JP2023052160A - Extracted fraction containing polymer condensed tannin obtained from ephedra herb extract or ephedrine alkaloid-free ephedra herb extract as well as manufacturing method and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To elucidate a novel active ingredient with an anti-cancer/anti-metastatic action, a pain-suppressing action, and an anti-influenza virus action, via c-Met kinase inhibitory action, from Ephedra herb extract or ephedrine alkaloid-free Ephedra herb extract (EFE) and establishing a manufacturing method for the ingredient, thereby providing a pharmaceutical that can be administered in high potency and in which the risk of developing side effects due to ingredients other than the active ingredient has been eliminated.

SOLUTION: Provided are an extract fraction containing a polymer condensed tannin with a weight average molecular weight of 45,000 or more derived from Ephedra herb extract or ephedrine alkaloid-free Ephedra herb extract (EFE), the extract fraction containing 30% or more of the polymer condensed tannin having a weight average molecular weight of 45,000 or more; a manufacturing method thereof; and a pharmaceutical composition containing the extract fraction.

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

Description

The present invention provides an extract fraction containing polymer condensed tannins derived from Ephedra extract or ephedrine alkaloids-free Ephedra Herb extract (EFE) and having a weight-average molecular weight of 45,000 or more, and a method for producing the same. It relates to a pharmaceutical composition comprising said extract fraction. The pharmaceutical composition of the present invention is useful as an anticancer drug, anti-metastatic drug, pain suppressant, and anti-influenza virus drug.

Tannin is a natural product extracted from plant stems, skins, leaves, fruits, etc., and refers to polyphenols that have the property of binding to proteins, basic compounds, etc. to make them insoluble. It is contained in familiar foods, luxury goods, and plants used for industrial purposes. Tannins include hydrolyzable tannins, which are easily hydrolyzed in the presence of acids and enzymes, and condensed tannins, which undergo condensation. Hydrolyzable tannins have relatively low molecular weights, and have been isolated from many plant materials such as crude drugs. Condensed tannins, on the other hand, have not been clearly identified. In 1989, Weinges named proanthocyanidins substances that generate anthocyanidins when colorless plant extracts are heated with acid. and the component is flavan 3-
3,4-diols or flavans were found to consist of multiple linked flavonoid units. It has been clarified that many of the fruit components that were conventionally called condensed tannins are proanthocyanidins, and many studies on proanthocyanidins have been conducted. Delphinidin, Proguibourtinidin, Profisetinidin, Prorobinetinidin, Proteracacidin, Promelacacidin, Proapigeninidin, Prolteolinidin (Proluteolinidin) and other chemical structures and pharmacological actions have been clarified. That is, proanthocyanidins are condensed or polymerized tannins present in various plants, and are a group of compounds condensed with flavan 3-ols or flavan 3,4-diols as structural units.

The chemical structure of proanthocyanidins is generally composed of flavan 3-ol or flavan 3,4-diol as a constituent unit at 4β → 6, 4β → 8, 4β → 8, 2β → O → 7, etc. It is a dimer or higher polymer polymerized by the bonding mode of (Non-Patent Documents 1 and 2). They are classified into several types based on their binding modes. One is proanthocyanidin B type in which two or more flavan 3-ol structures are linked via 4β→8 or 4β→6, and the other is proanthocyanidin B type in which the flavan 3-ol structure → 8-position and 2β-position → O → 7-position or 4β-position → 6-position and 2β-position → O
→ Proanthocyanidin A type with at least one bond at position 7 (Fig. 1
reference).

Condensed tannins have a wider range of molecular weights than hydrolyzed tannins, with large molecular weights reaching about 20,000 (Non-Patent Document 3).

Proanthocyanidins are known to exhibit various physiological activities, and their activities such as antitumor, anti-inflammatory, anti-aging, antioxidant, anti-allergic, antibacterial, and hair growth have been reported (Non-Patent Documents 4 and 5). ), but the structure-activity relationship between these physiological activities and the degree of polymerization of proanthocyanidins has not been clarified (Patent Document 1). In Patent Document 2, procyanidin A
Structural formulas are shown in which binding of monomeric units of type and procyanidin B type (catechol type only) occurs at the C-4 and C-8 positions. In Patent Document 3, a proanthocyanidin A type polymer and a proanthocyanidin B type (pyrogallol type, catechol type, monomer unit bonds occur at C-4 and C-8 positions) condensates/polymers Hepatic fibrosis inhibitory effect has been shown. In Patent Document 4, binding of proanthocyanidin B (pyrogallol type, catechol type) monomer units occurs at the C-4, C-6, and C-8 positions to form proanthocyanidin, which has urease inhibitory activity. are reporting.

Plants containing proanthocyanidins include, for example, apples, blueberries, elderberries, grapes, strawberries, red currants, cowberries, gooseberries, cranberries, salmonberries, bilberries, cassis, cherries, huckleberries, blackberries, plums, whittles. Berry, boysenberry, mulberry, raspberry, red currant, loganberry, persimmon, pine, oak, yam, barley, wheat, soybean, black soybean, cacao, adzuki bean, horse chestnut, peanut, ginkgo biloba, green tea, rhubarb, mahuang, Yangbai skin etc. are known (Patent Documents 5 and 6
).

Ephedra is the ground stem of Ephedra sinica Stapf, Ephedra intermedia Schrenk et CA Meyer or Ephedra equisetina Bunge (Ephedraceae). Ephedra is the most important crude drug that has been used since ancient times. Ephedra is defined by the Japanese Pharmacopoeia as containing 0.7% or more of total alkaloids (ephedrine and pseudoephedrine), and most of the pharmacological actions of Ephedra have been thought to derive from ephedrine alkaloids (Non-Patent Document 6). On the other hand, side effects of ephedra, such as palpitations, increased blood pressure, insomnia, and dysuria, are caused by the central nervous system and sympathetic nerve stimulating effects of ephedrine alkaloids, but it is considered difficult to separate the main effects and side effects of ephedra. However, the idea of removing ephedrine alkaloids from Ephedra to eliminate the side effects of Ephedra has never been thought of before.

The present inventors have found from previous studies that the non-alkaloid fraction of mahuang has an anti-cancer anti-metastatic effect mediated by hepatocyte growth factor (HGF) receptor c-Met kinase inhibitory action. We isolated Herbacetin glycosides from the non-alkaloid fraction of Ma Huang, and clarified that the non-sugar portion of Herbacetin has multikinase inhibitory activity, including c-Met kinase inhibitory activity. Among them, it was found that Herbacetin has a strong TrkA (NGF receptor) inhibitory action and has a pain suppressing action via NGF-TrkA signal inhibition (Patent Documents 7 and 8). These results suggested that some of the pharmacological effects of Ephedra were not dependent on ephedrine alkaloids, and thus the side effects could be removed from Ephedra. Therefore, when EFE was prepared by removing ephedrine alkaloids from mahuang extract (Non-Patent Document 7) (Patent Document 9), it was found that it has several pharmacological actions including pain suppressing action and anti-influenza virus action (Patent Document 9). (Non-Patent Document 8), and side effects such as insomnia, palpitations, and excitement have disappeared (Non-Patent Document 9). However, since EFE contains various ingredients, not to mention Ephedra extract, the bulk that can be taken normally is a limiting factor, and high-potency administration, which is expected to be more effective, could not be administered.

On the other hand, polymerized proanthocyanidins of low-molecular-weight proanthocyanidin A type or proanthocyanidin B type (pyrogallol type, catechol type) derived from Ephedra have already been reported (Non-Patent Documents 10, 11, 12). Furthermore, recently, an oligomer in which both proanthocyanidin A type and proanthocyanidin B type were polymerized was isolated from a 70% acetone extract of Ephedra huang (Non-Patent Document 13).

After treating the Ephedra-derived proanthocyanidin-containing liquid with an ultrafiltration membrane with a cutoff molecular weight of 5000 or less and/or a reverse osmosis membrane with a salt rejection rate of 30% or less, the fraction with a cutoff molecular weight of 500 to 5000 is A method for producing proanthocyanidins characterized by fractionation is known (Patent Document 6).

However, to date, there have been no reports of high-molecular-weight tannins with a weight-average molecular weight exceeding 20,000.

Method for purifying proanthocyanidin oligomer Patent No. 4582917 Cranberry extract and its production method Patent No. 5459699 Hepatic fibrosis inhibitor Patent No. 4822291 Urease inhibitor, pharmaceutical and food compositions containing the same, and method for measuring urease activity JP-A-2000-159669 Capsules JP 2016-69335 Manufacturing method of proanthocyanidins JP-B-6-31208 MET inhibitor composed of Ephedra as a component Patent No. 5786164 Multikinase inhibitor, anticancer agent, antimetastatic agent, drug resistance suppressor, pain suppressant and antipruritic agent JP2014-129341 Ephedrine alkaloid-removed mahuang extract and its production method and use PCT/JP2014/080605 (WO2015/076286)

"Steinegger Hensel Pharmacognosy [Part 1] Approaches to Chemistry and Pharmacology" (translated by Shuji Itokawa et al., published by Hirokawa Shoten Co., Ltd.) pp. 204-208 (1977) Porter L. J. , Flavans and proanthocyanidins, In: Harborne J.; B. (ed.), "The Flavonoids, Advances in Research Science 1986", Chapman & Hall, 1994, pp. 23-55) Seisuke Ohara, "Components and Uses of Tropical Forests (2) Tannin," Tropical Forestry, 39, 56-60, 1997 Bert Schwitters/Jacques Masquelier, "Bioprotective Substance OPC for the 21st Century" (translated by Ryo Sasaki, published by Fragrance Journal, 1997), pp. 50-135 Tomoya Takahashi, et al. , Journal of Investigative Dermatology, 112, 310-316, 1999 Masatoshi Harada, Pharmacology of Ephedra, Modern Oriental Medicine, 1(2), 34-39, 1980 Oshima, N., et al., J Nat Med, 70, 554-562, 2016 Hyuga, S., et al., J Nat Med, 70, 571-583, 2016 Hiroaki Takemoto et al., Abstracts of the 33rd Annual Meeting of the Society of Japanese and Chinese Medicine, Poster P4-5, p87, 2016 Molecules. 2013 May 6;18(5):5172-89. A-type proanthocyanidins from the stems of Ephedra sinica (Ephedraceae) and their antimicrobial activities. Zang X1, Shang M, Xu F, Liang J, Wang X, Mikage M , Cai S. Molecules. 2013 May 10;18(5):5326-34. Characterization of phenolic constituents from ephedra herb extract. Amakura Y1, Yoshimura M, Yamakami S, Yoshida T, Wakana D, Hyuga M, Hyuga S, Hanawa T, Goda Y . Recent Studies on Tannin Yakugakushi 103(2), 125-142, 1983 Molecules. 2017 August 6;22(8):1308 Characterization of proanthocyanidin oligomers of Ephedra sinica. Orejola J, Matsuo Y, Saito Y, Tanaka T. Kennedy, J.A., et al., J. Chromatogr. A 995, 99-107, 2003

A method for producing EFE by removing ephedrine alkaloids from mahuang extract was established, and c-Met
It has become possible to provide pharmaceuticals that have anti-cancer, anti-metastatic, pain-suppressing, and anti-influenza virus effects through kinase inhibitory action, and eliminate the side effects of ephedrine alkaloids. However, since EFE contains various ingredients, not to mention Ephedra extract, the bulk that can be taken normally is a limiting factor, and high-potency administration, which is expected to be more effective, could not be administered. As a result of careful examination of active ingredients in mahuang extract or EFE that have anti-cancer, anti-metastatic, pain-suppressing, and anti-influenza virus activities mediated by c-Met kinase inhibitory action, it was found to be one of the effective ingredients of mahuang. Herbacetin glycosides have been found, but they were not optimal as pharmaceuticals because they are not highly efficacious.

The present invention clarifies a novel active ingredient having anticancer activity, anti-metastatic activity, pain suppression activity, and anti-influenza virus activity mediated by c-Met kinase inhibitory action from mahuang extract or EFE, and a method for producing the ingredient. By establishing this, the purpose is to provide a drug that can be administered with a high potency and eliminates the risk of side effects due to ingredients other than the active ingredient. Furthermore, in the course of research related to the present invention, new efficacy effects of active ingredients (c-Met expression level lowering action and epidermal growth factor receptor (EGFR) phosphorylation inhibitory action and EGFR expression level lowering action) Action) was discovered, and the object is to provide it as a use of the present active ingredient.

The present inventors have made intensive studies to solve the above problems, and found that Ephedra extract or EFE
was extracted with water/n-butanol, and the water-soluble portion was fractionated by eluting with water and 20% to 100% methanol (MeOH) using a synthetic aromatic adsorbent Diaion HP-20 column to obtain 20% Analysis of the portion eluted with more than methanol revealed that this chemical structure polymerized at the C-4, C-6, and C-8 positions of procyanidin A type and procyanidin B type (pyrogallol type and catechol type) with a weight average molecular weight of 45, 000 or more macromolecules.

Furthermore, we clarified the structure of the obtained component group, and found that the extract fraction containing high molecular weight condensed tannins with a weight average molecular weight of 45,000 or more has c-Met kinase inhibitory activity, c-Met phosphorylation inhibitory activity, and c-Met inhibitory activity.
Discovered that it has anti-cancer/anti-metastatic effect, pain suppression effect, anti-cancer effect and anti-influenza virus effect through inhibition of EGFR phosphorylation and reduction of EGFR expression level. As a result, the present invention was completed.

That is, the present invention provides the following.
[Claim 1]
An extract fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more derived from mahuang extract or derived from ephedrine alkaloid-removed mahuang extract (EFE), wherein 30 Extracted fraction containing more than %.
[Claim 2]
Polymer condensed tannins, as extension units, are condensed mainly with proanthocyanidin B type catechol-type or pyrogallol-type flavan 3-ols, part of which contains proanthocyanidin A-type condensed tannin units, and terminal 2. The extract fraction according to claim 1, which mainly contains catechol-type or pyrogallol-type flavan 3-ols as units, and pyrogallol-type and catechol-type at a ratio of about 5:1.
[Claim 3]
Water-soluble fractions derived from Ephedra extract or Ephedrine alkaloid-removed Ephedra extract (EFE) are eluted by column chromatography using a mixed solvent of water and an alcohol selected from methanol or ethanol while increasing the alcohol concentration. 2. The method for producing an extract fraction according to claim 1, which comprises measuring the molecular weight of each obtained fraction to obtain an extract fraction containing polymer condensed tannins having a weight average molecular weight of 45,000 or more.
[Claim 4]
A pharmaceutical composition comprising the extract fraction according to claim 1 or 2.
[Claim 5]
5. The pharmaceutical composition according to claim 4, which is a pain suppressant.
[Claim 6]
5. The pharmaceutical composition according to claim 4, which is an anticancer/anti-metastatic drug against c-Met-expressing cancers.
[Claim 7]
5. The pharmaceutical composition according to claim 4, which is an anticancer drug against EGFR-expressing cancers.
"Claim 8"
5. The pharmaceutical composition according to claim 4, which is an anti-influenza virus drug.
[Claim 9]
A method for measuring the content of a pharmaceutical composition containing polymer condensed tannin as an active ingredient, using the extract fraction according to claim 1.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a pharmaceutical composition containing, as an active ingredient, an extract fraction containing a polycondensed tannin derived from mahuang extract or derived from EFE and having a weight average molecular weight of 45,000 or more. It is possible to provide anti-cancer/anti-metastatic drugs for cancers expressing -Met, anti-cancer drugs for cancers expressing EGFR, and pharmaceuticals with anti-influenza virus activity that can be administered with high potency. The extract fraction of the present invention is a high molecular weight fraction (weight average molecular weight of 45,000 or more)
proanthocyanidin A type and proanthocyanidin B type polymerized proanthocyanidins (condensed tannins, high molecular weight polyphenols).

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a drug that can be administered with a high potency and that eliminates the risk of developing side effects due to ingredients other than the active ingredient.

FIG. 2 shows the structures of proanthocyanidin A type and proanthocyanidin B type and the structures of their constituent units. H ₂ O fraction, 10% MeOH fraction, 20% MeOH fraction, 30% MeOH fraction, 40% MeOH fraction, 50% MeOH fraction, and water extract derived from mahuang extract FIG. 3 shows an HPLC chromatogram of MeOH fractions analyzed with an HPLC column. H ₂ O fraction, 10% MeOH fraction, 20% MeOH fraction, 30% MeOH fraction, 40% MeOH fraction, 50% MeOH fraction, and water extract derived from mahuang extract FIG. 13 shows structural analysis of MeOH fractions by ¹³ C-NMR. FIG. 2 shows HPLC chromatograms of H ₂ O fraction, 20% MeOH fraction, 40% MeOH fraction and MeOH fraction of EFE-derived water extract analyzed by HPLC column. FIG. 2 shows structural analysis by ¹³ C-NMR of H ₂ O fraction, 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction of EFE-derived water extract. FIG. 2 shows structural analysis by ¹³ C-NMR of high-molecular fractions of water extract-30% MeOH fraction and 40% MeOH fraction derived from mahuang extract. FIG. 4 shows the results of GPC analysis of 20% MeOH, 40% MeOH, and MeOH fractions of water extract. Decomposition products obtained by degrading phloroglucinol from 40% MeOH fraction of water extract: catechin, epicatechin, gallocatechin, epicatechin-(4→2)-phloroglucinol, gallocatechin-(4→2) )-phloroglucinol and prodelphinidin A-type-(4'→2)-phloroglucinol. The c-Met kinase inhibitory effects of n-hexane extract, ethyl acetate extract, n-butanol extract, and water extract obtained by dissolving mahuang extract in water were investigated using recombinant protein of c-Met kinase domain and synthetic substrate peptide. It is a figure which shows the result investigated by the in vitro assay using. FIG. 2 shows the results of testing the motility inhibitory effects of n-hexane extracts, ethyl acetate extracts, n-butanol extracts, and water extracts obtained by dissolving ephedra extract in water. H ₂ O fraction, 10% MeOH fraction, 20% MeOH fraction, 30% MeOH fraction, 40% MeOH fraction, obtained by adding water extract to Diaion HP-20 column FIG. 1 shows the results of testing the c-Met kinase inhibitory activity of 50% MeOH fractions and MeOH fractions. H ₂ O fraction, 10% MeOH fraction, 20% MeOH fraction, 30% MeOH fraction, 40% MeOH obtained by adding water extract derived from mahuang extract to Diaion HP-20 column Fig. 2 shows the results of testing the motility-suppressing action of cancer cells on fractions, 50% MeOH fractions, and MeOH fractions. H ₂ O fraction, 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction obtained by adding EFE and EFE-derived water extract to Diaion HP-20 column, mouse is a diagram showing the results of testing the inhibitory effect on formalin-induced pain using . For H ₂ O fraction, 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction obtained by adding EFE and water extract derived from EFE to Diaion HP-20 column, c Fig. 2 shows the results of testing for inhibitory action on -Met expression and tyrosine phosphorylation, and EGFR expression and tyrosine phosphorylation. FIG. 2 shows the pain-suppressing effects of H ₂ O fraction and 40% MeOH fraction obtained by adding EFE-derived water extract to Diaion HP-20 column on arthritis model mice. FIG. 4 is a diagram showing the results of GPC analysis of a polymer condensed tannin standard substance solution.

As one aspect, the present invention provides an extract fraction containing polymer condensed tannins having a weight-average molecular weight of 45,000 or more derived from an Ephedra extract or derived from an ephedrine alkaloid-removed Ephedra extract (EFE).

The Ephedra extract used as a raw material for obtaining the extract fraction of the present invention is a hot water extract of Ephedra in the Japanese Pharmacopoeia. Alternatively, it is an EFE obtained by removing the ephedrine alkaloids from the mahuang extract (Patent Document 9). EFE can be obtained by removing ephedrine from mahuang extract by cation exchange chromatography, followed by concentration and drying. As shown in the examples described later, the extraction fraction of the present invention can be obtained in the same manner when Ephedra extract is used as a raw material and when EFE is used as a raw material.

The extract fraction of the present invention is characterized by containing a polymer condensed tannin having a weight average molecular weight of 45,000 or more. The weight average molecular weight is the weight average molecular weight converted using standard polystyrene for GPC as a standard substance. The extract fraction of the present invention is polymerized proanthocyanidins of proanthocyanidin A type and proanthocyanidin B type (condensed tannins, high molecular weight polyphenols) having a weight average molecular weight of 45,000 or more.

The extract fraction of the present invention contains 30% or more, more preferably 40% or more, of polymer condensed tannins having a weight average molecular weight of 45,000 or more.

In the polymer condensed tannin contained in the extract fraction of the present invention, as an extension unit, catechol-type or pyrogallol-type flavan 3-ol is mainly condensed with proanthocyanidin B type, and a part thereof is proanthocyanidin A type Furthermore, as a terminal unit, it mainly contains catechol-type or pyrogallol-type flavan 3-ols, and contains pyrogallol-type and catechol-type at a ratio of about 5:1 (see FIG. 1).

As another aspect of the present invention, a water-soluble fraction derived from Ephedra extract or Ephedrine alkaloid-removed Ephedra extract (EFE) is subjected to column chromatography using a mixed solvent of water and an alcohol selected from methanol or ethanol. , The extraction of the present invention, which consists of sequentially eluting while increasing the alcohol concentration, measuring the molecular weight of each fraction obtained, and obtaining an extracted fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more. A method for producing a fraction is provided.

Specifically, Ephedra extract-derived or Ephedrine alkaloid-removed Ephedra extract (EFE)
is dissolved in water, sequentially distributed using an organic solvent with low polarity to an organic solvent with high polarity, and the resulting water extract is analyzed by column chromatography with a mixed solvent of water and an alcohol selected from methanol or ethanol. was sequentially eluted with increasing alcohol concentration, and the molecular weight of each resulting fraction was measured to obtain an extracted fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more.

Examples of organic solvents with low polarity include hexane, diethyl ether, and ethyl acetate, and examples of organic solvents with high polarity include n-butanol, ethanol, methanol, water, and the like. In column chromatography used for fractionation, styrene-divinylbenzene synthetic adsorbents such as Diaion HP-20 or other aromatic synthetic adsorbents can be used. For measuring the molecular weight of each fraction, viscosity method, gel permeation chromatography (GPC), mass spectrometry, osmotic pressure method and the like can be used. In the examples of the present application, the weight-average molecular weight was determined by GPC using standard polystyrene for GPC as a standard substance.

Specifically, Ephedra huang extract or EFE dissolved in water is sequentially partitioned with ethyl acetate and water-saturated n-butanol to obtain partitioned extracts of ethyl acetate extract, n-butanol extract and water extract. Among the obtained partitioned extracts, the water extract was found to be active.
, Mitsubishi Chemical) was used to fractionate by sequentially eluting with H ₂ O → 20% MeOH → 40% MeOH → MeOH in that order, H ₂ O fraction, 20% MeOH fraction, 40% MeOH fraction A fraction, a MeOH fraction, are obtained. As a result of measuring the activity of each fraction, 20% MeOH fraction, 40% MeOH fraction, c-Met kinase inhibitory effect on MeOH fraction,
It was found to have phosphorylation inhibitory action, expression level reduction action, cancer cell motility suppression action, EGFR phosphorylation inhibitory action, expression level reduction action, pain suppression action, and anti-influenza virus action. In addition, the 20% MeOH fraction, 40% MeOH fraction and MeOH fraction were polymer condensed tannins with a weight average molecular weight of 45,000 or more.

Therefore, it was clarified that the extract fraction of the present invention can be prepared by using 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction alone or in any combination. Based on these results, as an efficient preparation method, the above water extract is extracted with 100% MeOH, and the 100% MeOH fraction is further applied to a Sephadex LH-20 column, etc. to obtain a high molecular weight fraction. It is also possible to

Another aspect of the present invention provides a pharmaceutical composition containing an extract fraction containing the polymer condensed tannin. The pharmaceutical composition of the present invention is useful as a pain suppressant, an anti-cancer/anti-metastatic drug, and an anti-influenza virus drug, as shown in the examples below. In addition, the pharmaceutical composition of the present invention exhibits a similar effective pharmacological effect whether Ephedra extract is used as a raw material or EFE is used as a raw material.

The pain suppressant, anti-cancer/anti-metastatic drug, and anti-influenza virus drug according to the present invention are, for example, formulations for oral administration such as tablets, granules, fine granules, capsules, etc., and various formulations suitable for oral administration. It can be a simple liquid formulation, or a formulation for parenteral administration such as an injection. Oral administration is preferred.

In the case of preparations for oral administration, extract fractions containing polymer condensed tannins with a weight-average molecular weight of 45,000 or more are formulated together with pharmaceutical carriers in the form of tablets, granules, fine granules, capsules, and the like.

Excipients, binders, disintegrants, lubricants, plasticizers and the like can be used as pharmaceutical carriers. Examples of excipients that can be used include sucrose, sodium chloride, mannitol, lactose, glucose, starch, calcium carbonate, and the like. Examples of binders that can be used include water, ethanol, propanol, glucose solution, starch solution, gelatin solution, and the like. Examples of disintegrants that can be used include carboxymethylcellulose calcium, dry starch, sodium bicarbonate, and the like. Examples of lubricants that can be used include purified talc, stearate, boric acid powder, and polyethylene glycol. Examples of plasticizers that can be used include glycerin fatty acid esters, dioctyl phthalate, dibutyl phthalate, and castor oil.

Dispersing agents such as water-soluble polymers and surfactants can be used in the above formulations for oral administration. Examples of water-soluble polymers that can be used include hydroxypropylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone. Examples of surfactants that can be used include sodium lauryl sulfate and magnesium lauryl sulfate.

To prepare tablets, the extract fraction containing polymer condensed tannins with a weight-average molecular weight of 45,000 or more is made into tablets by a conventional method using the above formulation carriers. Granules or fine granules are produced by adding the above formulation carriers to the extracted fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more, followed by fluid bed granulation, high-speed stirring granulation, stirring fluid bed granulation, and centrifugation. It can be prepared by granulation by fluid granulation, extrusion granulation, or the like. Capsules are prepared by mixing with an inert pharmaceutical filler or diluent and filled into hard gelatin capsules or soft capsules.

Oral liquid preparations contain extract fractions containing high molecular weight condensed tannins with a weight average molecular weight of 45,000 or more, sweeteners (e.g. sucrose), preservatives (e.g. methylparaben, propylparaben), colorants, fragrances, etc. Prepared by mixing

Injectable formulations are prepared, for example, in the form of solutions, emulsions, or suspensions and are made isotonic with blood. Liquid, emulsion or suspension form formulations are prepared using, for example, aqueous media, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, and the like.

Additives commonly used in the art for injection preparations can be used as appropriate. For example, tonicity agents, stabilizers, buffers, preservatives, chelating agents, antioxidants and the like can be used. . Tonicity agents include, for example, glucose, sorbitol, mannitol and the like. Examples of stabilizers include sodium sulfite and the like. Buffers include, for example, borate buffers, phosphate buffers, citrate buffers and the like. Preservatives include, for example, p-oxybenzoate, benzyl alcohol and the like. Chelating agents include, for example, sodium edetate, sodium citrate and the like. Examples of antioxidants include sodium sulfite, sodium hydrogen sulfite and the like.

The dose of the pain suppressant, anticancer/anti-metastatic drug, and anti-influenza virus drug of the present invention is, for example, a single administration of an extract fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more. The dosage can be selected in the range of 0.5 mg to 500 mg, preferably 10 mg to 200 mg, per 1 kg of body weight. However, the pharmaceutical composition of the present invention is not limited to these doses.

Furthermore, as another aspect of the present invention, there is provided a content measurement method using the extract fraction of the present invention as one of the control methods for a pharmaceutical composition containing polymer condensed tannin as an active ingredient. Specifically, using the GPC analysis used to analyze the molecular weight of the polymer condensed tannin shown in the present invention,
The peak areas of polymer condensed tannins are obtained for each of a sample of unknown content and a standard substance of polymer condensed tannins, and the content of polymer condensed tannins in the sample can be calculated from the relative values of the peak areas. In addition, this content measurement method can also measure the content of polymer condensed tannins in mahuang extract and EFE. It can be used as one of the management methods when manufacturing anti-metastatic drugs and anti-influenza virus drugs.

The present invention will be described in more detail by examples, but the present invention is not limited to these. Various changes and modifications are possible for those skilled in the art, and these changes and modifications are also included in the present invention.
(Example 1) Fractionation and analysis of Ephedra extract or EFE Ephedra extract (300 g) was dissolved in water (5 L), n-hexane (4 L), ethyl acetate (12 L), water saturated n -butanol (12 L), n-hexane extract (92.3 mg),
Ethyl acetate extract (13.2 g), n-butanol extract (65.7 g) and water extract (161.1 g) were obtained. The c-Met kinase inhibitory activity and cancer cell motility inhibitory activity were determined for each partitioned extract derived from the Ephedra extract. As a result, n-butanol and water extracts were found to be active.

The water extract (90 g), which yielded a large amount and was found to be active, was further subjected to a Diaion HP-20 column chromatography (5.5 x 40 cm, Mitsubishi Chemical) for H ₂ O → 10% MeOH → 20% MeOH → Fractions were obtained by sequentially eluting with 30% MeOH → 40% MeOH → 50% MeOH → MeOH, and the H ₂ O fraction (47.9 g), 10% MeOH fraction (2.5 g), 20% MeOH fraction fraction (2.6 g), 30% MeOH fraction (7.0 g), 40% MeOH fraction (11.5 g), 50% MeOH fraction (5.1 g), MeOH fraction (2.6 g). A fraction was obtained. As a result of conducting c-Met kinase inhibitory activity and cancer cell motility inhibitory activity tests for each of the obtained fractions, 30% MeOH fraction, 40% MeOH fraction, and then 50% MeOH fraction A strong activity was observed in the substance.

Therefore, HPLC analysis was performed for the purpose of comparing the components contained in each fraction of the water extract. HPLC conditions are as follows.

Column: L-column ODS (2.1 ID x 150 mm) (National Institute for Chemical Evaluation and Research),
Column temperature: 40°C,
Flow rate: 0.3mL/min,
Measurement wavelength: 200-400 nm,
Mobile phase: (A) 0.1% formic acid-distilled water and (B) 0.1% formic acid-acetonitrile [Concentration gradient conditions (B in A): 0 → 30 min (0 → 50%), 30 → 35 min (50 → 85 %), 35→40 min (85%), 40→50 min (85→90%), 50→55 min (90→100%), 55→60 min (100%)].

As a result, broad peaks characteristic of condensed tannin oligomers were observed in fractions other than the H ₂ O fraction and the 10% MeOH fraction (Fig. 2).

Structural analysis was also performed on each fraction by ¹³ C-NMR. As for the NMR conditions, MeOH-d ₄ :D ₂ O (1:1) was used as a measurement solvent, and Bruker AVANCE500 (Bruker BioSpin) was used as an apparatus. As a result, a signal corresponding to the flavan 3-ol skeleton was commonly observed in fractions other than the H ₂ O fraction and the 10% MeOH fraction, and the fraction contributing to the activity was condensed tannin ( proanthocyanidin) oligomer fraction. A particularly strong signal was observed for the 30% MeOH fraction, 40% MeOH fraction, and 50% MeOH fraction, which showed strong activity. In addition, based on spectral analysis, all fractions had structures containing proanthocyanidin A type, and the signal intensity of catechol type was low, suggesting that pyrogallol type was abundant (Fig. 3).

Similarly, EFE (400 g) was dissolved in water (5 L), ethyl acetate (10 L), water saturated n-
It was partitioned sequentially with butanol (10 L) to obtain ethyl acetate extract (7.3 g), n-butanol extract (85.0 g) and water extract (311.0 g). A pain test was performed on each resulting partitioned extract from EFE. As a result, activity was recognized in the water extract. The active water extract (290 g) was further analyzed using Diaion HP-20 column chromatography (5.5 x 40 cm, Mitsubishi Chemical) in the order of H ₂ O → 20% MeOH → 40% MeOH → MeOH. Sequentially eluted and fractionated, H ₂ O fraction (190.6 g), 20% MeOH fraction (35.9 g), 40% MeOH fraction (41.7 g), MeOH fraction (13.9 g). Each fraction was obtained. A pain test was performed on each of the obtained fractions, and activity was observed in 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction.

Therefore, the same HPLC analysis was performed for the purpose of comparing the components contained in each fraction of the water extract. As a result, broad peaks characteristic of condensed tannin oligomers were observed in 20% MeOH fractions, 40% MeOH fractions, and MeOH fractions in which activity was recognized (Fig. 4).

Structural analysis of each fraction by ¹³ C-NMR revealed activity in 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction corresponding to flavan 3-ol skeleton. A signal was commonly observed, confirming that it was a condensed tannin (proanthocyanidin) oligomer fraction similar to the Ephedra extract-derived active fraction (Fig. 5).

In this example, the c-Met kinase inhibitory activity was determined by the method described in Examples 5 and 7, the cancer cell motility inhibitory activity was determined by the method described in Examples 6 and 8, and the pain test was performed by the method described in Example 9. I followed the method.
(Example 2) Molecular weight analysis of active fraction Since it was confirmed that the fraction contributing to the activity was the condensed tannin (proanthocyanidin) oligomer fraction, the active fraction was further subjected to molecular weight separation by ultrafiltration. did the painting. The 20% MeOH-insoluble part of the water extract-40% MeOH fraction derived from mahuang extract was centrifuged at 11400 xg for 10 minutes using Vivaspin 500 (GE Healthcare: 3, 5, 10, 30, 50, 100 KDa). 7 fractions [<3 KDa (approximately 10% yield), 3-5 KDa (approximately 4%), 5-10 KDa (approximately 5%), 10-30 KDa (approximately 6%), 30
~50 KDa (~2%), 50-100 KDa (~3%), >100 KDa (~70%)]. Each fraction was tested for c-Met kinase inhibitory activity, and as a result, activity was observed in the high-molecular-weight fraction of >100 KDa (Mahuang extract high-molecular-weight fraction). Structural analysis of this polymer fraction by ¹³ C-NMR confirmed that it was a similar condensed tannin (proanthocyanidin) oligomer fraction. Structural analysis was also performed on the >100 KDa polymer fraction of the water extract-30% MeOH fraction by ¹³ C-NMR (acetone-d ₆ :D ₂ O (1:1) as the measurement solvent). As a result, similar spectra were observed (Fig. 6).

GPC analysis was performed on the active fractions (Ephedra extract high-molecular weight fraction, EFE-derived water extract-20% MeOH, 40% MeOH, MeOH fractions). Using standard polystyrene for GPC as a standard substance, analysis was performed under the following conditions.

Column: TSK-gel Super AW4000 (6.0 ID x 150 mm) (Tosoh),
Column temperature: 30°C,
Flow rate: 0.3mL/min,
Measurement wavelength: 280 nm,
Mobile phase: N,N-dimethylformamide + 3M ammonium formate (0.5%)
As a result of the analysis, a high-molecular region peak showing a peak top at a retention time of 7.5 to 8.5 minutes was observed in all fractions (Fig. 7). Data processing was performed using Chromato-PRO-GPC, and the weight average molecular weight of each fraction was calculated. substance: 59042, MeOH fraction: 45004 (weight-average molecular weight converted using standard polystyrene for GPC as a standard substance, average value of values measured three times).
(Example 3) Analysis of partial structure A water extract-40% MeOH fraction derived from Ephedra extract, which is one of the active fractions, was subjected to phloroglucinol decomposition. As a result, catechin, epicatechin, gallocatechin, epicatechin-(4→2)-phloroglucinol, gallocatechin-(4→2)-phloroglucinol, prodelphinidin A-type-(4′→2)-phloroglucinol were confirmed as degradation products ( Figure 8).

Therefore, as extension units, catechol-type or pyrogallol-type flavan 3-ols are mainly condensed in B-type, and some of them contain A-type condensed-type tannin units. It was found to contain mainly flavan 3-ols. In addition, as a result of calculating the approximate abundance ratio of the pyrogallol type and the catechol type by HPLC analysis of the thiol-decomposed units, it was approximately 5:1. (See Figure 1)
(Example 4) Content of high-molecular-weight condensed tannin in EFE-derived extract fraction There is a report of fractionation by elution (Non-Patent Document 14). According to this method, the 70% acetone (acetone-water (7:3)) elution part is the polymer condensed tannin fraction, and the active fraction (EFE-derived water extract-20% MeOH, 40% MeOH, MeOH fraction ) was estimated. The results were about 32% for the 20% MeOH fraction, about 45% for the 40% MeOH fraction, and about 43% for the MeOH fraction.

(Example 5) c-Met kinase inhibitory action of partitioned Ephedra extract Ephedra extract dissolved in water was sequentially partitioned with n-hexane, ethyl acetate, and water-saturated n-butanol, and each fraction obtained ( n-hexane extract, ethyl acetate extract, n-butanol extract, and water extract) were investigated by an in vitro assay using a recombinant protein of the kinase domain of c-Met and a synthetic substrate peptide.

The assay conditions are as follows. 4 nM recombinant c-Met kinase domain in 40 mM Tris-HCl pH 7.5, 2 mM DTT, 7 mM MgCl ₂ , 10 μM ATP, 0.2 μg/mL Poly (Glu4, Tyr1) and 5 μg/ml of each fraction. A mL solution was prepared and incubated for 1 hour at room temperature. After stopping the reaction, the ADP-Glo reagent was used to measure the ADP amount-dependent luminol emission intensity with a luminometer (PerkinElmer, EnSpire) to calculate the c-Met kinase activity. As a result, it was revealed that the n-butanol extract and the water extract have a kinase inhibitory effect (Fig. 9).

(Example 6) Cancer cell motility inhibitory effect of partitioned extract of Ephedra extract The extracts (n-hexane extract, ethyl acetate extract, n-butanol extract, water extract) and cancer cell motility inhibitory effects were evaluated by transwell tests. The assay used MDA-MB-231 cells, a highly metastatic human breast cancer cell line that expresses c-Met and whose motility is induced by HGF.

The assay conditions are as follows. MDA-MB-231 cells (5×10 ⁴ cells/well) were suspended in Vehicle (2.5% EtOH-DMEM) or 2.5% EtOH-DMEM in which 25 μg/mL of each partitioned extract was dissolved, and added to the upper wells. A collagen-coated 24-well plate was used for the lower wells. 600 μL of 50 ng/mL HGF-DMEM was added to the lower wells. After culturing for 24 hours, the number of cells that migrated to the lower well was measured under a microscope and used as an index of motility. As a result, it was revealed that the n-butanol extract and the water extract have an inhibitory effect on the motility of cancer cells (Fig. 10). (Example 7) c-Met kinase inhibitory effect of Diaion HP-20 column fraction of water extract
From the results of the pain test in Example 9, it was found that the distributed extract showing pharmacological action by oral administration was the water extract, and the n-butanol extract did not show pharmacological action by oral administration. Therefore, the water extract obtained by partitioning the Ephedra extract was added to a Diaion HP-20 column and eluted sequentially with H ₂ O, 10%MeOH, 20%MeOH, 30%MeOH, 40%MeOH, 50%MeOH, and MeOH. A fine fraction was obtained. Each fraction was examined by an in vitro assay using a recombinant protein of the c-Met kinase domain and a synthetic substrate peptide.

The assay conditions are as follows. 4 nM recombinant c-Met kinase domain in 40 mM Tris-HCl pH 7.5, 2 mM DTT, 7 mM MgCl ₂ , 10 μM ATP, 0.2 μg/mL Poly (Glu4, Tyr1) and 5 μg/ml of each fraction. A mL solution was prepared and incubated for 1 hour at room temperature. After stopping the reaction, the ADP-Glo reagent was used to measure the ADP amount-dependent luminol emission intensity with a luminometer (PerkinElmer, EnSpire) to calculate the c-Met kinase activity. As a result, 20% MeOH fraction, 30% MeOH fraction, 40% MeOH fraction, 50% MeOH fraction and MeOH fraction of Diaion HP-20 column have c-Met kinase inhibitory activity. (Fig. 11).
(Example 8) Diaion HP-20 column fraction of water extract inhibits motility of cancer cells
From the results of the pain test in Example 9, it was found that the distributed extract showing pharmacological action by oral administration was the water extract, and the n-butanol extract did not show pharmacological action by oral administration. Therefore, the water extract obtained by partitioning the Ephedra extract was added to a Diaion HP-20 column and eluted sequentially with H ₂ O, 10%MeOH, 20%MeOH, 30%MeOH, 40%MeOH, 50%MeOH, and MeOH. A fraction was obtained. Each fraction was analyzed for its inhibitory effect on cancer cell motility induced by HGF.

The assay conditions are as follows. MDA-MB-231 cells (5×10 ⁴ cells/well) were suspended in Vehicle (DMEM) or DMEM in which 10 μg/mL of each fraction was dissolved, and added to the upper wells.
A collagen-coated 24-well plate was used for the lower wells. 600 μL of 50 ng/mL HGF-DMEM was added to the lower wells. After culturing for 24 hours, the number of cells that migrated to the lower well was measured under a microscope and used as an index of motility. As a result, 30% MeOH fraction, 40% MeOH fraction, and 50% MeOH fraction strongly inhibited HGF-induced motility (Fig. 12).
(Example 9) Suppressive effect on formalin-induced pain using mice (formalin test)
Diaion HP-20 column fractions of EFE partitioned extracts and water extracts were examined for inhibitory effects on formalin-induced pain.

4-week-old male ddY mice were brought in and acclimatized for 1 week or longer, and 4-8 mice per group were used. On the day of the start of the experiment, the animals were weighed and divided into a control group, an EFE-administered group, and each fraction-administered group (7 groups) so that each group had approximately the same weight. 10% DMSO (DMSO:Saline=1:9) was added to EFE and each fraction, and dissolved by ultrasonication. The control group was orally administered with 10% DMSO. The EFE group was orally administered 700 mg/kg EFE. Each fraction group was orally administered in an amount calculated from the weight yield of each fraction with respect to EFE 700 mg/kg. Six hours after administration of each sample, 10 μL of a 2.5% formalin solution was intradermally administered to the left hind paw. Immediately after administration, the mice were placed in cylindrical acrylic cages (height 20 cm x diameter 10 cm) and video-recorded for 45 minutes. Pain-related behavior was targeted for the first phase that occurred from immediately after administration to 5 minutes after administration and the second phase that occurred from 10 minutes to 45 minutes after administration, and the behavior time of licking the treated paw was measured. Statistical analysis was performed by Dunnett's tests on the pain behavior time of the control group and the test substance administration group, and the significance level was set to 5%.

The results are shown in FIG. As for the first phase, the sample-administered group did not affect pain-related behavior, so it was considered that there was no pain-suppressing effect on nociceptive pain. On the other hand, in phase 2, EFE group, water extract group, 20% MeOH fraction group, 40% MeOH fraction group, and MeOH fraction group showed less pain-related behavior than the control group, and inflammation. It was found to have an inhibitory effect on sexual pain. The water extract group showed a strong pain-suppressing effect at a lower dose than the EFE group, indicating that the pain-suppressing effect was concentrated in the water extract, that is, the specific activity was significantly higher. became. On the other hand, the n-butanol extract, which was found to be effective in vitro, did not show any efficacy in vivo.

This water extract was then fractionated on a Diaion HP-20 column. From Examples 7 and 8, 20% MeOH fraction, 30% MeOH fraction, 40% MeOH fraction, 50% MeOH fraction, and MeOH fraction showed c-Met kinase inhibitory action and motility. was found to have an inhibitory effect. Therefore, in order to increase the recovery of each fraction for animal testing, the water extract was processed by DiaionHP-20 into H ₂ O fraction, 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction. separated into As a result, it was clarified that the H ₂ O fraction had no pain-suppressing effect, while the 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction had activity. From the above, it was found that 20% MeOH fraction, 40% MeOH fraction and MeOH fraction have an analgesic effect on inflammatory pain.
(Example 10) Effects on c-Met expression and c-Met tyrosine phosphorylation, and EGFR expression and EGFR tyrosine phosphorylation
Diaion HP-20 column fractions of the EFE-derived water extract were analyzed for effects on c-Met and EGFR using human non-small cell lung cancer-derived H1993 cells overexpressing c-Met and EGFR.

Each fraction was dissolved in 10% DMSO-RPMI1640 medium to obtain a stock solution with a concentration of 2 to 4 mg/mL, and diluted with RPMI medium for use. The final concentration of DMSO in the medium was 0.125%. H1993 cells were treated with 100 μg/mL EFE, 50 μg/mL H ₂ O fraction, 25 μg/mL 20% MeOH fraction, 25 μg/mL 40% MeOH fraction, or 25 μg/mL MeOH fraction was added and incubated for 4 hours, the cells were lysed, and Western-blotting was performed using the centrifuged supernatant. The results are shown in FIG.

Since H1993 cells overexpress c-Met and EGFR, the receptor is phosphorylated by autophosphorylation even without HGF or EGF stimulation.
(1) Action on c-Met:
The H ₂ O fraction had no effect on c-Met expression levels and phosphorylation. 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction decreased c-Met expression level and inhibited phosphorylation. These effects were highest in the 40% MeOH fraction, showing almost the same effect as EFE. Therefore, c-Met
20% MeOH fraction, 40% MeOH fraction, and MeOH fraction had no effect on H ₂ O fraction. This was substantially consistent with the results of the pain test in (Example 9). (2) Action on EGFR:
The H ₂ O fraction had no effect on EGFR expression level and phosphorylation. 20% MeOH fraction, 40% MeOH fraction and MeOH fraction inhibited EGFR phosphorylation. On the other hand, the expression level of EGFR was decreased by EFE and 40% MeOH fractions. However, the 20% MeOH fraction and the MeOH fraction slightly decreased the expression level of EGFR.

These results revealed that 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction, but not H ₂ O fraction, had an inhibitory effect on EGFR phosphorylation. The result of the pain test in Example 9 and the action on c-Met in (1) above were almost consistent. On the other hand, it was found that the 40% MeOH fraction had the effect of reducing the expression level of EGFR.

In lung cancer, the leading cause of cancer death in Japan, the type for which EGFR tyrosine kinase inhibitor (EGFR-TKI), a molecular-targeted drug, is remarkably effective, prolongs survival, but develops resistance in a few years and relapses. c-Met overexpression has been reported as one of the causes of resistance acquisition (Seiji Yano, Experimental Medicine, 29, 203-208, 2011). In the case of EGFR-TKI resistance caused by c-Met overexpression, the combined use of c-Met inhibitors and EGFR-TKIs is considered to be effective, but c-Met inhibitors are yet to be commercialized. do not have.

20% MeOH fraction, 40% MeOH fraction, and MeOH fraction are c-Met overexpressed by lung cancer cells.
and inhibit EGFR phosphorylation, it may be useful in treating EGFR-TKI-resistant lung cancer patients due to c-Met overexpression. Furthermore, the 40% MeOH fraction not only inhibits the phosphorylation of overexpressed c-Met and EGFR, but also reduces their expression level, so it can be a more effective molecular target therapeutic drug. .
(Example 11) Pain suppression effect on arthritis model mice From Example 10, the 40% MeOH fraction has the highest efficacy, and from Examples 9-10, it is clear that the H ₂ O fraction has no efficacy. became. Therefore, the analgesic effects of these fractions on arthritis model mice were compared in vivo. The results are shown in FIG.

As experimental animals, 9-week-old female Balb/c mice were used. After the mice were brought in and acclimatized for about one week, 10 μL of Complete Freud's Adjuvant (CFA) 5 mg/mL was inoculated into the right hind footpad, and the mice were raised for 7 days and used as arthritis model mice. This is because mice on day 7 of CFA inoculation have significantly swollen ankle joints and are most sensitive to mechanical stimuli. 390 mg/kg H ₂ O fraction dissolved in 10% DMSO solution or 190 mg/kg 40% MeOH fraction was orally administered to arthritis model mice. In the control group, vehicle (10% DMSO solution) was administered to normal mice, and in the vehicle group, vehicle was administered to arthritis model mice. Six hours after administration of the test drug, the right paw von Frey test was performed, and the 50% paw withdrawal threshold (50% PWT) was calculated using the up-down method (SR Chapman et al, Journal of Neuroscience Method 53 (1994) 55-63). Statistical analysis was performed by Tukey's multiple comparison test after one-way analysis of variance. Analysis software uses Prism 7 (GraphPad Software Inc., San Diego, CA, USA), statistically significant level p < 0.05
was judged to be significantly different.

Oral administration of 40% MeOH fraction significantly increased 50% PWT and decreased sensitivity to mechanical stimulation compared to the vehicle group. On the other hand, oral administration of the H ₂ O fraction resulted in a 50% PWT similar to that in the vehicle group, and remained highly sensitive to mechanical stimuli. From these results, it was revealed that the 40% MeOH fraction exhibits pain-suppressing activity in arthritis model mice, but the H ₂ O fraction does not.
(Example 12) Anti-influenza virus action From the results of the formalin test in Example 9, the water extract of EFE was fractionated with Diaion HP-20, 20% MeOH fraction, 40% MeOH fraction, MeOH fraction Anti-influenza virus activity of these fractions was determined, since it was found that the substance had a pain-inhibiting effect. This measurement method utilizes the lysis of MDCK cells by influenza virus infection, and evaluates the anti-influenza virus activity at a sample concentration at which 50% of the cells survive. In addition, in order to confirm that the anti-influenza virus action was being measured, not the cytotoxicity, the concentration at which the cell viability was 50% was also measured at the same time.

For each sample, a 25 μg/mL solution was prepared with 10% FBS-MEM, and a 10-step two-fold dilution series was prepared to prepare a sample solution. A fluid containing influenza virus (A/WSN/33 (H1N1) strain) was diluted with 10% FBS-MEM to 1000 TCID ₅₀ /mL to prepare an influenza virus fluid. MDCK
Cells were suspended in 10% FBS-MEM and seeded at 3×10 ⁴ cells/100 μL per well of a 96-well culture plate. After culturing for 24 hours, the culture supernatant was removed, 100 μL of sample solution was added to each well, and 100 μL of influenza virus solution or 10% FBS-MEM was added. After culturing for 72 hours, the cells were stained with crystal violet and the absorbance of each well at 560 nm was measured using a microplate reader. A 4-parameter logistic analysis was performed on the added sample concentration and cell viability curve, and the 50% inhibitory concentration (IC ₅₀ value) from the influenza virus addition group,
The 50% cytotoxicity concentration (CC ₅₀ value) was determined from the non-influenza virus addition group (Table 1).

各試料のIC₅₀値は、CC₅₀値よりも低濃度であったことから、抗インフルエンザウイルス作用が測定できていることが確認された。また、20%MeOH分画物、40%MeOH分画物、MeOH分画物のIC₅₀値は、EFEのIC₅₀値と同程度であったことから、EFEの抗インフルエンザウイルス作用を示す活性成分は、20%MeOH分画物、40%MeOH分画物、MeOH分画物に存在することが明らかになった。
（実施例１３）高分子縮合型タンニン標準物質の調製
実施例１で得た水エキス-40%MeOH分画物について、さらにSephadex LH-20カラムクロマトグラフィー（GE Healthcare）を用いて精製した。カラムを50%MeOHで洗浄後、70%アセ
トンで溶出し、高分子縮合型タンニンを得た。溶出液を濃縮、凍結乾燥した後、約100 mgを精密に量り（100.0 mg）、50%ジメチルホルムアミド溶液10 mLを正確に加え、完全に溶解した。この溶液について、実施例２で示したGPC分析した結果、重量平均分子量は85642で、低分子量域にピークは確認されなかった（図１６）。この溶液約1 mLをバイアルに分注し、高分子縮合型タンニン標準物質溶液（10.0 mg/mL）とした。
（実施例１４）GPCを用いた高分子縮合型タンニン含量の測定
実施例２で示したGPCを用いた分子量分析法及び実施例１３で示した高分子縮合型タン
ニン標準物質を用いて、麻黄エキスの高分子縮合型タンニン含量を測定した。

Since the IC ₅₀ value of each sample was lower than the CC ₅₀ value, it was confirmed that the anti-influenza virus activity could be measured. In addition, the _IC50 values of the 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction were comparable to the _IC50 value of EFE, indicating that EFE has an anti-influenza virus effect. was found to exist in 20% MeOH, 40% MeOH and MeOH fractions.
(Example 13) Preparation of Polymer Condensed Tannin Standard Substance The water extract-40% MeOH fraction obtained in Example 1 was further purified using Sephadex LH-20 column chromatography (GE Healthcare). After washing the column with 50% MeOH, it was eluted with 70% acetone to obtain polymer condensed tannins. After concentrating and freeze-drying the eluate, about 100 mg was accurately weighed (100.0 mg), and 10 mL of a 50% dimethylformamide solution was accurately added to dissolve completely. As a result of the GPC analysis shown in Example 2 for this solution, the weight average molecular weight was 85642, and no peak was confirmed in the low molecular weight region (Fig. 16). About 1 mL of this solution was dispensed into a vial to obtain a polymeric condensed tannin standard substance solution (10.0 mg/mL).
(Example 14) Measurement of polymer condensed tannin content using GPC was measured for the content of polymeric condensed tannins.

Three lots of Ephedra huang extract were each dissolved in a 50% dimethylformamide solution to give a sample solution of 10.00 mg/mL. GPC analysis was performed under the conditions shown in Example 2 for each sample solution and polymer condensed tannin standard substance solution (10.0 mg/mL). Concerning the peak area of polymer condensed tannins 5 to 10 minutes after sample introduction, the content of polymer condensed tannins in 1 mL of the sample was determined by the following formula (Table 2).

Amount of polymer condensed tannin in 1 mL of sample solution (mg) = A _T /A _S x 10.00
A _T : Sample peak area
A _S : Peak area of standard substance
10.00: Amount of polymer condensed tannin in 1 mL of standard solution (mg)

Claims (9)

An extract fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more derived from mahuang extract or derived from ephedrine alkaloid-removed mahuang extract (EFE), wherein 30 Extracted fraction containing more than %. Polymer condensed tannins, as extension units, are condensed mainly with proanthocyanidin B type catechol-type or pyrogallol-type flavan 3-ols, part of which contains proanthocyanidin A-type condensed tannin units, and terminal 2. The extract fraction according to claim 1, which mainly contains catechol-type or pyrogallol-type flavan 3-ols as units, and pyrogallol-type and catechol-type at a ratio of about 5:1. Water-soluble fractions derived from Ephedra extract or Ephedrine alkaloid-removed Ephedra extract (EFE) are eluted by column chromatography using a mixed solvent of water and an alcohol selected from methanol or ethanol while increasing the alcohol concentration. 2. The method for producing an extract fraction according to claim 1, which comprises measuring the molecular weight of each obtained fraction to obtain an extract fraction containing polymer condensed tannins having a weight average molecular weight of 45,000 or more. A pharmaceutical composition comprising the extract fraction according to claim 1 or 2. 5. The pharmaceutical composition according to claim 4, which is a pain suppressant. 5. The pharmaceutical composition according to claim 4, which is an anticancer/anti-metastatic drug against c-Met-expressing cancer. 5. The pharmaceutical composition according to claim 4, which is an anticancer drug against EGFR-expressing cancers. 5. The pharmaceutical composition according to claim 4, which is an anti-influenza virus drug. A method for measuring the content of a pharmaceutical composition containing polymer condensed tannin as an active ingredient, using the extract fraction according to claim 1.

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