新生物, 表现为肿块。 肿瘤细胞在外部形态、 代谢和功能方面不 同于正常细胞, 多呈现持续性增殖。 据有效数据统计, 肿瘤是目前威胁全世界人类生命健康 的三大因素之首, 所以抗肿瘤药物的研发成为近年来的热点。  Tumors are the body's normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in the clonal dysplasia of new organisms, which appear as a mass. Tumor cells differ from normal cells in terms of external morphology, metabolism, and function, and exhibit persistent proliferation. According to effective statistics, tumors are the first of three major factors that threaten the health of human beings around the world. Therefore, the development of anti-tumor drugs has become a hot topic in recent years.
根据抗肿瘤药物的传统分类和研究进展, 可将常用的抗肿瘤药物分为以下几大类: 一、 细胞毒药物, 包括破坏 DNA结构和功能的药物(如环磷酰胺、 伊立替康)、 影响核酸生物合 成的药物 (如 5-氟尿嘧啶、 阿糖胞苷、 甲氨喋呤) 等; 二、 影响激素平衡的药物, 包括抗雌 激素类药物 (如托瑞米芬)、 抗雄激素类药物 (如比卡鲁胺) 和芳香化酶抑制剂 (如来曲唑) 等; 三、 其它及辅助药物, 包括机体免疫功能调节剂(如白介素、 干扰素)、 生物反应调节剂 (如埃罗替尼、 吉非替尼)、 细胞分化诱导剂 (如维甲酸、 亚砷酸)、 抗叶酸制剂 (力比泰)、 单克隆抗体 (如阿瓦斯汀) 和辅助镇痛、 止吐、 白细胞升高等药物。 但是, 以上药物或多或 少存在着副作用, 像细胞毒药物一般会引发患者的中重度消化系统反应 (如恶性呕吐、 口腔 炎)、 骨髓抑制 (如白细胞减少、 血小板减少) 和器官毒性 (如神经毒性、 肝肾毒性), 激素 平衡干扰药物则会引起患者的轻中度胃肠道反应、 生殖系统损伤甚至精神抑郁症状, 多数调 节剂和诱导剂等也会引发患者不同程度的皮肤反应和肝肾功能损害。例如伊立替康(Irinotecan Hydrochloride ) 常用于成人转移性大肠癌的治疗, 效果比较显著, 但是用药后, 20%的患者 出现胃肠道不良反应——严重腹泻, 78.7%的患者均出现过中性粒细胞减少症, 9%的患者出 现短暂严重的急性胆碱能综合征 (Bret Wallace等. Science, 330, 2010.; 孙祎等. 屮国药房, 18(35), 2007.; 吴育红等. 安徽医药, 14(10), 2010. ); 力比泰( Pemetrexed disodium , Alimta ) 是首个抗胸膜间皮瘤药物, 它可以引起患者的骨髓抑制, 包括中性粒细胞减少、 血小板 减少、 贫血或各类血细胞减少, 肝、 肾功能不全者忌用 (郑航等. 肿瘤防治研究, 34(4), 2007.; 王剑英等. 中外健康文摘, 12, 201 1. ) ; 阿瓦斯汀 ( Bevaciz b Avastin )是血管内 皮生长因子 VEGF的单克隆抗体, 通过结合 VEGF并防止其与内皮细胞表面的受体结 合, 减少了微血管的生成并抑制了转移病灶进展。 但是它的副作用很明显, 包括胃肠穿 孔、 伤口开裂综合症、 出血、 高血压危象、 肾病综合征和充血性心力衰竭等 (Eric O. Gamboa等. Clinical Colorectal Cancer, 9( 1 ), 2010 A Mailliez等. British Journal of Cancer, 103, 2010 Sanjaykumar Hapani等. Oncology, 79, 2010 )。 According to the traditional classification and research progress of anti-tumor drugs, commonly used anti-tumor drugs can be divided into the following categories: 1. Cytotoxic drugs, including drugs that destroy DNA structure and function (such as cyclophosphamide, irinotecan), Drugs that affect the biosynthesis of nucleic acids (such as 5-fluorouracil, cytarabine, methotrexate); Second, drugs that affect hormone balance, including anti-estrogen drugs (such as toremifene), anti-androgens Drugs (such as bicalutamide) and aromatase inhibitors (such as letrozole); Third, other and auxiliary drugs, including immune function regulators (such as interleukins, interferons), biological response modifiers (such as Ero Tini, gefitinib), cell differentiation inducers (such as retinoic acid, arsenic acid), antifolate preparations (Libitai), monoclonal antibodies (such as Avastin) and adjuvant analgesia, antiemetic, leukocyte elevation, etc. drug. However, these drugs have more or less side effects, such as cytotoxic drugs generally cause moderate to severe digestive system reactions (such as malignant vomiting, stomatitis), myelosuppression (such as leukopenia, thrombocytopenia) and organ toxicity (such as Neurotoxicity, liver and kidney toxicity), hormone balance interference drugs can cause mild to moderate gastrointestinal reactions, reproductive system damage and even depressive symptoms in patients, most regulators and inducers can also cause different degrees of skin reactions and Liver and kidney dysfunction. For example, Irinotecan Hydrochloride is commonly used in the treatment of adult metastatic colorectal cancer, but the effect is significant, but after administration, 20% of patients have gastrointestinal adverse reactions - severe diarrhea, 78.7% of patients have been neutral In neutropenia, 9% of patients develop transient acute cholinergic syndrome (Bret Wallace et al. Science, 330, 2010.; Sun Wei et al. Shuo Pharmacy, 18(35), 2007.; Wu Yuhong et al. Anhui Medicine, 14(10), 2010. ); Pemetrexed disodium (Alimta) is the first anti-pleural mesothelioma drug that causes bone marrow suppression in patients, including neutropenia, thrombocytopenia, anemia or each Hematopoietic reduction, liver and kidney dysfunction are hanged (Zheng Hang et al. Cancer Research, 34(4), 2007.; Wang Jianying et al. Chinese and Foreign Health Digest, 12, 201 1. ) ; Bevaciz b Avastin is a monoclonal antibody to vascular endothelial growth factor VEGF that binds to VEGF and prevents its binding to receptors on the surface of endothelial cells. , reduces microvascular production and inhibits the progression of metastatic lesions. However, its side effects are obvious, including gastrointestinal perforation, wound dehiscence syndrome, hemorrhage, hypertensive crisis, nephrotic syndrome and congestive heart failure (Eric O. Gamboa et al. Clinical Colorectal Cancer, 9( 1 ), 2010 A Mailliez et al. British Journal of Cancer, 103, 2010 Sanjaykumar Hapani et al. Oncology, 79, 2010).
相比之下, 抗肿瘤中药对机体的损害则较小, 抗肿瘤中药的作用机理主要涉及细胞毒作 用、 提高机体免疫力、 诱导肿瘤细胞凋亡及分化和抑制肿瘤血管新生等方面。 根据中药的功 效和作用特点, 可将抗肿瘤中药分成以下几大类: 一、 清热解毒类, 如黄芩、 大青叶; 二、 活血化瘀类, 如大黄、 丹参; 三、 扶正培本类, 如人参、 黄芪、 天冬; 四、 化痰散结类, 如 半夏、 瓜蒌、 天南星; 五、 利水化湿类, 如茯苓、 车前子、 翟麦; 六、 外用型药物, 如雄黄、 马钱子。 上述抗肿瘤中药中的部分有效成分, 像紫杉 ( Taxus brevifolic 中的紫杉醇 (taxol ) 常被人们用于抗肿瘤研究, 且取得了较好的疗效 (唐朝晖等. 中国临床康复, 27, 2006.; 江治 武等. 黑龙江科技信息, 7, 2010; Pluznilc, DH等. Cancer Research, 54 (15), 1994)。 因此, 关于 抗肿瘤中药有效成分的深入探索具有重要的理论研究意义和临床指导意义。 In contrast, anti-tumor Chinese medicines have less damage to the body. The mechanism of anti-tumor Chinese medicine mainly involves cytotoxicity, improving immunity, inducing tumor cell apoptosis and differentiation, and inhibiting tumor angiogenesis. According to the efficacy and action characteristics of traditional Chinese medicine, anti-tumor Chinese medicine can be divided into the following categories: 1. Detoxification, such as Astragalus and Daqingye; 2. Blood-activating and stasis-like, such as rhubarb and Salvia; 3. Fuzheng Peiben Such as ginseng, astragalus, asparagus; four, phlegm and stagnation, such as Pinellia, melon, Tiannanxing; 5, Lishui and dampness, such as scorpion, psyllium, buckwheat; 6, topical drugs, such as Realgar, horse money. Some of the active ingredients in the above anti-tumor Chinese medicines, such as taxol in Taxus br ev ifolic, are often used in anti-tumor research and have achieved good results (Tang Zhaohui et al. Chinese Clinical Rehabilitation, 27 , 2006.; Jiang Zhiwu et al. Heilongjiang Science and Technology Information, 7, 2010; Pluznilc, DH et al. Cancer Research, 54 (15), 1994). Therefore, the in-depth exploration of the active ingredients of anti-tumor Chinese medicine has important theoretical significance and clinical significance. Guiding significance.
然而, 由于大多数中药中的化合物成分复杂, 分离提纯困难, 质量控制受限且药效和作 用机理尚不清晰, 海量筛选抗肿瘤的有效成分非常不易, 所以关于抗肿瘤中药有效成分的研 究普遍存在着诸多不足和局限。 例如从紫杉中提取的紫杉醇是微管的特异性稳定剂, 可促进 微管的装配和保持微管稳定, 这些微管的积累干扰了细胞的各种功能, 特别是使细胞分裂停 止于有丝分裂期, 从而阻断了细胞的正常分裂, 发挥了抗癌的作用。 但是紫杉醇对患者的副 作用较大, 常见不良反应有过敏反应(发生率高达 39%)、骨髓抑制(严重中性粒细胞发生率 为 47%, 严重的血小板降低发生率为 5%)、 神经毒性 (周围神经病变发生率为 62%)、 心血 管毒性(发生率为 55%)、 胃肠道反应(恶心, 呕吐, 腹泻和黏膜炎发生率分别为 59% 43% 和 39%) 和脱发 (发生率高达 80%以上)等 (叶冬梅等. 中国药房, 29, 2011.; 马连顺等. 医 药世界, 3, 2009 Deny, WB等. Cancer Research, 58(6 1998. )。  However, due to the complexity of the compounds in most Chinese medicines, the difficulty in separation and purification, the limited quality control and the unclear efficacy and mechanism of action, it is very difficult to screen the anti-tumor active ingredients. Therefore, the research on the active ingredients of anti-tumor Chinese medicine is common. There are many shortcomings and limitations. For example, paclitaxel extracted from yew is a specific stabilizer for microtubules, which promotes the assembly of microtubules and maintains the stability of microtubules. The accumulation of these microtubules interferes with various functions of cells, especially the cell division stops at mitosis. Period, which blocks the normal division of cells and exerts an anti-cancer effect. However, paclitaxel has a significant side effect on patients. Common adverse reactions include allergic reactions (up to 39%), myelosuppression (47% for severe neutrophils, 5% for severe thrombocytopenia), and neurotoxicity. (The incidence of peripheral neuropathy was 62%), cardiovascular toxicity (55% incidence), gastrointestinal reactions (nausea, vomiting, diarrhea, and mucositis were 59% 43% and 39%, respectively) and hair loss ( The incidence rate is as high as 80% or more) (Ye Dongmei et al. Chinese Pharmacy, 29, 2011.; Ma Lianshun et al. Pharmaceutical World, 3, 2009 Deny, WB et al. Cancer Research, 58 (6 1998.).
因此, 药学研究人员仍在致力于开发新的抗肿瘤中药有效成分。  Therefore, pharmaceutical researchers are still working on the development of new anti-tumor Chinese medicine active ingredients.
白芍为毛茛科植物芍药 (Paeonia a琴 ora Pa!! 的根, 己知的药理作用包括镇痛、 镇静、 抗惊厥、 抗炎、 抗病原微生物和护肝, 还对免疫系统、 平滑肌有作用, 临床上可应用于抗癲 痫、 镇痫、 戒毒、 止眩晕, 治疗类风湿性关节炎、 细菌性痢疾及肠炎、 病毒性肝炎以及老年 性疾病, 还具有抗硫酸钡絮凝和溶解粘液的作用。 白芍的活性成分之一是芍药苷类化合物, 以芍药内酯苷(英文名为 Albiflorin ; 英文别名 Paeonilactone C ; 简称 AL )和芍药苷(英文名 为 Paeoniflorin ) 为代表。 本发明的目的在于提供芍药苷类化合物的新治疗 途, 为临床肿瘤 的预防和治疗提供新的选择。 发明内容 White peony is the root of Paeonia a piano ora Pa!! Known pharmacological effects include analgesia, sedation, anticonvulsant, anti-inflammatory, anti-pathogenic microorganisms and liver protection, and also on the immune system and smooth muscle. Function, clinically applicable to anti-epilepsy, epilepsy, detoxification, vertigo, treatment of rheumatoid arthritis, bacterial dysentery and enteritis, viral hepatitis and senile diseases, also anti-sulphate flocculation and dissolution of mucus One of the active ingredients of Radix Paeoniae Alba is a paeoniflorin compound, represented by paeoniflorin (English name: Albiflorin; English name Paeonilactone C; AL) and paeoniflorin (English name Paeoniflorin). It provides a new treatment for paeoniflorin compounds and provides new options for the prevention and treatment of clinical tumors. Summary of the invention
本发明的目的在于提供一类新型抗肿瘤药物——芍药苷类化合物, 该类化合物来源于中 药, 可用于临床上多种肿瘤的预防和 /或治疗。  The object of the present invention is to provide a novel antitumor drug, paeoniflorin, which is derived from a traditional Chinese medicine and can be used for the prevention and/or treatment of various tumors in the clinic.
为了实现上述发明目的, 本发明采用了如下的技术方案:  In order to achieve the above object, the present invention adopts the following technical solutions:
芍药苷类化合物或其药学上可接受的盐、 溶剂化物、 多晶型体、 对映体或外消旋混合物 在制备预防和 /或治疗肿瘤的药物中的用途。  Use of a paeoniflorin compound or a pharmaceutically acceptable salt, solvate, polymorph, enantiomer or racemic mixture thereof for the manufacture of a medicament for the prevention and/or treatment of a tumor.
优选的, 所述芍药苷类化合物选自结构式 1的芍药内酯苷和 /或结构式 2的芍药苷-  Preferably, the paeoniflorin compound is selected from the group consisting of paeoniflorin of structural formula 1 and/or paeoniflorin of structural formula 2 -
Figure imgf000004_0001
Figure imgf000004_0001
本发明所述用途中, 所述肿瘤为实体肿瘤。 In the use of the invention, the tumor is a solid tumor.
优选的, 所述肿瘤选自肠癌、 肺癌、 肝癌、 神经母细胞瘤、 脑瘤、 乳腺癌、 宫颈癌、 横 纹肌肉瘤、 鼻咽癌、 胰腺癌、 喉癌、 前列腺癌、 肾癌、 肾上腺皮质瘤、 皮肤癌、 黑色素瘤、 卵巢癌、 膀胱癌、 淋巴癌和胃癌中的一种或多种。  Preferably, the tumor is selected from the group consisting of colon cancer, lung cancer, liver cancer, neuroblastoma, brain tumor, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal carcinoma, pancreatic cancer, laryngeal cancer, prostate cancer, renal cancer, adrenal cortex One or more of tumor, skin cancer, melanoma, ovarian cancer, bladder cancer, lymphoma, and gastric cancer.
更优选的, 所述肿瘤选自肠癌、 肺癌、 神经母细胞瘤、 乳腺癌、 宫颈癌、 横纹肌肉瘤、 鼻咽癌、 胰腺癌、 喉癌、 前列腺癌、 肾癌和肾上腺皮质瘤中的一种或多种。  More preferably, the tumor is selected from the group consisting of a colon cancer, a lung cancer, a neuroblastoma, a breast cancer, a cervical cancer, a rhabdomyosarcoma, a nasopharyngeal cancer, a pancreatic cancer, a laryngeal cancer, a prostate cancer, a renal cancer, and an adrenocortical tumor. Kind or more.
本发明所述用途中, 一种优选的方案是: 所述芍药苷类化合物为芍药内酯苷, 所述肿瘤 选自肠癌、 胰腺癌、 喉癌、 宫颈癌、 乳腺癌和前列腺癌中的一种或多种。  In the use of the present invention, a preferred embodiment is: the paeoniflorin compound is a paeoniflorin, and the tumor is selected from the group consisting of intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, and prostate cancer. One or more.
本发明所述用途中, 另一种优选的方案是: 所述芍药苷类化合物为芍药苷, 所述肿瘤选 自喉癌、 宫颈癌、 鼻咽癌和胰腺癌中的一种或多种。  In another aspect of the use of the present invention, the paeoniflorin compound is paeoniflorin, and the tumor is selected from one or more of laryngeal cancer, cervical cancer, nasopharyngeal cancer, and pancreatic cancer.
本发明所述用途中, 所述肠癌选 fi结肠癌、 直肠癌、 大肠癌中的一种或多种。  In the use of the present invention, the intestinal cancer is selected from one or more of colon cancer, rectal cancer, and colorectal cancer.
本发明所述用途中, 所述预防肿瘤是指治疗癌前病变。 优选的, 所述预防肿瘤是指治疗 肠癌癌前病变, 预防重症肠炎转变为肠癌。  In the use of the present invention, the prevention of tumor refers to the treatment of precancerous lesions. Preferably, the preventing tumor refers to treating precancerous lesions of intestinal cancer and preventing the conversion of severe enteritis into intestinal cancer.
本发明所述用途中, 所述药物为临床上可接受的任一剂型。 优选的, 所述剂型包括经胃 肠道给药制剂和非经胃肠道给药制剂。更优选的,所述经胃肠道给药制剂选自散剂、片剂、 颗 粒剂、 胶囊剂、 滴丸、 乳剂或混悬剂; 所述非经胃肠道给药制剂选向注射剂、 喷雾剂、 栓剂、 灌注剂、 贴剂或软膏剂。  In the use of the invention, the medicament is in any of the clinically acceptable dosage forms. Preferably, the dosage form comprises a preparation for parenteral administration and a preparation for parenteral administration. More preferably, the preparation for parenteral administration is selected from the group consisting of a powder, a tablet, a granule, a capsule, a dropping tablet, an emulsion or a suspension; the parenteral preparation is selected as an injection, a spray Agent, suppository, infusion, patch or ointment.
在本发明所述用途中,芍药内酯苷和芍药苷可以分别作为所述预防和 /或治疗肿瘤药物的 唯一活性成分, 也可以共同作为所述预防和 /或治疗肿瘤药物的活性成分; 还可以分别或共同 与其它物质一起制备所述预防和 /或治疗肿瘤药物。 In the use of the present invention, paeoniflorin and paeoniflorin can be used as the prophylactic and/or therapeutic agent for tumors, respectively. The sole active ingredient may also be used together as an active ingredient of the preventive and/or therapeutic tumor drug; the preventive and/or therapeutic tumor drug may also be prepared separately or together with other substances.
体内外实验表明, 本发明的芍药苷类化合物能够显著抑制肠癌、 乳腺癌、 宫颈癌、 横纹 肌肉瘤、 鼻咽癌、 胰腺癌、 喉癌、 前列腺癌、 肺癌、 神经母细胞瘤、 肾癌和肾上腺皮质瘤等 多种肿瘤, 具有广谱的抗肿瘤功能; 芍药苷类化合物还可以治疗癌前病变, 有效阻断重度肠 炎向肠癌的转化, 对治疗肠癌癌前病变、 降低肠癌的发生率, 具有重要意义。 而且芍药苷和 芍药内酯苷的优势抗瘤谱存在一定差异, 芍药内酯苷尤其能够显著抑制肠癌、胰腺癌、 喉癌、 宫颈癌、 乳腺癌和前列腺癌, 芍药苷尤其能够显著抑制喉癌、 宫颈癌、 鼻咽癌和胰腺癌。 具 体来说,  In vitro and in vivo experiments have shown that the paeoniflorin compounds of the present invention can significantly inhibit intestinal cancer, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal cancer, pancreatic cancer, laryngeal cancer, prostate cancer, lung cancer, neuroblastoma, renal cancer and A variety of tumors such as adrenal cortex have broad-spectrum anti-tumor function; Paeoniflorin can also treat precancerous lesions, effectively block the conversion of severe enteritis to intestinal cancer, treat precancerous lesions of intestinal cancer, and reduce intestinal cancer. The incidence rate is of great significance. Moreover, there are some differences in the dominant anti-tumor spectrum of paeoniflorin and paeoniflorin. Paeoniflorin can significantly inhibit intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer and prostate cancer. Paeoniflorin can significantly inhibit laryngeal Cancer, cervical cancer, nasopharyngeal cancer and pancreatic cancer. Specifically,
1、 芍药苷类成分抑制肿瘤细胞增殖实验  1. Paeoniflorin inhibits tumor cell proliferation experiment
MTT检测结果显示, 芍药内酯苷对肠癌细胞 HT29、 肠癌细胞 CT26、 横纹肌肉瘤细胞 A204、 鼻咽癌细胞 CNE-2Z、 喉癌细胞 Hep2、 胰腺癌细胞 PANC1、 前列腺癌细胞 DU145、 宫颈癌细胞 Hela、 乳腺癌细胞 MDA231、 乳腺癌细胞 T47D和乳腺癌细胞 MCF7的增殖具有 显著抑制作用。 其中, 肠癌细胞 HT29、 胰腺癌细胞 PANC1、 喉癌细胞 Hep2、 宫颈癌细胞 Hela、 乳腺癌细胞 MDA231和前列腺癌细胞 DU145对芍药内酯苷尤其敏感。  MTT test results showed that paeoniflorin on intestinal cancer cell line HT29, intestinal cancer cell line CT26, rhabdomyosarcoma cell line A204, nasopharyngeal carcinoma cell CNE-2Z, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1, prostate cancer cell DU145, cervical cancer The proliferation of cell Hela, breast cancer cell MDA231, breast cancer cell T47D and breast cancer cell MCF7 has a significant inhibitory effect. Among them, intestinal cancer cells HT29, pancreatic cancer cells PANC1, laryngeal cancer cells Hep2, cervical cancer cells Hela, breast cancer cells MDA231 and prostate cancer cells DU145 are particularly sensitive to paeoniflorin.
MTT法检测结果显示,芍药苷对鼻咽癌细胞 CNE-2Z、喉癌细胞 Hep2、胰腺癌细胞 PANC1 和宫颈癌细胞 Hela和横纹肌肉瘤细胞 A204等多种肿瘤细胞的增殖具有显著抑制作用。其中, 鼻咽癌 CNE-2Z细胞、喉癌 Hep2细胞、胰腺癌 PANC1细胞和宫颈癌 Hela细胞对芍药苷尤其 敏感。  The results of MTT assay showed that paeoniflorin significantly inhibited the proliferation of nasopharyngeal carcinoma cells CNE-2Z, laryngeal carcinoma cells Hep2, pancreatic cancer cells PANC1, cervical cancer cells Hela and rhabdomyosarcoma cells A204. Among them, nasopharyngeal carcinoma CNE-2Z cells, laryngeal carcinoma Hep2 cells, pancreatic cancer PANC1 cells, and cervical cancer Hela cells are particularly sensitive to paeoniflorin.
2、 芍药苷类成分抑制肿瘤细胞迁移实验  2. Paeoniflorin inhibits tumor cell migration experiment
肿瘤细胞迁移检测实验结果显示, 芍药内酯苷对肠癌细胞 CT26、 宫颈癌细胞 Hela、 神 经母细胞瘤细胞 SY5Y、 乳腺癌细胞 MDA231、 乳腺癌细胞 T47D、 乳腺癌细胞 MCF7、 横纹 肌肉瘤细胞 A204、 肾癌 Wilms细胞 G401、鼻咽癌细胞 CNE2Z、前列腺癌细胞 DU145、 喉癌 细胞 Hep2、 肺癌细胞 A549、 肺癌细胞 LLC、 胰腺癌细胞 PANC1和肾上腺皮质瘤细胞 SW13 等的迁移具有显著抑制作用。 其中, 芍药内酯苷对宫颈癌细胞 Hela、 肠癌细胞 CT26、 乳腺 癌细胞 MDA231、 鼻咽癌细胞 CNE2Z和胰腺癌细胞 PANC1迁移的抑制作用尤其显著。  The results of tumor cell migration assay showed that paeoniflorin on intestinal cancer cell CT26, cervical cancer cell line Hela, neuroblastoma cell line SY5Y, breast cancer cell MDA231, breast cancer cell T47D, breast cancer cell MCF7, rhabdomyosarcoma cell A204, Renal cancer Wilms cell G401, nasopharyngeal carcinoma cell CNE2Z, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, lung cancer cell A549, lung cancer cell LLC, pancreatic cancer cell PANC1 and adrenocortical tumor cell SW13 have significant inhibition. Among them, paeoniflorin inhibited the migration of cervical cancer cell line Hela, intestinal cancer cell line CT26, breast cancer cell line MDA231, nasopharyngeal carcinoma cell line CNE2Z and pancreatic cancer cell line PANC1.
肿瘤细胞迁移检测实验结果显示,芍药苷对横纹肌肉瘤细胞 A204、鼻咽癌细胞 CNE-2Z、 肠癌细胞 CT26、 前列腺癌细胞 DU145、 喉癌细胞 Hep2、 胰腺癌细胞 PANC1和肾上腺皮质 瘤细胞 SW13 等的迁移具有显著抑制作用。 其中, 芍药 ϋ:对肠癌细胞 CT26、 鼻咽癌细胞 CNE2Z、 喉癌细胞 Hep2和肾上腺皮质瘤细胞 SW13的抑制作用尤其显著。 The results of tumor cell migration assay showed that paeoniflorin on rhabdomyosarcoma cell A204, nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1 and adrenocortical tumor cell SW13, etc. The migration has a significant inhibitory effect. Among them, paeoniflorin : the inhibitory effect on intestinal cancer cell CT26, nasopharyngeal carcinoma cell CNE2Z, laryngeal carcinoma cell Hep2 and adrenal cortical tumor cell SW13 was particularly significant.
3、 体内抗肿瘤作用  3, anti-tumor effect in vivo
裸鼠模型的体内实验结果显示,芍药内酯苷可以显著抑制 CT26肠癌、 LLC肺癌、 MDA231 乳腺癌和 Hela宫颈癌的^长和转移。特别地,芍药内酯苷对 CT26肠癌的抑制率可达 54.41%, 对 Hela宫颈癌的抑制率可达 52.3%。 In vivo experiments in nude mice showed that paeoniflorin can significantly inhibit the length and metastasis of CT26 intestinal cancer, LLC lung cancer, MDA231 breast cancer and Hela cervical cancer. In particular, the inhibition rate of paeoniflorin on CT26 intestinal cancer can reach 54.41%. The inhibition rate of Hela cervical cancer can reach 52.3%.
裸鼠模型的体内实验结果显示,芍药苷可以显著抑制 CNE-2Z鼻咽癌、 PANC1胰腺癌和 Hela宫颈癌细胞的生长和转移。  In vivo experiments in a nude mouse model showed that paeoniflorin significantly inhibited the growth and metastasis of CNE-2Z nasopharyngeal carcinoma, PANC1 pancreatic cancer, and Hela cervical cancer cells.
4、 预防肿瘤发生的作用  4, the role of prevention of tumorigenesis
采用芍药内酯苷处理基因突变剂氧化偶氮甲垸(AOM )诱导小鼠肠炎到原发肠癌转化的 实验结果显示, 芍药内酯苷在治疗肠癌癌前病变、 预防和阻断从重度肠炎到肠癌的转化过程 中发挥着显著的作用。 附图说明  The results of the experiment on the transformation of mouse enteritis to primary colorectal cancer induced by guanidinium lactone treatment of gene mutation agent AOM showed that paeoniflorin is effective in the treatment of precancerous lesions, prevention and blockade of intestinal cancer. Enteritis plays a significant role in the transformation of intestinal cancer. DRAWINGS
图 1 : 芍药内酯苷对肿瘤细胞迁移能力的影响 (Transwell检测)。 Figure 1: Effect of paeoniflorin on tumor cell migration (Transwell assay).
图 2: 芍药内酯苷对肿瘤细胞迁移能力的影响 (Scratch Analysis检测)。 Figure 2: Effect of paeoniflorin on tumor cell migration (Scratch Analysis).
图 3: 芍药苷对肿瘤细胞迁移能力的影响 (Transwell检测)。 Figure 3: Effect of paeoniflorin on tumor cell migration (Transwell assay).
图 4: 芍药内酯苷抗肿瘤的体内实验结果; 其中, Figure 4: In vivo results of anti-tumor effects of paeoniflorin;
4A: CT26肠癌裸鼠肿瘤生长曲线;  4A: tumor growth curve of CT26 intestinal cancer in nude mice;
4B: LLC肺癌裸鼠肿瘤生长曲线;  4B: tumor growth curve of LLC lung cancer nude mice;
4C: MDA231乳腺癌裸鼠肿瘤生长曲线;  4C: tumor growth curve of MDA231 breast cancer nude mice;
4D: Hela宫颈癌裸鼠肿瘤生长曲线。  4D: Tumor growth curve of Hela cervical cancer nude mice.
图 5: 芍药内酯苷对肠炎到肠癌转化的阻断作用实验结果: 其中, Figure 5: Blocking effect of paeoniflorin on enteritis-to-intestinal cancer transformation: Among them,
5A: 三组小鼠的结肠照片;  5A: photo of the colon of three groups of mice;
5B: 三组小鼠的结肠组织 HE染色结果。  5B: Results of HE staining of colon tissue in three groups of mice.
图 6: 芍药内酯苷和白芍总苷对肿瘤细胞迁移的影响比较 (Transwell检测)。 Figure 6: Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwell assay).
图 7: 芍药苷和白芍总苷对肿瘤细胞迁移的影响比较 (Transwell检测)。 具体实施方式 Figure 7: Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwell assay). detailed description
以下参照具体的实施例来说明本发明。 本领域技术人员能够理解, 这些实施例仅用于说 明本发明, 其不以任何方式限制本发明的范围。  The invention is described below with reference to specific embodiments. Those skilled in the art can understand that the examples are only intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
下述实施例中所用的芍药内酯苷和芍药苷购自上海鼎瑞化工有限公司(纯度均为 98%以 上), 白芍总苷胶囊 (帕夫林) 购自宁波立华制药有限公司。 实施例 1 芍药苷类化合物对肿瘤细胞增殖的影响  The paeoniflorin and paeoniflorin used in the following examples were purchased from Shanghai Dingrui Chemical Co., Ltd. (purity was 98% or more), and the total glucoside capsule (Pavlin) was purchased from Ningbo Lihua Pharmaceutical Co., Ltd. Example 1 Effect of Paeoniflorin on Tumor Cell Proliferation
1、 芍药内酯苷对肿瘤细胞增殖的影响 1. Effect of paeoniflorin on tumor cell proliferation
实验方案: 采 噻唑蓝 (3-(4, 5-二甲基噻唑 -2)-2, 5-二苯基四氮唑溴盐, MTT) 实验, 芍药内酯苷分别处理横纹肌肉瘤细胞 A204、 鼻咽癌细胞 CNE-2Z、 肠癌细胞 CT26、 肠癌细 胞 HT29、神经母细胞瘤细胞 SY5Y、喉癌细胞 Hep2、胰腺癌细胞 PANC 1、宫颈癌细胞 Hela、 乳腺癌细胞 MDA231、 乳腺癌细胞 T47D、 乳腺癌细胞 MCF7、 皮肤鳞癌细胞 A431、 肾癌 Wilms细胞 G401、前列腺癌细胞 DU145约 24小时;之后 MTT染色 4小时,二甲基亚砜 (DMSO) 溶解, 酶标仪 570 nm下测吸光光度值。 Experimental protocol: Triazolium blue (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, paeoniflorin treatment of rhabdomyosarcoma A204, Nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, intestinal cancer HT29, neuroblastoma cell SY5Y, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC 1, cervical cancer cell Hela, breast cancer cell MDA231, breast cancer cell T47D, breast cancer cell MCF7, skin squamous cell carcinoma A431, renal cancer Wilms Cell G401 and prostate cancer cell DU145 were for about 24 hours; after MTT staining for 4 hours, dimethyl sulfoxide (DMSO) was dissolved, and the absorbance value was measured at 570 nm.
实验结果: 发现芍药内酯苷对胰腺癌细胞 PANC1、 肠癌细胞 CT26、 肠癌细胞 HT29、 喉 癌细胞 Hep2、 宫颈癌细胞 Hela、 乳腺癌细胞 MDA231、 前列腺癌细胞 DU145、 鼻咽癌细胞 CNE2Z、横纹肌肉瘤细胞 A204、乳腺癌细胞 T47D和乳腺癌细胞 MCF7细胞的增殖都具有显 著的抑制作用。 其中, 芍药内酯苷对肠癌细胞 HT29、 胰腺癌细胞 PANC1、 喉癌细胞 Hep2、 宫颈癌细胞 Hela、 乳腺癌细胞 MDA231和前列腺癌细胞 DU145的作用最为敏感, 具体结果 见表 1。  Experimental results: Paeoniflorin was found to be pancreatic cancer cell PANC1, intestinal cancer cell line CT26, intestinal cancer cell line HT29, laryngeal cancer cell Hep2, cervical cancer cell line Hela, breast cancer cell MDA231, prostate cancer cell DU145, nasopharyngeal carcinoma cell CNE2Z, The proliferation of rhabdomyosarcoma cell A204, breast cancer cell T47D and breast cancer cell MCF7 cells all had significant inhibitory effects. Among them, paeoniflorin is most sensitive to the effects of intestinal cancer cells HT29, pancreatic cancer cells PANC1, laryngeal carcinoma cells Hep2, cervical cancer cells Hela, breast cancer cells MDA231 and prostate cancer cells DU145. The specific results are shown in Table 1.
表 1 芍药内酯苷对肿瘤细胞增殖的影响 (ic50) Table 1 Effect of paeoniflorin on tumor cell proliferation (ic 50 )
瘤株 芍药内酯苷  Tumor drug
A204 (横纹肌肉瘤细胞) 45 μ /ιυ1  A204 (rhabdomyosarcoma cells) 45 μ /ιυ1
CNE2Z (鼻咽癌细胞) 43 μ§ ηύ  CNE2Z (nasopharyngeal carcinoma cells) 43 μ§ ηύ
HT29 (肠癌细胞) 33 g/ml  HT29 (intestinal cancer cells) 33 g/ml
CT26 (肠癌细胞) 59 g/ml  CT26 (intestinal cancer cells) 59 g/ml
Hep2 (喉癌细胞) 36 g/ml  Hep2 (laryngeal cancer cell) 36 g/ml
PANC 1 (胰腺癌细胞) 30 μ^πιΐ  PANC 1 (pancreatic cancer cells) 30 μ^πιΐ
SY5Y (神经母细胞瘤细胞) 67 μ^ηιΐ  SY5Y (neuroblastoma cells) 67 μ^ηιΐ
DU145 (前列腺癌细胞) 41 μ^πιΐ  DU145 (prostate cancer cells) 41 μ^πιΐ
Hela (宫颈癌细胞) 39 g/ml  Hela (cervical cancer cells) 39 g/ml
MDA231 (乳腺癌细胞) 40 μ^η\\  MDA231 (breast cancer cells) 40 μ^η\\
T47D (乳腺癌细胞) 56 g/nil  T47D (breast cancer cells) 56 g/nil
MCF7 (乳腺癌细胞) 61 g/ml  MCF7 (breast cancer cells) 61 g/ml
A431 (皮肤鳞癌细胞) 92 g/ml  A431 (skin squamous cell carcinoma) 92 g/ml
G401 (肾癌 Wilms细胞) 108 g/ml  G401 (kidney cancer Wilms cell) 108 g/ml
2、 芍药苷对肿瘤细胞增殖的影响 2. Effect of paeoniflorin on tumor cell proliferation
实验方案: 采用噻唑蓝 (3-(4, 5-二甲基噻唑 -2)-2, 5-二苯基四氮唑溴盐, MTT) 实验, 芍药 ¾:分别处理横纹肌肉瘤细胞 A204、鼻咽癌细胞 CNE-2Z、肠癌细胞 CT26、肠癌细胞 HT29、 喉癌细胞 Hep2、 胰腺癌细胞 PANC 1、 乳腺癌细胞 MDA231、 宫颈癌细胞 Hela、 肾癌 Wilms 细胞 G401细胞和前列腺癌细胞 DU 145约 24小时,之后 MTT染色 4小时,二甲基亚砜( DMSO ) 溶解, 酶标仪 570 nm下测吸光光度值。 Experimental protocol: thiazolyl blue (3-(4,5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, peony 3⁄4 : treatment of rhabdomyosarcoma cells A204, nasal Pharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, intestinal cancer cell HT29, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC 1, breast cancer cell MDA231, cervical cancer cell Hela, renal cancer Wilms cell G401 cell and prostate cancer cell DU 145 Approximately 24 hours, followed by MTT staining for 4 hours, dimethyl sulfoxide (DMSO) Dissolved, the absorbance photometric value was measured at 570 nm.
实验结果: 发现芍药苷对喉癌细胞 Hep2、 宫颈癌细胞 Hela、 鼻咽癌细胞 CNE-2Z、 胰腺 癌细胞 PANC1和横纹肌肉瘤细胞 A204细胞的增殖都具有显著的抑制作用, 其中芍药苷对喉 癌细胞 Hep2、 宫颈癌细胞 Hda、 鼻咽癌细胞 CNE-2Z和胰腺癌细胞 PANCl细胞的作用最为 敏感, 具体结果见表 2。 表 2 芍药苷对肿瘤细胞增殖的影响 (IC5o) Experimental results: Paeoniflorin was found to have significant inhibitory effects on the proliferation of laryngeal carcinoma cell Hep2, cervical cancer Hela, nasopharyngeal carcinoma cell CNE-2Z, pancreatic cancer cell PANC1 and rhabdomyosarcoma cell A204 cells. The cells Hep2, cervical cancer cell Hda, nasopharyngeal carcinoma cell CNE-2Z and pancreatic cancer cell PANCl cells were most sensitive. The specific results are shown in Table 2. Table 2 Effect of paeoniflorin on tumor cell proliferation (IC 5 o)
细胞类型 芍药苷  Cell type paeoniflorin
A204细胞 (横纹肌肉瘤细胞) 67
Figure imgf000008_0001
A204 cells (rhizomatous muscle cells) 67
Figure imgf000008_0001
CNE-2Z细胞(鼻咽癌细胞) 50 μ^πιΐ  CNE-2Z cells (nasopharynx cancer cells) 50 μ^πιΐ
CT26细胞 (肠癌细胞) 82 πιΐ  CT26 cells (intestinal cancer cells) 82 πιΐ
Hep2细胞 (喉癌细胞) 44
Figure imgf000008_0002
Hep2 cells (laryngeal cancer cells) 44
Figure imgf000008_0002
肿瘤细胞 ώ Tumor cells ώ
PANC1细胞 (胰腺癌细胞) 53 g/ml  PANC1 cells (pancreatic cancer cells) 53 g/ml
Hela细胞 (宫颈癌细胞) 49 μ^πιΐ  Hela cells (cervical cancer cells) 49 μ^πιΐ
HT29细胞 (肠癌细胞) 96 g/ml  HT29 cells (intestinal cancer cells) 96 g/ml
MDA231细胞(乳腺癌细胞) \15μ^η\]  MDA231 cells (breast cancer cells) \15μ^η\]
G401细胞 (肾癌 Wilms细胞) 89 μ^ ιΐ  G401 cells (kidney cancer Wilms cells) 89 μ^ ιΐ
DU145细胞 (前列腺癌细胞) 97 g/ml 实施例 2 芍药苷类化合物对肿瘤细胞迁移的影响  DU145 cells (prostate cancer cells) 97 g/ml Example 2 Effect of paeoniflorin compounds on tumor cell migration
1、 芍药内酯苷对肿瘤细胞迁移的影响 (Transwell检测和 Scratch Analysis检测) 1. Effect of paeoniflorin on tumor cell migration (Transwell assay and Scratch Analysis assay)
实验方案:  Experimental program:
1)采用细胞迁移检测 (Transwell) 实验, 对 A549、 LLC、 Hela, SY5Y、 CT26、 T47D、 MCF7、 MDA23K A204、 G401、 CNE-2Z、 DU145、 Hep2、 PANCl和 SW13肿瘤细胞加药 芍药内酯苷 24小时后 (选用安全剂量 20 g/ml), 无水冰甲醇固定 20分钟, 结晶紫染色 15 分钟, 100倍光镜拍照, 检测细胞迁移的个数。  1) Using the cell migration assay (Transwell) assay, add steroidal lactones to A549, LLC, Hela, SY5Y, CT26, T47D, MCF7, MDA23K A204, G401, CNE-2Z, DU145, Hep2, PANCl and SW13 tumor cells. After 24 hours (using a safe dose of 20 g/ml), anhydrous ice-cold methanol was fixed for 20 minutes, crystal violet stained for 15 minutes, and 100 times light microscopy was taken to detect the number of cell migration.
2)采用细胞迁移检测实验 (Scratch Analysis), 对 Hela、 LLC、 A549、 SY5Y、 CT26、 MCF7、 MDA231和 T47D细胞加芍药内酯苷 6小时和 24小时 (选用安全剂量 20
Figure imgf000008_0003
), 40倍光镜拍照, 检测细胞间距。
2) Using the Scratch Analysis test, add HeLa lactones to Hela, LLC, A549, SY5Y, CT26, MCF7, MDA231 and T47D cells for 6 hours and 24 hours (using a safe dose of 20)
Figure imgf000008_0003
), taking a 40x light microscope to detect the cell spacing.
实验结果: 1) Transwell实验结果显示, 芍药内酯苷(AL)可以显著抑制肺癌细胞 A549、 肺癌细胞 LLC、 宫颈癌细胞 Hela、神经母细胞癤细胞 SY5Y、肠癌细胞 CT26、乳腺癌 T47D、 乳腺癌 MCF7、 乳腺癌 MDA231、 横纹肌肉瘤细胞 A204、 肾癌 Wilms细胞 G401、 鼻咽癌细 胞 CNE-2Z、 前列腺癌细胞 DU145、 喉癌细胞 Hep2、 胰腺癌细胞 PANC1和肾上腺皮质瘤细 胞 SW13细胞的迁移, 表现为给药组中细胞数目相对于对照组显著减少, 其中, 芍药内酯苷 对宫颈癌细胞 Hela、肠癌细胞 CT26、 乳腺癌细胞 MDA231、 鼻咽癌细胞 CNE2Z和胰腺癌细 胞 PANC1肿瘤细胞迁移的抑制作用尤其显著。 具体见图 1。 Experimental results: 1) Transwell results showed that paeoniflorin (AL) can significantly inhibit lung cancer cell A549, lung cancer cell LLC, cervical cancer cell Hela, neuroblast cell SY5Y, intestinal cancer cell CT26, breast cancer T47D, breast Cancer MCF7, breast cancer MDA231, rhabdomyosarcoma cell A204, renal cancer Wilms cell G401, nasopharyngeal carcinoma The migration of CNE-2Z, prostate cancer cell line DU145, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1 and adrenocortical tumor cell SW13 cells showed that the number of cells in the drug-administered group was significantly reduced compared with the control group, among them, paeoniflorin Inhibition of cervical cancer cell line Hela, intestinal cancer cell line CT26, breast cancer cell MDA231, nasopharyngeal carcinoma cell CNE2Z and pancreatic cancer cell PANC1 tumor cell migration is particularly significant. See Figure 1 for details.
2) Scratch Analysis实验结果显示, 相对于对照组而言, 芍药内酯苷给药组的划痕宽度相 对正常对照组均明显增大, 证明芍药内酯苷可以抑制所测肿瘤细胞的迁移。 具体见图 2。  2) The results of Scratch Analysis showed that compared with the control group, the scratch width of the paeoniflorin-administered group was significantly increased compared with the normal control group, which proved that paeoniflorin could inhibit the migration of the measured tumor cells. See Figure 2 for details.
综上所述, 实验结果提示芍药内酯苷对肿瘤细胞的迁移具有广泛的、 显著的抑制作用。  In summary, the experimental results suggest that paeoniflorin has a broad and significant inhibitory effect on tumor cell migration.
2、 芍药苷对肿瘤细胞迁移的影响 (Transwell检测) 2. Effect of paeoniflorin on tumor cell migration (Transwell assay)
实验方案: 采用细胞迁移检测 (Transwell ) 实验, 对 A204、 CNE-2Z、 CT26、 DU145、 Hep2、 PANC1和 SW13肿瘤细胞分别加入芍药苷作用 24小时(选用安全剂量 10 μΜ), 无水 冰甲醇固定 20分钟, 结晶紫染色 15分钟, 100倍光镜拍照并检测迁移细胞的个数。  Experimental protocol: Cell migration assay (Transwell) was used to add paeoniflorin to A204, CNE-2Z, CT26, DU145, Hep2, PANC1 and SW13 tumor cells for 24 hours (using a safe dose of 10 μΜ), fixed with anhydrous ice methanol. After 20 minutes, crystal violet staining for 15 minutes, photographing 100 times light microscopy and detecting the number of migrated cells.
实验结果: 芍药苷可以显著抑制横纹肌肉瘤细胞 Α204、 鼻咽癌细胞 CNE-2Z、 肠癌细胞 CT26、前列腺癌细胞 DU145、喉癌细胞 Hep2、胰腺癌细胞 PANC1和肾上腺皮质瘤细胞 SW13 肿瘤细胞迁移, 表现为给药组中细胞数目相对于对照组显著减少。 其中, 芍药苷对肠癌细胞 CT26、鼻咽癌细胞 CNE2Z、喉癌细胞 Hep2和肾上腺皮质瘤细胞 SW13的抑制作用尤其显著。 具体见图 3。 实施例 3 芍药苷类化合物体内抗肿瘤作用  Experimental results: Paeoniflorin can significantly inhibit the migration of rhabdomyosarcoma cell Α204, nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, prostate cancer cell DU145, laryngeal carcinoma cell Hep2, pancreatic cancer cell PANC1 and adrenal cortical tumor cell SW13. It was shown that the number of cells in the administration group was significantly reduced relative to the control group. Among them, paeoniflorin inhibited intestinal cancer cell line CT26, nasopharyngeal carcinoma cell line CNE2Z, laryngeal carcinoma cell line Hep2 and adrenocortical tumor cell line SW13. See Figure 3 for details. Example 3 Antitumor effect of paeoniflorin compounds in vivo
1、 芍药内酯苷体内抗肿瘤作用 1. Antitumor effect of paeoniflorin in vivo
实验方案: 采用瘤块接种技术, 在裸鼠右侧腋窝皮下接种实体瘤块, 共 CT26、 LLC, MDA231和 Hela四种。每种实体瘤移植模型裸鼠随机分为 3组: 对照组(指接种瘤块而不给 药)、 芍药内酯苷预先给药组(指接种瘤块的当天给药)和芍药内酯苷给药组(指成瘤体积达 70-100 mm3以上时给药), 每组 5-6只裸鼠。 Experimental protocol: The tumor block inoculation technique was used to inoculate solid tumor blocks under the skin of the right axilla of nude mice, including CT26, LLC, MDA231 and Hela. Nude mice of each solid tumor transplantation model were randomly divided into 3 groups: control group (inoculated with tumor mass without administration), pre-dosing group of paeoniflorin (referring to the day of inoculation of tumor block) and paeoniflorin The drug-administered group (administered when the tumor-forming volume reached 70-100 mm 3 or more), 5-6 nude mice per group.
芍药内酯苷预先给药组在接种瘤块当天腹腔注射给药, 剂量为 200mg/kg, 给药频率为 2 天一次,共给药 3周,给药结束后第二天处死小鼠。芍药内酯苷给药组,待瘤体积达 70-100 mm3 以上时, 腹腔注射芍药内酯苷, 剂量为 300 mg/kg, 给药频率为 2天一次, 共给药 2周, 给药 结束后第二天处死小鼠。 检测肿瘤体积和重量, 计算抑瘤率。 The pre-administration group of paeoniflorin was intraperitoneally administered on the day of inoculation of the tumor block at a dose of 200 m g/kg, the frequency of administration was once every 2 days for 3 weeks, and the mice were sacrificed the day after the end of the administration. . In the paeoniflorin administration group, when the tumor volume reached 70-100 mm 3 or more, the paeoniflorin was administered intraperitoneally at a dose of 300 mg/kg, and the administration frequency was once every 2 days for 2 weeks. Mice were sacrificed the day after the end. Tumor volume and weight were measured and the tumor inhibition rate was calculated.
实验结果: 在肠癌、 肺癌、 乳腺癌、 宫颈癌四种不同的肿瘤动物模型中, 芍药内酯苷预 先给药组、 芍药内酯苷给药组均能明显抑制肿瘤细胞的生长和转移, 尤其是对 CT26肠癌和 Hela宫颈癌模型的效果极为显著。 具体抗肿瘤结果见表 3A、 表 3B、 表 3C和表 3D。  Experimental results: In the four different tumor animal models of colon cancer, lung cancer, breast cancer and cervical cancer, the pre-administration group of paeoniflorin and the paeoniflorin administration group can significantly inhibit the growth and metastasis of tumor cells. Especially for the CT26 intestinal cancer and Hela cervical cancer models, the effect is extremely significant. The specific anti-tumor results are shown in Table 3A, Table 3B, Table 3C and Table 3D.
① CT26肠癌模型实验结果 (见图 4A ):  1 CT26 colon cancer model experimental results (see Figure 4A):
A. CT26肿瘤的牛.长在 12天左右即达到实验要求, 即体积达到 100mm3; B. 与对照组比较, 芍药内酯苷预先给药组、 给药组中肿瘤体积和重量均明显减小, 表明 芍药内酯苷对肠癌具有显著的预防和治疗作用。 A. CT26 tumor cattle. The length of the experiment is about 12 days, that is, the volume reaches 100mm 3 ; B. Compared with the control group, the tumor volume and weight of the paeoniflorin pre-administered group and the administered group were significantly reduced, indicating that paeoniflorin has significant preventive and therapeutic effects on intestinal cancer.
② LLC肺癌模型实验结果 (附图 4B): 2 LLC Lung Cancer Model Experimental Results (Fig. 4B) :
A. LLC肿瘤和 CT26肿瘤的生长速度相当, 体积大小达到 100 mm3均为 12天左右;The growth rate of A. LLC tumor and CT26 tumor is equivalent, and the volume reaches 100 mm 3 and is about 12 days;
B.与对照组比较, 芍药内酯苷预先给药组、给药组中肿瘤体积和重量均明显减小, 表明 芍药内酯苷具有一定的预防和治疗肺癌效果。 B. Compared with the control group, the tumor volume and weight in the pre-administration group and the drug-administered group were significantly reduced, indicating that the paeoniflorin has certain preventive and therapeutic effects on lung cancer.
③ MDA231乳腺癌模型实验结果 (附图 4C):  3 MDA231 breast cancer model experiment results (Figure 4C):
A. MDA231细胞为人源的, 相对于 LLC和 CT26肿瘤而言, 在裸鼠皮下成瘤时间较慢, 肿瘤生长速度也相对较慢, 体积大小达到 70 mm3约为 18天左右; A. MDA231 cells are human. Compared with LLC and CT26 tumors, the tumor formation time in nude mice is slower, and the tumor growth rate is relatively slow. The volume reaches 70 mm 3 and is about 18 days.
B. 与对照组比较, 芍药内酯苷预先给药组、给药组中肿瘤体积和重量均明显减小, 表明 芍药内酯苷具有一定的预防和治疗乳腺癌效果。  B. Compared with the control group, the tumor volume and weight of the paeoniflorin pre-administered group and the administered group were significantly reduced, indicating that paeoniflorin has certain preventive and therapeutic effects on breast cancer.
④ Hela宫颈癌模型实验结果 (附图 4D):  4 Hela cervical cancer model experimental results (Figure 4D):
A. Hela细胞为人源的, 相对于 LLC和 CT26肿瘤而言, 在裸鼠皮下成瘤时间较慢, 肿瘤 生长速度也相对较慢, 体积大小达到 70 mm3约为 20天左右; A. Hela cells are human. Compared with LLC and CT26 tumors, the tumor formation time in nude mice is slower, and the tumor growth rate is relatively slow. The volume reaches 70 mm 3 and is about 20 days.
B. 与对照组比较, 芍药内酯苷预先给药组、给药组中肿瘤体积和重量均明显减小, 表明 芍药内酯苷对宫颈癌具有显著的预防和治疗作用。  B. Compared with the control group, the tumor volume and weight of the paeoniflorin pre-administered group and the administered group were significantly reduced, indicating that paeoniflorin has significant preventive and therapeutic effects on cervical cancer.
芍药内酯苷体内抗肿瘤作用实验结果 (CT26肠癌模型)  Experimental results of anti-tumor effect of paeoniflorin in vivo (CT26 colon cancer model)
Figure imgf000010_0001
Figure imgf000010_0001
与对照组比, P < 0.01。 芍药内酯苷体内抗肿瘤作用实验结果 (LLC肺癌模型)  Compared with the control group, P < 0.01. Experimental results of anti-tumor effect of paeoniflorin in vivo (LLC lung cancer model)
Figure imgf000010_0002
Figure imgf000010_0002
: 与对照组比, Ρ < 0.01 ;  : compared with the control group, Ρ < 0.01 ;
*: 与对照组比, Ρ < 0.05„ 芍药内酯苷体内抗肿瘤作用实验结果 (MDA231乳腺癌模型) *: Compared with the control group, Ρ < 0.05 Experimental results of anti-tumor effect of paeoniflorin in vivo (MDA231 breast cancer model)
Figure imgf000011_0001
Figure imgf000011_0001
与对照组比, P < 0.01。 芍药内酯苷体内抗肿瘤作用实验结果 (Hela宫颈癌模型)  Compared with the control group, P < 0.01. Experimental results of anti-tumor effect of paeoniflorin in vivo (Hela cervical cancer model)
Figure imgf000011_0002
Figure imgf000011_0002
与对照组比, P < 0.01。  Compared with the control group, P < 0.01.
2、 芍药苷体内抗肿瘤作用  2, anti-tumor effect of paeoniflorin in vivo
实验方案: 选择芍药苷体外抗肿瘤的优势瘤种——胰腺癌 PANC1细胞和宫颈癌 Hela细 胞, 建立裸鼠皮下荷瘤模型。待肿瘤体积达到 70mm3后腹腔注射芍药苷, 剂量为 300 mg/kg, 给药频率为 2天一次, 共给药 2周, 以模型组为对照。 给药结束后第二天处死小鼠。 检测肿 瘤体积和重量, 计算抑瘤率。 Experimental protocol: The dominant tumors of paeoniflorin in vitro, pancreatic cancer PANC1 cells and cervical cancer Hela cells, were selected to establish a subcutaneous tumor-bearing model in nude mice. After the tumor volume reached 70 mm 3 , paeoniflorin was intraperitoneally injected at a dose of 300 mg/kg, and the frequency of administration was once every 2 days for 2 weeks. The model group was used as a control. Mice were sacrificed the day after the end of dosing. Tumor volume and weight were measured and the tumor inhibition rate was calculated.
实验结果: 在胰腺癌和宫颈癌两种肿瘤动物模型中, 芍药苷给药组均能明显抑制肿瘤细 胞的生长和转移, 表明芍药苷对胰腺癌和宫颈癌细胞均有显著抑制作用。 具体结果见表 4A 和表 4B。  Experimental results: In the two animal models of pancreatic cancer and cervical cancer, the paeoniflorin administration group can significantly inhibit the growth and metastasis of tumor cells, indicating that paeoniflorin has significant inhibitory effects on pancreatic cancer and cervical cancer cells. The specific results are shown in Table 4A and Table 4B.
表 4A 芍药苷体内抗肿瘤作用实验结果 (PANC 1胰腺癌模型)  Table 4A Experimental results of anti-tumor effects of paeoniflorin in vivo (PANC 1 pancreatic cancer model)
对照组 芍药苷给药组 剂量 (mg/kg) 300 mg/ g  Control group Paeoniflorin administration group dose (mg/kg) 300 mg/g
裸鼠体重 (g) 20.58 ± 0.49 20.43士 0.51 瘤重 (g) 2.11 ± 0.47 1.36士 0.28* 抑瘤率 (o/< 35.55 Body weight of nude mice (g) 20.58 ± 0.49 20.43 ± 0.51 tumor weight (g) 2.11 ± 0.47 1.36 ± 0.28 * Tumor inhibition rate (o/< 35.55)
与对照组比, P < 0.01 芍药苷体内抗肿瘤作用实验结果 (Hela宫颈癌模型)  Compared with the control group, P < 0.01 anti-tumor effect test results of paeoniflorin (Hela cervical cancer model)
对照组 芍药苷给药组  Control group paeoniflorin administration group
齐 U量 (mg/kg) 300 mg/Kg  U amount (mg/kg) 300 mg/Kg
裸鼠体重 (g) 20.94±0.58 20.77土 0.32  Nude mouse weight (g) 20.94 ± 0.58 20.77 soil 0.32
瘤重 (g) 1.78 ± 0.19 1.34 ± 0.25" 抑瘤率(%) 24.04  Tumor weight (g) 1.78 ± 0.19 1.34 ± 0.25" tumor inhibition rate (%) 24.04
与对照组比, P < 0.05。 实施例 4 芍药内酯苷对肠癌癌前病变的治疗作用  Compared with the control group, P < 0.05. Example 4 Therapeutic effect of paeoniflorin on precancerous lesions of colorectal cancer
实验方案: BALB/C小鼠分为 3组: 正常组, 模型组和芍药内酯苷干预组, 每组 5只。 模型组和干预组小鼠腹腔注射基因突变剂氧化偶氮甲烷 (AOM, 20 mg/ g)。 芍药内酯苷干 预组在 AOM注射的第 2天腹腔注射给药 (100 mg/Kg), 两天一次, 共 3次。 模型组无任何 药物处理。 AOM给药一周后用 3%硫酸葡聚糖钠 (DSS )取代正常饮用水, 小鼠连续饮用 7 天; 之后换回正常饮用水, 小鼠连续饮用 14天。 从 AOM注射、 饮用 DSS水到饮用正常水 14天的周期共重复 3次, 芍药内酯苷干预组在 3个周期中都正常给药。 正常组小鼠正常喂食 和饮水, 不加任何处理。 第 3周期结束时, 处死小鼠, 剖出肠组织, 制备病理切片, 进行 HE 染色。  Experimental protocol: BALB/C mice were divided into 3 groups: normal group, model group and paeoniflorin intervention group, 5 in each group. The model group and the intervention group were intraperitoneally injected with the gene mutation agent oxidized azomethane (AOM, 20 mg/g). The paeoniflorin dry group was administered intraperitoneally (100 mg/Kg) on the 2nd day of AOM injection, once every two days for 3 times. The model group did not have any drug treatment. One week after AOM administration, 3% sodium dextran sulfate (DSS) was used to replace normal drinking water. The mice were continuously consumed for 7 days; after that, they were returned to normal drinking water, and the mice were continuously consumed for 14 days. The cycle of AOM injection, drinking DSS water to drinking normal water for 14 days was repeated three times, and the paeoniflorin intervention group was normally administered in three cycles. Normal mice were fed and watered normally without any treatment. At the end of the third cycle, the mice were sacrificed, the intestinal tissues were dissected, pathological sections were prepared, and HE staining was performed.
实验结果: 外观显示, 正常小鼠的结肠粗细均匀, 黏膜没有充血。 相对于正常小鼠, 模 型组小鼠结肠黏膜充血明显, 肠道粗细不均, 且己经生成若干个肿瘤; 而芍药内酯苷干预组 小鼠结肠粗细较为均匀, 无明显出血点, 也未见肿瘤生成, 具体见附图 5A。  Experimental results: The appearance showed that the colon of normal mice was uniform in thickness and the mucosa was not congested. Compared with normal mice, the mice in the model group had obvious colonic mucosal congestion, uneven intestinal thickness, and several tumors had been formed. However, the mice in the paeoniflorin intervention group had a uniform colon thickness and no obvious bleeding points. See tumor formation, see Figure 5A for details.
病理切片 HE染色结果显示, 正常小鼠肠组织腺体结构完整且分布均勾, 无明显炎性细 胞浸润, 无黏膜下出血和溃疡现象。 模型组小鼠肠组织出现黏膜溃疡, 多个腺体缺失伴炎性 细胞浸润, 癌细胞浸润至肠黏膜层和黏膜下层, 呈现原位肠癌的典型特征。 而芍药内酯 :T 预组小鼠肠组织虽出现灶性炎性细胞浸润, 个别腺体缺失, 但无明显癌细胞浸润现象, 显示 肠炎的典型特征, 具体见附图 5B。 The HE staining results showed that the glandular structure of normal mouse gland was intact and distributed, no obvious inflammatory cell infiltration, no submucosal hemorrhage and ulceration. In the model group, mucosal ulcers appeared in the intestinal tissues of mice, multiple glandular deletions accompanied by inflammatory cell infiltration, and cancer cells infiltrated into the intestinal mucosa and submucosa, presenting typical features of intestinal cancer in situ. And the paeoniflorin : T pre-group mouse intestinal tissue showed focal inflammatory cell infiltration, individual gland loss, but no obvious cancer cell infiltration, showing the typical characteristics of enteritis, see Figure 5B.
结果显示, 芍药内酯苷在小鼠从 ffi度肠炎到肠癌的转化过程中发挥着有效的阻断作 ffl, 对治疗癌前病变具有重要作用。 实施例 5 芍药苷类成分与白芍总苷抑制肿瘤的效果比较 1 > 芍药内酯苷和白芍总苷对肿瘤细胞增殖的影响比较 (MTT) The results showed that paeoniflorin played an important role in the transformation of ffi degree enteritis to intestinal cancer, which plays an important role in the treatment of precancerous lesions. Example 5 Comparison of the effects of paeoniflorin components and total glucosides of paeony on tumor suppression 1 > Comparison of effects of paeoniflorin and total glucosides of paeony on tumor cell proliferation (MTT)
实验方案: 采用噻唑蓝 (3-(4, 5-二甲基噻唑 -2)-2, 5-二苯基四氮唑溴盐, MTT) 实验, 芍药内酯苷和白芍总苷分别处理 A204、 CNE-2Z、 CT26、 Hep2和 PANC l肿瘤细胞 24小时, 之后 MTT染色 4小时, 二甲基亚砜(DMSO )溶解, 酶标仪 570 nm下测吸光光度值。  Experimental protocol: thiazolyl blue (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, paeoniflorin and paeoniflorin treatment A204, CNE-2Z, CT26, Hep2 and PANC l tumor cells were used for 24 hours, then MTT staining for 4 hours, dimethyl sulfoxide (DMSO) was dissolved, and the absorbance value was measured at 570 nm.
实验结果: 发现芍药内酯苷和白芍总苷对横纹肌肉瘤细胞 A204、 鼻咽癌细胞 CNE-2Z、 肠癌细胞 CT26、 喉癌细胞 Hep2和胰腺癌细胞 PANCl的增殖都具有抑制作用, 但是芍药内 酯苷对这些肿瘤的 IC5o (半数抑制率)值全部低于白芍总苷, 说明芍药内酯苷对横纹肌肉瘤 细胞 A204、 鼻咽癌细胞 CNE-2Z、 结肠癌细胞 CT26、 喉癌细胞 Hep2和胰腺癌细胞 PANC l 肿瘤细胞增殖的抑制作用强于白芍总苷。 具体结果见表 5。 Experimental results: It was found that paeoniflorin and total glucosides of paeony inhibited the proliferation of rhabdomyosarcoma cell line A204, nasopharyngeal carcinoma cell line CNE-2Z, intestinal cancer cell line CT26, laryngeal carcinoma cell line Hep2 and pancreatic cancer cell line PANCl. The IC 5 o (half inhibition rate) values of lactones on these tumors were all lower than total glucosides of paeony, indicating that paeoniflorin on rhabdomyosarcoma cell A204, nasopharyngeal carcinoma cell CNE-2Z, colon cancer cell CT26, laryngeal carcinoma The inhibition of cell Hep2 and pancreatic cancer cell PANC l tumor cell proliferation is stronger than that of total glucosides of paeony. The specific results are shown in Table 5.
表 5芍药内酯苷和白芍总苷对肿瘤细胞增殖的影响 (IC50) Table 5 Effect of paeoniflorin and total glucosides of paeony on tumor cell proliferation (IC 50 )
瘤种 芍药内酯苷 白芍总苷  Paeoniflorin
A204 (横纹肌肉瘤细胞) 45 g/ml 61 g/ml  A204 (rhabdomyosarcoma cells) 45 g/ml 61 g/ml
CNE-2Z (鼻咽癌细胞) 43 g/ml 64 μ^πιΐ  CNE-2Z (Nasopharyngeal Carcinoma) 43 g/ml 64 μ^πιΐ
月中瘤细胞 CT26 (肠癌细胞) 59
Figure imgf000013_0001
67 g/ml
Mid-month tumor cell CT26 (intestinal cancer cells) 59
Figure imgf000013_0001
67 g/ml
Hep2 (喉癌细胞) 36 g/ml 39 μ^ι \  Hep2 (laryngeal cancer cell) 36 g/ml 39 μ^ι \
PANC1 (胰腺癌细胞) 30 μ§ πι\ 41 μ^πιΐ  PANC1 (pancreatic cancer cells) 30 μ§ πι\ 41 μ^πιΐ
2、 芍药内酯苷和白芍总苷对肿瘤细胞迁移的影响比较 (Transwdl ) 2. Comparison of effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwdl)
实验方案: 采用细胞迁移检测 (Transwell ) 实验, 对 A204、 CNE-2Z、 CT26、 DU145. Hep2、 PANCl和 SW13肿瘤细胞分别加入芍药内酯苷和白芍总苷作用 24小时 (选用安全剂 量 10 μΜ), 无水冰甲醇固定 20分钟, 结晶紫染色 15分钟, 100倍光镜拍照并检测迁移细胞 的个数。  Experimental protocol: Using the cell migration assay (Transwell) experiment, the A204, CNE-2Z, CT26, DU145. Hep2, PANCl and SW13 tumor cells were added with paeoniflorin and total glucosides for 24 hours (using a safe dose of 10 μΜ). ), anhydrous ice-methanol was fixed for 20 minutes, crystal violet stained for 15 minutes, photographed by a 100-fold light microscope and the number of migrated cells was detected.
实验结果: 芍药内酯苷和白芍总苷都可以显著抑制对横纹肌肉瘤细胞 Α204、鼻咽癌细胞 CNE-2Z、 肠癌细胞 CT26、 前列腺癌细胞 DU145、 喉癌细胞 HeP2、 胰腺癌细胞 PANC l和肾 上腺皮质瘤 SW13肿瘤细胞迁移, 表现为给药组中细胞数目相对于对照组显著减少; 但芍药 内酯苷对横纹肌肉瘤细胞 A204、鼻咽癌细胞 CNE-2Z、肠癌细胞 CT26、前列腺癌细胞 DU145、 喉癌细胞 Hep2、 胰腺癌细胞 PANC 1和肾上腺皮质瘤 SW13肿瘤细胞迁移的抑制作用全部强 过白芍总 ¾:。 具体见图 6。 Experimental results: Both paeoniflorin and total glucosides of paeoniflorin can significantly inhibit rhabdomyosarcoma cells 204, nasopharyngeal carcinoma cells CNE-2Z, intestinal cancer cells CT26, prostate cancer cells DU145, laryngeal cancer cells He P 2, pancreatic cancer cells The migration of PANC l and adrenocortical tumor SW13 tumor cells showed that the number of cells in the drug-administered group was significantly lower than that of the control group; but the drug of paeoniflorin on rhabdomyosarcoma cell A204, nasopharyngeal carcinoma cell CNE-2Z, intestinal cancer cell CT26, prostate cancer DU145, laryngeal carcinoma cells Hep2, inhibition of pancreatic cancer cell PANC 1 adrenal tumor cell migration and tumor SW13 stronger than all TGP ¾:. See Figure 6 for details.
3、 芍药苷和白芍总苷对肿瘤细胞增殖的影响比较 (MTT ) 3. Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell proliferation (MTT)
实验方案: 采用噻唑蓝 (3-(4, 5-二甲基噻唑 -2)-2, 5-二苯基四氮唑溴盐, MTT) 实验, 芍药苷和白芍总苷分别处理 CNE-2Z、 Hela和 HT29肿瘤细胞 24小时, MTT染色 4小时, 二 甲基亚砜 (DMSO) 溶解, 酶标仪 570 nm下测吸光光度值。 Experimental protocol: Using thiazolyl blue (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide, MTT) experiment, paeoniflorin and total glucosides of paeony treated CNE- 2Z, Hela and HT29 tumor cells for 24 hours, MTT staining for 4 hours, two Methyl sulfoxide (DMSO) was dissolved, and the absorbance value was measured at 570 nm.
实验结果: 发现芍药苷和白芍总苷对鼻咽癌细胞 CNE-2Z、 宫颈癌细胞 Hela和肠癌细胞 Experimental results: It was found that paeoniflorin and total glucosides of paeony on nasopharyngeal carcinoma cells CNE-2Z, cervical cancer cells Hela and intestinal cancer cells
HT29的增殖都具有抑制作用, 但是芍药苷对鼻咽癌细胞 CNE2Z、 宫颈癌细胞 Hela和肠癌细 胞 HT29的 IC5o (半数抑制率) 全部低于白芍总苷, 说明芍药苷对这几种肿瘤细胞增殖的抑 制作用强于白芍总苷。 具体结果见表 6。 The proliferation of HT29 has an inhibitory effect, but the IC 5 o (half inhibition rate) of paeoniflorin on nasopharyngeal carcinoma cell line CNE2Z, cervical cancer cell line Hela and intestinal cancer cell line HT29 is lower than total glucosides of paeony, indicating that paeoniflorin The inhibition of tumor cell proliferation is stronger than that of total glucosides of paeony. The specific results are shown in Table 6.
表 6 芍药苷和白芍总苷对肿瘤细胞增殖的影响 (IC50) Table 6 Effect of paeoniflorin and total glucosides of paeony on tumor cell proliferation (IC 50 )
mm. 苟药苷 白芍总苷  Mum. paeoniflorin
CNE-2Z (鼻咽癌细胞) 50 μ≠η\ 64 μ^τ \  CNE-2Z (nasopharynx cancer cells) 50 μ≠η\ 64 μ^τ \
肿瘤细胞 Hela (宫颈癌细胞) 49 μ^ηιΐ 55 μβ/ιη1 Tumor cell Hela (cervical cancer cell) 49 μ^ηιΐ 55 μβ /ιη1
ΗΤ29 (肠癌细胞) 96 μ^πιΐ 137 g/ml  ΗΤ29 (intestinal cancer cells) 96 μ^πιΐ 137 g/ml
4、 芍药苷和白芍总苷对肿瘤细胞迁移的影响比较 (Transwell ) 4. Comparison of the effects of paeoniflorin and total glucosides of paeony on tumor cell migration (Transwell)
实验方案: 采用细胞迁移检测 (Transwell ) 实验, 对 HT29、 DU145和 Hela肿瘤细胞分 别加入芍药苷和白芍总苷作用 24小时(选用安全剂量 10 μΜ), 无水冰甲醇固定 20分钟, 结 晶紫染色 15分钟, 100倍光镜拍照, 检测迁移细胞的个数。  Experimental protocol: Using the cell migration assay (Transwell) experiment, the HT29, DU145 and Hela tumor cells were added with paeoniflorin and total glucosides of paeony for 24 hours (using a safe dose of 10 μΜ), fixed with anhydrous ice methanol for 20 minutes, crystal violet The staining was performed for 15 minutes, and a 100-fold light microscope photograph was taken to detect the number of migrated cells.
实验结果: 芍药苷和白芍总苷都可以显著抑制肠癌细胞 ΗΤ29、 前列腺癌细胞 DU145和 宫颈癌细胞 Hela肿瘤细胞迁移, 表现为给药组中细胞数目相对于对照组显著减少; 但芍药苷 对肠癌细胞 HT29、前列腺癌细胞 DUM5和宫颈癌细胞 Hela肿瘤细胞迁移的抑制作用强过白 芍总苷。 具体见图 7。 ' 总结, 芍药内酯苷能够有效抑制肠癌、 肺癌、 胰腺癌、 喉癌、 宫颈癌、 乳腺癌、 前列腺 癌、 鼻咽癌、 横纹肌肉瘤、 肺癌、 祌经母细胞瘤、 肾癌和肾上腺皮质瘤的生长和转移, 特别 能够有效抑制肠癌、 胰腺癌、 喉癌、 宫颈癌、 乳腺癌和前列腺癌的生长和转移; 芍药苷能够 有效抑制喉癌、 宫颈癌、 鼻咽癌、 胰腺癌和横纹肌肉瘤的生长和转移, 特别能够有效抑制喉 癌、 宫颈癌、 鼻咽癌和胰腺癌的生长和转移。 芍药内酯苷和芍药苷治疗的优势瘤种存在一定 差异。 芍药内酯苷还能够有效治疗肠癌癌前病变, 明显阻断重度溃疡性肠炎向肠癌的转化。 因此, 体内、 外实验结果综合表明, 芍药苷类化合物能有效地抑制多种肿瘤的生长和转移, 具有广谱的抗肿瘤作用; 芍药苷类化合物还能够治疗肠癌癌前病变, 阻断重度肠炎向肠癌的 转化。  Experimental results: Both paeoniflorin and total glucosides of paeony can significantly inhibit the migration of intestinal cancer cells ΗΤ29, prostate cancer cells DU145 and cervical cancer Hela tumor cells, which showed that the number of cells in the drug-administered group was significantly reduced compared with the control group; The inhibitory effect on the migration of intestinal cancer cells HT29, prostate cancer cells DUM5 and cervical cancer Hela tumor cells is stronger than that of total glucosides of paeony. See Figure 7 for details. ' Summary, paeoniflorin can effectively inhibit intestinal cancer, lung cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, prostate cancer, nasopharyngeal carcinoma, rhabdomyosarcoma, lung cancer, sputum mesothelioma, kidney cancer and adrenal cortex Tumor growth and metastasis can effectively inhibit the growth and metastasis of colorectal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer and prostate cancer; paeoniflorin can effectively inhibit laryngeal cancer, cervical cancer, nasopharyngeal cancer, pancreatic cancer and The growth and metastasis of rhabdomyosarcoma is particularly effective in inhibiting the growth and metastasis of laryngeal, cervical, nasopharyngeal and pancreatic cancers. There are some differences in the dominant tumor species treated with paeoniflorin and paeoniflorin. Paeoniflorin can also effectively treat precancerous lesions of colorectal cancer and significantly block the conversion of severe ulcerative enteritis to intestinal cancer. Therefore, the results of in vitro and in vivo experiments show that paeoniflorin can effectively inhibit the growth and metastasis of a variety of tumors, and has a broad-spectrum anti-tumor effect; paeoniflorin compounds can also treat precancerous lesions of intestinal cancer, blocking severe The conversion of enteritis to intestinal cancer.

Claims

权 利 要 求 Rights request
1、 芍药苷类化合物或其药学上可接受的盐、溶剂化物、 多晶型体、对映体或外消旋混合物在 制备预防和 /或治疗肿瘤的药物中的用途。  A use of a paeoniflorin compound or a pharmaceutically acceptable salt, solvate, polymorph, enantiomer or racemic mixture thereof for the manufacture of a medicament for the prevention and/or treatment of a tumor.
2、根据权利要求 1所述的用途, 其特征在于: 所述芍药苷类化合物选自结构式 1的芍药内酯 苷和 /或结构式 2的芍药苷- The use according to claim 1, wherein the paeoniflorin compound is selected from the group consisting of paeoniflorin of structural formula 1 and/or paeoniflorin of formula 2 -
Figure imgf000015_0001
Figure imgf000015_0001
3、 根据权利要求 1或 2所述的用途, 其特征在于: 所述肿瘤为实体肿瘤。 3. Use according to claim 1 or 2, characterized in that the tumor is a solid tumor.
4、根据权利要求 1至 3中任一项所述的用途,其特征在于:所述肿瘤选自肠癌、肺癌、肝癌、 神经母细胞瘤、 脑瘤、 乳腺癌、 宫颈癌、 横纹肌肉瘤、 鼻咽癌、 胰腺癌、 喉癌、 前列腺癌、 肾癌、 肾上腺皮质瘤、 皮肤癌、 黑色素瘤、 卵巢癌、 膀胱癌、 淋巴癌和胃癌中的一种或多种; 优选的, 所述肿瘤选自肠癌、 肺癌、 神经母细胞瘤、 乳腺癌、 宫颈癌、 横纹肌肉瘤、 鼻 咽癌、 胰腺癌、 喉癌、 前列腺癌、 肾癌和肾上腺皮质瘤中的一种或多种。 The use according to any one of claims 1 to 3, wherein the tumor is selected from the group consisting of colon cancer, lung cancer, liver cancer, neuroblastoma, brain tumor, breast cancer, cervical cancer, rhabdomyosarcoma, One or more of nasopharyngeal carcinoma, pancreatic cancer, laryngeal cancer, prostate cancer, renal cancer, adrenal cortex, skin cancer, melanoma, ovarian cancer, bladder cancer, lymphoma, and gastric cancer; preferably, the tumor One or more selected from the group consisting of intestinal cancer, lung cancer, neuroblastoma, breast cancer, cervical cancer, rhabdomyosarcoma, nasopharyngeal cancer, pancreatic cancer, laryngeal cancer, prostate cancer, renal cancer, and adrenocortical tumor.
5、 根据权利要求 1所述的用途, 其特征在于: 所述芍药苷类化合物为芍药内酯苷, 所述肿瘤 选自肠癌、 胰腺癌、 喉癌、 宫颈癌、 乳腺癌和前列腺癌中的一种或多种。 5. The use according to claim 1, wherein: the paeoniflorin compound is a paeoniflorin, and the tumor is selected from the group consisting of intestinal cancer, pancreatic cancer, laryngeal cancer, cervical cancer, breast cancer, and prostate cancer. One or more.
6、根据权利要求 1至 5任一项所述的用途, 其特征在于: 所述肠癌选自结肠癌、 直肠癌和大 肠癌中的一种或多种。 The use according to any one of claims 1 to 5, characterized in that the intestinal cancer is selected from one or more of colon cancer, rectal cancer and colorectal cancer.
7、 根据权利耍求 1所述的用途, 其特征在于: 所述芍药苷类化合物为芍药苷, 所述肿瘤选自 喉癌、 宫颈癌、 鼻咽癌和胰腺癌中的一种或多种。 7. The use according to claim 1, wherein: the paeoniflorin compound is paeoniflorin, and the tumor is selected from one or more of laryngeal cancer, cervical cancer, nasopharyngeal cancer, and pancreatic cancer. .
8、根据权利要求 1至 3中任 -项所述的用途,其特征在于:所述预防肿瘤是指治疗癌前病变; 优选的, 所述预防肿瘤是指治疗肠癌癌前病变, 预防重症肠炎转变为肠癌。 The use according to any one of claims 1 to 3, characterized in that the prevention of tumor refers to the treatment of precancerous lesions; Preferably, the preventing tumor refers to treating precancerous lesions of intestinal cancer and preventing the conversion of severe enteritis into intestinal cancer.
9、根据权利要求 1至 8中任一项所述的用途, 其特征在于: 所述药物为临床上可接受的任一 剂型, 包括经胃肠道给药制剂和非经胃肠道给药制剂; Use according to any one of claims 1 to 8, characterized in that the medicament is in any clinically acceptable dosage form, including gastrointestinal administration and parenteral administration. Formulation
优选的, 所述经胃肠道给药制剂任选自散剂、 片剂、 颗粒剂、 胶囊剂、 滴丸、 乳剂或 混悬剂;  Preferably, the gastrointestinal administration preparation is selected from the group consisting of a powder, a tablet, a granule, a capsule, a dropping pill, an emulsion or a suspension;
优选的, 所述非经胃肠道给药制剂任选自注射剂、 喷雾剂、 栓剂、 灌注剂、 贴剂或软膏 剂。  Preferably, the parenteral administration preparation is selected from the group consisting of an injection, a spray, a suppository, a perfusion, a patch or an ointment.
10、 根据权利要求 1至 9任一项所述的用途, 其特征在于: 芍药内酯苷和芍药苷分别作为所 述预防和 /或治疗肿瘤药物的唯一活性成分,或共同作为所述预防和 /或治疗肿瘤药物的活性成 分, 或分别或共同与其它物质一起制备所述预防和 /或治疗肿瘤的药物。 The use according to any one of claims 1 to 9, characterized in that paeoniflorin and paeoniflorin are the sole active ingredients of the preventive and/or therapeutic tumor drug, respectively, or together as the prevention and / or treating the active ingredient of a tumor drug, or separately or together with other substances to prepare the drug for preventing and/or treating a tumor.
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