WO2005023278A1

WO2005023278A1 - A method of extracting glycyrrhizic flavone and the use of glycyrrhizic flavone for preparing anticarcinogen

Info

Publication number: WO2005023278A1
Application number: PCT/CN2003/000763
Authority: WO
Inventors: Deru Lu
Original assignee: Molecular Biology Limited
Priority date: 2003-09-09
Filing date: 2003-09-09
Publication date: 2005-03-17
Also published as: AU2003264314A1; CN1867347A

Abstract

The present invention disclosses a method of extracting glycyrrhizic flavone from glycyrrhiza and the use of extracted glycyrrhizic flavone for reparing anticarcinogen. The extracted glycyrrhizic flavone has obvious antineoplastic effect, and glycyrrhizic extractive sample is not cell toxin anticarcinogen and has little toxic side effect.

Description

Extraction method of licorice flavones and preparation of anticancer drugs

Technical field

The present invention relates to the field of plant extracts, and more particularly, to a method for extracting licorice flavones from licorice and the use of the extracted licorice flavones for preparing anticancer drugs. Background technique

The main chemical components of licorice are four types: triterpenes, flavones, alkaloids and polysaccharides. Among them, triterpenoids include glycyrrhizic acid, 24-hydroxyglycyrrhetinic acid, and the like. Flavonoids include licorice flavonoids, isoliquiritiflavone, glycyrrhizin, isoliquiritigenin and the like. Alkaloids are tetrahydroquinoline compounds, and polysaccharides are neutral polysaccharides.

For a long time, the development of active ingredients in glycyrrhizin mainly focused on glycyrrhizic acid and glycyrrhetic acid. So far, more than 10 drugs have been developed, and they have been used clinically for anti-hepatitis, anti-allergy, anti-inflammatory, and anti-ulcer.

At present, licorice flavonoids have not been well developed. Many licorice product factories have not used a large amount of "waste residue" containing licorice flavonoids after extracting glycyrrhizic acid.

In recent years, a great deal of research has been done on the pharmacological effects of licorice flavonoids. Although it has been mentioned that licorice flavonoids have some anti-mutagenic, antioxidant and anti-cancer effects, there have been no reports of direct inhibition of tumors and tumor spread. In addition, compared with glycyrrhizic acid, glycyrrhizin flavonoids are a class of compounds with similar structure and similar biological activity. Their monomers are very low in glycyrrhizin, it is difficult to obtain their monomers to study their pharmacological effects. The monomers are extracted and purified in large quantities for large-scale production.

In summary, the antitumor effect of glycyrrhizin flavonoids has not been reported, and there is no antitumor drug with glycyrrhizin as the main active ingredient. In addition, there is an urgent need in the art to develop drugs that can effectively treat tumors. Summary of the invention

The object of the present invention is to provide a new application of a medicine for treating tumors with licorice flavonoids.

Another object of the present invention is to provide a pharmaceutical composition for treating tumors containing glycyrrhizin. In a first aspect of the invention, a pharmaceutical composition is provided, which, based on the total weight of the composition, contains:

(a) 2-99% licorice flavonoids with a total flavonoid content of 50-99% by weight; and

-1-Confirm this (b) 1-95% pharmaceutically acceptable carrier.

In another preferred example, in the licorice flavonoid extract, the total flavonoid content of licorice is 55-90% by weight, and more preferably 60-80% by weight.

In another preferred example, the content of licorice flavonoid extract in the composition is 5-95%, preferably 10-90%, and more preferably 15-80%.

In another preferred example, the licorice flavonoid extract is prepared by the extraction method of the present invention. In another preferred embodiment, the pharmaceutical composition of the present invention is used for treating cancer.

In a second aspect of the present invention, a dietary supplement is provided, which contains a total flavonoid content of licorice that is

50-99% by weight of licorice flavonoid extract as essential ingredients: and the weight of essential ingredients is 5-99% of the total weight of the supplement, and the balance of the dietary acceptable carrier.

In a third aspect of the present invention, a method for preparing a pharmaceutical composition of the present invention is provided, which includes steps:

( _a ) extracting licorice powder with 2- 100 times the weight of 70 ± 15% ethanol at 5 ° C-reflux temperature to obtain an extraction solution;

(b) removing ethanol from the extract under reduced pressure to obtain an aqueous solution;

(c) alkalizing the aqueous solution to pH 9.5 ± 1;

(d) acidifying the aqueous solution to pH 2.5 ± 1;

(e) Leave it for 8-24 hours until precipitation occurs;

(f) filtering to obtain a filtered substance;

(g) purifying the filtrate through a polyamide column and eluting with 90-99% ethanol to obtain an eluate containing glycyrrhizin;

(h) removing ethanol under reduced pressure to obtain a licorice flavonoid extract having a total flavonoid content of 50-99% by weight;

(i) Mixing a licorice flavonoid extract with a carrier of a pharmaceutically acceptable carrier to form a pharmaceutical composition. In a fourth aspect of the present invention, a method for preparing a licorice flavonoid extract is provided, which includes the steps:

(a) extracting licorice powder with 2- 100 times the weight of 70 ± 15% ethanol at 5 ° C-reflux temperature to obtain an extraction solution;

(c) alkalizing the aqueous solution to pH 9.5 ± 1;

(d) acidifying the aqueous solution to pH 2.5 ± 1;

(e) Leave it for 8-24 hours until precipitation occurs;

(f) filtering to obtain a filtered substance;

(g) Purify the filtrate through a polyamide column and elute with 90-99% ethanol to obtain a wash containing glycyrrhizin Dehydration

(h) Ethanol is removed under reduced pressure to obtain a licorice flavonoid extract having a total flavonoid content of 50-99% by weight. In another preferred example, the amount of ethanol in step (a) is 5-20 times the weight of licorice powder, and the ethanol concentration is 70 ± 10%; and the standing time in step (e) is 10-16 hours. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a high performance liquid chromatography separation spectrum of the total flavonoids of licorice. Of which I is glycyrrhizin; II isoliquiritigenin; II is glycyrrhizin; IV isoliquiritigenin; V is glycyrrhizin coumarin; VI isoliquirizin coumarin; VII glycyrrhizin A; VIII glycyrrhol; IX isoliquiritin.

Figure 2 is a standard HPLC separation spectrum of total flavonoids in licorice. Among them 1 glycyrrhizin; 2 iso glycyrrhizin; 3 glycyrrhizin; 4 iso glycyrrhizin; 5 glycyrrhizin coumarin; 6 iso glycyrrhizin coumarin; 7 glycyrrhizin A; 8 glycyrrhizin; 9 iso glycyrrhizin. detailed description

After extensive and in-depth research, the inventor has developed a process for preparing total flavonoids of licorice. The process has two characteristics. One is that the method is simple and has high yield. It can produce large amounts of total flavonoids from licorice and has industrial production value. Second, the total flavonoid content in the product is more than 60%, and the glycyrrhizin (such as glycyrrhizin, isoliquiritigenin) with higher biological activity is higher in content.

Further tests showed that the total flavonoids of licorice had a very effective antitumor effect. In mice S180 and B16 experimental lung metastasis models, tumor inhibition rates of more than 50% can be seen, and in turn, medium and low doses also showed a tumor inhibition rate of more than 40%, and the dose-effect relationship was not obvious. The body weight of tumor-bearing mice increased significantly before and after the experiment. The experimental results suggest that the licorice extract sample is not an anti-tumor cytotoxic drug, and has little toxicity except for some reactions in the digestive tract. As used herein, the term "essential component" or "active ingredient" refers to a licorice extract with a flavonoid content of greater than 50% by weight, such as 50-99%, preferably 55-90% by weight, more preferably 60-80% by weight. Extract.

As used herein, the terms "glycyrrhizin total flavonoid extract" and "glycyrrhiza flavonoid extract" are used interchangeably, and both refer to those extracted from licorice with a licorice flavonoid content greater than 50wt ° /. Extract. Generally, the total flavonoid extract of licorice comprises 2-99% of the total weight of the pharmaceutical composition, preferably 5-95%, more preferably 10-90%, and most preferably at least 20-80%

As used herein, the term "consisting essentially of" refers to being in a composition other than containing the necessary components or In addition to the active ingredient, minor ingredients and / or impurities that do not affect the active ingredient may be contained. For example, sweeteners may be included to improve taste, antioxidants to prevent oxidation, and other additives commonly used in the art.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent and includes various excipients and diluents. The term refers to pharmaceutical carriers that are not essential active ingredients per se and are not excessively toxic after administration. Suitable vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). A pharmaceutically acceptable carrier in the composition may contain a liquid, such as water , Saline, glycerin, and ethanol. In addition, auxiliary substances may be present in these carriers, such as wetting or emulsifying agents, pH buffering substances, and the like.

Non-essential ingredients from licorice flavonoids, as well as other non-essential ingredients (such as other auxiliary medicines), are also included in the definition of a pharmaceutically acceptable carrier.

In another aspect of the present invention, a method for preparing a pharmaceutical composition or dietary supplement of the present invention is provided.

The pharmaceutical composition or dietary supplement of the present invention can be made into any conventional preparation form by a conventional method, preferably a tablet or capsule preparation. The main advantages of the invention are:

(a) The extraction method has high efficiency, and the flavonoid content in the total flavonoid product of licorice is high.

(b) The antitumor effect of total flavonoids of licorice is strong.

(c) The toxic and side effects of total flavonoids in licorice are small. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions specified in the following examples are generally based on conventional conditions or conditions recommended by the manufacturer. Example 1

Extraction of total flavonoids from licorice

Glycyrrhiza uralensis was purchased from Linxian County, Shanxi Province. After drying, crushing and sieving with 60 meshes, licorice powder was obtained, and then 100 g of it was extracted with 70% ethanol for five times. The first time was 180 ml / l hour. The second to fifth times were 130ml / 40minutes, the combined extracts were collected under reduced pressure to recover an alcohol-free water solution, alkalized with 5% Na ₂ C0 ₃ to a pH of 9.5, with 5% HC1 Acidified to a pH of 2.5, left for 12 hours until precipitation occurred, and filtered with suction to obtain a filtrate and a filtrate. The filtrate was washed with water to obtain crude flavonoids of licorice.

The crude flavonoids of glycyrrhizae were applied to a polyamide column (30-60 mesh), washed with water to remove impurities, and then eluted with 95% ethanol. The ethanol was recovered under reduced pressure to obtain higher purity licorice total flavones.

The filtrate obtained from the total flavonoids was filtered by suction, and extracted with n-butanol 1-4 times (the n-butanol was washed with water to remove sugar impurities), and the total triterpene acid component was recovered under reduced pressure.

This method simultaneously obtains flavonoids and triterpene acids. Example 2

Identification of Flavonoids in Total Flavonoids of Licorice by High Performance Liquid Chromatography

In this example, the components in the total flavonoid product of licorice obtained in Example 1 were analyzed by a conventional method.

A. Instruments and reagents:

Agilent HPLC: Model 1100, G1312A Quaternary Pump, 1100 Series Manual Sampler, 1100 Series Variable Length Detector, Agilent ChemStation, Agilent Laser Printer.

B. Chromatographic column:

Zorbax ODS 15cm X 4. 6mm I. D. (Agi lent)

C> Reagent

Yiqing: HPLC-grade (Sigma) water is three times distilled

Acetic acid: Analytical alcohol Shanghai Lingfeng Chemical Reagent Co., Ltd.

D. Separation and determination conditions

Zorbax ODS column, CH ₃ CN-3% H0AC (¾0) linear gradient elution program, flow rate gradient, UV detection wavelength switching.

E. Results

With reference to the prior art method for the determination of nine flavonoids and coumarins in licorice, this example uses the same Zorbax ODS 15cm X 4. 6mm ID column, the solvent is CH ₃ CN-3% H0AC and measurement The wavelength is switched between 310-365nm.

The separation spectrum of the total flavonoids of licorice measured by high performance liquid chromatography is shown in FIG. 2, and the spectrum of FIG. 1 shows that there are 9 monomers such as glycyrrhizin, isoliquiritin, glycyrrhizin, isoliquiritin, and coumarin. This result is basically the same as the prior art (Figure 1), but the retention time is slightly different because the column efficiency is not exactly the same as that of the pre-column. Example 3 Determination of Total Flavonoids in Licorice

In this example, the licorice flavonoid content in the total flavonoid product of licorice obtained in Example 1 was analyzed by a conventional method.

The determination of total flavonoids in crude drugs is generally quantified by visible spectrophotometry, such as alumina and aluminum nitrate colorimetric methods, and also by ultraviolet spectrophotometry. The company uses UV spectrophotometry to quantify the total flavonoids in licorice.

A. Instrument

1600 UV-visible spectrophotometer (Japan Shimaidzu)

B. Reagent

Anhydrous ethanol AR Shanghai Zhenxing Chemical Plant

Methanol NPLC EMERK

C, Rutin standard solution

Rutin: China Pharmaceutical and Biological Products Inspection Institute

Rutin standard solution: Weigh accurately 4.86mg rutin (dried at 120 ° C) into a 100ml volumetric flask with a 100,000th scale and add a methanol solution to the volume to obtain 48.6ug / ml.

D. Results

As for the total flavonoid product obtained in Example 1, as determined by the above method, the content was about 60% (55%, 62%, 71%). This shows that the total flavonoid content obtained by this extraction process is high. Example 4

Acute Toxicity of Total Flavonoids in Licorice

The total flavonoids of glycyrrhizae prepared in Example 1 were used, and the acute toxicity test of the total flavones of glycyrrhizae was performed by a conventional method. Methods as below:

Kunming mice were randomly divided into groups of 20 males and females, divided into five groups. Five doses of enema were 5g / k _g , 4 g / kg, 3.2 g / kg, 2. 56 g / kg, 2. 05 g / kg body weight. Observe the immediate response after administration, and record the weight of the mice within two weeks. Finally, the Blds method was used to calculate the LD _5Q value of the samples.

The results showed that it was not very toxic except for some reactions in the digestive tract (Table A). Table A. Results of Acute Toxicity Test of Glycyrrhiza uralensis Root Extract by Single Gavage to Kunming Mice. Sex Distribution, Number of Animals, Death Distribution (Days), Mortality, LD ₅₀ Mean Animal Weight

g / kg (only) 12345678910—14% (95% CL) (g) g / kg start / end

5 10 10000000000-0 0 3.25 19.8 Male 4 10 6100000000-0 70 (2.91-3.86) 19.8 / 26.3

3.2 10 3100000000-0 0 50 20.0 / 26.6

2.56 10 0 100000000—0 10 19.9 / 26.7

2.05 10 0000000000—0 0 19.8 / 26.4

5 10 10000000000-0 0 3.14 20.4 Male 4 10 7100000000-0 80 (2.72-3.62) 20.2 / 23.5

3.2 10 5100000000—0 60 20.0 / 24.0

2.56 10 1100000000—0 20 20.0 / 24.2

2.05 10 0000000000—0 0 20.1 / 24.3

5 20 20000000000-0 0 3.24

Male and female 4 20 13100000000-0 75 (2.93-3.59)

Each half 3.2 20 8210000000-0 0 55

2.56 20 1200000000-0 0 15

2.05 20 0000000000-0 0 Example 5

Inhibitory effect of licorice total flavonoids on tumors

The total flavonoids of glycyrrhizae prepared in Example 1 were taken, and the inhibition of tumors of the total flavones of glycyrrhizae was performed by a conventional method.

Methods as below:

Efficacy test of S180 sarcoma subcutaneously inoculated with solid tumors: Take vigorously growing solid tumor mice, remove the tumors under aseptic conditions, prepare l_2 X 10Vml cell suspension by homogenization, and inoculate each host with 0 . 2ml / head, the next day, the high, middle and low dose groups were 2, 0.5 and 0.25g / kg positive control cyclophosphamide (STX) 30mg / kg, the negative control group was given the corresponding solvent The animals were sacrificed about ten days after administration, and tumors were dissected and weighed. The tumor suppression rate was calculated according to the following formula: Tumor suppression rate = [(average tumor weight in the control group-average tumor weight in the administration group) / average tumor in the control group Heavy] X 100%.

Tail vein inoculation model B 16: taken under sterile conditions on B16 mouse melanoma tumor cells were cultured in logarithmic growth _phase: prepared at approximately 2.5 X 10 ⁵ cell suspension in C ₅₇ BL / 6 mice were inoculated with the tail vein 0. 2ml Only one animal was given the same dosing schedule as the S180 sarcoma experiment the next day. After three weeks, the animals in each group were sacrificed, and the lungs of the mice in each group were dissected out. The tumor suppression rate was calculated according to the following formula: Tumor suppression rate = [(average tumor weight in the control group-average tumor weight in the administration group) / average tumor weight in the control group] X 100%. The results are shown in Tables B and C. The mouse S180 tumor suppression test and B16 experimental lung metastasis model test show that the total flavonoids of licorice root prepared in Example 1 are very toxic and have a tumor inhibition rate and metastasis inhibition rate of more than 50%, which can be used as anticancer drugs and are particularly suitable for inhibition Tumor recurrence and metastasis after surgery and after chemoradiation are suitable for various malignant tumors.

Table B. Antitumor effect of licorice extract samples on mouse S180 sarcoma (sc)

Compared with negative control *** P <0.01 Table C. Antitumor effect of licorice extract sample on mouse B16 melanosarcoma (iv)

Compared with the negative control *** P <0.01 Example 6

Preparation of drugs

The licorice flavonoid extract prepared in Example 1 was added, and an appropriate amount of auxiliary materials was added as shown in Table 1. The capsule filling machine was used to fill capsules as an oral preparation or make tablets. Table 1

Examples 7-10

Extraction of total flavonoids from licorice

The method of Example 1 was repeated, except that some parameters were changed as shown in Table 2:

Table 2

In Examples 7-10, a licorice flavonoid extract having a total flavonoid content of 55-65% by weight was also obtained. All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

Rights request

What is claimed is: 1. A pharmaceutical composition, characterized in that, based on the total weight of the composition, it contains: (a) a glycyrrhiza flavonoid extract having a total flavonoid content of 2-99% glycyrrhizin of 50-99% by weight; and

5 (b) 1-95% pharmaceutically acceptable carrier.

The pharmaceutical composition according to claim 1, wherein in the licorice flavonoid extract, the total flavonoid content of licorice is 55-90% by weight.

The pharmaceutical composition according to claim 1, characterized in that in the licorice flavonoid extract, the total flavonoid content of licorice is 60-80% by weight.

The pharmaceutical composition II according to claim 1, wherein the content of licorice flavonoid extract in the composition is from 5 to 95%.

5. The pharmaceutical composition according to claim 1, wherein the licorice flavonoid extract is prepared by a method comprising the following steps:

( _a ) extracting licorice powder with 2 to 100 times the weight of 70 ± 15% ethanol at 5 ° C-reflux temperature to obtain an extract solution;

(c) alkalizing the aqueous solution to pH 9.5 ± 1;

(d) acidifying the aqueous solution to pH 2.5 ± 1;

(e) Leave it for 8-24 hours until precipitation occurs;

Ό (f) filtering to obtain a filtered substance;

(g) performing a polyamide column purification on the filter and eluting with 90-99% ethanol to obtain an eluate containing glycyrrhizin;

(h) Ethanol is removed under reduced pressure to obtain a licorice flavonoid extract having a total flavonoid content of 50-99% by weight.

The pharmaceutical composition according to claim 1, which is used for treating cancer.

5 7. A dietary supplement, characterized in that it contains a licorice flavonoid extract with a total flavonoid content of 50-99% by weight as an essential component: and the weight of the essential component accounts for 5-99% of the total weight of the supplement, And the rest of the dietary acceptable carrier.

The method for preparing a pharmaceutical composition according to claim 1, characterized in that it comprises the steps of:

(c) alkalizing the aqueous solution to pH 9.5 ± 1; (d) acidifying the aqueous solution to pH 2.5 ± 1;

(e) Leave it for 8-24 hours until precipitation occurs;

(f) filtering to obtain a filtered substance;

(i) A licorice flavonoid extract is mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

9. A method for preparing a licorice flavonoid extract, comprising the steps of:

( _a ) extracting licorice powder with 2 to 100 times the weight of 70 ± 15% ethanol at 5 ° C_reflux temperature to obtain an extraction solution;

(c) alkalizing the aqueous solution to pH 9.5 ± 1;

(d) Acidify the aqueous solution to pH 2.5 ± 1;

(e) Leave it for 8-24 hours until precipitation occurs;

(f) filtering to obtain a filtered substance;

(h) The ethanol was removed under reduced pressure to obtain a licorice flavonoid extract having a total flavonoid content of 50-99% by weight.

10. The method according to claim 9, wherein the amount of ethanol in step (a) is 5 to 20 times the weight of licorice powder, and the ethanol concentration is 70 ± 10%; and the standing time of step (e) is 10- 16 hours.