WO2009074065A1

WO2009074065A1 - Use of daphne primeverose genkwanine and daphne plants

Info

Publication number: WO2009074065A1
Application number: PCT/CN2008/073216
Authority: WO
Inventors: Wenhua Huang; Quan Pu
Original assignee: Wenhua Huang; Quan Pu
Priority date: 2007-12-10
Filing date: 2008-11-27
Publication date: 2009-06-18
Also published as: CN101456886A; CN101456886B

Abstract

Use of Daphne primeverose Genkwanine (DPG) of formula (I) or alcohol extract of Daphne plants containing it, Daphne plants containing it in manufacture of a medicament in treatment of leukaemia. Pharmaceutical compositions containing Daphne primeverose Genkwanine of formula (I).

Description

瑞香樱草糖-芫花黄素和瑞香属植物的用途技术领域 Use of Ruixiang primrose - safflor yellow and ruthenium

本发明涉及植物药物领域。更具体地涉及瑞香属植物和从瑞香属植物中提取的瑞香樱草糖-芫花黄素（Daphnepr imeverosyl-Genkwanine , DPG)的用途。背景技术 The invention relates to the field of botanical drugs. More specifically, it relates to the use of the genus Durian and the Daphnepr imeverosyl-Genkwanine (DPG) extracted from the genus Durian. Background technique

随着医学的发展与进步，白血病的治疗水平也有了很大提高，人们不仅仅满足于病人的完全缓解，而致力于最终使病人长期无病存活乃至痊愈的研究，目前白血病的治疗方法有化疗、中西医结合治疗、和造血干细胞移植等。 With the development and progress of medicine, the treatment level of leukemia has also been greatly improved. People are not only satisfied with the complete remission of patients, but also committed to the study of patients who have long-term disease-free survival and even recovery. Currently, the treatment of leukemia is chemotherapy. , integrated Chinese and Western medicine treatment, and hematopoietic stem cell transplantation.

化疗是使用化学制剂杀灭白血病细胞，使病人达到长期存活乃至治愈的目的。白血病的类型很多，对不同的白血病类型应选择不同的化疗方案。目前，化疗的成功率较低，失败的原因有化疗期内因感染和出血引起早期死亡，或白血病细胞耐药而无效果。 Chemotherapy uses chemical agents to kill leukemia cells, so that patients can achieve long-term survival and even cure. There are many types of leukemia, and different chemotherapy regimens should be chosen for different leukemia types. At present, the success rate of chemotherapy is low. The cause of failure is early death due to infection and bleeding during chemotherapy, or leukemia cells are resistant and have no effect.

对于中西医结合治疗，无论是单纯中医中药治疗，还是中西医药联合使用，效果都不是十分理想。 For the combination of traditional Chinese and Western medicine treatment, whether it is pure Chinese medicine treatment or the combination of Chinese and Western medicine, the effect is not very satisfactory.

造血干细胞移植（HSCT)可以说是一个最根本的治疗白血病的方法。但它存在很大的风险，主要有两点：一是 HSCT 中存在许多移植相关并发症，二是 HSCT后仍有白血病复发问题：主要的移植相关并发症有：肝静脉闭塞病，其发病率为 25%，死亡率为 80%，移植物抗宿主病，发病率 10%-80%。 HSCT 后白血病复发率大约为 15%-50%。 Hematopoietic stem cell transplantation (HSCT) is arguably the most fundamental method of treating leukemia. However, it has great risks. There are two main points: First, there are many transplant-related complications in HSCT, and second, there is still leukemia recurrence after HSCT: The main transplant-related complications are: Hepatic vein occlusion, its incidence 25%, mortality rate is 80%, graft versus host disease, incidence rate is 10%-80%. The recurrence rate of leukemia after HSCT is about 15%-50%.

因此，本领域迫切需要提供一种可有效治疗白血病等克隆性恶性血液病的物质和方法。发明内容 Therefore, there is an urgent need in the art to provide a substance and a method which are effective for treating clonal hematological diseases such as leukemia. Summary of the invention

本发明旨在提供如式 I的化合物，或含有式 I化合物的植物提取物，或含有式 I化合物的原药材的新用途。 The present invention is intended to provide a compound of formula I, or a plant extract containing a compound of formula I, or a novel use of a prodrug containing a compound of formula I.

本发明的第二个目的，是提供一种药物组合物。 A second object of the invention is to provide a pharmaceutical composition.

本发明的第三个目的，是提供一种治疗白血病的方法。在本发明的第一方面，提供了一种如式 I 所示的瑞香樱草糖-芫花黄素 (Daphne primeverosyl-Genkwanine, DPG)，或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物的醇提物，或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物药材在制备治疗白血病的药物中的用途， A third object of the invention is to provide a method of treating leukemia. In a first aspect of the invention, there is provided a fragrant primrose-baicalin as shown in Formula I (Daphne primeverosyl-Genkwanine, DPG), or an alcoholic extract of the genus Raytheon of the scent of scented scented scutellaria, as shown in Formula I, or a scented scented sage The use of flavin's medicinal herbs in the preparation of a medicament for treating leukemia,

在另一优选例中，所述的白血病选自耐药性急性白血病，慢性粒细胞白血病，骨髓增生异常综合症 (MDS)，或终末期多发性骨髓瘤 (Kahler's disease)。 In another preferred embodiment, the leukemia is selected from the group consisting of drug-resistant acute leukemia, chronic myeloid leukemia, myelodysplastic syndrome (MDS), or end stage multiple myeloma (Kahler's disease).

在另一优选例中，所述的瑞香属植物选自金边瑞香（/¾p/7/j_e odora Thunb) , Tcit (Daphne genkwaSieb. et Zucc) , 白瑞香 (Z?ap ?/je papyracea Wall, ex Steud)，云 Daphne y議 arwnsis H F. Zhou)；更优选瑞香属植物的全草粉末。在另一优选例中，所述的醇提物选自甲醇或乙醇提取物。 In another preferred embodiment, the genus genus is selected from the group consisting of Phnom Penh scent (/3⁄4p/7/j _e odora Thunb), Tcit (Daphne genkwa Sieb. et Zucc), and white scent (Z?ap?/je papyracea Wall, Ex Steud), Cloud Daphne y arwnsis H F. Zhou); More preferably, the whole grass powder of the genus Durian. In another preferred embodiment, the alcohol extract is selected from the group consisting of methanol or ethanol extract.

在另一优选例中，所述的醇提物由下述步骤获得： In another preferred embodiment, the alcohol extract is obtained by the following steps:

(a)将瑞香属植物全草粉末用甲醇和 /或乙醇提取获得。 (a) Extracting the genus whole plant powder with methanol and/or ethanol.

在另一优选例中，在步骤（a)后还包括步骤： In another preferred embodiment, after step (a), the method further comprises the steps of:

(b)将步骤（a)得到的醇提物用氯仿浸泡、离心得到沉淀物；和 (b) immersing the alcohol extract obtained in the step (a) with chloroform and centrifuging to obtain a precipitate;

(c)将沉淀物用水漂洗后得到醇提物。 (c) The precipitate is rinsed with water to obtain an alcohol extract.

在另一优选例中，所述的药物为选自口服胶囊，粉剂，片剂，酊剂，贴剂，膏剂，汤药，注射剂，缓释剂，药酒，或口服液的剂型。在本发明的第二方面，提供了一种药物组合物，它含有- (i) 作为活性成分的如式 I 所示的瑞香樱草糖 -芫花黄素 In another preferred embodiment, the medicament is a dosage form selected from the group consisting of an oral capsule, a powder, a tablet, an elixir, a patch, a plaster, a soup, an injection, a sustained release, a medicinal liquor, or an oral solution. In a second aspect of the invention, there is provided a pharmaceutical composition comprising - (i) as a living ingredient, a fragrant primrose flavonoid as shown in formula I

(Daphneprimeverosyl-Genkwanine , DPG) , 或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物的醇提物，或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物药材；和 (Daphneprimeverosyl-Genkwanine, DPG), or an alcoholic extract of the genus Raytheon of the scent of scented scented scutellaria, as shown in Formula I, or a scent of scented sage Medicinal herbs of the genus

替换页（细则第 26条) Replacement page (Article 26)

(i i)药学上可接受的载体。 (i i) a pharmaceutically acceptable carrier.

在另一优选例中，所述活性成分的重量为占有组合物总重量的 1-99%。 In another preferred embodiment, the active ingredient has a weight of from 1 to 99% by weight based on the total weight of the composition.

在另一优选例中，所述的瑞香属植物选自金边瑞香（Z¾p ?/7e odora Thunb) , 完花 (Z¾pA/?e genkwaSieb. et Zucc)，白瑞香 (Zfep we papyracea Wall, ex Steud)，或云南瑞香（Dap/me yunnar nsis H. F. Zhou)；所述的醇提物选自甲醇或乙醇提取物。在本发明的第三方面，提供了一种制备治疗白血病的药物的方法，它包括步骤：将如式 I所示的瑞香樱草糖-芫花黄素（Daphne primeverosyl-Genkwanine, DPG) , 或含有如式 I 所示的瑞香樱草糖-芫花黄素的瑞香属植物的醇提物，或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物药材和药学上可接受的载体混合得到。在本发明的第四方面，提供了一种治疗白血病的方法，所述的方法是对患者施用如式 I 所示的瑞香樱草糖 - 芫花黄素 (Daphnepr imeverosyl-Genkwanine , DPG)，或含有如式 I所示的瑞香樱草糖 -芫花黄素的瑞香属植物的醇提物，或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物药材。据此，本发明提供了一种可有效治疗白血病等克隆性恶性血液病的物质和方法。附图说明 In another preferred embodiment, the genus of the genus genus is selected from the group consisting of Z3⁄4p?/7e odora Thunb, the flower (Z3⁄4pA/?e genkwaSieb. et Zucc), and the Zfep we papyracea Wall (ex Steud) , or Dap/me yunnar nsis HF Zhou; the alcohol extract is selected from the group consisting of methanol or ethanol extract. In a third aspect of the invention, a method of preparing a medicament for treating leukemia, comprising the steps of: administering Daphne prime verosyl-Genkwanine (DPG) as shown in Formula I, or An alcoholic extract of a genus of the genus eucalyptus containing the scented scent of scented scutellaria as shown in Formula I, or a medicinal material of medicinal medicinal material containing ruthenium scutellaria and scutellarin as shown in Formula I Acceptable carriers are obtained by mixing. In a fourth aspect of the invention, there is provided a method of treating leukemia, which comprises administering to a patient a Daphnepr imeverosyl-Genkwanine (DPG) as shown in Formula I, or An alcoholic extract of the genus Durian of the scented scented scutellaria- safflorin as shown in Formula I, or a medicinal material of the genus eucalyptus containing the scented sage of the formula I. Accordingly, the present invention provides a substance and a method for effectively treating clonal hematological diseases such as leukemia. DRAWINGS

图 1显示了 L1210小鼠白血病细胞系的情况。 Figure 1 shows the condition of the L1210 mouse leukemia cell line.

图 2显示了 DPG对 L 1210小鼠白血病细胞系的影响。替换页（细则第 26条) 图 3显示了人胚正常成纤维细胞（HLF)的情况。 Figure 2 shows the effect of DPG on the L 1210 mouse leukemia cell line. Replacement page (Article 26) Figure 3 shows the condition of human embryonic normal fibroblasts (HLF).

图 4显示了 DPG对人胚正常原纤维细胞（HLF)的影响。具体实施方式 Figure 4 shows the effect of DPG on human embryonic normal fibroblasts (HLF). detailed description

发明人经过广泛而深入的研究，意外地发现瑞香属植物的全草对人类恶性血液病有病理学上的阳性效应。进一步地，发明人发现可以从上述的瑞香属植物中提取得到一种化学结构式如式 I的化合物（5-0- β -OH-樱草糖 -芫花黄素， Daphne priraeverosyl-Genkwanine , DPG)，它对白血病细胞具有明显的抗增殖和促凋亡作用，能够抑制白血病细胞的生长。 After extensive and intensive research, the inventors have unexpectedly discovered that the whole plant of the genus Durian has a pathological positive effect on human malignant hematological diseases. Further, the inventors have found that a compound of the formula I (5-0-β-OH- primrose- safflorin, Daphne priraeverosyl-Genkwanine, DPG) can be extracted from the above-mentioned genus Durian. It has obvious anti-proliferative and pro-apoptotic effects on leukemia cells and can inhibit the growth of leukemia cells.

如本文所用， "全草" 是指瑞香属植物的全草，包括植物的根、茎、枝、叶，所述的瑞香属植物优选金边瑞香 Daphne odora Thunb) ,芫花 Daphne genkwaSieb. e t Zucc)，白瑞香 {Daphne papyracea Wall, ex Steud)，或云南瑞香 (Z¾pj¾/7e yunnanensis H. F. Zhou)。 As used herein, "whole grass" refers to the whole grass of the genus Durian, including the roots, stems, branches, and leaves of the plant, and the genus Daphne odora Thunb), Daphne genkwa Sieb. et Zucc) , Daphne papyracea Wall, ex Steud, or Z3⁄4pj3⁄4/7e yunnanensis HF Zhou.

如本文所用， "全草粉末" 或 "全草粉剂" 可互换使用，是指将瑞香属植物的全草晒干后，粉碎成 50-200 目的颗粒物，优选 70- 150 目，更优选 90- 120 目。 As used herein, "whole grass powder" or "whole grass powder" is used interchangeably to mean that after drying the whole plant of the genus Durian, it is pulverized into 50-200 mesh particles, preferably 70-150 mesh, more preferably 90. - 120 mesh.

如本文所用，所述的 "瑞香属植物药材" 可以是瑞香属植物的全株，也可以是所述植物的局部，如根、茎、枝、叶、花、和 /或果实等。本发明提供的瑞香属植物全草粉末可用于治疗白血病，使用方法为每曰口服 3次，每次 2剂 /50kg体重，每剂 1克，每日总量 6克。医生可根据疾病和病人的具体情况作相应的调整。本发明提供瑞香属植物的提取物，所述的提取物选自甲醇或乙醇提取 As used herein, the "Russian medicinal material" may be a whole plant of the genus Durian, or may be a part of the plant, such as roots, stems, branches, leaves, flowers, and/or fruits. The Durian plant whole grass powder provided by the invention can be used for treating leukemia, and the method is as follows: oral administration 3 times per dose, 2 doses / 50 kg body weight per dose, 1 gram per dose, 6 grams per day. The doctor can make adjustments according to the disease and the specific conditions of the patient. The present invention provides an extract of the genus Durian, the extract being selected from the group consisting of methanol or ethanol extraction.

-4- 替换页（细则第 26条) 物。可以使用本领域常规的方法获得，一种优选的方法是在全草粉末中加入乙醇，回流提取、离心后，取乙醇萃取液，减压抽干，得到固体粉末。一种更佳的方法是在全草粉末中加入乙醇，回流提取、离心后，取乙醇萃取液，减压抽干，得到固体粉末；加入氯仿，浸泡过夜，次日离心，弃去氯仿；再加入氯仿洗涤一次，离心取沉淀物；减压抽干氯仿，得到固体，用蒸馏水漂洗两次，烘干得到精制提取物（DPG)。 -4- Replacement page (Article 26) Things. It can be obtained by a method conventional in the art, and a preferred method is to add ethanol to the whole grass powder, reflux extracting, centrifugation, taking an ethanol extract, and vacuum-drying to obtain a solid powder. A better method is to add ethanol to the whole grass powder, reflux extraction, centrifugation, take the ethanol extract, and vacuum dry to obtain a solid powder; add chloroform, soak overnight, centrifuge the next day, discard the chloroform; The mixture was washed once with chloroform, and the precipitate was centrifuged; chloroform was evaporated under reduced pressure to give a solid, which was rinsed twice with distilled water and dried to give purified extract (DPG).

本发明提供的醇提物和 /或 DPG可用于治疗白血病，使用方法为每日 3次，每次 2剂 /50kg体重，每剂 10毫克醇提物或 DPG，每日总量 60毫克。医生可根据疾病和病人的具体情况作相应的调整。本发明所治疗的白血病包括耐药性急性白血病，慢性粒细胞白血病，骨髓增生异常综合症和终末期多发性骨髓瘤 (Kahler's dise_ase)。本发明的天然瑞香属植物属瑞香科，桃金娘目，约 70种，分布于欧洲、北非和亚洲温带和亚热带地区至大洋洲，我国有 35 种，大部产西南部和西北部，他处少见，有些种类的靭皮纤维为制纸的原料，有些供观赏。 The alcohol extract and/or DPG provided by the present invention can be used for the treatment of leukemia by using 3 times a day, 2 doses per 50 kg body weight, 10 mg alcohol extract or DPG per dose, and a total daily dose of 60 mg. The doctor can make adjustments according to the disease and the specific circumstances of the patient. The leukemias treated by the present invention include drug-resistant acute leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and end-stage multiple myeloma (Kahler's dise _ase ). The natural genus of the genus Rutaceae belongs to the genus Rutaceae, about 70 species, distributed in the temperate and subtropical regions of Europe, North Africa and Asia to Oceania. There are 35 species in China, most of which are in the southwest and northwest. Rarely, some types of bast fiber are the raw materials for papermaking, and some are for viewing.

所属科瑞香科在我国主要品种种名如下： The names of the main varieties of Keruiaceae in China are as follows:

尖瓣瑞香 acu tiloba Rehd. Sharp scented acu tiloba Rehd.

澄黄瑞香 apAoe auran thiaca Diels 澄黄瑞香 apAoe auran thiaca Diels

藏东瑞香 bholua Buch. -Ham. ex D. Don Tibetan fragrant bholua Buch. -Ham. ex D. Don

fe ^ ¾ ^ Daphne bre vi tuba H. F. Zhou Fe ^ 3⁄4 ^ Daphne bre vi tuba H. F. Zhou

⁷J^N R† Jffii ^ Daphne championi i Ben th. ⁷ J ^N R† Jffii ^ Daphne championi i Ben th.

少花瑞香 apAoe depaupera ta H. F. Zhou 少花瑞香 apAoe depaupera ta H. F. Zhou

峨眉瑞香 Daphne emeiensis C. Y. Chang Ph眉瑞香 Daphne emeiensis C. Y. Chang

啮蚀瓣瑞香 Daphne erosiloba C. Y. Chang The pharyngeal scent Daphne erosiloba C. Y. Chang

白脉瑞香 apAoe esquirol i i Le vi. 白脉瑞香 apAoe esquirol i i Le vi.

短瓣瑞香 Daphne feddei Le vi. Short-mouthed fragrant Daphne feddei Le vi.

J 11 H ¾ ^ Daphne gemma ta E. Pri tz. J 11 H 3⁄4 ^ Daphne gemma ta E. Pri tz.

HDaphne genkwa Sieb. e t Zucc. HDaphne genkwa Sieb. e t Zucc.

绢毛瑞香 Daphne holosericea (Diels) Hamaya Ph毛瑞香 Daphne holosericea (Diels) Hamaya

If ill Jffit ^ Daphne leishanensis H. F. Zhou ^ IH ¾ ^ Daphne longi loba ta (Lecomte) Turrill If ill Jffit ^ Daphne leishanensis HF Zhou ^ IH 3⁄4 ^ Daphne longi loba ta (Lecomte) Turrill

大花瑞香 macran tha Ludlow Dahua Ruixiang macran tha Ludlow

瘦叶瑞香 Daphne modes ta Rehd. Daphne modes ta Rehd.

长梗瑞香 apAoe peduncula ta H. F. Zhou Long stalk fragrant apAoe peduncula ta H. F. Zhou

紫花瑞香 apAoe purpurascens S. C. Huang 紫花瑞香 apAoe purpurascens S. C. Huang

H 0† Jffit Daphne re tusa Hemsl. H 0† Jffit Daphne re tusa Hemsl.

口彖果瑞香 rhynchocarpa C. Y. Chang 彖果果香 rhynchocarpa C. Y. Chang

甘青瑞香 Daphne tangutica Maxim. Gan Qingrui Daphne tangutica Maxim.

细花瑞香 apAoe tenui flora Bur. Fine flower fragrant apAoe tenui flora Bur.

fi' ^n ^ Daphne triparti te H. F. Zhou Fi' ^n ^ Daphne triparti te H. F. Zhou

西畴瑞香 apAoe xichouensis H. F. Zhou 西域瑞香 apAoe xichouensis H. F. Zhou

本发明优选的品种是金边瑞香 ( aphne odora Thunb)，芫花 (J)a_Phne genkwaSieb. e t Zucc) , ί=| ¾ ^ (Daphne papyracea Wall, ex Steud) , 或云南瑞香 ( apAoe yunnanensis H. F. Zhou) _α A preferred variety of the invention is aphne odora Thunb, J花(J) a _P hne genkwaSieb. et Zucc) , ί=| 3⁄4 ^ (Daphne papyracea Wall, ex Steud), or apAoe yunnanensis HF Zhou ) _α

本发明提到的上述特征，或实施例提到的特征可以任意组合。本案说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。本发明的主要优点在于： The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, may be arbitrarily combined. All of the features disclosed in the present specification can be used in combination with any of the compositions, and the various features disclosed in the specification can be substituted for any alternative feature that provides the same, equal or similar purpose. Therefore, unless otherwise stated, the disclosed features are only a general example of equivalent or similar features. The main advantages of the invention are:

1、本发明提供的治疗白血病的方法可逆转，延长或阻止白血病的复发； 1. The method for treating leukemia provided by the present invention can be reversed to prolong or prevent the recurrence of leukemia;

2、本发明提供的治疗白血病的方法可防止白血病多药耐药； 2. The method for treating leukemia provided by the invention can prevent multidrug resistance of leukemia;

3、本发明提供的治疗白血病的方法毒副作用小。 3. The method for treating leukemia provided by the present invention has small toxic and side effects.

4、本发明所提供的治疗多发性骨髓瘤的方法，不仅可诱使取得缓解，且止痛效果明显。下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则所有的百分比和份数按重量计。 4. The method for treating multiple myeloma provided by the invention can not only induce remission, but also has obvious analgesic effect. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, Otherwise all percentages and parts are by weight.

除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。实施例 1 Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only. Example 1

制备全草粉末 I Preparation of whole grass powder I

采用金边瑞香 Daphne odora Thunb ±蓴，晒干后，经粉碎成 100 目颗粒物，得到全草粉末 I。实施例 2 Using Daphne odora Thunb ± 金, after drying, it is pulverized into 100 mesh granules to obtain whole grass powder I. Example 2

制备全草粉末 II Preparation of whole grass powder II

采用芫花 ¼7 ?/76> genkwa Sieb. et Z"cc全草，晒干后，经粉碎成 100 目颗粒物，得到全草粉末 Π。实施例 3 Using 芫花 1⁄47 ?/76> genkwa Sieb. et Z"cc whole grass, after drying, pulverized into 100 mesh particles to obtain whole grass powder Π. Example 3

制备全草粉末 III Preparation of whole grass powder III

采用白瑞香 papyracea Wall, ex S e .全草，晒干后，经粉碎成 100 目颗粒物，得到全草粉末 III。实施例 4 Using Bairuxiang papyracea Wall, ex S e. whole grass, after drying, it is pulverized into 100 mesh particles to obtain whole grass powder III. Example 4

制备全草粉末 IV Preparation of whole grass powder IV

^ 云 Daphne yu醒 sis H. F. Zhou, 晒干后，经粉碎成 100 目颗粒物，得到全草粉末 IV。实施例 5 ^ Cloud Daphne yu wake up sis H. F. Zhou, after drying, it is pulverized into 100 mesh particles to obtain whole grass powder IV. Example 5

制备醇提物 I Preparation of alcohol extract I

称取 50克全草粉末 I，加入 200毫升乙醇，回流提取 2小时。离心，取乙醇萃取液，减压抽干，得到固体粉末即醇提物 I。实施例 6 制备醇提物 II 50 g of whole grass powder I was weighed, 200 ml of ethanol was added, and reflux extraction was carried out for 2 hours. After centrifugation, the ethanol extract was taken and dried under reduced pressure to obtain a solid powder, i.e., alcohol extract I. Example 6 Preparation of alcohol extract II

称取 50克全草粉末 I I，加入 200毫升甲醇，回流提取 2小时。离心，取甲醇萃取液，减压抽干，得到固体粉末即醇提物 I I。实施例 7 50 g of whole grass powder I I was weighed out, and 200 ml of methanol was added thereto, followed by reflux extraction for 2 hours. After centrifugation, the methanol extract was taken and dried under reduced pressure to give a solid powder, i.e., an alcohol extract. Example 7

制备醇提物 III Preparation of alcohol extract III

称取 50克全草粉末 I I I，加入 200毫升甲醇，回流提取 2小时。离心，取甲醇萃取液，减压抽干，得到固体粉末即醇提物 I I I。实施例 8 50 g of whole grass powder I I I was weighed out, and 200 ml of methanol was added thereto, followed by reflux extraction for 2 hours. After centrifugation, the methanol extract was taken and dried under reduced pressure to give a solid powder, i.e., an alcohol extract. Example 8

制备醇提物 IV Preparation of alcohol extract IV

称取 50克全草粉末 IV，加入 200毫升乙醇，回流提取 2小时。离心，取乙醇萃取液，减压抽干，得到固体粉末即醇提物 IV。实施例 9 50 g of whole grass powder IV was weighed out, added with 200 ml of ethanol, and refluxed for 2 hours. After centrifugation, the ethanol extract was taken and dried under reduced pressure to give a solid powder, that is, an alcohol extract IV. Example 9

制备 DPG (No. 1) Preparation DPG (No. 1)

称取 50克全草粉末 I，加入 200毫升乙醇，回流提取 2小时。离心，取乙醇萃取液，减压抽干，得到固体粉末即醇提物 I。 50 g of whole grass powder I was weighed out, and 200 ml of ethanol was added thereto, followed by reflux extraction for 2 hours. After centrifugation, the ethanol extract was taken and dried under reduced pressure to give a solid powder, i.e., an alcohol extract.

加入 50毫升氯仿，浸泡过夜。次日离心，弃去氯仿；再加入 20毫升氯仿洗涤一次，离心取沉淀物。减压抽干氯仿，得到固体，用蒸馏水漂洗两次，烘干得到精制提取物（DPG (No. 1) )。实施例 10 Add 50 ml of chloroform and soak overnight. After the next day, the mixture was centrifuged, and chloroform was discarded; the mixture was washed once with 20 ml of chloroform, and the precipitate was centrifuged. The chloroform was evaporated under reduced pressure to give a solid, which was rinsed twice with distilled water and dried to give a purified extract (DPG (No. 1)). Example 10

制备 DPG (No. 2) Preparation DPG (No. 2)

称取 50克全草粉末 I I，加入 200毫升甲醇，回流提取 2小时。离心，取甲醇萃取液，减压抽干，得到固体粉末即醇提物 I I。 50 g of whole grass powder I I was weighed out, and 200 ml of methanol was added thereto, followed by reflux extraction for 2 hours. After centrifugation, the methanol extract was taken and dried under reduced pressure to give a solid powder, i.e., an alcohol extract.

加入 50毫升氯仿，浸泡过夜。次日离心，弃去氯仿；再加入 20毫升氯仿洗涤一次，离心取沉淀物。减压抽干氯仿，得到固体，用蒸馏水漂洗两次，烘干得到精制提取物 (DPG (No. 2) )。实施例 11 制备 DPG (No. 3) Add 50 ml of chloroform and soak overnight. After centrifugation the next day, chloroform was discarded; the mixture was washed once with 20 ml of chloroform, and the precipitate was centrifuged. The chloroform was drained under reduced pressure to give a solid, which was rinsed twice with distilled water and dried to give a purified extract (DPG (No. 2)). Example 11 Preparation of DPG (No. 3)

称取 50克全草粉末 I I I，加入 200毫升甲醇，回流提取 2小时。离心，取甲醇萃取液，减压抽干，得到固体粉末即醇提物 I I I。 50 g of whole grass powder I I I was weighed out, and 200 ml of methanol was added thereto, followed by reflux extraction for 2 hours. After centrifugation, the methanol extract was taken and dried under reduced pressure to give a solid powder, i.e., an alcohol extract.

加入 50毫升氯仿，浸泡过夜。次日离心，弃去氯仿；再加入 20毫升氯仿洗涤一次，离心取沉淀物。减压抽干氯仿，得到固体，用蒸馏水漂洗两次，烘干得到精制提取物 (DPG (No. 3) )。实施例 12 Add 50 ml of chloroform and soak overnight. After the next day, the mixture was centrifuged, and chloroform was discarded; the mixture was washed once with 20 ml of chloroform, and the precipitate was centrifuged. The chloroform was drained under reduced pressure to give a solid, which was rinsed twice with distilled water and dried to give a purified extract (DPG (No. 3)). Example 12

制备 DPG (No. 4) Preparation DPG (No. 4)

称取 50克全草粉末 IV，加入 200毫升乙醇，回流提取 2小时。离心，取乙醇萃取液，减压抽干，得到固体粉末即醇提物 IV。 50 g of whole grass powder IV was weighed out, added with 200 ml of ethanol, and refluxed for 2 hours. After centrifugation, the ethanol extract was taken and dried under reduced pressure to give a solid powder, that is, an alcohol extract IV.

加入 50毫升氯仿，浸泡过夜。次日离心，弃去氯仿；再加入 20毫升氯仿洗涤一次，离心取沉淀物。减压抽干氯仿，得到固体，用蒸馏水漂洗两次，烘干得到精制提取物 (DPG (No. 3) )。实施例 13 Add 50 ml of chloroform and soak overnight. After the next day, the mixture was centrifuged, and chloroform was discarded; the mixture was washed once with 20 ml of chloroform, and the precipitate was centrifuged. The chloroform was drained under reduced pressure to give a solid, which was rinsed twice with distilled water and dried to give a purified extract (DPG (No. 3)). Example 13

有效成分的鉴定 Identification of active ingredients

标准品 DPG购自 SIGMA, 采用从 4个不同种但同一属的瑞香植物提取的 DPG精制品，即实施例 9-12制得的 DPG (No. 1-4)，进行薄板层析分析。 Standard DPG was purchased from SIGMA, and DPG refined products extracted from four different species of the same genus of scented plants, i.e., DPG (No. 1-4) obtained in Examples 9-12, were subjected to thin plate chromatography analysis.

薄板层析的具体条件： Specific conditions for thin plate chromatography:

硅胶 G板，点样后，层析液：正丁醇：醋酸：水 = 4 : 1： 1。 Silica gel G plate, after spotting, chromatography: n-butanol: acetic acid: water = 4 : 1: 1.

发现层析斑点呈单一性，而且迁移位置与对照品一致，分子量与对照品完全一致。 The chromatographic spots were found to be singular, and the migration position was consistent with the control, and the molecular weight was completely consistent with the control.

化学性质分析：标准品和 4个不同种但同一属的瑞香植物提取的 DPG精制品，即实施例 9- 12制得的 DPG (No. 1-4)对比，均呈细粒针状结晶粉末，不溶于***、氯仿或水，但溶于甲醇或乙醇。性质完全相同。实施例 14 Chemical analysis: The standard and four different species of DPG extracts extracted from the genus of the same genus, that is, the DPG (No. 1-4) obtained in Example 9-12, are fine needle-like crystal powders. , insoluble in ether, chloroform or water, but soluble in methanol or ethanol. The nature is exactly the same. Example 14

临床药理学初步观察 Preliminary observation of clinical pharmacology

使用实施例 1 制得的全草粉剂 I 治疗慢性粒细胞白血病加速期患者（志愿者） 2例，一例无效，另一例获部分缓解。骨髓白血病原始细胞从 18%降至 7. 5%。当时的给药方法是：口服全草粉剂 I，每次 l g，每天 3 次，连用 14 天为 1疗程。 Two patients with chronic myeloid leukemia accelerated phase (volunteers) were treated with whole grass powder I prepared in Example 1, one case was ineffective, and the other was partially relieved. Myeloid leukemia blast cells dropped from 18% 7. 5%. The method of administration at that time was: Oral whole grass powder I, lg every time, 3 times a day, for 14 days for a course of treatment.

用实施例 1 制得的全草粉剂 I 分别治疗难治和复发性急性白血病 3 例 (ALL2例， AGL1例）（志愿者），内 1例 AGL获部分缓解； 1例 ALL好转， 1例 ALL无效。 The whole grass powder I prepared in Example 1 was used to treat refractory and relapsed acute leukemia in 3 cases (ALL2 cases, AGL1 cases) (volunteers), 1 case of AGL was partially relieved; 1 case of ALL improved, 1 case of ALL invalid.

结果表明，瑞香全草粉剂已显示出对人类恶性血液病有病理学上的阳性效应，对耐药性急性白血病和慢性粒细胞白血病服用后有抗原始细胞效应，白血病细胞明显减少，血象改善，获部分或完全缓解。 The results showed that Ruixiang whole grass powder has been shown to have a pathological positive effect on human hematological malignancies, and has anti-primary cell effect on drug-resistant acute leukemia and chronic myeloid leukemia, leukemia cells are significantly reduced, blood picture is improved, Partial or complete relief.

对终末期多发性骨髓瘤（Kahler' s 病）不仅可以止痛，且血象改善， I g 单克隆副蛋白下降。实施例 15 End-stage multiple myeloma (Kahler's disease) not only relieves pain, but also improves blood levels, and I g monoclonal paraprotein decreases. Example 15

醇提物的效果 Effect of alcohol extract

采用实施例 5制备的醇提取物 I进行细胞试验，观察对 L 1210小鼠白血病细胞系的体外生物效应。发现醇提取物 I可明显的促凋亡作用，能够抑制白血病细胞的生长。 The alcohol extract I prepared in Example 5 was subjected to a cell test to observe the in vitro biological effect on the L 1210 mouse leukemia cell line. It was found that alcohol extract I can significantly promote apoptosis and inhibit the growth of leukemia cells.

同时，还采用采用 HLF (人胚正常原纤维细胞）进行试验，发现醇提取物 I 对正常人细胞无抑制作用。实施例 16-18 At the same time, experiments using HLF (human embryonic normal fibroblasts) were also found to have no inhibitory effect on normal human cells. Example 16-18

醇提物的效果 Effect of alcohol extract

用实施例 6-8制备的醇提物 I I-IV重复实施例 15 中的细胞试验，同样发现醇提物 I I-IV具有明显的促凋亡作用，能够抑制白血病细胞的生长，且对于正常细胞无影响。实施例 19 The cell assay in Example 15 was repeated using the alcohol extracts I I-IV prepared in Examples 6-8, and it was also found that the alcohol extract I I-IV had a significant pro-apoptotic effect and inhibited the growth of leukemia cells, and Normal cells have no effect. Example 19

DPG的作用 The role of DPG

采用实施例 9制备的 DPG (No. 1)进行细胞试验，观察对 L1210小鼠白血病细胞系的体外生物效应。 The cell test was carried out using DPG (No. 1) prepared in Example 9, and the in vitro biological effect on the L1210 mouse leukemia cell line was observed.

其中图 1为对照：不加 DPG， 37 °C培养 3天； Figure 1 is a control: without DPG, cultured at 37 °C for 3 days;

图 2为添加 2ug/ml DPG , 37 °C培养 3天。发现 DPG可明显的促凋亡作用，能够抑制白血病细胞的生长。 Figure 2 shows the addition of 2 ug/ml DPG and incubation at 37 °C for 3 days. It was found that DPG can significantly promote apoptosis and inhibit the growth of leukemia cells.

同时，还采用采用 HLF (人胚正常成纤维细胞）进行试验，发现 DPG对正常人细胞无抑制作用（图 3， 4)。 At the same time, experiments using HLF (human embryo normal fibroblasts) were found to have no inhibitory effect on normal human cells (Fig. 3, 4).

采用实施例 9制备的 DPG (No. 1)治疗终末期多发性骨髓瘤，骨髓瘤细胞从 37%降至 4. 5%。给药方法是：口服全 DPG (No. 1)，每次 10毫克，每天 3次，连用 14天为 1疗程。 Ig单克隆副蛋白（M-蛋白）恢复正常，止骨痛明显。实施例 20-22 DPG (No. 1) prepared in Example 9 was used to treat end-stage multiple myeloma, and myeloma cells were reduced from 37% to 4.5%. The method of administration is: Oral DPG (No. 1), 10 mg each time, 3 times a day, for 14 days for 1 course of treatment. Ig monoclonal paraprotein (M-protein) returned to normal, and the bone pain was obvious. Example 20-22

DPG的作用 The role of DPG

将实施例 10-12制备的 DPG (No. 2-4)重复进行实施例 19中的体外细胞试验，结果表明，标准品和 4个不同种但同一属的瑞香植物提取的 DPG精制品 DPG (No. 1 -4)具有相同的抑制白血病生长的作用，且作用剂量一致。实施例 23 The DPG (No. 2-4) prepared in Examples 10-12 was repeatedly subjected to the in vitro cell test in Example 19, and the results showed that the standard and four different species but the same genus of the scented plant extract DPG refined product DPG ( No. 1 -4) has the same effect of inhibiting the growth of leukemia, and the dosage is uniform. Example 23

初步毒理学研究 Preliminary toxicology study

大白鼠亚急性毒性实验 Subacute toxicity test in rats

取全草粉剂 I，喂食白鼠，剂量为 0. 5mg/kg/天，连服 15天，证明无毒性。 Take whole grass powder I, and feed the white rats at a dose of 0.5 mg/kg/day for 15 days, which proves to be non-toxic.

DPG (No. 1 )喂食白鼠，剂量为 0. 5mg/kg/天，连服 15天，同样证明无毒性。 DPG (No. 1) was fed a white rat at a dose of 0.5 mg/kg/day for 15 days, which also proved to be non-toxic.

小白鼠急性毒性实验 Acute toxicity test in mice

取高浓度悬液（全草粉剂 1)，剂量同上， 1次给予服用，证明无毒性。 DPG (No. 1 )喂食白鼠，剂量为 0. 5mg/kg/天，连服 15天，同样证明无毒性。 Take a high concentration suspension (whole grass powder 1), the same dose as above, one dose, proved non-toxic. DPG (No. 1) was fed a white rat at a dose of 0.5 mg/kg/day for 15 days, which also proved to be non-toxic.

在以上大鼠实验中，证明瑞香全草粉剂单一剂量超过临床使用的每天剂量的数千倍也未见明显急性毒性效应。实施例 24-26 In the above rat experiments, it was confirmed that the single dose of Ruixiang whole grass powder exceeded the thousands of times of the daily dose used in clinical use, and no obvious acute toxic effect was observed. Example 24-26

初步毒理学研究 Preliminary toxicology study

用实施例 2-4及实施例 10-12制备得到的全草粉剂 I I-IV和 DPG (No. 2-4) 重复实施例 23的试验，结果类似。实施例 27 The whole grass powders I I-IV and DPG (No. 2-4) prepared by using Examples 2-4 and Examples 10-12 were repeated for the test of Example 23, and the results were similar. Example 27

药物组合物配方一： Pharmaceutical composition Formulation one:

全草粉剂 I 1克 Whole grass powder I 1 g

姜黄素 0.2克 Curcumin 0.2 g

赋型剂 0.3克 Excipient 0.3 g

压成片剂制成配方二： Compressed into tablets Formula 2:

醇提物 I 100毫克 Alcohol extract I 100 mg

姜黄素 0.2克 Curcumin 0.2 g

赋型剂 0.3克 Excipient 0.3 g

压成片剂制成配方三： Compressed into tablets Formula 3:

醇提物 ΠΙ 100毫克 Alcohol extract ΠΙ 100 mg

青蒿素 0.2克 Artemisinin 0.2 g

赋型剂 0.3克 Excipient 0.3 g

压成片剂制成配方四： Compressed into tablets Formulation 4:

DPG(No.2) 10毫克 DPG (No. 2) 10 mg

青蒿素 0.2克 Artemisinin 0.2 g

赋型剂 0.3克 Excipient 0.3 g

压成片剂制成配方五： Compressed into tablets Formulation 5:

DPG(No.3) 10毫克 DPG (No. 3) 10 mg

溶解于 150微升异丙醇（助溶剂）注射水 2毫升 Dissolved in 150 μl of isopropanol (cosolvent) 2 ml of water for injection

过滤除菌得到注射剂以上配方给大约 10名患有耐药性急性白血病，慢性粒细胞白血病， MDS 和 Kahler's 病的志愿者使用后，病情都有改善。 Filtration and sterilization to obtain an injection The above formula was improved after about 10 volunteers with drug-resistant acute leukemia, chronic myeloid leukemia, MDS and Kahler's disease.

在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entirety in their entireties in the the the the the the the the In addition, it is to be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the present invention.

Claims

权利要求 Rights request

1.一种如式 I所示的瑞香樱草糖 -芫花黄素，或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物的醇提物，或含有如式 I 所示的瑞香樱草糖-芫花黄素的瑞香属植物药材在制备治疗白血病的药物中的用途， 1. An alcoholic extract of scented scented scutellaria, scutellarin, or scented scented scutellin as shown in Formula I, or containing I. The use of the scent of scented scented scutellaria scutellaria, a medicinal material of sassafras, for the preparation of a medicament for treating leukemia,

2.如权利要求 1 所述的用途，其特征在于，所述的白血病选自耐药性急性白血病，慢性粒细胞白血病，骨髓增生异常综合症，或终末期多发性骨髓瘤。 The use according to claim 1, wherein the leukemia is selected from the group consisting of drug-resistant acute leukemia, chronic myeloid leukemia, myelodysplastic syndrome, or end-stage multiple myeloma.

3.如权利要求 1所述的用途，其特征在于，所述的瑞香属植物选自金边瑞香，芫花，白瑞香，或云南瑞香；优选瑞香属植物的全草粉末。 The use according to claim 1, characterized in that the genus of the genus genus is selected from the group consisting of Phnom Penh, scented scent, scented scent, or Yunnan scented scent;

4.如权利要求 1 所述的用途，其特征在于，所述的醇提物选自甲醇或乙醇提取物。 The use according to claim 1, wherein the alcohol extract is selected from the group consisting of methanol or ethanol extract.

5.如权利要求 1所述的用途，其特征在于，所述的醇提物由下述步骤获得： The use according to claim 1, wherein the alcohol extract is obtained by the following steps:

(a)将瑞香属植物全草粉木用甲醇和 /或乙醇提取获得。 (a) Extracting the genus eucalyptus whole wood flour with methanol and/or ethanol.

6.如权利要求 1 所述的用途，其特征在于，所述的药物为选自口服胶囊，粉剂，片剂，酊剂，贴剂，膏剂，汤药，注射剂，缓释剂，药酒，或口服液的剂型。 The use according to claim 1, wherein the drug is selected from the group consisting of an oral capsule, a powder, a tablet, an elixir, a patch, a plaster, a soup, an injection, a sustained release, a medicinal liquor, or an oral solution. Formulation.

7.—种药物组合物，其特征在于，它含有： 7. A pharmaceutical composition characterized in that it comprises:

(i)作为活性成分的如式 I所示的瑞香樱草糖-芫花黄素，或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物的醇提物，或含有如式 I所示的瑞香樱草糖-芫花黄素的瑞香属植物药材；和 (i) as an active ingredient, a scent of scented scented flavonoids of the formula I, or an alcoholic extract of a genus of the genus of the genus eucalyptus, as shown in formula I, or a medicinal material of the genus eucalyptus containing the fragrant primrose flavonoid as shown in Formula I;

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8.如权利要求 7 所述的药物组合物，其特征在于，所述活性成分的重量为占有组合物总重量的 1-99%。 The pharmaceutical composition according to claim 7, wherein the active ingredient has a weight of from 1 to 99% by weight based on the total weight of the composition.

9.如权利要求 Ί 所述的药物组合物，其特征在于，所述的瑞香属植物选自金边瑞香，芫花，白瑞香，或云南瑞香；所述的醇提物选自甲醇或乙醇提取物。 The pharmaceutical composition according to claim ,, wherein the genus genus is selected from the group consisting of Phnom Penh, scented scent, scented scent, or Yunnan scent; the alcohol extract is selected from the group consisting of methanol or ethanol. Things.

10.—种制备治疗白血病的药物的方法，其特征在于，它包括步骤：将如式 I所示的瑞香樱草糖 -芫花黄素，或含有如式 I所示的瑞香樱草糖 -芫花黄素的瑞香属植物的醇提物，或含有如式 I 所示的瑞香樱草糖-芫花黄素的瑞香属植物药材和药学上可接受的载体混合。 10. A method of preparing a medicament for treating leukemia, characterized in that it comprises the steps of: adding fragrant primrose-baicalin as shown in formula I, or containing ruthenium sorbitol as shown in formula I - An alcoholic extract of the genus eucalyptus of the genus eucalyptus, or a medicinal material of the genus eucalyptus containing the scented scented scutellaria- flavonoid as shown in Formula I, and a pharmaceutically acceptable carrier.

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