CN112022892B

CN112022892B - Application of organic extract of plant of Cirsium

Info

Publication number: CN112022892B
Application number: CN201910477365.6A
Authority: CN
Inventors: 何建明
Original assignee: Zhejiang Wolwo Bio Pharmaceutical Co Ltd
Current assignee: Zhejiang Wolwo Bio Pharmaceutical Co Ltd
Priority date: 2019-06-03
Filing date: 2019-06-03
Publication date: 2022-06-07
Anticipated expiration: 2039-06-03
Also published as: WO2020244493A1; CN112022892A

Abstract

The invention discloses an organic extract of thistle plants, which is preferably obtained by extracting one or more of ethanol, acetone, ethyl acetate, dichloromethane, trichloromethane, petroleum ether, n-hexane and cyclohexane; preferably, the extract of the thistle plant is an extract of the root part of the thistle plant. In addition, the invention also discloses a thistle plant extract, which has a characteristic ultraviolet absorption peak with the peak-appearing time of 35-40 minutes, wherein the peak-appearing time is detected by high performance liquid chromatography at the wavelength of 254 nm. The invention also relates to the application of the extract in the preparation of anti-mycobacterium tuberculosis drugs or tuberculosis treatment drugs.

Description

Application of organic extract of plant of Cirsium

Technical Field

The invention relates to the field of pharmacy, in particular to an organic extract of thistle plants and application and a composition thereof.

Background

Mycobacterium tuberculosis can infect the lung leading to tuberculosis and can also infect extrapulmonary organs such as the intestine, peritoneum, kidney, bladder, ureter, pleura, bone, joint, brain, meninges, reproductive system, etc. Extrapulmonary tuberculosis is commonly seen in people with low immunity. Pulmonary tuberculosis includes primary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis and secondary pulmonary tuberculosis. The primary pulmonary tuberculosis is also called as primary tuberculosis, which is often seen in children, and the classical pathological changes include tuberculous inflammation of primary foci of the lung, draining lymphatic vessels and pulmonary portal or mediastinal lymph nodes, which are jointly called as primary syndrome. Tuberculosis patients commonly have low fever, night sweat, hypodynamia and weight loss, high fever can appear when the focus is rapidly developed, and other symptoms are different due to different infection parts: tuberculosis patients often suffer from cough, expectoration, hemoptysis, chest pain and shortness of breath, and allergic reaction and anergic tuberculosis can also occur. Patients with intestinal tuberculosis are often infected orally, namely patients with open pulmonary tuberculosis or patients with laryngeal tuberculosis swallow sputum containing mycobacterium tuberculosis. It is usually found in the ileocecal region, which can cause abdominal pain, diarrhea, constipation, abdominal mass, intestinal obstruction, fistula, abscess, acute perforation of intestine, etc. Tuberculous peritonitis is often manifested by abdominal pain, ascites, a pliable feeling of the abdominal wall or abdominal mass, and diarrhea.

Currently, the first line drugs for tuberculosis include isoniazid, rifampicin, diazinamide, streptomycin, ethambutol, and the like. However, these drugs are generally resistant and have severe liver damage. Therefore, there is an urgent need in the art to find a natural drug against mycobacterium tuberculosis to overcome the limitations of the existing drugs.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides an extract of thistle genus plant, wherein the extract of thistle genus plant is an organic phase extract; preferably, the organic extract is an organic extract obtained by extraction using one or more of ethanol, acetone, ethyl acetate, dichloromethane, chloroform, petroleum ether, n-hexane, and cyclohexane; preferably, the extract of the thistle plant is an extract of the root part of the thistle plant.

In one or more embodiments, the thistle plant is selected from the group consisting of thistle (Cirsium japonica), thistle (Cirsium maackii), echium sativum (Cirsium chinensis), echium setosum (Cirsium setosum), setaria japonica (Cirsium linearium), and thistle (Cirsium tianmushanicum).

In another aspect of the present invention, there is provided a thistle extract characterized by having a characteristic uv absorption peak with a peak time of between 35-40 minutes, wherein the peak time is determined by hplc at a wavelength of 254 nm.

In one or more embodiments, the extract has characteristic uv absorption peaks with peak times of 35.8 ± 0.3 minutes, 36.6 ± 0.3 minutes, and 37.7 ± 0.3 minutes, respectively; more preferably, the extract has characteristic ultraviolet absorption peaks with peak times of 35.8 ± 0.2 min, 36.6 ± 0.2 min and 37.7 ± 0.2 min, respectively; more preferably, the extract has characteristic UV absorption peaks with peak times of 35.8 + -0.1 min, 36.6 + -0.1 min and 37.7 + -0.1 min, respectively.

In one or more embodiments, the extract further has one or more of characteristic uv absorbance peaks with a peak time of 37.0 ± 0.3 minutes, preferably 37.0 ± 0.2 minutes, more preferably 37.0 ± 0.1 minutes, and a peak time of 38.9 ± 0.3 minutes, preferably 38.9 ± 0.2 minutes, more preferably 38.9 ± 0.1 minutes;

in one or more embodiments, the extract also has a characteristic uv absorbance peak with a peak time between 30 and 35 minutes, and/or a characteristic uv absorbance peak with a peak time between 40 and 45 minutes; preferably, the extract has a characteristic ultraviolet absorption peak with a peak time of 34.2 ± 0.3 minutes, preferably 34.2 ± 0.2 minutes, more preferably 34.2 ± 0.1 minutes, a characteristic ultraviolet absorption peak with a peak time of 32.5 ± 0.3 minutes, preferably 32.5 ± 0.2 minutes, more preferably 32.5 ± 0.1 minutes, a characteristic ultraviolet absorption peak with a peak time of 31.5 ± 0.3 minutes, preferably 31.5 ± 0.2 minutes, more preferably 31.5 ± 0.1 minutes, a characteristic ultraviolet absorption peak with a peak time of 42.0 ± 0.3 minutes, preferably 42.0 ± 0.2 minutes, more preferably 42.0 ± 0.1 minutes, and one or more of the characteristic ultraviolet absorption peaks with a peak time of 43.8 ± 0.3 minutes, preferably 43.8 ± 0.2 minutes, more preferably 43.8 ± 0.1 minutes;

wherein the detection conditions of the high performance liquid chromatography are as follows:

column temperature: at a temperature of 35 c,

a detector: an ultraviolet detector is arranged on the base plate,

detection wavelength: the wavelength of the light beam is 254nm,

a chromatographic column: phenomenex luna 4.6X 250mm, 5 μm,

sample introduction amount: 1-20 mu L of the mixture is prepared,

mobile phase: 0.1% glacial acetic acid water solution, acetonitrile,

flow rate of mobile phase: 1mL/min of the reaction solution is added,

gradient elution conditions:

time/minute	Acetonitrile/volume percent	0.1% glacial acetic acid solution/volume percent
			0	12	88
8	40	60
			18	70	30
55	100	0

In one or more embodiments, the extract has characteristic uv absorption peaks with peak times as shown below: 35.8. + -. 0.1 min, 36.6. + -. 0.1 min and 37.7. + -. 0.1 min, preferably 34.2. + -. 0.1 min, 35.8. + -. 0.1 min, 36.6. + -. 0.1 min, 37.0. + -. 0.1 min, 37.7. + -. 0.1 min and 42.0. + -. 0.1 min, more preferably 31.5. + -. 0.1 min, 32.5. + -. 0.1 min, 34.2. + -. 0.1 min, 35.8. + -. 0.1 min, 36.6. + -. 0.1 min, 37.0. + -. 0.1 min, 37.7. + -. 0.1 min, 38.9. + -. 0.1 min, 42.0. + -. 0.1 min and 43.8. + -. 0.1 min; or characteristic ultraviolet absorption peaks with the peak-out time as shown below: 35.8. + -. 0.2 min, 36.6. + -. 0.2 min and 37.7. + -. 0.2 min, preferably 34.2. + -. 0.2 min, 35.8. + -. 0.2 min, 36.6. + -. 0.2 min, 37.0. + -. 0.2 min, 37.7. + -. 0.2 min and 42.0. + -. 0.2 min, more preferably 31.5. + -. 0.2 min, 32.5. + -. 0.2 min, 34.2. + -. 0.2 min, 35.8. + -. 0.2 min, 36.6. + -. 0.2 min, 37.0. + -. 0.2 min, 37.7. + -. 0.2 min, 38.9. + -. 0.2 min, 42.0. + -. 0.2 min and 43.8. + -. 0.2 min; or has characteristic ultraviolet absorption peaks with the peak-off times as shown below: 35.8. + -. 0.3 minutes, 36.6. + -. 0.3 minutes and 37.7. + -. 0.3 minutes, preferably 34.2. + -. 0.3 minutes, 35.8. + -. 0.3 minutes, 36.6. + -. 0.3 minutes, 37.0. + -. 0.3 minutes, 37.7. + -. 0.3 minutes and 42.0. + -. 0.3 minutes, more preferably 31.5. + -. 0.3 minutes, 32.5. + -. 0.3 minutes, 34.2. + -. 0.3 minutes, 35.8. + -. 0.3 minutes, 36.6. + -. 0.3 minutes, 37.0. + -. 0.3 minutes, 37.7. + -. 0.3 minutes, 38.9. + -. 0.3 minutes, 42.0. + -. 0.3 minutes and 43.8. + -. 0.3 minutes.

In one or more embodiments, the thistle extract has characteristic uv absorbance peaks with peak times as shown below: 35.6 + -0.1 min, 36.6 + -0.1 min, 37.8 + -0.1 min, 38.9 + -0.1 min, 42.0 + -0.1 min and 43.7 + -0.1 min, optionally with characteristic UV absorption peaks at 38.2 + -0.1 min and 46.8 + -0.1 min.

In one or more embodiments, the thistle extract has characteristic uv absorbance peaks with peak times as shown below: 34.2 + -0.1 min, 35.9 + -0.1 min, 36.6 + -0.1 min, 36.9 + -0.1 min and 37.6 + -0.1 min, optionally having characteristic absorption peaks at 32.9 + -0.1 min, 33.2 + -0.1 min, 34.5 + -0.1 min and 38.1 + -0.1 min.

In one or more embodiments, the thistle plant extract has characteristic uv absorbance peaks for uv with peak times as shown below: 31.5 + -0.1 min, 32.5 + -0.1 min, 34.3 + -0.1 min, 35.8 + -0.1 min, 36.6 + -0.1 min, 37.0 + -0.1 min and 37.6 + -0.1 min, optionally 29.9 + -0.1 min, 30.3 + -0.1 min, 31.0 + -0.1 min, 32.0 + -0.1 min, 33.2 + -0.1 min, 33.9 + -0.1 min, 35.2 + -0.1 min, 35.7 + -0.1 min, 36.1 + -0.1 min, 40.0 + -0.1 min, 40.7 + -0.1 min and 41.2 + -0.1 min.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an extract according to any one of the embodiments herein and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition consists of a therapeutically effective amount of the extract according to any of the embodiments herein and a pharmaceutically acceptable carrier.

The invention also provides application of the extract in any embodiment of the invention in preparing anti-mycobacterium tuberculosis drugs or tuberculosis treatment drugs.

In one or more embodiments, the mycobacterium tuberculosis is selected from the group consisting of human mycobacterium tuberculosis and mycobacterium bovis; more preferably, the mycobacterium tuberculosis is selected from drug-resistant human mycobacterium tuberculosis.

In one or more embodiments, the tuberculosis is caused by mycobacterium tuberculosis; more preferably, the tuberculosis is selected from tuberculosis or extrapulmonary tuberculosis; still preferably, the tuberculosis is pulmonary tuberculosis, hepatic tuberculosis, gastric tuberculosis or intestinal tuberculosis.

The invention also provides a method of treating tuberculosis, the method comprising administering to a patient a therapeutically effective amount of an extract according to any one of the embodiments of the invention, or administering to a patient a pharmaceutical composition according to any one of the embodiments of the invention.

In one or more embodiments, the tuberculosis is caused by mycobacterium tuberculosis; more preferably, the mycobacterium tuberculosis is selected from the group consisting of human mycobacterium tuberculosis and mycobacterium bovis; still more preferably, the mycobacterium tuberculosis is selected from drug-resistant human mycobacterium tuberculosis.

In one or more embodiments, the tuberculosis is selected from tuberculosis or extrapulmonary tuberculosis; more preferably, the tuberculosis is pulmonary tuberculosis, hepatic tuberculosis, gastric tuberculosis or intestinal tuberculosis.

The present invention also provides a method for preparing a thistle extract, comprising the steps of extracting a thistle with an organic solution; preferably, the organic solution is selected from one or more of ethanol, acetone, ethyl acetate, dichloromethane, chloroform, petroleum ether, n-hexane and cyclohexane.

In one or more embodiments, the extraction is performed using the root locus of a plant of the genus Cirsium.

Drawings

FIG. 1 shows the HPLC profile of the ethanol extract of roots of example 4.

FIG. 2 shows the HPLC chromatogram of the root petroleum ether extract of example 4.

FIG. 3 shows an HPLC chromatogram of the C01-34 component in example 4.

FIG. 4 shows the HPLC profile of the C01-84 component in example 4.

FIG. 5 shows the HPLC profile of the C01-159 component in example 4.

Detailed description of the preferred embodiments

In the present specification, the components referred to or the preferred components thereof may be combined with each other to form a new technical solution, if not specifically stated.

In the present specification, all embodiments and preferred embodiments mentioned herein may be combined with each other to form a new technical solution, if not specifically stated.

In the present description, all the technical features mentioned herein as well as preferred features may be combined with each other to form new technical solutions, if not specifically mentioned.

In the present specification, the sum of the contents of the components in the composition is 100% if not otherwise stated.

In the present specification, the sum of the parts of the components in the composition may be 100 parts by weight, if not indicated to the contrary.

In this specification, unless otherwise stated, the numerical range "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "0 to 5" indicates that all real numbers between "0 to 5" have been listed herein, and "0 to 5" is only a shorthand representation of the combination of these numbers.

In this specification, unless otherwise stated, the range of integer values "a-b" represents a shorthand representation of any combination of integers between a and b, where a and b are both integers. For example, an integer numerical range of "1-N" means 1, 2 … … N, where N is an integer.

In this specification, unless stated otherwise, "combinations thereof" means multi-component mixtures of the elements described, for example two, three, four and up to the maximum possible multi-component mixtures.

The term "a" or "an" as used herein means "at least one" if not otherwise specified.

All percentages (including weight percentages) stated in this specification are based on the total weight of the composition, unless otherwise specified.

The "ranges" disclosed herein are in the form of lower and upper limits. There may be one or more lower limits, and one or more upper limits, respectively. The given range is defined by the selection of a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this manner are inclusive and combinable, i.e., any lower limit can be combined with any upper limit to form a range. For example, ranges of 60-120 and 80-110 are listed for particular parameters, with the understanding that ranges of 60-110 and 80-120 are also contemplated. Furthermore, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5.

Specifically, the invention provides an organic extract of a plant of the genus Cirsium. The plant of the genus Cirsium is a plant of the family Compositae, and includes plants of the genus Cirsium well known in the art, including but not limited to Cirsium (Cirsium japonicum), Cirsium japonicum (Cirsium maackii), Cirsium setosum (Cirsium chinensis), Cynara cardunculus (Cirsium setosum), Cirsium linearis (Cirsium Lineare), and Cynara scolymus (Cirsium tianmushanicum). In a preferred embodiment of the invention, the thistle plant is a thistle plant growing in Shanghai, Zhejiang, Jiangsu and Anhui provinces, more preferably in Yiwu, Zhejiang. The part of the plant of the genus Cirsium used for extraction may preferably be the root.

Herein, the organic extract refers to an extract obtained by extraction with an organic solvent, and is usually located in an organic phase. Organic solvents suitable for use in the present invention include, but are not limited to, any one or more of ethanol, acetone, ethyl acetate, dichloromethane, chloroform, petroleum ether, n-hexane, and cyclohexane. Preferred organic solvents are one or more of ethanol, petroleum ether, n-hexane and cyclohexane. When a water-soluble organic solvent is used, the concentration of the organic solvent may be 20 to 100% by mass, preferably 40 to 100% by mass, more preferably 60 to 100% by mass, most preferably 80 to 100% by mass. For example, the extraction may be performed using ethanol or an aqueous ethanol solution having a concentration of 20 to 100% by mass, preferably 40 to 100% by mass, more preferably 60 to 100% by mass, most preferably 80 to 100% by mass.

In a preferred embodiment of the invention, said thistle extract is an ethanol extract, petroleum ether extract, n-hexane extract or cyclohexane extract of the roots of thistle.

In some embodiments of the present invention, the present invention provides a thistle extract characterized by having a characteristic ultraviolet absorbance peak with a peak time between 35 and 40 minutes, wherein the peak time is determined by High Performance Liquid Chromatography (HPLC) at a wavelength of 254 nm. Preferably, the extract has characteristic UV absorption peaks with peak times of 35.8 + -0.3 min, 36.6 + -0.3 min and 37.7 + -0.3 min; more preferably, the extract has characteristic ultraviolet absorption peaks with peak times of 35.8 ± 0.2 min, 36.6 ± 0.2 min and 37.7 ± 0.2 min, respectively; more preferably, the extract has characteristic UV absorption peaks with peak times of 35.8 + -0.1 min, 36.6 + -0.1 min and 37.7 + -0.1 min, respectively. Optionally, the extract also has characteristic UV absorption peak with peak time of 37.0 + -0.3 min, preferably 37.0 + -0.2 min, more preferably 37.0 + -0.1 min, and one or more of characteristic UV absorption peaks with peak time of 38.9 + -0.3 min, preferably 38.9 + -0.2 min, more preferably 38.9 + -0.1 min. Optionally, the extract also has a characteristic uv absorbance peak with a peak time between 30 and 35 minutes, and/or a characteristic uv absorbance peak with a peak time between 40 and 45 minutes. Preferably, the extract has a characteristic ultraviolet absorption peak with a peak time of 34.2 ± 0.3 minutes, preferably 34.2 ± 0.2 minutes, more preferably 34.2 ± 0.1 minutes, a characteristic ultraviolet absorption peak with a peak time of 32.5 ± 0.3 minutes, preferably 32.5 ± 0.2 minutes, more preferably 32.5 ± 0.1 minutes, a characteristic ultraviolet absorption peak with a peak time of 31.5 ± 0.3 minutes, preferably 31.5 ± 0.2 minutes, more preferably 31.5 ± 0.1 minutes, a characteristic ultraviolet absorption peak with a peak time of 42.0 ± 0.3 minutes, preferably 42.0 ± 0.2 minutes, more preferably 42.0 ± 0.1 minutes, and one or more of the characteristic ultraviolet absorption peaks with a peak time of 43.8 ± 0.3 minutes, preferably 43.8 ± 0.2 minutes, more preferably 43.8 ± 0.1 minutes.

In some preferred embodiments, the thistle plant extract of the present invention has characteristic uv absorbance peaks with peak times as shown below: 35.8. + -. 0.1 min, 36.6. + -. 0.1 min and 37.7. + -. 0.1 min, preferably 34.2. + -. 0.1 min, 35.8. + -. 0.1 min, 36.6. + -. 0.1 min, 37.0. + -. 0.1 min, 37.7. + -. 0.1 min and 42.0. + -. 0.1 min, more preferably 31.5. + -. 0.1 min, 32.5. + -. 0.1 min, 34.2. + -. 0.1 min, 35.8. + -. 0.1 min, 36.6. + -. 0.1 min, 37.0. + -. 0.1 min, 37.7. + -. 0.1 min, 38.9. + -. 0.1 min, 42.0. + -. 0.1 min and 43.8. + -. 0.1 min; alternatively, the characteristic uv absorption peaks with peak-off times as shown below: 35.8. + -. 0.2 min, 36.6. + -. 0.2 min and 37.7. + -. 0.2 min, preferably 34.2. + -. 0.2 min, 35.8. + -. 0.2 min, 36.6. + -. 0.2 min, 37.0. + -. 0.2 min, 37.7. + -. 0.2 min and 42.0. + -. 0.2 min, more preferably 31.5. + -. 0.2 min, 32.5. + -. 0.2 min, 34.2. + -. 0.2 min, 35.8. + -. 0.2 min, 36.6. + -. 0.2 min, 37.0. + -. 0.2 min, 37.7. + -. 0.2 min, 38.9. + -. 0.2 min, 42.0. + -. 0.2 min and 43.8. + -. 0.2 min; alternatively, the characteristic uv absorption peaks with peak-off times as shown below: 35.8. + -. 0.3 min, 36.6. + -. 0.3 min and 37.7. + -. 0.3 min, preferably 34.2. + -. 0.3 min, 35.8. + -. 0.3 min, 36.6. + -. 0.3 min, 37.0. + -. 0.3 min, 37.7. + -. 0.3 min and 42.0. + -. 0.3 min, more preferably 31.5. + -. 0.3 min, 32.5. + -. 0.3 min, 34.2. + -. 0.3 min, 35.8. + -. 0.3 min, 36.6. + -. 0.3 min, 37.0. + -. 0.3 min, 37.7. + -. 0.3 min, 38.9. + -. 0.3 min, 42.0. + -. 0.3 min and 43.8. + -. 0.3 min.

It will be appreciated that the thistle extract of the invention may also be a purified extract, e.g. an extract purified by preparative High Performance Liquid Chromatography (HPLC), or specific fractions collected by suitable methods, e.g. separation and purification using column chromatography with silica gel as a packing material.

As shown herein, the purified extract, in its various fractions, is likely to have the biological activity (anti-mycobacterium tuberculosis activity) described herein. Therefore, the thistle plant extract of the present invention can be a mixture of different organic solvent extracts in any ratio, or a mixture of different fractions obtained by purifying the same organic solvent extract in any ratio, or a mixture of fractions obtained by purifying and an organic solvent extract not purified in any ratio.

In some embodiments, the thistle plant extract of the present invention has characteristic uv absorbance peaks with peak times as shown below: 35.6 + -0.1 min, 36.6 + -0.1 min, 37.8 + -0.1 min, 38.9 + -0.1 min, 42.0 + -0.1 min and 43.7 + -0.1 min, optionally with characteristic UV absorption peaks at 38.2 + -0.1 min and 46.8 + -0.1 min.

In some embodiments, the thistle extract of the present invention has characteristic uv absorbance peaks with peak times as shown below: 34.2 + -0.1 min, 35.9 + -0.1 min, 36.6 + -0.1 min, 36.9 + -0.1 min and 37.6 + -0.1 min, optionally with characteristic absorption peaks at 32.9 + -0.1 min, 33.2 + -0.1 min, 34.5 + -0.1 min and 38.1 + -0.1 min.

In some embodiments, the thistle plant extract of the present invention has uv characteristic uv absorbance peaks with peak times as shown below: 31.5 + -0.1 min, 32.5 + -0.1 min, 34.3 + -0.1 min, 35.8 + -0.1 min, 36.6 + -0.1 min, 37.0 + -0.1 min and 37.6 + -0.1 min, optionally 29.9 + -0.1 min, 30.3 + -0.1 min, 31.0 + -0.1 min, 32.0 + -0.1 min, 33.2 + -0.1 min, 33.9 + -0.1 min, 35.2 + -0.1 min, 35.7 + -0.1 min, 36.1 + -0.1 min, 40.0 + -0.1 min, 40.7 + -0.1 min and 41.2 + -0.1 min.

In a preferred embodiment, such extracts are those purified by preparative High Performance Liquid Chromatography (HPLC).

Herein, unless otherwise indicated, the characteristic uv absorption peaks described herein are detected using High Performance Liquid Chromatography (HPLC) detection conditions as described below:

column temperature: at a temperature of 35 c,

a detector: an ultraviolet detector is arranged on the base plate,

detection wavelength: the wavelength of the light source is 254nm,

and (3) chromatographic column: phenomenex luna 4.6X 250mm, 5 μm,

sample injection amount: 1-20 mu L of the mixture is prepared,

mobile phase: 0.1% glacial acetic acid water solution, acetonitrile,

flow rate of mobile phase: 1mL/min of the reaction solution is added,

gradient elution conditions:

It will be appreciated that although the examples herein specifically disclose methods for purifying extracts of the genus Cirsium and give the corresponding time-to-peak for some of the extracts, extracts purified from organic solvent extracts of the genus Cirsium using other purification techniques and tested by the test methods described herein to obtain time-to-peak within the ranges described herein should also fall within the ranges described herein.

The extract of the invention can be used for resisting mycobacterium tuberculosis and is used for treating tuberculosis caused by the mycobacterium tuberculosis. Herein, Mycobacterium tuberculosis is preferably selected from human Mycobacterium tuberculosis and Mycobacterium bovis, and more preferably drug-resistant human Mycobacterium tuberculosis. Herein, tuberculosis is selected from pulmonary tuberculosis or extrapulmonary tuberculosis. Preferably, the tuberculosis is pulmonary tuberculosis, hepatic tuberculosis, gastric tuberculosis or intestinal tuberculosis.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an extract of a plant of the genus Cirsium as described herein and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition consists of a therapeutically effective amount of the extract of the plant of the genus Cirsium and a pharmaceutically acceptable carrier.

In the present specification, unless otherwise specified, the term "comprising … …" means that the pharmaceutical composition may also contain any other components, which may be present in any amount, as long as the component present in this amount is acceptable to the human body and does not negatively affect the activity of the active ingredient (the extract of the thistle genus plant) in the pharmaceutical composition of the present invention.

In the present specification, unless otherwise specified, the term "pharmaceutically acceptable carrier" shall be compatible with the active ingredient (the extract of the plant of the genus thistle) in the pharmaceutical composition of the present invention, i.e. able to be blended therewith without a substantial decrease in the efficacy of the drug under normal circumstances. As "pharmaceutically acceptable carriers" there may be included, but are not limited to: sugars such as lactose, glucose, sucrose, trehalose; starches, such as corn starch, potato starch; cellulose or its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose; powdered gum tragacanth; gelatin; talc powder; petrolatum; solid lubricants, such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, cocoa butter; alcohols, such as ethanol, propylene glycol, glycerol, sorbitol, mannitol, polyethylene glycol; aminocaproic acid, alginic acid; emulsifiers, such as tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting; an antioxidant; a pharmaceutically acceptable preservative; pyrogen-free water; isotonic saline solution; buffers, and the like, or combinations thereof.

The term "therapeutically effective amount" means that the active ingredient (extract of said thrips genus plant) in said pharmaceutical composition may act to treat a disease, such as tuberculosis.

Preferably, the pharmaceutical composition is administered via the digestive tract, via the respiratory tract, transdermally, transmucosally, or by injection. The subject to which the pharmaceutical composition is administered is a primate, more preferably a human. The dosage form of the pharmaceutical composition is a dosage form for administration through the alimentary canal, a dosage form for administration through the respiratory tract, a dosage form for transdermal administration, a dosage form for administration through mucosa and a dosage form for injection; more preferably, the dosage form of the pharmaceutical composition is selected from oral liquid, tablets, capsules, powder, granules, pills, eye drops, nasal drops, suppositories, dripping pills, liniments, ointments, patches, pastes, sprays, aerosols, powder inhalation solutions (suspensions), injections, powder injections, water injections, large infusion dosage forms and the like.

The application also provides the use of the thistle plant extract described herein in the preparation of an anti-mycobacterium tuberculosis medicament or a medicament for the treatment of tuberculosis, or the use of the thistle plant extract in the treatment of tuberculosis. Preferably, the tuberculosis is caused by mycobacterium tuberculosis. More preferably, the mycobacterium tuberculosis is selected from the group consisting of human mycobacterium tuberculosis and mycobacterium bovis; still more preferably, the Mycobacterium tuberculosis is selected from drug-resistant human Mycobacterium tuberculosis. Preferably, the tuberculosis is selected from pulmonary tuberculosis or extrapulmonary tuberculosis; more preferably, the tuberculosis is pulmonary tuberculosis, hepatic tuberculosis, gastric tuberculosis or intestinal tuberculosis.

In one embodiment of the application, the subject of said use or method is a primate (e.g. monkey or human), preferably a human.

The invention also provides a method for preparing the thistle plant extract, which comprises the step of extracting the thistle plants by adopting an organic solution. As previously mentioned, organic solvents suitable for use in the present invention include, but are not limited to, any one or more of ethanol, acetone, ethyl acetate, dichloromethane, chloroform, petroleum ether, n-hexane, and cyclohexane. Preferred organic solvents are one or more of ethanol, petroleum ether, n-hexane and cyclohexane. When a water-soluble organic solvent is used, the concentration of the organic solvent may be 20 to 100% by mass, preferably 40 to 100% by mass, more preferably 60 to 100% by mass, most preferably 80 to 100% by mass. For example, ethanol or an aqueous ethanol solution having a concentration of 20 to 100% by mass, preferably 40 to 100% by mass, more preferably 60 to 100% by mass, most preferably 80 to 100% by mass may be used.

The thistle plants suitable for use in the method of the invention may be any of a variety of thistle plants known in the art, including but not limited to thistle (Cirsium japonicum), wild thistle (Cirsium maackii), green thistle (Cirsium chinensis), echinacea (Cirsium setosum), wireflower thistle (Cirsium Lineare) and Hangzhou thistle (Cirsium tianmushanicum). In a preferred embodiment of the invention, the invention uses thistle plants grown in Shanghai, Zhejiang, Jiangsu and Anhui provinces, more preferably in Yiwu, Zhejiang. In a preferred embodiment, the present invention uses roots of plants of the genus Cirsium for extraction.

Before extraction, dried thistle can be pulverized by a conventional method and then soaked with an organic solvent. In general, the volume to mass ratio of organic solvent to plant of the genus Cirsium may be in the range of 5:1 to 40:1, preferably in the range of 10:1 to 30: 1. The amount of the organic solvent to be used may be appropriately adjusted depending on the kind of the organic solvent. The soaking time may be in the range of 4 to 48 hours, preferably in the range of 8 to 36 hours, more preferably in the range of 12 to 24. The soaking time can be properly adjusted according to the kind and the amount of the organic solvent and the amount of the thistle plants. When soaking, the mixture may be appropriately stirred as necessary. After soaking, a leaching solution is obtained, i.e. the thistle plant extract described herein. The leachate is obtained by using means such as centrifugation and/or filtration. Of course, as is well known to those skilled in the art, percolation may also be used to obtain the leaching solution gradually while soaking and extracting.

In some embodiments, the process of the invention further comprises the step of purifying the leachate. For example, purification can be performed using preparative High Performance Liquid Chromatography (HPLC).

In some embodiments, column chromatography may be used for purification, such as separation and purification using silica gel as a packing material, and fractions having the above-described peak time may be collected to form the extract of the present invention alone or in combination.

In a preferred embodiment, the present invention uses petroleum ether as a solvent to extract the roots of the thrips genus plant. Preferably, the volume mass ratio of the petroleum ether to the thistle plants is 10:1 to 30:1, in the above range. After the petroleum ether extract of the thistle plant is obtained, column chromatography separation is carried out on the thistle plant. In a particularly preferred embodiment, the column chromatography (e.g. using silica gel packing) is performed with petroleum ether and a petroleum ether/ethyl acetate gradient. Wherein, during elution, the usage amount of the petroleum ether can be 1-2 BV; the petroleum ether/ethyl acetate gradient was 20: 1. 15: 1. 10: 1. 5:1 and 1:1, the dosage of which can be respectively 1.5-2.5BV, 2.5-3.5BV and 1.5-2.5 BV. Collecting 0.02-0.1BV of each fraction, and separately or combining the fractions to obtain the extract of the plant of the genus Cirsium of the present invention.

The invention also includes the thistle plant extract prepared by the method and the medical application thereof, such as the various applications mentioned above.

The present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are given for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: preparation of thistle plant extract

1. Root ethanol extract

The root of Cirsium japonicum (Cirsium japonicum) purchased from Yiwu Zhejiang is cleaned, naturally dried and crushed. Loading the pulverized root into a glass column with a sieve, adding 5 times (L/kg) of ethanol, soaking for 12 hr, percolating at room temperature at uniform speed, and continuously supplementing fresh ethanol. The ratio of the total used solvent volume (L) to the mass of the crude drug (kg) was 20: 1. mixing the percolate extractive solutions, concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain root ethanol extract.

2. Root water extract

The root of Cirsium japonicum (Cirsium japonicum) purchased from Yiwu Zhejiang is cleaned, naturally dried and crushed. Placing pulverized root into round-bottom flask, adding 10 times (L/kg) of water, heating at 100 deg.C under reflux, and filtering to obtain extractive solution. Concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain root water extract.

3. Root Petroleum ether extract

The root of Cirsium japonicum (Cirsium japonicum) purchased from Yiwu Zhejiang is cleaned, naturally dried and crushed. Loading the pulverized root into a glass column with a sieve, adding 5 times (L/kg) of petroleum ether, soaking for 12 hr, percolating at room temperature at uniform speed, and continuously supplementing fresh petroleum ether. The ratio of the total used solvent volume (L) to the mass of the crude drug (kg) was 20: 1. mixing the percolate extractive solutions, concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain root petroleum ether extract.

4. Root n-hexane extract

The root of Cirsium japonicum (Cirsium japonicum) purchased from Yiwu Zhejiang is cleaned, naturally dried and crushed. Charging the pulverized root into a round-bottom flask, adding 10 times (L/kg) of n-hexane, stirring at normal temperature and low speed for 12 hours, and filtering to obtain an extracting solution; adding n-hexane 10 times (L/kg), stirring at normal temperature and low speed for 12 hr, and filtering to obtain extractive solution. Mixing extractive solutions, concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain n-hexane extract.

5. Root cyclohexane extract

The root of Cirsium japonicum (Cirsium japonicum) purchased from Yiwu Zhejiang is cleaned, naturally dried and crushed. Loading the pulverized root into a glass column with a sieve, adding 5 times (L/kg) of cyclohexane, soaking for 12 hr, percolating at room temperature at uniform speed, and continuously supplementing fresh cyclohexane. The ratio of the total used solvent volume (L) to the mass of the crude drug (kg) was 20: 1. mixing the percolate extractive solutions, concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain the extract.

6. Ethanol extract of stem

Collecting stem of Cirsium japonicum (Cirsium japonicum) from Yiwu Zhejiang, cleaning, air drying, and pulverizing. Loading the pulverized stem into a glass column with a sieve, adding 5 times (L/kg) of ethanol, soaking for 12 hr, percolating at room temperature at uniform speed, and continuously supplementing fresh ethanol. The ratio of the total used solvent volume (L) to the mass of the crude drug (kg) was 20: 1. mixing the percolate extractive solutions, concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain stem ethanol extract.

7. Stem water extract

Collecting stem of Cirsium japonicum (Cirsium japonicum) from Yiwu Zhejiang, cleaning, air drying, and pulverizing. Placing the pulverized stem into round-bottom flask, adding 10 times (L/kg) of water, heating at 100 deg.C, reflux extracting, and filtering to obtain extractive solution. Concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain stem water extract.

8. Leaf petroleum ether extract

The method comprises collecting Cirsium japonicum (Cirsium japonicum) from Yiwu Zhejiang, cleaning, naturally air drying, and pulverizing. Loading the crushed leaves into a glass column with a sieve, adding 5 times (L/kg) of petroleum ether, soaking for 12 hours, then maintaining uniform speed, percolating and extracting at room temperature, and continuously supplementing fresh petroleum ether. The ratio of the total used solvent volume (L) to the mass of the crude drug (kg) was 20: 1. and mixing the percolation extract, concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain the leaf petroleum ether extract.

9. Leaf water extract

The method comprises collecting Cirsium japonicum (Cirsium japonicum) from Yiwu Zhejiang, cleaning, naturally air drying, and pulverizing. Placing pulverized leaves into round-bottom flask, adding 10 times (L/kg) of water, heating at 100 deg.C under reflux, and filtering to obtain extractive solution. Concentrating under reduced pressure to obtain solvent-free extract, and weighing to obtain leaf water extract.

Example 2: preparation of a Petroleum Ether extract subcomponent

And (3) chromatographic separation: the root petroleum ether extract obtained in example 1 was separated by column chromatography on silica gel (200-300 mesh), eluting with petroleum ether (1.2BV) and a petroleum ether/ethyl acetate gradient (20:1(2BV), 15:1(2BV), 10: 1(2BV), 5:1(3BV), 1:1(2BV)), and 0.049BV was collected for each fraction to give a total of 249 fractions.

Merging and concentrating: respectively combining 1-22, 23-27, 28-33, 34-83, 84-158, 159-177, 178-248 and 249 flow parts, and concentrating under reduced pressure to obtain components C01-1, C01-23, C01-28, C01-34, C01-84, C01-159, C01-178 and C01-249.

Example 3: antituberculous Activity test

Preparing a solution: the extracts or sub-fractions obtained in example 1 and example 2 were dissolved in DMSO to prepare a mother liquor of 25.6 mg/mL. On the day of experiment, the mother liquor was diluted 50 times with 7H9+ 10% ADC liquid medium (7H9 medium from Beijing Sorleuba science and technology Co., Ltd.; ADC enrichment broth from Shanghai Guanguan bioengineering Co., Ltd.) to prepare working solution.

Preparing a seed receiving plate: adding 100 mu L of 7H9+ 10% ADC liquid culture medium into each well of a 96-well plate, adding 100 mu L of compound working solution into the 1 st column, sucking 100 mu L of liquid from the 1 st column, adding the liquid into the 2 nd column, sucking 100 mu L to the 3 rd column after the liquid is uniformly blown, sequentially carrying out gradient dilution for 2 times, and removing 100 mu L from each well of the 11 th column to obtain the inoculation plate.

Preparing bacterial inoculation liquid: the mycobacterium tuberculosis is from research institute of tuberculosis breast tumor in Beijing. The colonies growing for 3-4 weeks on the neutral Roche medium were scraped with a disposable sterile inoculating loop and transferred to a bacteria grinding bottle with sterilized glass beads and 100. mu.L of 5% Tween-80. The cells were shaken and homogenized by a vortex shaker for 30 seconds and then allowed to stand for 15 minutes. Sterile physiological saline was added until the turbidity of the obtained bacterial suspension reached 1McFarland (Macleya turbidity), and diluted 20-fold with 7H9+ 10% ADC liquid medium to obtain a bacterial inoculum.

Bacterial inoculation and culture: after adding bacterial inoculum to each well of an inoculation plate containing 100. mu.L of different concentrations of compound, and incubating at 37 ℃ for 9 days, 20. mu.L of Alamar Blue (Almary Blue) was added to each well and incubated for 24 hours.

MIC value reading: the Minimum Inhibitory Concentration (MIC) is the minimum drug concentration which can completely inhibit the discoloration of Alamar Blue (Almar Blue) by visual observation, or the minimum drug concentration generated by reduced Alamar Blue with the inhibition rate of more than 90% by fluorescence detection (Ex/Em, 530nm/590 nm).

And (3) calculating an inhibition rate: inhibition rate%.

The results of the anti-tuberculosis susceptibility test are shown in table 1. The activity of the root extract of Cirsium is much higher than that of the stem and leaf extract, and the stem and leaf extract has almost no anti-tuberculous activity (MIC > 128 μ g/mL). The activity of the root organic phase extract of the plant of genus Cirsium is much greater than that of the root aqueous phase extract. Wherein, part of the sub-components obtained by the separation of the root petroleum ether extract by silica gel column chromatography has stronger antitubercular activity, and the antitubercular activity of the components C01-34, C01-84 and C01-159 is better. Particularly, for the drug-resistant strain (M3), the MICs of the marketed positive drugs streptomycin and ethambutol are respectively more than 32 mug/mL and more than 160 mug/mL, namely, the extract of the thistle plants has remarkable advantages.

Table 1: petroleum ether fraction antitubercular Activity (MIC, μ g/mL)

Example 4: high Performance Liquid Chromatography (HPLC) profiling

HPLC chromatogram determination was performed on the ethanol extract, petroleum ether extract, C01-34, C01-84 and C01-159 fractions (obtained in example 1) of the roots, which had a better anti-tubercular effect.

1. Chromatographic conditions are as follows:

equipment: shimadzu LC-20A high performance liquid chromatograph

Column temperature: 35 deg.C

A detector: SPD-20AV ultraviolet detector

Detection wavelength: 254nm

A chromatographic column: phenomenex luna 4.6X 250mm, 5 μm

Sample introduction amount: 20 μ L

Mobile phase: a (0.1% glacial acetic acid in water), B (acetonitrile)

Flow rate of mobile phase: 1mL/min

Gradient elution conditions:

time (minutes)	B (vol%)	A (vol%)
			0	12	88
8	40	60
			18	70	30
55	100	0

2. And (3) detection results:

the HPLC detection results are shown in FIG. 1-FIG. 5 and Table 2-Table 6.

Table 2: HPLC peak results of root ethanol extract

Table 3: HPLC peak results of root Petroleum Ether extract

Table 4: HPLC peak result of C01-34 component

Peak #	Retention time (minutes)	Area of	Height	Area (%)	Height (%)
						1	35.681	1367752	117857	3.35	3.897
2	36.646	4923542	365106	12.059	12.074
						3	37.878	326112	29027	0.799	0.96
4	38.274	434852	38610	1.065	1.277
						5	38.926	4940657	269386	12.101	8.908
6	42.054	20792974	1553895	50.929	51.385
						7	43.788	6862902	571415	16.81	18.896
8	46.808	1178599	78730	2.887	2.603
						Total of		40827389	3024026	100	100

Table 5: HPLC peak result of C01-84 component

Peak #	Retention time (minutes)	Area of	Height	Area (%)	Height (%)
						1	32.953	378876	32121	4.536	4.662
2	33.283	118617	11293	1.42	1.639
						3	34.217	1862267	166814	22.296	24.213
4	34.507	183183	18915	2.193	2.745
						5	35.917	762467	64538	9.129	9.368
6	36.6	884790	78284	10.593	11.363
						7	36.986	1996637	144028	23.905	20.905
8	37.694	1939589	154345	23.222	22.403
						9	38.15	225910	18612	2.705	2.701
Total of		8352337	688951	100	100

Table 6: HPLC peak results of C01-159 component

Peak #	Retention time (minutes)	Area of	Height	Area (%)	Height (%)
						1	29.993	788938	52159	1.786	1.727
2	30.376	1391002	91550	3.150	3.031
						3	31.013	603079	36863	1.366	1.220
4	31.502	3406533	233149	7.713	7.719
						5	32.032	546416	29342	1.237	0.971
6	32.545	6200899	388165	14.040	12.851
						7	33.249	1134082	55405	2.568	1.834
8	33.923	1345130	91446	3.046	3.028
						9	34.354	1460842	71987	3.308	2.383
10	35.205	1875686	92416	4.247	3.060
						11	35.750	2591330	176131	5.867	5.831
12	35.860	2947609	298668	6.674	9.888
						13	36.146	5590577	439389	12.658	14.547
14	36.685	1490338	86874	3.374	2.876
						15	37.041	8280789	649573	18.750	21.505
16	37.649	1656504	71904	3.751	2.381
						17	40.063	1404068	62822	3.179	2.080
18	40.767	980713	55184	2.221	1.827
						19	41.277	470779	37495	1.066	1.241
Total of		44165315	3020521	100.000	100.000

The inventors believe that the extract with an anti-tubercular active ingredient has a characteristic uv absorption peak at a wavelength of 254nm with a peak-off time centered at 30-50 minutes, preferably 30-45 minutes, more preferably 35-40 minutes, combined with factors such as the common peak-off time, peak-off area, etc. Further, the extract with anti-tubercular active ingredient has characteristic uv absorption peak off-time at 254nm wavelength of: 35.8. + -. 0.3 min, 36.6. + -. 0.3 min, 37.7. + -. 0.3 min, preferably 34.2. + -. 0.3 min, 35.8. + -. 0.3 min, 36.6. + -. 0.3 min, 37.0. + -. 0.3 min, 37.7. + -. 0.3 min, 42.0. + -. 0.3 min, more preferably 31.5. + -. 0.3 min, 32.5. + -. 0.3 min, 34.2. + -. 0.3 min, 35.8. + -. 0.3 min, 36.6. + -. 0.3 min, 37.0. + -. 0.3 min, 37.7. + -. 0.3 min, 38.9. + -. 0.3 min, 42.0. + -. 0.3 min, 43.8. + -. 0.3 min.

While specific examples of the invention have been described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. It is, therefore, intended that the appended claims cover all such modifications that are within the scope of this present invention.

Claims

1. Application of organic extract of root part of plant of genus Cirsium in preparing medicine for resisting Mycobacterium tuberculosis or treating tuberculosis; wherein the plant of the genus Cirsium is Cirsium japonicum (Cirsium japonicum Kimura.) (Cirsium japonicum Kimura)Cirsium japonicum) (ii) a Wherein the organic extract is prepared by adopting the following method: extracting root part of the plant of the genus Cirsium with petroleum ether to obtain petroleum ether extract of the root part of the plant of the genus Cirsium, and separating the petroleum ether extract by column chromatography; wherein, during column chromatography separation, petroleum ether and petroleum ether/ethyl acetate are used for gradient elution; during elution, the usage amount of petroleum ether is 1.2 BV; the petroleum ether/ethyl acetate gradient was 20: 1. 15: 1. 10: 1. 5:1 and 1:1 in an amount of 2BV, 3BV and 2BV, respectively, collecting 0.049BV for each fraction, combining 34 th to 83 th fractions, 84 th to 158 th fractions or 159 th to 177 th fractions, respectively, to obtain the organic extract.

2. The use according to claim 1, wherein the mycobacterium tuberculosis is selected from the group consisting of human mycobacterium tuberculosis and m.

3. The use of claim 2, wherein the mycobacterium tuberculosis is drug-resistant human mycobacterium tuberculosis.

4. The use according to claim 1, wherein the tuberculosis is caused by mycobacterium tuberculosis.

5. The use according to claim 1, wherein the tuberculosis is selected from tuberculosis or extrapulmonary tuberculosis.

6. The use according to claim 1, wherein the tuberculosis is pulmonary tuberculosis, hepatic tuberculosis, gastric tuberculosis or intestinal tuberculosis.

7. The use of claim 1, wherein the organic extract is characterized by having a characteristic uv absorption peak with a peak time of between 35 and 40 minutes, wherein the peak time is determined by high performance liquid chromatography at a wavelength of 254 nm; wherein, the detection conditions of the high performance liquid chromatography are as follows:

column temperature: at a temperature of 35 c,

a detector: an ultraviolet detector is arranged on the base plate,

detection wavelength: the wavelength of the light beam is 254nm,

and (3) chromatographic column: phenomenex luna 4.6X 250mm, 5 μm,

sample introduction amount: 1-20 mu L of the mixture is prepared,

mobile phase: 0.1% glacial acetic acid water solution, acetonitrile,

flow rate of mobile phase: 1mL/min of the reaction solution is added,

gradient elution conditions:

。

8. the use as claimed in claim 7, wherein the organic extract has characteristic UV absorption peaks with peak times of 35.8 ± 0.3 min, 36.6 ± 0.3 min and 37.7 ± 0.3 min, respectively.

9. The use according to claim 8, wherein the 159-177 fractions further have characteristic UV absorption peaks with a peak off time of 37.0 ± 0.3 minutes.

10. The use according to claim 8, wherein the 34 th to 83 th fractions further have characteristic UV absorption peaks with a peak off time of 38.9 ± 0.3 minutes.

11. The use as claimed in claim 7, wherein the organic extract has characteristic UV absorption peaks with peak times of 35.8 ± 0.2 min, 36.6 ± 0.2 min and 37.7 ± 0.2 min, respectively.

12. The use according to claim 11, wherein the 159-177 fractions further have characteristic uv absorption peaks with a peak off time of 37.0 ± 0.2 minutes.

13. The use according to claim 11, wherein the 34 th to 83 th fractions further have characteristic uv absorption peaks with a peak off time of 38.9 ± 0.2 minutes.

14. The use according to claim 7, wherein the 159-177 fractions have characteristic UV absorption peaks with peak times of 35.8 ± 0.1 min, 36.6 ± 0.1 min and 37.7 ± 0.1 min, respectively.

15. The use according to claim 14, wherein the 159-177 fractions further have characteristic uv absorption peaks with a peak off time of 37.0 ± 0.1 minutes.

16. The use according to claim 8, wherein the 34 th to 83 th fractions have characteristic UV absorption peaks with a peak off time of 38.9 ± 0.1 minutes.

17. The use according to claim 7, wherein the 84-158 th and 159-177 th fractions each further have a characteristic UV absorption peak with a peak time of between 30 and 35 minutes.

18. The use according to claim 17, wherein the 159-177 fractions have characteristic uv absorption peaks with peak times of 34.2 ± 0.3, 32.5 ± 0.3 and 31.5 ± 0.3 minutes.

19. The use according to claim 17, wherein the 159-177 fractions have characteristic uv absorption peaks with peak times of 34.2 ± 0.2 minutes, 32.5 ± 0.2 minutes and 31.5 ± 0.2 minutes.

20. The use according to claim 7, wherein the 34 th to 83 th fractions and 159 th to 177 th fractions each further have a characteristic UV absorption peak with a peak time of between 40 and 45 minutes.

21. The use according to claim 20, wherein the 34 th to 83 th fractions further have characteristic uv absorption peaks with a peak off time of 42.0 ± 0.3 minutes and 43.8 ± 0.3 minutes, respectively.

22. The use of claim 20, wherein the 34 th to 83 th fractions further have characteristic uv absorption peaks with a peak off time of 42.0 ± 0.2 minutes and 43.8 ± 0.2 minutes, respectively.

23. The use of claim 20, wherein the 34 th to 83 th fractions further have characteristic uv absorption peaks with a peak off time of 42.0 ± 0.1 minutes and 43.8 ± 0.1 minutes, respectively.

24. The use according to claim 7,

the 34 th to 83 th fractions have characteristic ultraviolet absorption peaks with peak emergence times as follows: 35.6 +/-0.1 minutes, 36.6 +/-0.1 minutes, 37.8 +/-0.1 minutes, 38.9 +/-0.1 minutes, 42.0 +/-0.1 minutes, 43.7 +/-0.1 minutes, 38.2 +/-0.1 minutes and 46.8 +/-0.1 minutes;

the 84 th to 158 th fractions have characteristic ultraviolet absorption peaks with the peak-off time as shown below: 34.2 +/-0.1 minutes, 35.9 +/-0.1 minutes, 36.6 +/-0.1 minutes, 36.9 +/-0.1 minutes, 37.6 +/-0.1 minutes, 32.9 +/-0.1 minutes, 33.2 +/-0.1 minutes, 34.5 +/-0.1 minutes and 38.1 +/-0.1 minutes;

the 159 th to 177 th fractions have characteristic ultraviolet absorption peaks with peak-off times as shown below: 31.5 + -0.1 min, 32.5 + -0.1 min, 34.3 + -0.1 min, 35.8 + -0.1 min, 36.6 + -0.1 min, 37.0 + -0.1 min, 37.6 + -0.1 min, 29.9 + -0.1 min, 30.3 + -0.1 min, 31.0 + -0.1 min, 32.0 + -0.1 min, 33.2 + -0.1 min, 33.9 + -0.1 min, 35.2 + -0.1 min, 35.7 + -0.1 min, 36.1 + -0.1 min, 40.0 + -0.1 min, 40.7 + -0.1 min, and 41.2 + -0.1 min.