JP2020172491A - Ephedra extract stripped of ephedrine alkaloids, method for producing the same and use of the same - Google Patents

Abstract

To provide an ephedra extract with increased safety, stripped of ephedrine alkaloids, which are substances to cause side effects, a method for producing the ephedra extract, and an anticancer/antimetastatic agent, a pain inhibitor, and an anti-influenza virus drug that include the ephedra extract as an active ingredient.SOLUTION: Ephedra extract stripped of ephedrine alkaloids are obtained by passing aqueous solution of liquid and/or material extracted from Ephedra through an acidic cation exchange resin and recovering the fractions that have not been adsorbed and have passed through.SELECTED DRAWING: None

Description

The present invention relates to a safer ephedrine alkaloid-removing ephedra extract, a method for producing the extract, and an anticancer / antimetastasis drug, a pain suppressant, and an anti-influenza virus drug containing the same as an active ingredient.

Ephedra is the above-ground stem of the Ephedra sinica Stapf, Ephedra intermedia Schrenk et C. A. Meyer or Ephedra equisetina Bunge (Ephedraceae). Mao is the most important raw medicine that has been used since ancient times, and as Chinese herbal medicines (Chinese prescription) that contain mao as a constituent raw medicine, Kakkonto, Uyakujunkito, Eppito, Eppikajutsuto, Kaishusanryo, Kaishanryo, Kakkonto, Kakkonto Kakkonto (Kakkonto Kasenkyushini), Kakkonto Kajutsuto, Kakkonto Hangeto, Kanzo Maoto, Katsura Maoto, Katsura Maoto Kakkonto, Kakkonto, Kakkonto, Kakkonto, Makyokansekito, Kakkonto, Makyokansekito, Kakkonto, Kakkonto, Kakkonto, Kakkonto Okakuhanto, Kobokumaoto, Gokoto, Gokonichinto, Goshakusanryo, Saikakkonto, Shoseiryuto, Shozokumeito, Shinpitou, Zokumeito, Dai Seiryuto, Kakkonto, Kakkonto, Boufutsushosanryo, Maoto, Maoubushi Saishinto, Makyokansekito, Makyokansekito, Makyokanto, Yakanmaoto, Kakkonto, Kakkonto, Reitaku Kakkonto is an example. In addition, there are currently 16 Chinese medicine prescriptions that include mao as a constituent raw medicine in the health insurance listed Chinese medicine prescriptions: Eppikajutsuto, Kakkonto, Kakkonto Kagawa Kyushinto. Tokasenkyushini, Kakkontojutsutsuto, Keishima Mao Hanto, Gokoto, Gokoto Sanryo, Shoseiryuto, Shinpitou, Zokumeito, Kakkonto, Maoto U), Mao-bushi Saishinto, Makyokansekito, Makyokansekito, Makyo-kakkonto, Makyo-kakkonto ).

Ephedra alkaloids ((-)-ephedrine, (+)-pseudoephedrine,) are famous as the active ingredient of Ephedra, and in the Japanese Pharmacopoeia, Ephedra sinica Stapf, Ephedra intermediaSchrenk et CA Meyer or Ephedra equisetina Bunge (Ephedraceae). ) Is quantified as a dried ground stem, it is specified to contain 0.7% or more of total alkaloids (ephedrine and pseudoephedrine). Central nervous system stimulant action, sympathetic nerve stimulant action, sweating action, antitussive action, anti-inflammatory action, and antiallergic action are known for mao (Non-Patent Document 1), and these pharmacological actions are considered to be derived from ephedrine alkaloids. (Non-Patent Document 2). Furthermore, Ephedra is used for various symptoms of body pain, and the analgesic effect of Ephedra is explained by the anti-inflammatory effect of pseudoephedrine (Non-Patent Document 3). On the other hand, as new pharmacological actions of mao, the present inventors have exhibited a cancer metastasis inhibitory effect through a cancer cell motility inhibitory effect and an antitumor effect through a cancer cell growth inhibitory effect in mice. It was clarified from the in vivo analysis using (Non-Patent Document 4), and its molecular mechanism is due to the suppression of the HGF-Met-Akt signal by Met inhibition, which is a receptor for hepatocyte growth factor (HGF). (Patent Document 1, Non-Patent Document 5). Since these effects were not observed with the ephedrine alkaloid alone, as a result of searching for the active ingredient, a flavonoid compound Herbacetin glycoside was found (Non-Patent Document 6). However, the content of herbacetin glycosides is as low as about 0.005%, and this component alone cannot explain the new pharmacological action of Ephedra, and it is considered that a plurality of components are involved (Non-Patent Document 7). Therefore, in order to effectively utilize the medicinal properties of Ephedra, it is desirable to use Ephedra extract as it is as a drug.

Since maoto has sympathetic and central nervous system stimulants, it is contraindicated in principle for people with a history of angina, myocardial infarction, or hypertension. In addition, it should be used with caution for the elderly. In addition, people with weak gastrointestinal tracts can cause abdominal pain due to loss of appetite and acute gastric mucosal lesions. In addition, attention should be paid to palpitation, agitation, dysuria, insomnia, rash, etc. (Non-Patent Document 8). These side effects are believed to be derived from ephedrine alkaloids (Non-Patent Document 9). In the United States, Ephedra was used as a supplement, but in response to a fatal accident due to improper use such as overdose, the FDA has sold all ephedrine alkaloids such as Ephedra. It stipulates a ban and alerts consumers (2004 Ephedra Ban). The Land Report, which was the basis for this, scrutinized over 10,000 side effect reports, including a huge number of papers and hundreds of deaths reported to the FDA, and palpitations and nausea with Ephedra preparations. It is concluded that there are mild to moderate side effects such as vomiting (Non-Patent Document 10). In Japan, Ephedra is classified as a material used exclusively as a drug, and those approved as a drug are allowed to be used, but in the medical field, it is serious depending on the constitution and medical condition of the patient. At present, there are concerns that side effects may occur.

Until now, it has been believed that the medicinal properties of Ephedra are almost entirely due to ephedrine alkaloids. The basis for this is that the de-alkaloid ephedra extract described in Non-Patent Document 2 has lost all the medicinal properties of ephedra. From this, it is considered difficult to separate the main action and side effects of Ephedra, and there has never been an idea to remove ephedrine alkaloids from Ephedra in order to eliminate the side effects of Ephedra. However, as mentioned above, the inventors have found a useful medicinal effect that does not depend on ephedrine alkaloids in Ephedra, and by selectively removing ephedrine alkaloids, which are the causative agents of side effects, as highly safe drugs. I came up with the idea that I could provide Ephedra extract.

"MET Inhibitor Containing Ephedra" Japanese Patent Application No. 2009-86363 (March 31, 2009)

Ephedra's medicinal properties / pharmacology, Contemporary Oriental Medicine, 15 (4), 551-554, 1994. Pharmacology of Ephedra, Modern Oriental Medicine, 1 (2), 34-39, 1980. Positioning and type of analgesics in Chinese herbs, pain and clinical practice, 5 (3), 262-268, 2005. Basic research on the use of Kampo medicines to protect against cancer recurrence and metastasis, J. Trad. Med., 30 (1), 19-26, 2013. Ephedrae herba, a major component of maoto, inhibits the HGF-induced motility of human breast cancer MDA-MB-231 cells through suppression of c-Met tyrosine phosphorylation and c-Met expression. J. Trad. Med., 28, 128-138 , 2011. Characterization of phenolic constituting from Ephedra Herba extract. Molecules, 18, 5326-5334, 2013. Research on ingredients contained in Ephedra extract, Annual Meeting of the Pharmaceutical Society of Japan 133 Years Meeting No. 2, 185, 2013. Toshihiko Hanawa, "Chinese Medicine Lessons", Supplement, p46-47, Kanahara Publishing, 2003 Haller CA, Benowitz NL., Adverse cardiovascular and central nervous system events associated with dietary supplements containing ephedra alkaloids, N Engl J Med. 2000 Dec 21; 343 (25): 1833-8. Final Rule Declaring Dietary Supplements Containing Ephedrine Alkaloids Adulterated Because They Present an Unreasonable Risk. Federal Register: 69 (28), pp 6787-6854, 2004

The present invention provides a method for producing a highly safe ephedrine alkaloid-removing maoto extract by removing ephedrine alkaloids from ephedra extract while retaining some of the medicinal properties of ephedra, and an anti-treatment using the same as an active ingredient. The purpose is to provide anti-metalytic agents, pain-suppressing agents and anti-influenza virus agents.

Non-Patent Document 2 describes a method for preparing a de-alkaloid ephedra extract by removing alkaloids by treating ephedra with NH ₄ OH and ether. However, the de-alkaloid ephedra extract produced by this method contains a high concentration (0.33-0.5%) of residual total alkaloids, and further, NH ₄ OH treatment causes a chemical change in the structure of plant components. (Makoto Ohara, Journal of the Wood Society, 55, 59-68, 2009; D. Ferreira et al., J. Chem. Soc. Perkin Trans. 1, 203-208, 1990), artificial different from the original Ephedra extract It is considered that it contains various components. The author of Non-Patent Document 2 also mentions this matter. In addition, the de-alkaloid ephedra extract described in Non-Patent Document 2 is a production method that affects the medicinal effect of ephedra because all the pharmacological effects of ephedra have been lost, and is a pharmaceutical product obtained by removing ephedrine alkaloids from ephedra. It is an inappropriate method to use as.

The present inventors have made extensive studies to overcome problems such as high alkaloid content of dealkaloid ephedra extract, chemical structural changes of the contained components, and loss of drug efficacy, and to solve the above problems. , We have succeeded in developing a mild manufacturing method that easily removes ephedrine alkaloids from Ephedra extract without affecting the structure of the components contained in Ephedra extract and maintaining its medicinal properties. Furthermore, it was found that the obtained ephedrine alkaloid-removing ephedra extract retains anti-cancer / anti-metastasis activity, and also has pain-suppressing activity and anti-influenza virus activity, thereby completing the present invention. I arrived.

That is, the present invention more specifically provides the following (1) to (9).
(1) Ephedra alkaloid-removed ephedra extract obtained by removing ephedrine alkaloid from ephedra extract.
(2) The ephedrine alkaloid-removing ephedra extract according to (1), which contains ephedrine alkaloid in an amount of 0.23% or less.
(3) The ephedrine alkaloid-removing ephedra extract according to (1), which contains ephedrine alkaloid in an amount of 0.023% or less.
(4) The ephedrine alkaloid-removing ephedra extract according to (1), which contains ephedrine alkaloid in an amount of 0.05 ppm (detection limit) or less.
(5) The ephedrine alkaloid-removed ephedra extract according to any one of (1) to (4), which contains components contained in the ephedra extract / or the aqueous solution of the extract that are not adsorbed on the cation exchange resin.
(6) A Chinese herbal preparation containing the ephedrine alkaloid-removing hemp yellow extract according to any one of (1) to (5).
(7) The method for producing an ephedrine alkaloid-removed ephedra extract according to any one of (1) to (5), which comprises removing the ephedrine alkaloid from the ephedra extract and / or the extract by ion exchange chromatography.
(8) An anticancer / antimetastasis agent containing the ephedrine alkaloid-removing ephedra extract according to any one of (1) to (5) as an active ingredient.
(9) A pain-suppressing agent containing the ephedrine alkaloid-removing ephedra extract according to any one of (1) to (5) as an active ingredient.
(10) An anti-influenza virus drug containing the ephedrine alkaloid-removing hemp yellow extract according to any one of (1) to (5) as an active ingredient.

The method of the present invention is extremely capable of removing ephedrine alkaloids to 0.23% or less, 0.023% or less, or 0.05 ppm (detection limit) or less simply by passing the hemp yellow extract through a certain cation exchange column. It is a simple and useful manufacturing method. In addition, the ephedrine alkaloid-depleted ephedra extract of the present invention suppresses the inhibitory activity of Met kinase, which has been clarified as the mechanism of anti-metalytic action of ephedra, to the same extent as the original ephedra extract, and further, Met high-expressing human lung cancer cells Cell proliferation of strain H1975 was suppressed in a concentration-dependent manner, similar to the original Ephedra extract. From these results, it is shown that the production method of the present invention can remove the ephedrine alkaloid while maintaining the MET-inhibiting anticancer / antimetastasis effect of the ephedrine extract, and the ephedrine alkaloid-removed hemp yellow extract is used as an active ingredient. It has become clear that we can provide anti-cancer and anti-metastasis drugs.

Since maoto (a Chinese herbal medicine with maoto as the main ingredient) is frequently used as a therapeutic agent for arthralgia, we focused on the analgesic effect of ephedrine alkaloid-removing maoto extract and evaluated it by a pain test (formalin test) using mice. As a result, oral administration of ephedrine alkaloid-removed Ephedra extract significantly suppressed pain and showed a higher pain-suppressing effect than Ephedra extract. Until now, it was thought that the analgesic effect of Ephedra was derived from pseudoephedrine, so this result was a discovery that overturned the previous idea of Ephedra. Therefore, it has been clarified that the present invention can provide a pain-suppressing agent containing ephedrine alkaloid-removing ephedra extract as an active ingredient.

Since maoto, one of the maoto agents, is used for the treatment of the early stage of influenza, it was evaluated in an influenza virus infection test using MDCK cells whether ephedrine alkaloid-depleted maoto extract could suppress influenza virus infection. As a result, the ephedrine alkaloid-free Ephedra extract suppressed influenza virus infection to the same extent as the original Ephedra extract. Therefore, it was clarified that an anti-influenza virus drug containing ephedrine alkaloid-removing hemp yellow extract as an active ingredient can be provided.

From the above, the ephedrine alkaloid-removing hemp yellow extract of the present invention is expected to be used as a safer anticancer / antimetastasis drug, pain suppressant and antiinfluenza virus drug. In addition, ephedrine alkaloid-removing ephedra extract is a new type of cancer treatment that can treat cancer and cancer pain at the same time.

Since maoto is widely used as a constituent crude drug of herbal medicine, it is possible to provide a new herbal preparation with higher safety by replacing maoto, which is a herbal medicine containing maoto, with ephedrine alkaloid-removed maoto extract.

For example, when maoto is used as an anti-cancer / anti-metastatic drug, it can be expanded to include patients who are sensitive to conventional maoto while imparting new indications.

Further, for example, it can be applied to the use of joint pain in the elderly. Joint pain in the elderly is one of the causes of poor quality of life (QOL), most of which is caused by osteoarthritis. Such joint pain accounts for about 10% of the causes of need for support and care for the elderly, and is the top cause of need for support (Matsui, Y., National Center for Geriatrics & Gerontology, 42, 1). -4, 2013). Treatment of arthralgia is mainly non-steroidal anti-inflammatory drugs (NSAIDs), but long-term administration is difficult due to side effects such as gastrointestinal disorders, and analgesics to replace NSAIDs are required. Recently, a pharmaceutical company developed an antibody drug with analgesic activity and conducted a clinical trial, but the trial was canceled due to worsening osteoarthritis in some patients (Yamaguchi, A, Drug Delivery). System, 26 (5), 457-460, 2011). This exacerbation of symptoms is thought to be due to a rapid increase in patient behavior due to a strong analgesic effect. On the other hand, Chinese herbal medicines containing maoto (Kakkonto, Maoyokanto, Eppikajutsuto, Koshijinto, etc.) are effective in treating joint pain, and their analgesic effect is mild, so the patient's abruptness. It is unlikely to cause an increase in activity and does not cause exacerbation of osteoarthritis. However, due to the side effects of maoto (palpitations, increased blood pressure, insomnia, dysuria, etc.), administration of maoto-containing Chinese herbs to the elderly requires caution. By using a Chinese herbal preparation that replaces maoto of these Chinese herbs with ephedrine alkaloid-removed maoto extract, it is possible to safely control joint pain in the elderly without causing exacerbation of osteoarthritis and improve QOL. Can contribute to.

In addition, clinical studies have reported that maoto, which is used to treat early influenza, has the same therapeutic effect on influenza as the Western drug oseltamivir (Nabeshima, S., et al., J. Trad). . Med., 27, 148-156, 2010; Hiroshi Kimoto, Therapeutics, 40, 385-388, 2006), due to the side effects of maoto, it is necessary to refrain from administration to elderly people and patients with maoto sensitivity. However, if maoto is used by replacing maoto with ephedrine alkaloid-removed maoto extract, it can be administered to elderly patients and patients sensitive to maoto. In addition, maoto was a Chinese herbal medicine that was difficult to administer for a long time due to the side effects of maoto, but maoto using ephedrine alkaloid-removing maoto extract can be used to prevent influenza infection in people at high risk of infection. Target administration can be performed for a long period of time.

According to the present invention, it has been clarified that the ephedrine alkaloid-removing maoto extract has an action as an anticancer / antimetalytic drug, a pain suppressant, and an antiinfluenza virus drug possessed by the maoto extract, and is a causative substance of side effects. It was confirmed that ephedrine alkaloids can be selectively removed and used as a highly safe drug. The present invention is an extremely important basic technique for providing an ephedrine alkaloid-free ephedra extract as a pharmaceutical product. It is expected that the present invention will be used in the clinical field.

The HPLC chart of the Ephedra extract and the ephedrine alkaloid-removed Ephedra extract before and after the purification by ion exchange chromatography described in Example 4 is shown. The fingerprints of the ephedra extract and the ephedrine alkaloid-removed ephedra extract before and after ion exchange chromatography purification described in Example 5 are shown. The LC-MS chart of the ephedra extract and the ephedrine alkaloid-removed ephedra extract before and after ion exchange chromatography purification described in Example 6 is shown. The MET kinase inhibitory action of the ephedra extract and the ephedrine alkaloid-removed ephedra extract described in Example 7 is shown. It shows the inhibitory effect of the ephedra extract and the ephedrine alkaloid-depleted ephedra extract described in Example 8 on the proliferation of human non-small cell lung cancer H1975 cells. The pain-suppressing action of the ephedra extract and the ephedrine alkaloid-removing ephedra extract described in Example 9 is shown. The effect of suppressing the influenza virus infection of the ephedra extract and the ephedrine alkaloid-removed ephedra extract described in Example 10 is shown. The effect of suppressing the influenza virus infection of the ephedra extract and the ephedrine alkaloid-removed ephedra extract described in Example 10 is shown.

Ephedra extract The ephedra used in the present invention uses the above-ground stems of the Ephedra sinica Stapf, Ephedra intermedia Schrenk et CA Meyer or Ephedra equisetina Bunge (Ephedraceae). Raw, dried or processed rhizomes are available.

The ephedra extraction step performed in the production method of the present invention can be performed based on any of the well-known methods. As the extraction solvent, water or hot water, hot water, an alcohol solvent, and other organic solvents such as acetone can be used. Examples of the alcohol solvent include methanol, ethanol, propanol, isopropanol, butanol, isobutanol and the like. These solvents may be used alone or in combination.

The amount of the extraction solvent is preferably 2-100 parts by weight with respect to the dry weight of Ephedra. The extraction temperature is preferably 4-98 ° C. The extraction time is preferably 30 minutes-2 hours. The extraction method can be any method such as stirring extraction, immersion extraction, countercurrent extraction, ultrasonic extraction, and supercritical extraction.

The obtained extract, the filtrate obtained by filtering the extract, the concentrate obtained by concentrating the filtrate, and the dried product obtained by drying the concentrate are obtained as the following cations. Although it may be subjected to a purification step by exchange chromatography, it is preferable to roughly separate the constituents before that. For example, fine solid components can be easily removed by filtering with an appropriate filter or centrifuging to prevent troubles such as column clogging when performing the next chromatography. Can be done.

In Example 1 described later, the ephedrine alkaloid in the ephedra extract thus obtained was 4.74% (ephedrine was 3.19%, pseudoephedrine was 1.55%).

Ephedrine alkaloid-removed ephedra extract The ephedrine alkaloid-removed ephedra extract can be obtained by removing ephedrine by cation exchange chromatography and concentrated drying to obtain ephedrine alkaloid-removed ephedra extract. Ephedrine alkaloids free Mao extract is sometimes abbreviated as EFM (ephedrine alkaloids free Mao extract).

Studies were conducted to determine suitable ion exchange chromatography fillers for the production of the ephedrine alkaloid-free Ephedra extract of the present invention. After treating the ephedra extract with various ion exchange resins, the ephedrine alkaloids contained in the extract were analyzed by TLC and HPLC. The ion exchange resins examined were 13 types of cation exchange resins, 1 type of amphoteric ion exchange resins, and 8 types of anion exchange resins, for a total of 22 types. As a result, it was found that the ion effect resin suitable for removing the ephedrine alkaloid is a cation exchange resin. Therefore, after treating the ephedra extract with the weakly acidic cation exchange resins WK10, WK11, WK20, WK40L, FPC3500, and the strongly acidic cation exchange resins SK104, SK110, SK1B, UBK530, UBK12, PK216, IR120B, and 106H. The ephedrine alkaloid content contained in the extract was quantified by HPLC. The results are shown in the table below.

From the above results, the cation exchange column suitable for producing the ephedrine alkaloid-removed ephedra extract is as follows. For the production of EFM with an ephedrine alkaloid content of 0.23% or less, as a column filler from weakly acidic cation exchange resins WK20, strongly acidic cation exchange resins SK104, SK110, SK1B, UBK530, PK216, IR120B, FPC3500, and 1060H. You can choose. For the production of EFM with an ephedrine alkaloid content of 0.023% or less, select from weakly acidic cation exchange resins WK20, strongly acidic cation exchange resins SK104, SK110, SK1B, UBK530, PK216, IR120B, and 106H as column fillers. Can be done. Further, for the production of EFM having an ephedrine alkaloid content of 0.05 ppm or less, the strongly acidic cation exchange resin PK216 can be selected as the column packing material, and from Examples 2 and 3, the strongly acidic cation exchange resins SK1B and IR120B are used. Can also be selected.

The specific method of performing chromatography is any of the methods well known to those skilled in the art.

The drying method can be any method such as vacuum drying, freeze drying, and spray drying. Excipients such as dextrin may be added if necessary.

The ephedrine alkaloid-removing hemp yellow extract of the present invention is an ephedrine alkaloid-removing hemp yellow extract obtained by removing ephedrine alkaloids from the ephedrine extract, and preferably contains ephedrine alkaloid (total of ephedrine and pseudoephedrine) in an amount of 0.23%% or less, more preferably. Contains ephedrine alkaloids in an amount of 0.023 %% or less, more preferably in an amount of 0.05 ppm (detection limit) or less.

The Japanese Pharmacy stipulates that Ephedra contains 0.7% or more of total alkaloids (ephedrine and psoid ephedrine) in the dried crude drug, and this amount is approximately 2.3% to 3.5% when converted to Ephedra extract. Since it is calculated to include the above, the ephedrine alkaloid-free Ephedra extract of the present invention is clearly distinguished from the currently used Ephedra extract.

The conversion of the total alkaloid content in the above-mentioned ephedra extract is performed in non-patent documents (Ichiro Narukawa "Kampo Claims-Modern Science and Kampo Preparations" p162-163, Kenyukan Co., Ltd., published in 1991) and Example 1. Required from. When Ephedra extract is prepared from Ephedra of the Japanese Pharmacopoeia used for manufacturing EFM, almost all ephedrine alkaloids contained in Ephedra are transferred to Ephedra extract. However, since the yield of Ephedra extract obtained from Ephedra is 20% to 30%, the total alkaloids contained in Ephedra extract are concentrated 3.3 to 5 times. Therefore, the value of 2.3% to 3.5% or more, which is obtained by multiplying 0.7% or more of the total alkaloid of Ephedra specified by the Japanese Pharmacopoeia by 3.3 to 5 times, is the specified value of the total alkaloid of Ephedra extract.

The alkaloid content of this EFM was determined from the following non-patent documents. When making a placebo of Chinese herbal medicine, 10% of the active drug is mixed (Katsutoshi Terasawa, edited by Toshiaki Kita "EBM Chinese medicine" p8, March 20, 2003, Ishiyaku Publications, Inc.), 10 minutes If the content is 1 or less, it is considered that the possibility that the pharmacological action appears is extremely low. Since Ephedra extract contains 2.3% to 3.5% or more of ephedrine alkaloids, it is considered that the possibility of side effects of ephedrine alkaloids is extremely low by setting it to 0.23% to 0.35% or less, which is one tenth of that. .. In the present invention, it is set to 0.23% or less in order to clearly distinguish it from the de-alkaloid ephedra extract (residual alkaloid content 0.33% to 0.5%) of Non-Patent Document 2. 0.023% or less is specified to further increase the certainty. Further, more preferably, it is 0.05 ppm or less (detection limit or less) shown in Example 1.

Ephedrine Alkaloid-Removing Maoto Extract-Containing Chinese Medicine Mao is widely used as a constituent crude drug of Chinese herbal medicine. It is possible to provide a safer Chinese herbal preparation. For example, Makyokansekito, which is used to treat joint pain, is composed of mao, apricot kernel, yokuinin, and licorice, so it can be used as a constituent crude drug other than mao, that is, an extract prepared from apricot kernel, yokuinin, and licorice. By adding the ephedrine alkaloid-removing mao extract, makyokansekito with few side effects can be prepared well.

In addition, maoto used in the early stages of influenza is composed of mao, katsura, apricot kernel, and licorice. Therefore, by adding ephedrine alkaloid-removed maoto extract to the extract prepared from katsura, apricot kernel, and licorice, maoto with few side effects Hot water can be made.

Pharmaceutical Compositions The present invention further provides a composition comprising an ephedrine alkaloid-removing hemp yellow extract, which can be used as an anticancer / antimetastasis agent, a pain suppressant, or an antiinfluenza virus agent, food, nutrition. It can be in the form of supplements or medicines.

The ephedrine alkaloid-removing ephedra extract used in the composition preferably has the above-mentioned ephedrine alkaloid content of 0.23% or less or 0.023% or less. More preferably, the ephedrialkaloid content is 0.05 ppm (detection limit) or less.

The administration method when the composition of the present invention is a pharmaceutical composition is not particularly limited, but a dosage form that can be orally administered is preferable. The pharmaceutical composition of the present invention can be in various dosage forms.

For example, for oral administration, tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, alcoholic beverages, syrups, extracts and elixirs can be used. , Not limited to these. In addition, various pharmaceutically acceptable carriers can be added to the preparation.

For example, excipients, binders, disintegrants, lubricants, flavoring agents, coloring agents, sweetening agents, flavoring agents, solubilizing agents, suspending agents, emulsifiers, coating agents, vitamin C, antioxidants. Can include, but is not limited to.

The dose of the pharmaceutical composition of the present invention can be used in the range of 100 mg to 3 g as a daily dose for adults when converted to the ephedrine alkaloid-free ephedra extract. When converted to ephedrine alkaloid-removed ephedra extract based on the general dose of ephedra extract, it can be estimated to be 740 mg to 770 mg per day for adults, but since the side effects caused by ephedrine alkaloid are excluded, administration It is possible to increase the amount.

Of course, it can be individually changed according to the age, body weight, symptom, administration route, administration period, treatment course, etc. of the person to be administered.

The daily dose can also be administered in several divided doses. It can also be administered in combination with other pain suppressants or anticancer agents.

The compositions of the present invention can also be in the form of foods or dietary supplements. For example, by blending ephedrine alkaloid-removed ephedra extract into a raw material, it can be formed into noodles, bread, candy, jelly, cookies, soup, and healthy beverages.

In addition to ephedrine alkaloid-removed hemp yellow extract, such foods and dietary supplements include inorganic components such as iron and calcium, various vitamins, dietary fiber such as oligosaccharides and chitosan, proteins such as soybean extract, and lecithin. Sugars such as lipids, sucrose, and lactose can be added.

Anti-cancer / anti-metastasis drug The type of cancer for which the composition of the present invention can be used as an anti-cancer / anti-metastasis drug is mainly MET-expressing cancer. Since MET is expressed on the surface of various cancer cells such as gastric cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, osteosarcoma, and brain tumor, it can be used for the treatment of these cancers. The HGF-c-Met signal promotes cancer cell proliferation, motility, cell dispersal, survival, and angiogenesis, and is the key to cancer cell proliferation, infiltration, and metastasis. Therefore, the composition of the present invention is also effective as a cancer recurrence / metastasis preventive agent.

Pain suppressant Diseases in which the composition of the present invention can be used as a pain suppressant include muscle pain, arthralgia, low back pain, rheumatism, gout, and cancerous pain.

Anti-influenza virus drug The composition of the present invention is effective in preventing influenza virus infection and treating the initial stage of infection.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto.

Example 1: Preparation of Ephedra extract.
The dry raw material of Ephedra was pulverized by a mixer, 500 mL of water was added to 50 g of the pulverized product, and the mixture was extracted at 95 ° C. for 1 hour with stirring. Solid-liquid separation was performed, and the extract was centrifuged at 3000 rpm for 10 minutes. The obtained supernatant was concentrated under reduced pressure at 60 ° C. and dried under reduced pressure at 60 ° C. overnight to obtain 9.6 g of Ephedra extract.

Example 2: Preparation of ephedrine alkaloid-removing ephedra extract using ion exchange resin SK1B.
The dry raw material of Ephedra was pulverized by a mixer, 500 mL of water was added to 50 g of the pulverized product, and the mixture was extracted at 95 ° C. for 1 hour with stirring. Solid-liquid separation was performed, the extract was centrifuged at 3000 rpm for 10 minutes, and the obtained supernatant was passed through a 25 mL strong acid cation exchange resin SK1B (manufactured by Mitsubishi Chemical Corporation). The passing solution was adjusted to pH = 5.2 with 5% LVDS ₃ and concentrated under reduced pressure at 60 ° C., and dried under reduced pressure at 60 ° C. overnight to obtain 6.3 g of ephedrine alkaloid-removed ephedra extract.

Example 3: Preparation of ephedrine alkaloid-removing ephedra extract using ion exchange resin IR120B.
The dry raw material of Ephedra was pulverized by a mixer, 500 mL of water was added to 50 g of the pulverized product, and the mixture was extracted at 95 ° C. for 1 hour with stirring. Solid-liquid separation was performed, the extract was centrifuged at 3000 rpm for 10 minutes, and the obtained supernatant was passed through a 25 mL strong acid cation exchange resin IR120B (manufactured by Organo Corporation). The passing solution was adjusted to pH = 5.2 with 5% LVDS ₃ and concentrated under reduced pressure at 60 ° C., and dried under reduced pressure at 60 ° C. overnight to obtain 6.3 g of ephedrine alkaloid-removed ephedra extract.

Example 4: Comparison of the contents of ephedrine contained in Ephedra extract (Example 1) and ephedrine alkaloid-removed Ephedra extract (Example 2 and Example 3) by high performance liquid chromatography (HPLC).
The HPLC charts of Ephedra extract (Example 1) and ephedrine alkaloid-removed Ephedra extract (Example 2) are shown in FIG.
Analytical condition column: SHISEIDO AG120 4.6 × 150mm 5μ
Mobile phase: Sodium lauryl sulfate solution (1 → 128) / acetonitrile / phosphoric acid mixed solution (640: 360: 1)
Temperature: 45 ° C
Detection: Ultraviolet absorptiometer (measurement wavelength: 210 nm)
Flow velocity: 0.6 mL / min (adjusted so that ephedrine is around 14 minutes)
Sample: 10 mg each of dried ephedra extract or ephedrine alkaloid-removed ephedra extract was taken, 1 mL of methanol was added and mixed, and 20 μL of each was injected.
Standard solution: 20 μL each of ephedrine standard solution (1.0 mg / 50% methanol 10 mL) and pseudoephedrine standard solution (1.0 mg / 50% methanol 10 mL) was injected.

The results obtained are shown in Table 1 below. From these results, it was clarified that ephedrine alkaloids (ephedrine and pseudoephedrine) can be removed from the ephedra extract to the detection limit (0.05 ppm) or less by column chromatography using the strong acid type cation exchange resin SK1B or IR120B.

Example 5: Comparison of composition components of Ephedra extract (Example 1) and ephedrine alkaloid-removed Ephedra extract (Example 2) by three-dimensional high performance liquid chromatography (3D-HPLC).
Analytical condition column: TSK-GEL 80TS 4.6 × 250mm 5μ
Mobile phase: (A) 0.05M ammonium acetate (pH 3.6)
(B) Acetonitrile 0 minutes: Solution B 10% → 60 minutes: Solution B 100%
Temperature: 40 ° C
Detection: Photodiode Array (PDA)
Flow velocity: 1.0 mL / min Sample: 10 mg each of dried Ephedra extract or ephedrine alkaloid-removed Ephedra extract was taken, 1 mL of methanol was added and mixed, and 20 μL of each was injected.
Standard solution: 20 μL each of ephedrine standard solution (1.0 mg / 10 mL methanol) and pseudoephedrine standard solution (1.0 mg / 10 mL methanol) was injected.

The 3D-HPLC fingerprint is shown in Figure 2.

As a result of comparing the composition components of the ephedrine alkaloid-removed ephedrine extract and the ephedrine alkaloid-removed ephedrine extract by 3D-HPLC, the peak of ephedrine alkaloid disappeared in the ephedrine alkaloid-removed ephedrine extract, but the pattern of other components was almost the same as that of the ephedrine alkaloid extract. ..

Example 6: Composition analysis and comparative study of Ephedra extract (Example 1) and ephedrine alkaloid-removed Ephedra extract (Example 2) by LC / MS Analytical condition column: Inertsil ODS-3 (2.1 × 150 mm I.D. 5 mm) )
Mobile phase: 0.1% HCOOH in water (A) −0.1% HCOOH in MeOH (B) in a gradient mode: 5% B (0-10 min) → 75% B (70 min)
Temperature: 40 ° C
Detection 1: PDA detector (200-400 nm)
Detection 2: MS detector
Interface, ESI positive / negative; ESI source voltage, 4.0 kV; capillary voltage, 10V; source temperature, 300 ° C; sheath gas flow rate, 50; auxiliary gas flow rate, 25; scan range, m / z 150-2000; mass resolution, 30,000 full width.
Flow velocity: 0.2 mL / min
Sample :); injection volume, 1 μL at 5 mg / mL

The LC / MS chart is shown in Figure 3.

As a result of comparing the compositional components of the ephedrine alkaloid-removed ephedrine extract and the ephedrine alkaloid-removed ephedrine extract by LC / MS, it was confirmed that the peaks of l-ephedrine, pseudoephedrine, methylephedrine, and norephedrine disappeared in the ephedrine alkaloid-removed ephedra extract ( Figure 3).

Example 7: MET kinase inhibitory effect of Ephedra extract (Example 1) and ephedrine alkaloid-removed Ephedra extract (Example 2)
The MET kinase activity was indexed by the amount of ADP produced by ATP consumption by the recombinant MET kinase domain using the Poly E4Y1 peptide as a substrate. Ephedra extract or ephedrine alkaloid-depleted ephedra extract (2 lots) in a mixture of reaction buffer containing Poly E4Y1 peptide and ATP in the recombinant MET kinase domain is 1 μg / mL, 5 μg / mL, and 10 μg / mL, respectively. And incubated for 1 hour at room temperature. After the reaction was stopped, the luminol emission intensity depending on the amount of ADP was measured using the ADP-Glo reagent, and the relative MET kinase activity was examined.

As a result, it was clarified that the ephedrine alkaloid-removed Ephedra extract inhibits the MET kinase action to the same extent as the Ephedra extract. The MET kinase inhibition curves of Ephedra extract (Example 1) and ephedrine alkaloid-removed Ephedra extract (Example 2) are shown in FIG.

Example 8: Inhibitory effect of Ephedra extract (Example 1) and ephedrine alkaloid-removed Ephedra extract (Example 2) on the proliferation of human lung cancer-derived H1975 cells
H1975 cells were purchased from the US Biological Resources Bank (ATCC). H1975 cells were suspended in 10% FCS-RPMI medium and sprinkled in 2 x 10 ³ cells / 100 μL per well of 96-well culture plate. After culturing overnight, the culture supernatant was removed, and Ephedra extract and ephedrine alkaloid-free Ephedra extract (3 lots) were added at 50, 100, 150, and 200 μg / mL, respectively, and the control was 10% FCS-RPMI medium. Added. After culturing for 72 hours, 10 μL of Cell counting kit-8 was added to each well, and after culturing for 4 hours, the absorbance (450 nm) of each well was measured with a plate reader. Four wells were measured for each concentration of each extract, and the relative cell number was calculated from the average value (Fig. 5).

As a result, it was clarified that the ephedrine alkaloid-removed ephedra extract suppresses the proliferation of human lung cancer-derived H1975 cells in the same manner as the ephedra extract.

Example 9: Suppressive effect of ephedra extract (Example 1) and ephedrine alkaloid-removed Ephedra extract (Example 2) on pain in animal tests Forty-eight 4-week-old male ICR mice were used as experimental animals. Weights were measured on the day of the start of the experiment, and the groups were divided into a control group, an ephedra alkaloid-removed ephedra extract 2 group (daily doses were 350 and 700 mg / kg, respectively) so that each group had almost the same weight. Distilled water for injection was added to the ephedra extract and the ephedrine alkaloid-removed ephedra extract, and the mixture was suspended at 37 ° C. for 30 minutes with stirring. The suspension was vortexed immediately before oral administration to homogenize and then administered in a sonde. On the 1st and 2nd days, the single dose was 175 mg / kg and 375 mg / kg, and the dose was administered twice a day (9:00 and 17:00). On the third day, only one dose of 350 mg / kg or 700 mg / kg was administered in the morning.

Six hours after the final administration in the morning of the third day, 20 μL of a 2.5% formalin solution was administered subcutaneously to the sole of the foot. Immediately after administration, the mice were placed in a cylindrical plastic container (inner diameter 116 mm, height 152 mm) and videotaped for 45 minutes. Pain-related behaviors were targeted at the first phase that occurred immediately after administration to 10 minutes and the second phase that occurred from 15 minutes to 30 minutes, and the action time of licking or biting the foot of the treatment at regular intervals was measured. For statistical analysis, the pain behavior time of the control group and the test substance administration group was performed by Dunnett's tests, and the significance level was set to 5%.

As a result, Ephedra extract significantly suppressed the pain-related behavior of the second phase when 700 mg / kg was administered. Ephedrine alkaloid-removed ephedra extract significantly suppresses phase 2 pain-related behavior in a concentration-dependent manner at both doses of 350 mg / kg and 700 mg / kg, and has a more pain-suppressing effect than ephedrine-removed ephedra extract. It was expensive (Fig. 6). From the above results, it was clarified that the ephedrine alkaloid-removed Ephedra extract has a higher pain-suppressing effect than the Ephedra extract.

Example 10: Influenza virus infection inhibitory action of hemp yellow extract (Example 1) and ephedrine alkaloid-removed hemp yellow extract (Example 2) The assay utilizes the lysis of MDCK cells by influenza virus infection to pigment the remaining cells. Detected by staining with.

Ephedra extract and ephedrine alkaloid-removed ephedra extract were each prepared with 10% FBS-MEM into a solution of 200 μg / mL, and a 2-fold dilution series of 10 steps was prepared as a sample solution. A solution containing influenza virus (A / WSN / 33 (H1N1) strain) was diluted with 10% FBS-MEM to 1000 TCID ₅₀ / mL to prepare an influenza virus solution. MDCK cells were suspended in 10% FBS-MEM and seeded at 3x10 ⁴ cells / 100 μL per well of 96-well culture plate. After culturing for 24 hours, the culture supernatant was removed, 100 μL of the sample solution was added to each well, and 100 μL of influenza virus solution or 10% FBS-MEM was further added. After culturing for 72 hours, the cells were stained with crystal violet, and the absorbance of each well at 560 nm was measured with a microplate reader.

As a result, in the series to which 10% FBS-MEM was added, no decrease in the number of cells was observed in any of the extracts in the sample concentration range of 1.56 to 25 μg / mL. Therefore, the cytotoxicity was observed in this addition concentration range. It was confirmed that it was not shown (Fig. 7B). On the other hand, in the series to which the influenza virus solution was added, it was observed that the cells were lysed in this sample concentration range in a concentration-dependent manner (Fig. 7A). The approximate expression of the four-parameter logistic curve was calculated, and the IC ₅₀ value was calculated from the parameters. As a result, it was 8.6 μg / mL for the ephedra extract and 8.3 μg / mL for the ephedrine alkaloid-free ephedra extract. From the above results, it was clarified that the ephedrine alkaloid-removed Ephedra extract inhibits influenza virus infection to MDCK cells to the same extent as the Ephedra extract.

Claims (14)

A method for producing an ephedrine alkaloid-free hemp yellow extract, which comprises removing ephedrine and pseudoephedrine from a hot water extract and / or hot water extract of ephedrine by cation exchange chromatography. .. The production method according to claim 1, wherein an ephedra extract from which ephedrine alkaloids have been removed is produced until the ephedrine alkaloid content is 0.05 ppm or less. The production method according to claim 1 or 2, wherein a strong acid type cation exchange resin is used as a filler for ion exchange chromatography. The production method according to claim 3, wherein the strong acid type positive exchange ion resin SK104, SK110, SK1B, UBK530, UBK12, PK216, IR120B, or 1060H is used. The production method according to claim 3, wherein the strong acid type positive exchange ion resin PK216, SK1B or IR120B is used. Ephedrine alkaloid-removed ephedrine alkaloid-removed ephedrine alkaloid extract from hemp yellow extract, in which an aqueous solution of hot water extract / or hot water extract of hemp yellow is passed through an acidic cation exchange resin and passed without being adsorbed. The ephedrine alkaloid-removing hemp yellow extract obtained by collecting minutes. The ephedrine alkaloid-removing ephedra extract according to claim 6, wherein the content of the ephedrine alkaloid is 0.23% or less, where the ephedrine alkaloid is the sum of ephedrine and pseudoephedrine. The ephedrine alkaloid-removing ephedra extract according to claim 7, wherein the content of the ephedrine alkaloid is 0.023% or less. The ephedrine alkaloid-removing ephedra extract according to claim 7, wherein the ephedrine alkaloid content is 0.05 ppm or less. A Chinese herbal preparation, food or dietary supplement containing the ephedrine alkaloid-free hemp yellow extract according to any one of claims 6 to 9. An anticancer / antimetastasis agent containing the ephedrine alkaloid-removing ephedra extract according to any one of claims 6 to 9 as an active ingredient. An anti-influenza virus agent containing the ephedrine alkaloid-removing ephedra extract according to any one of claims 6 to 9 as an active ingredient. A pain suppressant containing ephedrine alkaloid-removing ephedra extract as an active ingredient. The pain suppressant according to claim 13, wherein the ephedrine alkaloid-removing ephedra extract has an ephedrine alkaloid content of 0.05 ppm or less, wherein the ephedrine alkaloid is the total of ephedrine and pseudoephedrine.