WO2004009106A1 - Preparation extracted from stephania tetrandra s. moor and the use thereof - Google Patents

Preparation extracted from stephania tetrandra s. moor and the use thereof Download PDF

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Publication number
WO2004009106A1
WO2004009106A1 PCT/CN2002/000505 CN0200505W WO2004009106A1 WO 2004009106 A1 WO2004009106 A1 WO 2004009106A1 CN 0200505 W CN0200505 W CN 0200505W WO 2004009106 A1 WO2004009106 A1 WO 2004009106A1
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Prior art keywords
preparation
acid
tetrandrine
powder
solid
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PCT/CN2002/000505
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French (fr)
Chinese (zh)
Inventor
Zhimin Wang
Zuguang Ye
Aihua Liang
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Institute Of Chinese Materia Medica, China Academy Of Traditional Chinese Medicine
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Application filed by Institute Of Chinese Materia Medica, China Academy Of Traditional Chinese Medicine filed Critical Institute Of Chinese Materia Medica, China Academy Of Traditional Chinese Medicine
Priority to PCT/CN2002/000505 priority Critical patent/WO2004009106A1/en
Priority to AU2002318715A priority patent/AU2002318715A1/en
Priority to CNB028293363A priority patent/CN1282466C/en
Publication of WO2004009106A1 publication Critical patent/WO2004009106A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/59Menispermaceae (Moonseed family), e.g. hyperbaena or coralbead
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47Quinolines; Isoquinolines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/02Antineoplastic agents specific for leukemia

Definitions

  • the invention relates to a preparation prepared from powder and self-protection and its use.
  • the present invention also relates to one of the main ingredients of the preparation, the use of tetrandrine in the preparation of a medicament for increasing the level of white blood cells. Background technique
  • Tumors are one of the diseases with a high mortality rate in humans, and their incidence is increasing year by year.
  • Chemotherapy and radiation therapy are currently the main methods for clinical treatment of tumors.
  • the resistance to chemotherapeutic drugs can make tumor tissues regain their sensitivity to chemotherapeutic drugs, or can make chemotherapeutic drugs easily enter cells and increase the cumulative concentration of chemotherapeutic drugs in cells, or they have antitumor effects, and Chemotherapy drugs work synergistically to enhance sensitization chemotherapy.
  • Fangfangji is the dried root of Fangfangchi (Stephenia tetrandra S. Moor), which is also known as Fangfang and downed arch. Beginning in "Shen Nong's Materia Medica”. The taste is bitter and cold. Go to the bladder and lung meridians. It has the effects of diuresis and swelling, expelling wind and relieving pain. For water Swelling athlete's foot, unfavorable urination, eczema, sore, rheumatic pain, hypertension.
  • the total alkaloids in tetrandrine account for about 0.5-5% of the root, mainly including tetrandrine (also known as tetrandrine, tetrandrine), tetrandrine (fangchinoline, also known as tetrandrine), Normantine, haggingchin C, menisine, menisidine, cyclanoline, berbamine, oxidized tetrandrine Oxofangchirine, stephanthrine, fenfangjine A, B, C, D, (+) — 2—N—methyl tetrandrine, (+) — 2—N—methyl tetrandrine ( (+) -N-methyltetrandrine), 2,2'- ⁇ , ⁇ -dimethylchlorotrandrine and the like.
  • tetrandrine and tetrandrine are predominant in the roots, accounting for 50-80% of the total alkaloids.
  • Tetrandrine is used clinically to treat silicosis, hypertension, and coronary heart disease.
  • the body alkaloids of Fangfang total alkaloids have been shown to have analgesic effects (Zhao Yi, Fangyanhusuo, scopolamine components, analgesic effect, Chinese Medical Journal, 1955, 41 (10), 931-935).
  • tetrandrine can reverse the resistance of chemotherapy-resistant drugs such as vincristine, doxorubicin and other tumor cells (KB cells, etc.) in vitro (Ye Switzerlanduang et al.
  • the present inventors devoted themselves to the development of new pharmacological uses of alkaloids. As a result, it was found that from the powder of Fangfang and its genus or Batumi iMenispermum dauricum DC.) Or hair Extracts from plants such as Cydea barbata (Wall.) Miers), which mainly contain tetrandrine and tetrandrine have chemosensitization, radiosensitization, and increase leukocyte levels and antiallergic effects. In addition, the inventors of the present invention also found that tetrandrine has an effect of increasing leukocyte levels.
  • step (1) filtering the liquid in step (1), drying and pulverizing the solid to obtain powder 1;
  • step (3) the alcohol solution in step (3) is concentrated, left, and filtered to obtain solid 2;
  • the preparation mainly contains tetrandrine of molecular formula (I) and tetrandrine of molecular formula (II).
  • the molar ratio of the two is 9: 1 to 6: 4, and the total content of the two is greater than 50%.
  • the preparation of the present invention can be used for the preparation of a chemotherapeutic sensitization, a radiotherapy sensitization, an increase in the level of white blood cells, or an antiallergic drug.
  • tetrandrine has the effect of increasing the level of white blood cells in the body, so it can be used to prepare drugs that increase the level of white blood cells.
  • the experimental research of the present inventors has shown that the preparation of the present invention has a more effective effect of reversing the multidrug resistance of tumor tissues to chemotherapeutic drugs than the application of tetrandrin alone, and the effect of increasing the concentration of chemotherapeutic drugs in tumor cells Combined with chemotherapeutic drugs, it has a sensitizing or synergistic effect on chemotherapeutic drugs; the preparation of the present invention is used in advance, and then radiation treatment is performed on tumor patients, showing that the preparation has the effect of improving the comprehensive curative effect of radiation therapy without any occurrence The toxic and side effects of which are better for squamous cell carcinoma; the preparation of the present invention, tetrandrine and its salt have a strong effect on increasing the level of leukocytes in low levels of leukocytes of immunosuppressed animals, and the increase range is 100 The
  • the preparation of the present invention is obtained by the following method:
  • step (1) filtering the liquid of step (1), drying and pulverizing the solid to obtain powder 1;
  • step (3) the alcohol solution in step (3) is concentrated, left, and filtered to obtain solid 2;
  • the preparation mainly contains tetrandrine of molecular formula (I) and tetrandrine of molecular formula (II), and the molar ratio of the two is 9.8: 0.2-6: 4, and the total content of the two is more than 50%, preferably The ratio is 9.5: 0.5 to 8: 2, more preferably 9: 1 to 8: 2, and most preferably 8.5: 1.5 to 6.5: 3.5.
  • the method further comprises the following steps:
  • step (5) The precipitate obtained in step (5) is dissolved in alcohol, and an equivalent amount of a pharmaceutically acceptable acid is added to form a salt, and the mixture is allowed to stand and filtered.
  • a pharmaceutically acceptable acid is preferably 0.1 to 4.5, more preferably From 1.75 to 2.5, preferred inorganic acids include, for example, hydrobromic acid, hydrochloric acid, and sulfuric acid; preferred organic acids include, for example, methanesulfonic acid, tartaric acid, maleic acid, and citric acid. The most preferred acid is hydrochloric acid.
  • the dilute acid used in step (1) of the method is preferably dilute sulfuric acid; the base used is preferably calcium oxide and calcium hydroxide.
  • the preparation of the present invention may also contain berbamine, oxotetrandrine, oxofangchinoline, and the like.
  • the quantitative analysis of each component in the preparation of the present invention can be performed by a method known to those skilled in the art, and usually chromatography is used, such as high performance liquid chromatography.
  • the preparations of the present invention have various pharmacological activities, and tetrandrine has the effect of increasing the body's white blood cell level, so they can be used to prepare drugs with corresponding pharmacological activities, which are used for chemosensitization , Radiation sensitization, elevated white blood cell levels, or anti-allergy.
  • the pharmaceutical preparation can be prepared according to methods known to those skilled in the art, and can be For tablets, pills, powders, suspensions, emulsions, syrups, capsules, sterile injection solutions and other common dosage forms.
  • the pharmaceutical preparation of the present invention can be administered by various routes including oral, transdermal, subcutaneous, intravenous and intramuscular administration, and the dosage is usually in the range of 0.02-1.5 g / day depending on the weight and symptoms of the subject to be administered.
  • the supernatant is decanted and discarded
  • the lower turbid liquid was filtered with a filter cloth to obtain about 2 kg of precipitate, dried, crushed (10-40 mesh sieve), heated under reflux with 95% ethanol, poured out of the alcohol solution, concentrated, left, filtered, and the obtained solid was dissolved in In ammonia-based ethanol, it is left to precipitate, and the obtained crystals are dissolved in ethanol, a predetermined amount of acid is added, and the solution is allowed to stand and filtered to obtain the desired preparation.
  • Example 2 Ten grams of the solid obtained from ammoniacal ethanol was taken and recrystallized from acetone to obtain 5 grams of white needle-like crystals of tetrandrine with a purity of more than 98%.
  • Example 2 Ten grams of the solid obtained from ammoniacal ethanol was taken and recrystallized from acetone to obtain 5 grams of white needle-like crystals of tetrandrine with a purity of more than 98%.
  • mice Preparation and Tetrandrine's whitening effect on cyclophosphamide-induced low leukocytes in mice.
  • the mice were injected intraperitoneally with an antitumor drug cyclophosphamide 75mg / kg.
  • the number of white blood cells in peripheral blood decreased significantly.
  • the preparation obtained in Example A was orally or intramuscularly injected once a day for 3 consecutive days. The results are shown in Table 1, Table 2 and Table. 3.
  • the tetrandrin and the preparation can obviously inhibit the leukocyte decrease caused by cyclophosphamide, which shows that it has a certain attenuating effect on the toxic and side effects of the low leukocyte caused by cyclophosphamide.
  • mice were inoculated with S 18 .
  • the preparation obtained in Example A was orally or intramuscularly injected, and the animals received 6Q Co irradiation (500 rad, 1 minute 56 seconds) on the second day after the administration, and then continued the administration.
  • the non-administration group was the model group, and the non-irradiated normal control group was set up.
  • the results showed that the peripheral blood leukocytes of the model group were significantly lower than normal after irradiation, and the preparation obtained in Example 1 A 25 mg kg and 100 mg / kg was orally administered to the peripheral blood on the 3rd and 7th day after radiotherapy, respectively.
  • When administered by injection it also has a certain degree of promotion effect on peripheral blood leukocytes, and the promotion rate is as high as 59%.
  • the results are shown in Table 4.
  • tumor cells are resistant to anti-tumor drugs.
  • the drug concentration in tumor cells is reduced, so that the tumor cells cannot be inhibited.
  • Grow. We use the sensitive strain S 18 .
  • the preparation obtained in Example A and daunorubicin are administered in combination, the daunorubicin concentration in drug-resistant cells can be significantly increased, which is close to the drug concentration level in sensitive cells.
  • the results are shown in the table. 8. It can be seen that the preparation can increase the concentration of anticancer drugs in tumor cells, so that it can reach the level of inhibiting the growth of tumor cells.
  • the analgesic effect of the preparation obtained in A of Example 1 was observed using a warm-induced pain model and a chemical peritonitis model.
  • the results showed that: in the mouse tail-flick test, the preparation was given once by intramuscular injection at doses of 5 mg / kg and 10 mg / kg, both of which could prolong the warm-induced tail tail latency of mice, indicating that the preparation caused pain by warming Has a certain analgesic effect.
  • the inhibitory effect of the preparation on chemical peritonitis pain was observed using a model of writhing response caused by acetic acid.
  • the MTT method was used to determine the in vitro half inhibitory concentration of the preparation on various tumor cells. The results are shown in Table 11.
  • Example 10 Give the lung cancer patient the preparation obtained in Example A in Example 1 100-300 mg / times intravenously or intravenously 2 hours before radiotherapy, or take a small dose of radiation treatment 2 days after taking the preparation tablet in advance, each time the tumor volume is radiated 150-160 rads, 5 times a week, with a total radiation of 1500-3000 rads; 5 days of medication, 5 days of discontinuation, tablets of its preparation taken orally during the discontinuation period, 300 mg / time, 300- 600 mg / day.
  • the total effective rate for the treatment of advanced lung cancer is more than 60%, and all patients have no obvious side effects of radiotherapy.
  • Example 10 The total effective rate for the treatment of advanced lung cancer is more than 60%, and all patients have no obvious side effects of radiotherapy.
  • the administration group was injected with the preparation obtained in Example 1 A intraperitoneally 30 minutes before inflammation. Evans blue was injected intravenously. After 6 hours, the balloon was washed with Hank's solution, the cell fluid was collected, the number of cells was counted, and the OD of the cystic fluid was measured with a spectrophotometer.
  • Example 12 Dissolve 50 g of the preparation obtained in Example 1 with water for injection, add 2 g of sodium sulfite and water for injection to 1000 ml, and filter with No. 3 vertical melting glass funnel or other filter media (such as ultra-microfiltration membranes) To clear, potting, sterilization, you can. Each pack contains 50 nig of preparation.
  • Example 12 Dissolve 50 g of the preparation obtained in Example 1 with water for injection, add 2 g of sodium sulfite and water for injection to 1000 ml, and filter with No. 3 vertical melting glass funnel or other filter media (such as ultra-microfiltration membranes) To clear, potting, sterilization, you can. Each pack contains 50 nig of preparation.
  • Example 12 Dissolve 50 g of the preparation obtained in Example 1 with water for injection, add 2 g of sodium sulfite and water for injection to 1000 ml, and filter with No. 3 vertical melting glass funnel or other filter media (such as ultra-microfiltration membranes) To clear, potting, sterilization, you can. Each
  • Example 13 To 300 g of the preparation obtained in Example A was added an appropriate amount of lactose and microcrystalline fiber. Vitamine, polyethylene glycol 3000, sodium carboxymethyl starch, dry compressed granules, add an appropriate amount of magnesium stearate, press into 1000 tablets, sterilize, and coat with film. Each tablet contains 300 mg of preparation.
  • Example 13 To 300 g of the preparation obtained in Example A was added an appropriate amount of lactose and microcrystalline fiber. Vitamine, polyethylene glycol 3000, sodium carboxymethyl starch, dry compressed granules, add an appropriate amount of magnesium stearate, press into 1000 tablets, sterilize, and coat with film. Each tablet contains 300 mg of preparation.
  • Example 13 To 300 g of the preparation obtained in Example A was added an appropriate amount of lactos
  • Example 14 300 g of the S IJ preparation obtained in Example 1 was added with an appropriate amount of auxiliary materials such as lactose, microcrystalline cellulose and the like, and then pressed and granulated, and then sterilized and gelatinized to make 1000 granules. Each capsule contains 300 mg of preparation.
  • auxiliary materials such as lactose, microcrystalline cellulose and the like

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Abstract

The present invention relates to a preparation extracted from Stephania tetrandra S. Moor and its use for manufacture of medecine for enhancing the sensitivity or effect of chemotherapy, enhancing the sensitivity of radiotherapy, improving the level of leukocytes in body, and having antineoplastic and antianaphylaxis effects. Said preparation is mainly composed of tetrandrine and fangchinoline with the ratio of 9:1 to 8:2 (moles), which is obtained by the following process: (1) the powder or piece of Stephania tetrandra S. Moor is extracted with dilute acid with the cool acid extract being collected; and washed with water with the aqueous extract being collected; then the cool acid extract and aqueous extract are combined, adjusted with base pH 10 to 12, kept overnight; (2) the fluid of Step (1) is filtered, the solid dried, pulverized to give Powder 1; (3) Powder 1 is heated under reflux in alcohol for 30 min.; (4) the alcohol extract from Step (3) is concentrated, kept, filtered to give Solid 2; (5) Solid 2 is dissolved in warm ammoniac ethanol, kept for crystallization. The present invention also relates to the use of tetrandrine and the pharmaceutically acceptable salt thereof for manufacture of medecine for improving the level of leukocytes in body.

Description

自粉防己提取的制备物及其用途  Preparation from self-protecting powder and anti-self extraction and use thereof
技术领域 Technical field
本发明涉及一种自粉防己提取的制备物及其用途。 另外, 本发 明还涉该制备物的主要成分之一, 汉防己甲素在制备用于升高白细 胞水平的药物中的用途。 背景技术  The invention relates to a preparation prepared from powder and self-protection and its use. In addition, the present invention also relates to one of the main ingredients of the preparation, the use of tetrandrine in the preparation of a medicament for increasing the level of white blood cells. Background technique
肿瘤是人类死亡率很高的疾病之一, 且其发病率逐年升高。 化 学治疗和放射治疗是目前临床上治疗肿瘤的主要手段。 目前的抗肿 瘤药物, 特别是细胞毒类药物, 大多是免疫抑制剂, 患者长时间应 用容易产生多药耐药性, 从而导致治疗失败, 因此, 有必要寻找化 疗增敏药物, 它们能够消除机体对化疗药物产生的耐药性, 使肿瘤 组织重新恢复对化疗药物的敏感性, 或者能够使化疗药物易于进入 细胞内部而提高化疗药物在细胞内的累积浓度, 或者其本身具有抗 肿瘤作用, 与化疗药物协同作用而增敏化疗; 在放射治疗中, 为了 达到治疗目的通常需要大剂量的放射, 这使得患者因免疫系统受到 抑制而难以承受, 因此, 有必要研制能提高肿瘤组织对放射的敏感 性的药物, 使低剂量的放射治疗也能够达到治疗目的。 另外, 化学 治疗和放射治疗通常均导致严重的毒副作用, 主要表现为白细胞水 平降低及恶心、 呕吐、 脱发等, 白细胞水平降低会增加患者各种感 染的机率, 因此, 有必要寻找能够降低化疗和放疗的毒副作用, 特 别是升高白细胞水平的药物。  Tumors are one of the diseases with a high mortality rate in humans, and their incidence is increasing year by year. Chemotherapy and radiation therapy are currently the main methods for clinical treatment of tumors. Current anti-tumor drugs, especially cytotoxic drugs, are mostly immunosuppressants. Patients are prone to multi-drug resistance after prolonged application, which leads to treatment failure. Therefore, it is necessary to find chemotherapy-sensitizing drugs that can eliminate the body The resistance to chemotherapeutic drugs can make tumor tissues regain their sensitivity to chemotherapeutic drugs, or can make chemotherapeutic drugs easily enter cells and increase the cumulative concentration of chemotherapeutic drugs in cells, or they have antitumor effects, and Chemotherapy drugs work synergistically to enhance sensitization chemotherapy. In radiation therapy, large doses of radiation are usually required to achieve therapeutic purposes, which makes patients unbearable due to the suppression of the immune system. Therefore, it is necessary to develop a method that can increase the sensitivity of tumor tissues to radiation. Sexual drugs enable low-dose radiation therapy to achieve therapeutic goals. In addition, chemotherapy and radiation therapy usually cause severe toxic side effects, mainly manifested in reduced white blood cell levels and nausea, vomiting, hair loss, etc., lower white blood cell levels increase the probability of various infections in patients, so it is necessary to find ways to reduce chemotherapy and Toxic effects of radiotherapy, especially drugs that increase white blood cell levels.
粉防己为防己科植物粉防己 iStephania tetrandra S. Moor) 的 干燥根, 又称为汉防己、 倒地拱等。 始载于 《神农本草经》 。 性味 苦、 寒。 归膀胱、 肺经。 具有利水消肿, 祛风止痛之功效。 用于水 肿脚气, 小便不利, 湿疹疮毒, 风湿痹痛, 高血压症。 粉防己中的 总生物碱约占根的 0.5— 5 % , 主要包括汉防己甲素 (tetrandrine, 又 称汉防己碱, 粉防己碱) 、 汉防己乙素 (fangchinoline,又称防己诺 林碱, 去甲汉防己碱) 、 汉防己丙素 (hanfangchin C) 、 木防己素 甲(menisine)、木防己素乙(menisidine)、轮环藤酚碱 (cyclanoline)、 小檗胺 ( berbamine ) 、 氧化防己碱 ( oxofangchirine ) 、 防己菲碱 (stephanthrine) 、 fenfangjine A, B, C, D、 (+) — 2— N—甲基防己 诺林碱、 (+)— 2— N—甲基汉防己碱((+) -N-methyltetrandrine )、 2,2'— Ν,Ν—二氯甲基汉防己碱等。 在上述生物碱中, 在根中以汉防 己甲素和汉防己乙素为主, 占总生物碱的 50—80 %。 Fangfangji is the dried root of Fangfangchi (Stephenia tetrandra S. Moor), which is also known as Fangfang and downed arch. Beginning in "Shen Nong's Materia Medica". The taste is bitter and cold. Go to the bladder and lung meridians. It has the effects of diuresis and swelling, expelling wind and relieving pain. For water Swelling athlete's foot, unfavorable urination, eczema, sore, rheumatic pain, hypertension. The total alkaloids in tetrandrine account for about 0.5-5% of the root, mainly including tetrandrine (also known as tetrandrine, tetrandrine), tetrandrine (fangchinoline, also known as tetrandrine), Normantine, hanfangchin C, menisine, menisidine, cyclanoline, berbamine, oxidized tetrandrine Oxofangchirine, stephanthrine, fenfangjine A, B, C, D, (+) — 2—N—methyl tetrandrine, (+) — 2—N—methyl tetrandrine ( (+) -N-methyltetrandrine), 2,2'- Ν, Ν-dimethylchlorotrandrine and the like. Among the above-mentioned alkaloids, tetrandrine and tetrandrine are predominant in the roots, accounting for 50-80% of the total alkaloids.
汉防己甲素在临床上用于治疗矽肺、 高血压、 冠心病等。 防己 总生物碱的体^^试验研究表明具有镇痛作用 (赵一, 汉防己延胡索 膺碱成分的镇痛作用, 中华医学杂志, 1955, 41 ( 10) , 931 -935 ) 。 我们发表了汉防己甲素在体外试验中能够逆转耐化疗药物, 如耐长 春新碱、 阿霉素等的肿瘤细胞(KB细胞、 等) 的抗药性(叶 祖光等, 汉防己甲素对阿霉素或长春新碱耐药株人癌细胞的逆转抗 药性作用, 中国中药杂志, 1996; 21 ( 6 ) : 369-371 ) 、 逆转机体 对抗疟药, 如氯喹等的抗药性(Ye Zu-guang et al The potentiating action of tetrandrine in combinaton with c loroquine against chloroquine- sensitive and resistant falciparum malaria Biochem. Biophys. Res. Tetrandrine is used clinically to treat silicosis, hypertension, and coronary heart disease. The body alkaloids of Fangfang total alkaloids have been shown to have analgesic effects (Zhao Yi, Fangyanhusuo, scopolamine components, analgesic effect, Chinese Medical Journal, 1955, 41 (10), 931-935). We have published that tetrandrine can reverse the resistance of chemotherapy-resistant drugs such as vincristine, doxorubicin and other tumor cells (KB cells, etc.) in vitro (Ye Zuguang et al. Reversal of drug resistance of human cancer cells by voxel or vincristine-resistant strains, Chinese Journal of Chinese Materia Medica, 1996; 21 (6): 369-371), reverse the body's resistance to malaria drugs, such as chloroquine (Ye Zu-guang et al The potentiating action of tetrandrine in combinaton with c loroquine against chloroquine- sensitive and resistant falciparum malaria Biochem. Biophys. Res.
Commim. 1989;165:758-65 ) 、 和长春新碱、 阿霉素体外合用, 具有 抗肿瘤作用 ( Sun Ai-Xu, Ye Zu-Guang et al Synergistic anticancer effects of tetrandrine conbined with doxorubicin or vincristine in vitro 中国药理学报, 1999; 20 ( 1 ) : 69-73 ) 。 发明内容 Commim. 1989; 165: 758-65), combined with vincristine and doxorubicin, have antitumor effects (Sun Ai-Xu, Ye Zu-Guang et al Synergistic anticancer effects of tetrandrine conbined with doxorubicin or vincristine in vitro Chinese Journal of Pharmacology, 1999; 20 (1): 69-73). Summary of the Invention
本发明人致力于开发生物碱的新的药理学用途, 结果发现, 从 粉防己及其同属植物或蝙蝠葛 iMenispermum dauricum DC. ) 或毛 叶轮环藤 Cydea barbata (Wall.) Miers)等植物中提取的, 主要含 有汉防己甲素和汉防己乙素的制备物具有化疗增敏、 放疗增敏以及 升高白细胞水平和抗过敏的作用。 另外, 本发明的发明者还发现了 汉防己甲素具有升高白细胞水平的作用。 The present inventors devoted themselves to the development of new pharmacological uses of alkaloids. As a result, it was found that from the powder of Fangfang and its genus or Batumi iMenispermum dauricum DC.) Or hair Extracts from plants such as Cydea barbata (Wall.) Miers), which mainly contain tetrandrine and tetrandrine have chemosensitization, radiosensitization, and increase leukocyte levels and antiallergic effects. In addition, the inventors of the present invention also found that tetrandrine has an effect of increasing leukocyte levels.
本发明的一个目的是提供从粉防己及其同属植物或蝙蝠葛或毛 叶轮环藤等植物中提取的制备物, 其是通过以下方法得到的: It is an object of the present invention to provide a preparation extracted from the powder of Fangshen and its genus, or plants such as Batata or Hairy Leaf Ring Vine, which is obtained by the following method:
(1)将粉防己粉末或饮片用稀酸提取, 收集酸冷浸液; 再用水浸洗, 收集水液; 合并酸液和水液, 加入碱调节 pH至 10— 12, 放置过夜;(1) Extract the powder of tangle powder or decoction with dilute acid, and collect the acid cold infusion; then rinse with water to collect the water; combine the acid and water, add alkali to adjust the pH to 10-12, and leave it overnight;
(2)过滤步骤 (1)的液体, 将固体干燥并粉碎 ·, 得到粉末 1 ; (2) filtering the liquid in step (1), drying and pulverizing the solid to obtain powder 1;
(3)将粉末 1用醇加热回流 30分钟提取;  (3) extract powder 1 by heating under reflux with alcohol for 30 minutes;
(4)将步骤 (3)的醇液浓缩、 放置、 过滤得到固体 2;  (4) the alcohol solution in step (3) is concentrated, left, and filtered to obtain solid 2;
(5)将固体 2溶于热氨性乙醇中, 放置沉淀。  (5) The solid 2 is dissolved in hot ammoniated ethanol, and the precipitate is left to stand.
所述制备物主要含有分子式 (I) 的汉防己甲素和分子式 (II) 的汉防己乙素, 二者的摩尔比为 9: 1一 6:4, 二者的总含量大于 50%。 The preparation mainly contains tetrandrine of molecular formula (I) and tetrandrine of molecular formula (II). The molar ratio of the two is 9: 1 to 6: 4, and the total content of the two is greater than 50%.
Figure imgf000005_0001
Figure imgf000005_0001
本发明的制备物可以用于制备化疗增敏、 放疗增敏、 升高白细 胞水平或抗过敏的药物。  The preparation of the present invention can be used for the preparation of a chemotherapeutic sensitization, a radiotherapy sensitization, an increase in the level of white blood cells, or an antiallergic drug.
另外, 汉防己甲素具有升高机体白细胞水平的作用, 因而可以 用于制备升高白细胞水平的药物。 本发明者的试验研究表明, 本发明的制备物具有比单独应用汉 防己甲素更高效的逆转肿瘤组织对化疗药物产生的多药耐药性, 提 高化疗药物在肿瘤细胞中的浓度的作用, 与化疗药物合用, 对化疗 药物具有增敏或增效作用; 预先使用本发明的制备物, 接着对肿瘤 患者进行放射治疗, 显示该制备物具有提高放射治疗的综合疗效的 作用, 而不出现任何的毒副作用, 其中对鳞癌效果更好; 本发明的 制备物、 汉防己甲素及其盐对免疫抑制动物的白细胞水平低下均具 有较强的升高白细胞水平的作用, 升高幅度在 100%以上; 本发明的 制备物还具有一定的抗肿瘤作用, 对各种肿瘤的抑制率在 30— 80% 的范围内。 具体实施方式 . In addition, tetrandrine has the effect of increasing the level of white blood cells in the body, so it can be used to prepare drugs that increase the level of white blood cells. The experimental research of the present inventors has shown that the preparation of the present invention has a more effective effect of reversing the multidrug resistance of tumor tissues to chemotherapeutic drugs than the application of tetrandrin alone, and the effect of increasing the concentration of chemotherapeutic drugs in tumor cells Combined with chemotherapeutic drugs, it has a sensitizing or synergistic effect on chemotherapeutic drugs; the preparation of the present invention is used in advance, and then radiation treatment is performed on tumor patients, showing that the preparation has the effect of improving the comprehensive curative effect of radiation therapy without any occurrence The toxic and side effects of which are better for squamous cell carcinoma; the preparation of the present invention, tetrandrine and its salt have a strong effect on increasing the level of leukocytes in low levels of leukocytes of immunosuppressed animals, and the increase range is 100 The preparation of the present invention also has a certain anti-tumor effect, and the inhibition rate on various tumors is in the range of 30-80%. detailed description.
本发明的制备物是通过以下方法得到的:  The preparation of the present invention is obtained by the following method:
(1)将粉防己粉末或饮片用稀酸提取至少 24小时, 收集酸提取液; 再用水浸洗,收集水液;合并酸液和水液,加入碱调节 pH至 10— 12, 放置过夜;  (1) Extract the powder of tetrum powder or decoction pieces with dilute acid for at least 24 hours, and collect the acid extract; then dip it in water to collect the water solution; combine the acid solution and the water solution, add alkali to adjust the pH to 10-12, and leave it overnight;
(2)过滤步骤 (1)的液体, 将固体干燥并粉碎, 得到粉末 1 ;  (2) filtering the liquid of step (1), drying and pulverizing the solid to obtain powder 1;
(3)将粉末 1用醇加热回流 30分钟提取;  (3) extract powder 1 by heating under reflux with alcohol for 30 minutes;
(4)将步骤 (3)的醇液浓缩、 放置、 过滤得到固体 2;  (4) the alcohol solution in step (3) is concentrated, left, and filtered to obtain solid 2;
(5)将固体 2溶于热氨性乙醇中, 放置得到沉淀。  (5) The solid 2 is dissolved in hot ammoniated ethanol and left to obtain a precipitate.
所述制备物主要含有分子式 (I) 的汉防己甲素和分子式 (II) 的汉防己乙素, 二者的摩尔比为 9.8:0.2— 6:4, 二者的总含量大于 50%, 优选比例为 9.5:0.5— 8:2, 更优选为 9:1一 8:2, 最优选为 8.5:1.5 —6.5:3.5。 The preparation mainly contains tetrandrine of molecular formula (I) and tetrandrine of molecular formula (II), and the molar ratio of the two is 9.8: 0.2-6: 4, and the total content of the two is more than 50%, preferably The ratio is 9.5: 0.5 to 8: 2, more preferably 9: 1 to 8: 2, and most preferably 8.5: 1.5 to 6.5: 3.5.
Figure imgf000007_0001
Figure imgf000007_0001
优选地, 所述方法另外包括以下步骤:  Preferably, the method further comprises the following steps:
(6)将步骤 (5)得到的沉淀溶于醇, 加入等当量的药学可接受的酸使 之成盐,并放置、过滤,其中药学可接受的酸的 pKa优选为 0.1— 4.5, 更优选为 1.75— 2.5, 优选的无机酸包括, 例如, 氢溴酸、 盐酸和硫 酸; 优选的有机酸包括, 例如, 甲磺酸、 酒石酸、 马来酸、 柠檬酸。 最优选的酸为盐酸。  (6) The precipitate obtained in step (5) is dissolved in alcohol, and an equivalent amount of a pharmaceutically acceptable acid is added to form a salt, and the mixture is allowed to stand and filtered. The pKa of the pharmaceutically acceptable acid is preferably 0.1 to 4.5, more preferably From 1.75 to 2.5, preferred inorganic acids include, for example, hydrobromic acid, hydrochloric acid, and sulfuric acid; preferred organic acids include, for example, methanesulfonic acid, tartaric acid, maleic acid, and citric acid. The most preferred acid is hydrochloric acid.
所述方法的步骤 (1)中所用的稀酸优选为稀硫酸; 所用的碱优选 为氧化钙和氢氧化钙。  The dilute acid used in step (1) of the method is preferably dilute sulfuric acid; the base used is preferably calcium oxide and calcium hydroxide.
除汉防己甲素和汉防己乙素外, 本发明的制备物还可能含有小 檗胺 (berbamine) 、 氧化汉防己甲素 (oxotetrandrine) 、 氧化汉防 己乙素 ( oxofangchinoline ) 等。  In addition to tetrandrine and tetrandrine, the preparation of the present invention may also contain berbamine, oxotetrandrine, oxofangchinoline, and the like.
本发明的制备物中各成分的定量分析可以用本领域技术人员已 知的方法进行, 通常用色谱法, 例如高效液相色谱法。  The quantitative analysis of each component in the preparation of the present invention can be performed by a method known to those skilled in the art, and usually chromatography is used, such as high performance liquid chromatography.
如上所述, 本发明的制备物具有多种药理活性, 汉防己甲素具 有升高机体白细胞水平的作用, 因而可以将它们用于制备具有相应 药理活性的药物, 所述药物用于化疗增敏、 放疗增敏、 升高白细胞 水平、 或抗过敏。  As mentioned above, the preparations of the present invention have various pharmacological activities, and tetrandrine has the effect of increasing the body's white blood cell level, so they can be used to prepare drugs with corresponding pharmacological activities, which are used for chemosensitization , Radiation sensitization, elevated white blood cell levels, or anti-allergy.
药物制剂的制备可根据本领域技术人员已知的方法进行, 可以 为片剂、 丸剂、 粉剂、 混悬剂、 乳剂、 糖浆剂、 胶囊剂、 无菌注射 溶液剂等各种常见剂型。 The pharmaceutical preparation can be prepared according to methods known to those skilled in the art, and can be For tablets, pills, powders, suspensions, emulsions, syrups, capsules, sterile injection solutions and other common dosage forms.
本发明的药物制备物可以通过各种途径给予, 包括口服、 经皮、 皮下、 静脉内和肌内给予, 根据给予对象的体重及症状, 给予剂量 通常在 0.02—1.5克 /天的范围内。  The pharmaceutical preparation of the present invention can be administered by various routes including oral, transdermal, subcutaneous, intravenous and intramuscular administration, and the dosage is usually in the range of 0.02-1.5 g / day depending on the weight and symptoms of the subject to be administered.
以下实施例用于进一步说明本发明, 但它们并非试图在任何方 面限制本发明的范围。 实施例 1  The following examples serve to further illustrate the invention, but they are not intended to limit the scope of the invention in any way. Example 1
制备物、 汉防己甲素和汉防己乙素的制备 Preparation, preparation of tetrandrine and tetrandrine
A. 制备物  A. Preparation
向 10千克粉防己粗粉末或约 1一 2毫米厚的饮片中加入 20升 1% 的稀硫酸提取 24小时, 收集酸冷浸液, 剩余物用水 10升浸泡 30分 钟, 收集水液; 如此的酸冷浸 -水洗处理循环进行 3次, 合并各次的 酸冷浸液和水液, 向其中加入氧化钙或氢氧化钙调至 pH 10- 12, 放置过夜, 倾去上清液并弃去, 以滤布过滤下部的浑浊液, 得到约 2千克沉淀, 干燥, 粉碎 (10— 40 目筛) , 以 95%乙醇加热回流, 倾出醇液, 浓缩、 放置, 过滤, 将所得固体溶于氨性乙醇中, 放置 使沉淀, 将所得晶体溶于乙醇中, 加入定量的酸, 放置, 过滤, 即 得到期望的制备物。  Add 20 liters of powder of tangleproof powder or approximately 1.2 mm thick piece of decoction to add 20 liters of 1% dilute sulfuric acid for extraction for 24 hours, collect the acid cold soaking solution, and soak the remaining 10 liters of water for 30 minutes to collect the water liquid; The acid cold soaking-washing treatment cycle is performed 3 times, and the acid cold soaking solution and the water solution are combined each time, and calcium oxide or calcium hydroxide is added thereto to adjust the pH to 10-12, and the mixture is left overnight. The supernatant is decanted and discarded The lower turbid liquid was filtered with a filter cloth to obtain about 2 kg of precipitate, dried, crushed (10-40 mesh sieve), heated under reflux with 95% ethanol, poured out of the alcohol solution, concentrated, left, filtered, and the obtained solid was dissolved in In ammonia-based ethanol, it is left to precipitate, and the obtained crystals are dissolved in ethanol, a predetermined amount of acid is added, and the solution is allowed to stand and filtered to obtain the desired preparation.
B. 汉防己甲素 B. tetrandrine
取从氨性乙醇得到的固体 10克, 用丙酮重结晶, 得到 5克白 色针状结晶汉防己甲素, 其纯度在 98%以上。 实施例 2  Ten grams of the solid obtained from ammoniacal ethanol was taken and recrystallized from acetone to obtain 5 grams of white needle-like crystals of tetrandrine with a purity of more than 98%. Example 2
制备物及汉防己甲素对环磷酰胺所致的小鼠白细胞低下的升白作用 给小鼠一次性腹腔注射抗肿瘤药环磷酰胺 75mg/kg, 三天后外 周血白细胞数显著下降, 给小鼠注射环磷酰胺的同一天开始口服或 肌肉注射实施例 1中 A所得的制备物, 每天 1次, 连续 3天, 结果 列于表 1、 表 2和表 3, 可以看到, 该汉防己甲素和制备物可明显抑 制环磷酰胺引起的白细胞下降, 说明对环磷酰胺引起的白细胞低下 的毒副作用有一定的减毒作用。 Preparation and Tetrandrine's whitening effect on cyclophosphamide-induced low leukocytes in mice. The mice were injected intraperitoneally with an antitumor drug cyclophosphamide 75mg / kg. The number of white blood cells in peripheral blood decreased significantly. On the same day that cyclophosphamide was injected into mice, the preparation obtained in Example A was orally or intramuscularly injected once a day for 3 consecutive days. The results are shown in Table 1, Table 2 and Table. 3. It can be seen that the tetrandrin and the preparation can obviously inhibit the leukocyte decrease caused by cyclophosphamide, which shows that it has a certain attenuating effect on the toxic and side effects of the low leukocyte caused by cyclophosphamide.
表 1. 肌肉注射汉防己甲素和制备物对环磷酰胺诱导的白细胞减少 症的升白作用 (n=10)
Figure imgf000009_0001
表 3. 制备物对环磷酰胺化疗后骨髓中有核细胞比例的影响(n=10)
Figure imgf000009_0002
Table 1. Intramuscular injection of tetrandrine and preparations against leukopenia induced by cyclophosphamide (n = 10)
Figure imgf000009_0001
Table 3. Effect of preparations on the proportion of nucleated cells in bone marrow after cyclophosphamide chemotherapy (n = 10)
Figure imgf000009_0002
注: 与模型组比较 *P<0.05, **P<0.01 实施例 3 Note: Compared with model group * P <0.05, ** P <0.01 Example 3
制备物对 6QCo放疗小鼠的升白作用 Whitening effect of the preparation on 6Q Co radiotherapy mice
小鼠接种 S18。肉瘤后, 分别口服或肌肉注射实施例 1中 A所得 的制备物,给药后第 2天动物接受 6QCo照射(500拉德, 1分 56秒), 随后继续给药, 设只接受照射而不给药者为模型组, 另设不照射的 正常对照组。 结果表明, 模型组于照射后外周血白细胞较正常明显 降低, 口服给予实施例 1中 A所得的制备物 25 mg kg和 100 mg/kg, 分别于放疗后第 3天和第 7天对外周血有显著的升白细胞作用。 注 射给药时也在一定程度上对外周血白细胞有一定的提升作用, 提升 率最高达 59%, 结果列于表 4。 Mice were inoculated with S 18 . After the sarcoma, the preparation obtained in Example A was orally or intramuscularly injected, and the animals received 6Q Co irradiation (500 rad, 1 minute 56 seconds) on the second day after the administration, and then continued the administration. The non-administration group was the model group, and the non-irradiated normal control group was set up. The results showed that the peripheral blood leukocytes of the model group were significantly lower than normal after irradiation, and the preparation obtained in Example 1 A 25 mg kg and 100 mg / kg was orally administered to the peripheral blood on the 3rd and 7th day after radiotherapy, respectively. Has a significant leukocyte effect. When administered by injection, it also has a certain degree of promotion effect on peripheral blood leukocytes, and the promotion rate is as high as 59%. The results are shown in Table 4.
表 4. 制备物对 0QCo放疗小鼠的升白作用 Table 4. Whitening effect of the preparation on 0Q Co radiotherapy mice
Figure imgf000010_0001
Figure imgf000010_0001
注: *Ρ<0.05, **Ρ<0.01 (与模型组比较) 实施例 4  Note: * P <0.05, ** P <0.01 (compared with model group) Example 4
制备物对阿霉素治疗耐药 S18。肿瘤的增敏作用 Preparation of adriamycin treatment resistant S 18. Tumor sensitization
将体内诱导的耐阿霉素 S18Q腹水瘤细胞在体外进行培养, 然后 加入阿霉素; 结果显示: 这种肿瘤细胞对阿霉素明显耐药, 与敏感 株318。腹水瘤细胞相比, 耐药的肿瘤细胞抗性倍数为 62倍, 而在加 入阿霉素的同时加入实施例 1中 A所得的制备物, 则抗性倍数明显 下降,甚至其耐药性几乎完全逆转 (在制备物的浓度为 Ι.Ο^.ΟμιηοΙ 1 时), 结果见表 5。 表 5. 制备物对阿霉素治疗耐药 S18Q肿瘤的增敏作用 Doxorubicin-resistant S 18Q ascites tumor cells induced in vivo were cultured in vitro, and then doxorubicin was added; the results showed that this tumor cell was significantly resistant to doxorubicin and was sensitive to 3 18 . Compared with ascites tumor cells, the resistance multiple of drug-resistant tumor cells is 62 times. However, when doxorubicin is added at the same time as the preparation obtained in Example A, the resistance multiple is significantly reduced, and even the resistance is almost Completely reversed (when the concentration of the preparation was 1.0 ^. 0 μιηοΙ 1 ), the results are shown in Table 5. Table 5. Sensitization of preparations to doxorubicin-resistant S 18Q tumors
Figure imgf000011_0001
Figure imgf000011_0001
注:制备物对 S18Q/S的 IC5。为 5.64±0.94μπιο1丄 -1,对8180/入(11"的1( 5。为2.14 ±0.74μπιο1丄-1 实施例 5 Note: Preparation of IC 5 for S 18Q / S. It is 5.64 ± 0.94μπιο1 丄-1 , for 8 180 / in (11 "of 1 ( 5. Is 2.14 ± 0.74μπιο1 丄-1) Example 5
制备物对 Slso/Adr的耐药性的逆转活性 Reversal activity of preparation against S lso / Adr
给予 S1SQ耐阿霉素株阿霉素肌肉注射治疗时, 阿霉素对肿瘤没 有明显的抑制作用, 阿霉素治疗组小鼠的存活时间与未治疗组小鼠 的存活时间相近, 而在给予阿霉素的同时口服或肌肉注射给予实施 例 1 中 A所得的制备物, 可使荷瘤小鼠存活时间明显延长, 说明该 制备物可逆转 S18Q/Adr的耐药性, 结果列于表 6和表 7。 表 6. 制备物对 S18Q/Adr的耐药性的逆转活性 (口服给予制备物) When S 1SQ doxorubicin resistant doxorubicin was administered intramuscularly, doxorubicin did not significantly inhibit tumors. The survival time of mice in the doxorubicin treatment group was similar to that of mice in the untreated group. Oral or intramuscular injection of doxorubicin given the preparation obtained in Example 1 can significantly prolong the survival time of tumor-bearing mice, indicating that the preparation can reverse the drug resistance of S 18Q / Adr. The results are listed in Table 6 and Table 7. Table 6. Preparations for reversing S 18Q / Adr drug resistance (oral preparations)
Figure imgf000011_0002
Figure imgf000011_0002
注: *P<0.05, **P<0.01,与同剂量组的阿霉素单独用药组相比。 表 7.制备物对 S180/Adr的耐药性的逆转活性(腹腔注射给予制备物) Note: * P <0.05, ** P <0.01, compared with doxorubicin alone group in the same dose group. Table 7. Reversal activity of preparations against S 180 / Adr resistance (preparation by intraperitoneal injection)
Figure imgf000012_0001
Figure imgf000012_0001
**P<0.01,与阿霉素单独用药组相比。 实施例 6  ** P <0.01, compared with doxorubicin alone group. Example 6
制备物对细胞内柔红霉素累积浓度的测定  Determination of accumulated concentration of daunorubicin in cells
肿瘤细胞对抗肿瘤药耐药的主要原因之一是由于肿瘤细胞上的 一种 P糖蛋白的过度表达, 用抗肿瘤药治疗时, 致使肿瘤细胞内的 药物浓度下降, 以致于不能抑制肿瘤细胞的生长。 我们用敏感株 S18。 和耐药株 S18Q肿瘤细胞进行试验, 加入柔红霉素以后, 用流式细胞 仪测定细胞内代表柔红霉素药物浓度的荧光强度, 发现耐药肿瘤细 胞中的荧光强度比敏感株细胞低约 1倍, 实施例 1中 A所得的制备 物与柔红霉素联合给予时, 可使耐药细胞内的柔红霉素浓度明显提 高, 接近敏感细胞内药物浓度水平, 结果列于表 8, 可以看到, 该 制备物能提高瘤细胞内抗癌药物浓度, 使其达到抑制肿瘤细胞生长 的水平。 One of the main reasons why tumor cells are resistant to anti-tumor drugs is due to the overexpression of a P glycoprotein on tumor cells. When treated with anti-tumor drugs, the drug concentration in tumor cells is reduced, so that the tumor cells cannot be inhibited. Grow. We use the sensitive strain S 18 . Test with drug-resistant strain S 18Q tumor cells. After adding daunorubicin, the fluorescence intensity of the daunorubicin drug concentration in the cell was measured by flow cytometry. It was found that the fluorescence intensity in the drug-resistant tumor cells was higher than that of the sensitive strain cells. It is about 1 times lower. When the preparation obtained in Example A and daunorubicin are administered in combination, the daunorubicin concentration in drug-resistant cells can be significantly increased, which is close to the drug concentration level in sensitive cells. The results are shown in the table. 8. It can be seen that the preparation can increase the concentration of anticancer drugs in tumor cells, so that it can reach the level of inhibiting the growth of tumor cells.
表 8. 制备物对细胞内柔红霉素累积浓度  Table 8. Cumulative intracellular daunorubicin concentration of preparations
组别 平均荧 平均荧 两次平均值 光强度 光强度  Group Average Fluorescence Average Twice Average Light Intensity Light Intensity
敏感株 对照 4.13 5.41 4.77  Sensitive strain Control 4.13 5.41 4.77
柔红霉素 (20μΜ) 162.18 174.81 168.5±8.93* 抗性株 对照 10.12 8.08 9.1  Daunorubicin (20 μM) 162.18 174.81 168.5 ± 8.93 * resistant strain control 10.12 8.08 9.1
柔红霉素 (20μΜ) 94.89 97.24 96.07±1.66 柔红霉素 (20μΜ)+制备物 (ΙΟμΜ) 187.99 170.83 179.41±12.13 柔红霉素 (20μΜ)+制备物 (5μΜ) 146.08 164.80 155.44+13.24 注: *P<0.05 (与敏感株对照比较) , #P<0.05 (与抗性 ξ对照比较) 实施例 7 Daunorubicin (20 μM) 94.89 97.24 96.07 ± 1.66 Daunorubicin (20 μM) + Preparation (10 μM) 187.99 170.83 179.41 ± 12.13 Daunorubicin (20 μM) + Preparation (5 μM) 146.08 164.80 155.44 + 13.24 Note: * P <0.05 (compared to the control of sensitive plants), #P <0.05 (compared to the control of resistant ξ) Example 7
制备物的镇痛作用  Analgesic effect of the preparation
用温热致痛模型和化学性腹膜炎模型观察了实施例 1中 A所得 的制备物的镇痛作用。 结果表明: 在小鼠甩尾试验中, 肌肉注射给 予该制备物一次, 剂量为 5mg/kg和 10mg/kg, 均可延长小鼠温热致 里尾潜伏期, 说明该制备物对温热致痛有 定的镇痛作用。 用醋酸 引起的扭体反应模型观察了制备物对化学腹膜炎疼痛的抑制作用; 结果表明: 肌肉注射该制备物 12.5— 50 mg kg可明显延长扭体出现 的潜伏期, 减少扭体次数, 说明该制备物对化学刺激引起的疼痛有 明显的镇痛作用, 结果列于表 9和表 10。  The analgesic effect of the preparation obtained in A of Example 1 was observed using a warm-induced pain model and a chemical peritonitis model. The results showed that: in the mouse tail-flick test, the preparation was given once by intramuscular injection at doses of 5 mg / kg and 10 mg / kg, both of which could prolong the warm-induced tail tail latency of mice, indicating that the preparation caused pain by warming Has a certain analgesic effect. The inhibitory effect of the preparation on chemical peritonitis pain was observed using a model of writhing response caused by acetic acid. The results showed that intramuscular injection of the preparation 12.5-50 mg kg significantly prolonged the incubation period of writhing and reduced the number of writhing, indicating that the preparation The compounds have obvious analgesic effects on pain caused by chemical stimulation. The results are shown in Tables 9 and 10.
表 9. 制备物对小鼠热甩尾的影响 (n=10)  Table 9. Effects of preparations on thermal tail flick in mice (n = 10)
Figure imgf000013_0001
Figure imgf000013_0001
注: *P<0.05, **P<0.01 (与对照组比较) 表 10. 制备物对小鼠醋酸扭体的影响 (n=10)  Note: * P <0.05, ** P <0.01 (compared with control group) Table 10. Effect of preparations on mouse acetate torsion (n = 10)
Figure imgf000013_0002
Figure imgf000013_0002
注: *Ρ<0·05, **Ρ<0·01 (与对照组比较) 实施例 8  Note: * P <0 · 05, ** P <0 · 01 (compared with control group) Example 8
制备物的体内、 体外抗肿瘤作用  Preparation of in vivo and in vitro antitumor effects
50 mg/kg (肌肉注射) 制备物对艾氏腹水癌小鼠的存活时间较 模型组明显延长, 延长率为 161.0%。 50 mg / kg (intramuscular) preparation compared with the survival time of Ehrlich ascites cancer mice The model group was significantly prolonged, with an elongation rate of 161.0%.
利用 MTT法进行测定, 计算出制备物对各种肿瘤细胞的体外半 数抑制浓度。 其结果列于表 11。  The MTT method was used to determine the in vitro half inhibitory concentration of the preparation on various tumor cells. The results are shown in Table 11.
表 11. 制备物对不同瘤株的半数抑制浓度 IC5Q (μΜ . Ι 1 ) Table 11. Preparations with inhibitory concentrations IC 5Q (μM. Ι 1 ) for different tumor strains
Figure imgf000014_0001
实施例 9
Figure imgf000014_0001
Example 9
制备物的放疗增效作用 Radiotherapy synergism of preparation
在放射治疗前 2小时静推或静滴给予肺癌患者实施例 1中 Α所 得的制备物 100—300 mg/次, 或者预先服用制备物片剂 2天后进行 小剂量放射治疗, 每次放射肿瘤量为 150— 160拉德, 每周 5次, 总 放射量在 1500—3000拉德; 用药 5天, 停药 5天, 在停药期间口服 其制备物的片剂, 300 mg/次, 300— 600 mg/日。 治疗晚期肺癌的总 有效率达 60 %以上, 全部患者没有明显的放疗副作用出现。 实施例 10  Give the lung cancer patient the preparation obtained in Example A in Example 1 100-300 mg / times intravenously or intravenously 2 hours before radiotherapy, or take a small dose of radiation treatment 2 days after taking the preparation tablet in advance, each time the tumor volume is radiated 150-160 rads, 5 times a week, with a total radiation of 1500-3000 rads; 5 days of medication, 5 days of discontinuation, tablets of its preparation taken orally during the discontinuation period, 300 mg / time, 300- 600 mg / day. The total effective rate for the treatment of advanced lung cancer is more than 60%, and all patients have no obvious side effects of radiotherapy. Example 10
制备物的抗炎作用 Anti-inflammatory effect of the preparation
给大鼠背部正中皮下注射空气形成气囊, 6天后向气囊中注射 角叉菜胶 25 mg/kg致炎, 给药组分别于致炎前 30分钟腹腔注射实 施例 1中 A所得的制备物,并静脉注射伊文思蓝, 6小时后,用 Hank's 液洗涤气囊, 收集细胞液, 计数细胞数, 并用分光光度计测定囊液 OD值。 结果表明给予该制备物后, 囊内的伊文思蓝渗出明显减少 ·, 白细胞数显著降低, 表明该制备物对角叉菜胶致炎后引起的毛细血 管通透性增高有明显的抑制作用。 给予制备物后, 渗出液中的白细 胞数明显降低, 说明该制备物对炎性反应具有抑制作用, 结果列于 表 12。 Air was injected subcutaneously into the back of the rat to form a balloon, and carrageenan 25 mg / kg was injected into the balloon 6 days later to induce inflammation. The administration group was injected with the preparation obtained in Example 1 A intraperitoneally 30 minutes before inflammation. Evans blue was injected intravenously. After 6 hours, the balloon was washed with Hank's solution, the cell fluid was collected, the number of cells was counted, and the OD of the cystic fluid was measured with a spectrophotometer. The results showed that after administration of the preparation, the exudation of Evans blue in the capsule was significantly reduced, and the number of white blood cells was significantly reduced, indicating that the preparation has a significant inhibitory effect on the increase in capillary permeability caused by carrageenan-induced inflammation. . White fines in exudate after administration of preparation The number of cells was significantly reduced, indicating that the preparation had an inhibitory effect on the inflammatory response. The results are shown in Table 12.
表 12. 制备物的抗炎作用  Table 12. Anti-inflammatory effects of the preparations
Figure imgf000015_0001
Figure imgf000015_0001
*Ρ<0.05, **Ρ<0.01 (与对照组比较) 实施例 10  * P <0.05, ** P <0.01 (compared with control group) Example 10
制备物的抗过敏作用  Antiallergic effect of the preparation
大鼠足趾注射天花粉致敏 4周后, 再次注射天花粉溶液时, 大 鼠肺阻力急剧增加, 增高率达 95%, 肺顺应性降低 93%, 如果预先 雾化吸入实施例 1中 Α所得制备物, 15分钟后, 再注射天花粉时, 肺阻力只增加 4%, 肺顺应性降低 3%, 说明该制备物能抑制大鼠过 敏性哮喘。 实施例 11  Four weeks after sensitization with trichosanthin injection in the toes of rats, the lung resistance of the rats increased sharply with 95% increase in lung resistance and 93% reduction in lung compliance when the trichosantamine solution was injected again. After 15 minutes, when injected with trichosanthin, the lung resistance increased by only 4%, and the lung compliance decreased by 3%, indicating that the preparation can inhibit allergic asthma in rats. Example 11
制备物针剂的制备  Preparation of preparations for injection
将 50 g实施例 1中 A所得的制备物加注射用水溶解, 加 2 g亚 硫酸钠及注射用水至 1000 ml, 用 3号垂熔玻璃漏斗或其他滤器滤材 (如:超微过滤膜等)过滤至澄明,灌封,灭菌,即可。每支含 50 nig 制备物。 实施例 12  Dissolve 50 g of the preparation obtained in Example 1 with water for injection, add 2 g of sodium sulfite and water for injection to 1000 ml, and filter with No. 3 vertical melting glass funnel or other filter media (such as ultra-microfiltration membranes) To clear, potting, sterilization, you can. Each pack contains 50 nig of preparation. Example 12
制备物片剂的制备  Preparation of preparation tablets
向 300 g实施例 1中 A所得的制备物中加入适量乳糖、 微晶纤 维素、 聚乙二醇 3000、 羧甲基淀粉钠, 干压制粒后, 加硬脂酸镁适 量, 压制成 1000片, 灭菌, 包薄膜衣。 每片含 300 mg制备物。 实施例 13 To 300 g of the preparation obtained in Example A was added an appropriate amount of lactose and microcrystalline fiber. Vitamine, polyethylene glycol 3000, sodium carboxymethyl starch, dry compressed granules, add an appropriate amount of magnesium stearate, press into 1000 tablets, sterilize, and coat with film. Each tablet contains 300 mg of preparation. Example 13
制备物胶囊的制备 Preparation of preparation capsules
向 300 g实施例 1中 A所得 S IJ备物中加入适量乳糖、 微晶纤 维素等辅料干压制粒后,灭菌,装胶 ;,制成 1000粒。每粒含 300 mg 制备物。 实施例 14  300 g of the S IJ preparation obtained in Example 1 was added with an appropriate amount of auxiliary materials such as lactose, microcrystalline cellulose and the like, and then pressed and granulated, and then sterilized and gelatinized to make 1000 granules. Each capsule contains 300 mg of preparation. Example 14
制备物和植物药复方制备工艺 Preparation and compound medicine preparation process
1. 将 100 g黄芪提取物(含黄酮和皂甙) , 200 g实施例 1中 A 所得的制备物混合, 加入乳糖、 微晶纤维素、 硬脂酸镁适量, 制粒, 装胶囊和 /或压片, 制成 1000粒或 1000片。  1. Mix 100 g of Astragalus membranaceus extract (containing flavonoids and saponins) and 200 g of the preparation obtained in Example 1 A, add lactose, microcrystalline cellulose, magnesium stearate in an appropriate amount, granulate, capsule, and / or Press into tablets to make 1000 tablets or 1000 tablets.
2. 将 20 g人参提取物(含多糖和皂甙) 、 20 g丹参的醇冷浸提 取物、 200 g实施例 1 中 A所得的制备物混合, 加入乳糖、 微晶纤 维素、硬脂酸镁适量, 制粒, 装胶囊和 /或压片, 制成 1000粒或 1000 片。  2. Mix 20 g of ginseng extract (containing polysaccharides and saponins), 20 g of cold-soaked extract of salvia miltiorrhiza, 200 g of the preparation obtained in Example 1 A, and add lactose, microcrystalline cellulose, and magnesium stearate. Appropriate amount, granulated, filled with capsules and / or compressed into 1000 or 1000 tablets.

Claims

权利要求 Rights request
1 . 一种自粉防己提取的制备物, 其主要含有汉防己甲素和汉 防己乙素, 其中汉防己甲素和汉防己乙素的比例为 9.8:0.2至 8:2 (摩 尔) , 优选为 9:1至 8:2, 二者的含量总和大于 50%, 所述制备物通 过以下方法得到: What is claimed is: 1. A preparation prepared from powdered tetrandrine, which mainly contains tetrandrine and tetrandrine, wherein the ratio of tetrandrine and tetrandrine is 9.8: 0.2 to 8: 2 (mole), preferably It is 9: 1 to 8: 2, and the total content of the two is greater than 50%. The preparation is obtained by the following method:
(1)将粉防己粉末或饮片用稀酸,优选为稀硫酸提取,收集酸冷浸液; 再用水浸洗, 收集水液; 与酸冷浸液合并, 加入碱, 优选为氧化钙 或氢氧化钙, 调节 pH至 10— 12, 放置过夜;  (1) Extract the powder of tetrum powder or decoction with dilute acid, preferably dilute sulfuric acid, and collect the acid cold soaking solution; then dip it in water to collect the water solution; combine with the acid cold soaking solution, add alkali, preferably calcium oxide or hydrogen Calcium oxide, adjust the pH to 10-12, and leave it overnight;
(2)过滤步骤 (1)的液体, 将固体干燥并粉碎, 得到粉末 1 ;  (2) filtering the liquid of step (1), drying and pulverizing the solid to obtain powder 1;
(3)将粉末 1用醇加热回流 30分钟提取;  (3) extract powder 1 by heating under reflux with alcohol for 30 minutes;
(4)将步骤 (3)的醇液浓缩、 放置、 过滤得到'固体 2;  (4) the alcohol solution of step (3) is concentrated, left, and filtered to obtain 'solid 2;
(5)将固体 2溶于碱性乙醇液中, 加热溶解, 放置, 收集沉淀; (5) dissolve the solid 2 in an alkaline ethanol solution, dissolve it by heating, place it, and collect the precipitate;
(6)将步骤 (5)得到的沉淀溶于醇, 加入等当量的药学可接受的酸使 之成盐, 并放置、 过滤。 (6) The precipitate obtained in step (5) is dissolved in an alcohol, an equivalent amount of a pharmaceutically acceptable acid is added to make a salt, and it is left to stand and filtered.
2. 权利要求 1 中的制备物, 其中所述方法的步骤 (6)所使用的 药学可接受的酸的 pKa在 0.1— 4.5的范围内,优选 pKa在 1.75— 2.5 的范围内。  2. The preparation according to claim 1, wherein the pKa of the pharmaceutically acceptable acid used in step (6) of the method is in the range of 0.1-4.5, preferably the pKa is in the range of 1.75-2.5.
3. 权利要求 2 的制备物, 其中的药学可接受的酸选自盐酸、 磷酸、 马来酸、 酒石酸、 柠檬酸、 富马酸、 牛磺酸和苹果酸。  3. The preparation of claim 2, wherein the pharmaceutically acceptable acid is selected from the group consisting of hydrochloric acid, phosphoric acid, maleic acid, tartaric acid, citric acid, fumaric acid, taurine and malic acid.
4. 权利要求 1一 3 中的任一权利要求的制备物在制备化疗增敏 或增效药物中的用途。  4. Use of the preparation according to any one of claims 1 to 3 in the preparation of a chemosensitization or synergistic drug.
5. 权利要求 4 的用途, 其中所述药物通过逆转肿瘤组织对抗 肿瘤药的多药耐药性而实现化疗增敏或增效, 其中所述多药耐药性 由选自以下的抗肿瘤药诱导: 阿霉素、 长春新碱、 紫杉醇、 顺铂、 顺氯氨铂、 柔红霉素、 三尖杉酯碱、 羟基喜树碱。 其中所述药物是 通过对抗肿瘤药的增效作用而实现化疗增敏或增效的。  5. The use of claim 4, wherein the drug achieves chemotherapy sensitization or enhancement by reversing the multidrug resistance of tumor tissue antitumor drugs, wherein the multidrug resistance is selected from the following antitumor drugs Induction: doxorubicin, vincristine, paclitaxel, cisplatin, cisplatin, daunorubicin, harringtonine, hydroxycamptothecin. Wherein, the drug realizes the sensitization or enhancement of chemotherapy through the synergistic effect of antitumor drugs.
6. 权利要求 5 的用途, 其中所述抗肿瘤药选自阿霉素、 长春 新碱、 长春碱、 紫杉醇、 顺铂、 顺氯氨铂、 柔红霉素、 三尖杉酯碱、 博莱霉素和阿糖胞苷。 6. The use according to claim 5, wherein the antineoplastic agent is selected from the group consisting of doxorubicin, changchun Nerine, vinblastine, paclitaxel, cisplatin, cisplatin, daunorubicin, harringtonine, bleomycin, and cytarabine.
7. 权¾要求 1一 3 中的任一权利要求的制备物在制备放疗增敏 药物中的用途。  7. Use of a preparation according to any one of claims 1 to 3 in the manufacture of a medicament for enhancing the sensitivity of radiotherapy.
8. 权利要求 1一 3 中的任一权利要求的制备物在制备用于升高 机体白细胞水平的药物中的用途。  8. Use of the preparation of any one of claims 1 to 3 in the manufacture of a medicament for increasing the level of white blood cells in the body.
9. 权利要求 1一 3 中的任一权利要求的制备物在制备抗肿瘤药 物中的用途。  9. Use of a preparation according to any one of claims 1 to 3 for the preparation of an antitumor drug.
10. 汉防己甲素或其药学可接受的盐在制备用于升高机体白细 胞水平的药物中的用途。  10. Use of tetrandrine or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing the level of white blood cells in the body.
PCT/CN2002/000505 2002-07-18 2002-07-18 Preparation extracted from stephania tetrandra s. moor and the use thereof WO2004009106A1 (en)

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