WO1995031993A1

WO1995031993A1 - The extract from plants of the genus taxus and use thereof

Info

Publication number: WO1995031993A1
Application number: PCT/CN1995/000045
Authority: WO
Inventors: Ming Guo
Original assignee: Ming Guo
Priority date: 1994-05-24
Filing date: 1995-05-22
Publication date: 1995-11-30
Also published as: CN1112433A; AU2521095A

Abstract

The present invention discloses the extract from barks, roots, stems and/or leaves of plants of the genus Taxus, and an anticancer pharmaceutical composition containing said extract.

Description

FPCH 951604P Taxus plant extract and application thereof

The present invention relates to a Taxus plant extract and a preparation method and application thereof. More specifically, the present invention relates to an extract obtained from the bark, root, and / or stem and leaf of a Taxus plant. Its preparation method and its application as an anticancer drug.

Background technique

¹⁹⁷¹ Wani et isolated from Taxus brevifolia skin (T. brevifolia) in the Taxol, KB cells and demonstrate that it has remarkable cytotoxic effect, have high activity against P388 leukemia and P.1534, and Can inhibit the growth of W256 sarcoma, S180 and lung cancer (see Waniv MC et al., J. Am. Chem. Soc, 1971, 93: 2325). After more than 20 years of extensive research, people have isolated about 100 taxane diterpenes and their alkaloids from Taxus plants, all of which have different degrees of anticancer activity, of which the activity of paclitaxel is the most Strong. 02% Although paclitaxel has excellent anti-cancer activity, the United States FDA has approved the treatment of ovarian cancer in December 1992, but due to limited resources of Taxus in the United States, and the total amount of paclitaxel in its skin is less than 0.02% Coupled with the difficulty of extraction and separation, the amount of pure paclitaxel obtained is very small, so its price is quite expensive and it has not been possible to expand its application. People have been pinning their hopes on chemical synthesis, but so far they have not advised. Disclosure of invention

One of the objects of the present invention is to provide an extract from a yew plant and a preparation method thereof.

Another object of the present invention is to provide an extract which combines the extract and can be widely used. Anticancer drug composition and preparation method thereof.

Pharmacists have been focusing on the separation of single active ingredients of Taxus in the past. This is extremely difficult to isolate, and the amount of pure products obtained is very small, which cannot be used in large quantities. Nearly 100 pure The compounds, most of which are taxanes and their alkaloids, all have varying degrees of anticancer activity. Based on the above facts, the inventor fully extracted the active ingredients in the yew plant with a solvent according to the compound theory of traditional Chinese medicine, and applied it directly as a compound mixture. The results were surprisingly found: In addition to the extraction rate being more than 100 times higher than when separating a pure single substance, this extract also had unexpected anticancer activity comparable to paclitaxel, thus completing the present invention.

Therefore, the present invention provides a yew plant extract, which is characterized by:

(1) at least two or more anticancer active ingredients are combined in the extract; and

(2) The extract has excellent anticancer activity.

The extract of the present invention is extracted from the bark, roots, stems and leaves of a yew plant, or a mixture thereof by a specific method. The yew plants include plants of all species of the yew family, such as yew yunnan, yew tibet, yew southern, yew northeast, yew chinese, yew european, yew Pacific , Japanese yew and yew. Yunnan yew, yew tibet and yew pacific are preferred.

The anticancer active ingredient incorporated in the extract of the present invention includes two parts, known and unknown. The known part includes all effective compounds that have been isolated so far. Examples of representative compounds are: Taxol, Paclitaxel B (cephalmanine), Yunnaxane, and Taxus yunanensi) and so on. The unknown section includes all active compounds that have not yet been isolated and discovered.

The extract of the invention has excellent anti-cancer activity. The hair was measured by a standard method The extract of Ming Ming has a concentration of 0.01, 0.1, 1, 10 and 100 g / ml against nasopharyngeal squamous carcinoma cell line (KB), cervical squamous carcinoma cell line (Hela), and gastric adenocarcinoma cell line (MGC80- 3) Anti-cancer activity in vitro with mouse leukemia cell line (P388), and compared with clinically effective vincristine and colchicine at the same concentration (cells with no significant anticancer activity of vinblastine and colchicine) Strains were not measured). Surviving cancer cells were picked up 48 hours after drug action, and the percentage inhibition rate was calculated. The results are shown in Table 1 (the average of the three results).

Table ①: Antipruritic activity of the extract of the present invention on in vitro cultured ¾ cells

"A" means not imitated The results in Table 1 indicate that the anticancer spectrum of the extract of the present invention is broader than that of vinblastine and colchicine; its anticancer activity against KB, MGC80-3 P388 is comparable to that of vincristine, and the anticancer activity against Hela is similar to that of colchicine .

In addition, the extract of the present invention has the following advantages over paclitaxel:

A. The extraction process is simple and does not require complicated separation of pure substances, thus greatly saving time and costs;

B. The extraction rate is 100 times higher than that of paclitaxel, which can not only save resources, but also provide a large number of drugs conveniently;

C. The application of paclitaxel must be higher than 97%, and its total amount in yew plants is less than 0.02%. It is quite difficult to meet the requirements of clinical application; and the extract of the present invention only requires paclitaxel binding amount of not less than ^2.25%, and anticancer activity of the present invention thus extracts a low binding amount of 97% paclitaxel equivalent paclitaxel (taxol

KB cell line's EDT ¾ 5.5 x 10 " ⁵ ^ mol / L, that is 47 ^ g / ml);

D. The solubility of the extract of the present invention is significantly better than that of paclitaxel.

The present invention also provides a method for preparing an extract of Taxus spp., Which comprises fully extracting the bark, roots, and / or stems and leaves of Taxus spp. With a solvent, then evaporating the solvent, and performing necessary treatment on the obtained residue, An extract of the present invention is prepared. The solvents include: ice; aliphatic hydrocarbons, such as hexane, cyclohexane, petroleum ether, etc .; aromatic hydrocarbons, such as benzene, toluene, xylene, etc .; halogenated hydrocarbons, such as dichloromethane, chloroform, and tetrachloride. Carbon, dichloroethane, dichloroethylene, etc .;-alcohols such as methanol, ethanol, etc .; ethyl acetate, acetone, ether, tetrahydrofuran, dioxane, dimethylformamide and dimethylsulfoxide, etc .; and A mixture of two or more of the above solvents in any ratio. The extraction solvent used in the method of the present invention can be arbitrarily selected as long as it can sufficiently extract the anti-cancer active ingredients. Taxus bark, roots and / or stems and leaves are preferably crushed into

0 ~ 2mm 的 pellets, preferably 0.5 ~ 2. Omm particles.

'' In a preferred embodiment, the method of the invention comprises the following steps:

(1) crushing the bark, roots, and / or stems and leaves of Taxus into 0.5 to 2 mm particles;

( ² ) soaking the crushed particles with 80 ~ 95% ethanol;

(3) filtering out the soaked ethanol solution and evaporating the solvent to obtain a residue;

(4) treating the residue with a mixture of water and dichloromethane and separating the dichloromethane layer, and distilling off the solvent to obtain a crude extract;

(5) The crude extract was washed with n-hexane and methanol in sequence, then dissolved in dichloromethane and filtered, and evaporated to obtain a residue;

(6) The residue is eluted with diethyl ether and ethyl acetate, and the eluent is separately evaporated to obtain two parts of a yellow-brown solid, which are combined to obtain the extract of the present invention. If necessary, the extract may be subjected to silica gel column chromatography and / or activated carbon. \

Another aspect of the present invention is to provide an anti-cancer pharmaceutical composition comprising an anti-cancer effective amount of the Taxus plant extract of the present invention and a pharmaceutically acceptable carrier.

The invention also provides a method for preparing an anticancer pharmaceutical composition of the invention, which method comprises dispersing an effective amount of the extract of the invention in a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers refer to all non-toxic solid, semi-solid and liquid diluents or excipients commonly used in pharmacy that do not adversely affect the extracts of the invention. Examples of them include-water, ethanol, ethylene glycol, propanol, glycerol, polyethylene glycol, peanut oil, castor oil, sorbitol, starch, retinoid cellulose, sodium carboxymethyl cellulose, talc, etc., or they mixture.

In the pharmaceutical composition of the present invention, a lubricant, a disintegrant, a flavoring agent, a pigment, a solubilizer, a dispersant, an emulsifier, a surfactant, a pH adjuster, and the like may be added. The pharmaceutical composition of the present invention can be formulated in the form of seed preparations commonly used in the pharmaceutical field, and examples thereof include tablets, powders, granules, soft or hard gelatin capsules, pills, lozenges, suppositories, emulsions, soluble waves or Among them, injections are preferred.

The pharmaceutical composition of the present invention can be used for preventing and treating various cancers, such as ovarian cancer, breast cancer, endometrial cancer, colon cancer and the like. 'The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally, such as intravenously. The dosage used is based on the patient's age, gender, response to the drug, and severity of the condition. The daily dose for adults is usually 10 mg ~: LOOOmg, preferably 100mg ~ 500mg.

It is clear to the professionals that the invention can make many changes without departing from the spirit of the invention, but all these changes are included in the scope of the invention.

The following examples further explain the invention without limiting it.

Example 1

50 kg of yew yunnan bark crushed into lmm particles was soaked with 450 kg of 85% medical ethanol for 7 days, and the ethanol was filtered out, and the solid was further soaked with 300 kg of 85% ethanol for 3 days. The second ethanol wave was filtered, and the procedure was repeated. 5k _go Two times of mashing 1 time, 3 times of ethanol extraction waves combined, evaporated to remove the solvent under reduced pressure, to obtain a residual paste 9. 5k _go

The paste was extracted with a mixture of 19 kg of water and 19 kg of dichloromethane. The organic layer was separated and the dichloromethane was evaporated to give a residual solid. The solid was washed with 1.2 kg of n-hexane and 1.2 kg of methanol, respectively. The residue was dissolved in dichloromethane, filtered, and the filtrate was evaporated. The remaining yellow-brown solid was extracted with ether and ethyl acetate. and acetic acid - ethyl extracts were evaporated to give 250g and 300g respectively yellow powdery solid, which is the combined extract of the present invention, a total of ⁵ 5 0g, the extraction rate of 1.10% (by weight of crude drug).

Example 2

• According to the method of Example 1, 50 kg of Tibetan yew was used as a raw material to prepare the present invention 08%。 The extract of 540g, the extraction rate is Ί. 08%.

Example 3

99%。 According to the method of Example 1, using 1kg Pacific yew bark as the original family, 9.9 g of the extract of the present invention was prepared, the extraction rate was 0.99%.

Example 4

96%。 According to the method of Example 1, using 1kg Canadian yew skin as a raw material, an extract of the present invention 9.6g was obtained, the extraction rate was 0.96%, wherein the combined amount of paclitaxel was 0.96%.

Example 5

'' 10g of the extract of the present invention is dissolved in a mixed solvent of 1 liter of ethylene glycol, ethanol and water (5: 2: 3), filtered, and sealed in a 2ml ampoule. Injection, 10 mg of active ingredient per ml.

Example 6

The pharmaceutical composition tablet of the present invention is prepared with the following ingredients:

mg / tablet

Extract 20

Vitamin C cellulose 30

Starch 40

Polyvinyl ketone

(10% water soluble wave) 4 sodium carboxymethyl starch 4. 5

镆 stearic acid 0.5

Talcum powder 1 lOOmg The extract, starch and retinoid cellulose were sieved through a ⁴ 5 mesh sieve and thoroughly mixed. The water-soluble waves of polyvinyl alkane were mixed with it, and then passed through a 14 mesh sieve. The obtained granules were 50-6 (18 after TC drying). added to the granules after mesh screen. carboxymethyl starch, talc, magnesium stearate through a 60 mesh sieve and, after mixing, are compressed tablets, each weighing lOOmg, wherein the co-extract 20m _g.

Claims

Claim

1. An extract obtained from the bark, roots, and / or stems and leaves of a yew plant, characterized by:

(1) at least two or more anticancer active compounds are combined in the extract; and

(2) The extract has excellent anticancer activity.

2. The extract of claim 1, which is extracted from the roots and / or stems and leaves of any of the following taxus plants: yew yunnan, yew tibetan, yew south, yew northeast, yew chinese Yew, Taxus chinensis, Pacific yew, Canadian yew, Japanese yew and yew. ·

3. The extract of claim 1, which is extracted from the bark of Taxus yunnanensis, Taxus tibetiana or Taxus chinensis.

4. The extract of any one of claims 1 to 3, wherein the combined anticancer active compounds include at least one or more of the following compounds; paclitaxel, paclitaxel B, yew taxol Yunnan, and Yunnan Taxene.

5. A method for preparing the extract according to claim 1, comprising fully extracting the bark, roots, and / or stems and leaves of Taxus spp. With a solvent, then evaporating the solvent, and performing necessary treatment on the residue with an appropriate solvent, wherein The solvent includes water, ethanol, methanol, dichloromethane, chloroform, diethyl ether, petroleum ether, n-hexane, ethyl acetate, acetone, or any mixture of two or more thereof.

6. An anticancer pharmaceutical composition comprising an anticancer effective amount of the extract of any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

7. The application of the anticancer drug composition of claim 6 in the prevention and treatment of various cancers. 't

8. Application of the anticancer pharmaceutical composition of claim 6 in the prevention and treatment of ovarian cancer, breast cancer, endometrial cancer and colon cancer.

9. A method for preparing an anticancer pharmaceutical composition according to claim 6, which comprises combining anticancer drugs with The effective amount of the bifurcation requires that the extract of any one of 1 to 3 be dispersed in a pharmaceutically acceptable carrier.

10. Application of the extract of any one of claims 1 to 3 in the preparation of anticancer drugs