Ephedrine alkaloid-free ephedra extract, its manufacturing method, and uses

Abstract

To provide a method for producing an ephedra extract stripped of ephedrine alkaloids with high safety, by removing ephedrine alkaloids from ephedra extract while retaining some of the medicinal effects of ephedra, as well as an antineoplastic / antimetastatic agent, a pain inhibitor, and an anti-influenza virus drug that include the ephedra extract as an active ingredient.SOLUTION: A production method includes removing ephedrine and pseudoephedrine from hot water extract and / or dried extract from ephedra, through cation exchange chromatography.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a more highly safe ephedrine alkaloid-removed ephedra extract, a method for producing the same, and an anticancer / antimetastatic agent, a pain inhibitor, and an anti-influenza virus agent containing the same as an active ingredient.

Background Art

[0002] Ephedra sinica Stapf, Ephedra intermedia Schrenk et CA Meyer, or Ephedra equisetina Bunge (Ephedraceae) are the above-ground stems of plants in the Ephedra family. Ephedra has been used since ancient times and is one of the most important herbal medicines. Herbal medicines (prescriptions) that contain ephedra as a constituent ingredient include Uzuto, Uyakujunkitou, Eppito, Eppi-kajutsuto, Kaishusanryo, Kagaisanryo, Kakonto, and Kakonto-ka-senkyu-shin'i. (Kakkontoukasenkyushin'i), Kakkonkajutsubutō, Kakkonkahangeto, Kanzōmaōtō, Keikyōsōsōōshinbutō, Keishishakuyakuchimotō, Keishinieppiittō, Keishimaōka Kuhanto), Koboku Maoto, Gokoto, Gokonichinto, Goshakusanryo, Saikatsugekitou, Shoseiryuto, Shozokumeito, Shinpitou, Zokumeito, Daiseiryuto, Dokkatsukakkonto Examples include Dokkatsukakonto, Bofutsushosanryo, Maoto, Maobushisaishinto, Makyokansekito, Makyoyokukanto, Sheganmaoto, Yokuinintou, and Reitakutsuukito.Furthermore, among the Kampo prescriptions currently covered by health insurance, there are 16 prescriptions that contain ephedra as a constituent herb: Eppi-kajutsu-to, Kakkon-to, Kakkon-to-ka-senkyu-shin'i, Kakkon-kajutsu-bu-to, Keishi-ma-o-kakuhan-to, Goko-to, Goseki-sanryo Examples include Yakusanryo, Shoseiryuto, Shinpitou, Zokumeito, Bofutsushosanryo, Maoto, Maobushisaishinto, Makyokansekito, Makyoyokukanto, and Yokuinintou.

[0003] The active ingredients of ephedra are well-known ephedrine alkaloids ((-)-ephedrine, (+)-pseudoephedrine), and in the Japanese Pharmacopoeia, Ephedra sinica Stapf, Ephedra intermedia Schrenk et CA Meyer or Ephedra equisetina Bunge (Ephedraceae) are listed as above ground. When the dried stems are quantified, it is stipulated that they contain 0.7% or more total alkaloids (ephedrine and pseudoephedrine). Ephedra is known to have central nervous system stimulant effects, sympathetic nervous system stimulant effects, diaphoretic effects, antitussive effects, anti-inflammatory effects, and anti-allergic effects (Non-Patent Literature 1), and these pharmacological effects have been thought to originate from ephedrine alkaloids (Non-Patent Literature 2). Furthermore, ephedra is used for various symptoms of physical pain, and its analgesic effect is explained by the anti-inflammatory effect of pseudoephedrine (Non-Patent Literature 3). On the other hand, the present inventors have investigated new pharmacological effects of ephedra, such as an inhibitory effect on cancer metastasis through the inhibition of cancer cell motility and an antitumor effect through the inhibition of cancer cell proliferation, using mice in in vivo studies. Analysis revealed (Non-Patent Document 4) that the molecular mechanism involves hepatocyte growth factor (HGF). We reported that this is due to the suppression of the HGF-Met-Akt signaling pathway by inhibiting the receptor Met (Patent Document 1, Non-Patent Document 5). These effects were not observed with ephedrine alkaloids alone. Since this was not done, a search for the active ingredient led to the discovery of the flavonoid compound herbacetin glycoside (Non-Patent Literature 6). However, the herbacetin glycoside content was low, at approximately 0.005%, and this component alone cannot explain the novel pharmacological effects of ephedra, suggesting that multiple components are involved (Non-Patent Literature 7). Therefore, in order to effectively utilize the medicinal effects of ephedra, it is desirable to use ephedra extract as a pharmaceutical product.

[0004] Ephedra has sympathetic and central nervous system stimulating effects, so it is generally contraindicated in people with a history of angina pectoris, myocardial infarction, or hypertension. Furthermore, it should be used with caution in elderly patients. In addition, people with weak digestive systems may experience loss of appetite or abdominal pain due to acute gastric mucosal lesions. Other side effects to watch out for include palpitations, excitement, urinary dysfunction, insomnia, and rash (Non-patent Literature 8). These side effects are thought to be due to ephedrine alkaloids. (Non-patent document 9). In the United States, ephedra was used as a supplement, but excessive intake Following fatalities resulting from improper use, such as in the handling of ephedra, the FDA has issued a warning regarding ephedra, including ephedra. The 2004 Ephedra Ban stipulated that all products containing ephedra alkaloids should be prohibited from sale, and consumers should be warned. The RAND Report, which formed the basis of this ban, meticulously examined a vast number of papers and over 10,000 adverse event reports, including more than 100 deaths, submitted to the FDA, and concluded that ephedra preparations have mild to moderate side effects such as palpitations, nausea, and vomiting (Non-Patent Literature 10). In Japan, ephedra is classified as an ingredient used exclusively as a pharmaceutical, and only those approved as pharmaceuticals are permitted for use. However, in the medical field, there are concerns that serious side effects may occur depending on the patient's constitution and medical condition.

[0005] Until now, it has been believed that almost all of the medicinal effects of ephedra were due to ephedrine alkaloids. This belief stemmed from the fact that the dealkaloidized ephedra extract described in Non-Patent Document 2 had lost all of the medicinal effects of ephedra. From this, it was thought that it would be difficult to separate the main effects and side effects of ephedra, and the idea of ​​removing ephedrine alkaloids from ephedra to eliminate its side effects had never been considered before. However, as mentioned above, the inventors discovered useful medicinal effects in ephedra that do not depend on ephedrine alkaloids, and came to the conclusion that by selectively removing ephedrine alkaloids, which are the cause of side effects, they could provide ephedra extract as a highly safe pharmaceutical product. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] "MET inhibitor containing ephedra as an ingredient" Japanese Patent Application No. 2009-86363 (March 31, 2009) [Non-patent literature]

[0007] [Non-Patent Document 1] Medicinal effects and pharmacology of ephedra, Modern Oriental Medicine, 15(4), 551-554, 1994. [Non-Patent Document 2] The pharmacology of ephedra, Modern Oriental Medicine, 1(2), 34-39, 1980. [Non-Patent Document 3] The position and types of analgesics in traditional Chinese medicine, Pain and Clinical Practice, 5(3), 262-268, 2005. [Non-Patent Document 4] Basic research on the use of Kampo medicines to protect against cancer recurrence and metastasis, J. Trad. Med., 30(1), 19-26, 2013. [Non-Patent Document 5] 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. [Non-Patent Document 6] Characterization of phenolic constitutes from Ephedra Herba extract. Molecules, 18, 5326-5334, 2013. [Non-Patent Document 7] Research on the components contained in ephedra extract, Abstracts of the Annual Meeting of the Pharmaceutical Society of Japan, 133rd Annual Meeting, No. 2, 185, 2013. [Non-Patent Document 8] "Lessons in Traditional Chinese Medicine Practice" by Hisahiko Hanawa, enlarged edition, pp. 46-47, Kinbara Publishing, 2003. [Non-Patent Document 9] 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. [Non-Patent Document 10] Final Rule Declaring Dietary Supplements Containing Ephedrine Alkaloids Adulterated Because They Present an Unreasonable Risk. Federal Register: 69 (28), pp 6787-6854, 2004 [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to provide a method for producing a highly safe ephedrine alkaloid-removed ephedra extract by removing ephedrine alkaloids from ephedra extract while retaining some of the medicinal effects of ephedra, and to provide an anticancer / antimetastatic agent, pain suppressant, and anti-influenza virus agent that uses this as an active ingredient. [Means for solving the problem]

[0009] Non-patent document 2 describes how alkaloids can be obtained by treating ephedra with NH4OH and ether. A method for removing alkaloids and preparing dealkaloid ephedra extract is described. However, the dealkaloid ephedra extract prepared by this method contains a high concentration (0.33-0.5%) of residual total alkaloids. It contains, and furthermore, NH4OH treatment causes chemical changes in the structure of plant components (large Makoto Hara, Journal of the Japan Wood Research Society, 55, 59-68, 2009; D. Ferreira et al., J. Chem. Soc. Perkin Trans. 1, 203-208, 1990), contains artificial components different from the original ephedra extract. This is considered to be the case. The author of Non-Patent Document 2 also mentions this matter. Furthermore, the dealkaloidized ephedra extract described in Non-Patent Document 2 had lost all of the pharmacological effects of ephedra, and therefore this manufacturing method affects the medicinal effects of ephedra. It is an inappropriate method for removing ephedrine alkaloids from ephedra and using it as a pharmaceutical product.

[0010] In order to overcome the problems such as the high alkaloid content of de-alkaloid ephedra extract, the chemical structural changes of the contained components, and the disappearance of medicinal effects, the inventors of the present invention have conducted intensive research. As a result, they have successfully developed a mild production method that can simply remove ephedrine alkaloids from ephedra extract without affecting the structure of the contained components and while maintaining the medicinal effects. Furthermore, it has been found that the obtained ephedra extract with ephedrine alkaloids removed retains the anti-cancer and anti-metastasis effects, and in addition, also retains the pain inhibitory effect and the anti-influenza virus effect, thus completing the present invention.

[0011] That is, more specifically, the present invention provides the following (1) to (9). (1) Ephedra extract with ephedrine alkaloids removed, obtained by removing ephedrine alkaloids from ephedra extract. (2) The ephedra extract with ephedrine alkaloids removed according to (1), containing ephedrine alkaloids in an amount of 0.23% or less. (3) The ephedra extract with ephedrine alkaloids removed according to (1), containing ephedrine alkaloids in an amount of 0.023% or less. (4) The ephedra extract with ephedrine alkaloids removed according to (1), containing ephedrine alkaloids in an amount of 0.05 ppm (detection limit) or less. (5) The ephedra extract with ephedrine alkaloids removed according to any one of (1) to (4), containing components that are not adsorbed by cation exchange resin among the components contained in the aqueous solution of the extract and / or extract of ephedra. (6) A traditional Chinese medicine preparation containing the ephedra extract with ephedrine alkaloids removed according to any one of (1) to (5). (7) A method for producing the ephedra extract with ephedrine alkaloids removed according to any one of (1) to (5), characterized by removing ephedrine alkaloids from the extract and / or extract of ephedra by ion exchange chromatography. (8) An anti-cancer and anti-metastasis drug containing the ephedra extract with ephedrine alkaloids removed according to any one of (1) to (5) as an active ingredient. (9) An analgesic drug containing, as an active ingredient, an ephedrine alkaloid-removed ephedra extract as described in any one of (1) to (5). (10) An anti-influenza virus drug containing, as an active ingredient, an ephedrine alkaloid-removed ephedra extract as described in any one of (1) to (5).

Advantages of the Invention

[0012] The method of the present invention can remove ephedrine alkaloids to 0.23% or less, or 0.023% or less, or 0.05 ppm (detection limit) or less, simply by passing ephedra extract through a certain type of cation exchange column. It is an extremely simple and useful manufacturing method. Further, the ephedrine alkaloid-removed ephedra extract of the present invention inhibits the inhibitory activity of Met kinase, which has been clarified as the mechanism of the anti-metastatic action of ephedra, to the same extent as the original ephedra extract, and furthermore, inhibits the cell proliferation of the Met-high expressing human lung cancer cell line H1975 in a concentration-dependent manner similar to the original ephedra extract. From these results, it is shown that the manufacturing method of the present invention can remove ephedrine alkaloids while retaining the anti-cancer and anti-metastatic actions mediated by MET inhibition of ephedra extract, and it has become clear that an anti-cancer and anti-metastatic drug containing, as an active ingredient, an ephedrine alkaloid-removed ephedra extract can be provided. Since ephedra agents (Chinese herbal medicines with ephedra as the main drug) are also frequently used as therapeutic agents for joint pain, the analgesic effect of ephedrine alkaloid-removed ephedra extract was focused on and evaluated 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 inhibitory effect than ephedra extract. Since the analgesic effect of ephedra has been considered to be derived from pseudoephedrine until now, this result is a discovery that overturns the previous understanding of ephedra. Therefore, it has become clear that the present invention can provide an analgesic drug containing, as an active ingredient, an ephedrine alkaloid-removed ephedra extract. The manufacturing method of the present invention can remove ephedrine alkaloids while retaining the anti-cancer and anti-metastatic actions mediated by MET inhibition of ephedra extract, and it has become clear that an anti-cancer and anti-metastatic drug containing, as an active ingredient, an ephedrine alkaloid-removed ephedra extract can be provided. Since ephedra agents (Chinese herbal medicines with ephedra as the main drug) are also frequently used as therapeutic agents for joint pain, the analgesic effect of ephedrine alkaloid-removed ephedra extract was focused on and evaluated 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 inhibitory effect than ephedra extract. Since the analgesic effect of ephedra has been considered to be derived from pseudoephedrine until now, this result is a discovery that overturns the previous understanding of ephedra. Therefore, it has become clear that the present invention can provide an analgesic drug containing, as an active ingredient, an ephedrine alkaloid-removed ephedra extract.

[0013] Since ephedra agents (Chinese herbal medicines with ephedra as the main drug) are also frequently used as therapeutic agents for joint pain, the analgesic effect of ephedrine alkaloid-removed ephedra extract was focused on and evaluated 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 inhibitory effect than ephedra extract. Since the analgesic effect of ephedra has been considered to be derived from pseudoephedrine until now, this result is a discovery that overturns the previous understanding of ephedra. Therefore, it has become clear that the present invention can provide an analgesic drug containing, as an active ingredient, an ephedrine alkaloid-removed ephedra extract.

[0014] Since Ma Huang Tang, one of the ephedra preparations, is used to treat the early stages of influenza, we evaluated whether ephedrine alkaloid-removed ephedra extract could suppress influenza virus infection using an influenza virus infection test with MDCK cells. As a result, ephedrine alkaloid-removed ephedra extract suppressed influenza virus infection to the same extent as the original ephedra extract. Therefore, it became clear that an anti-influenza virus drug with ephedrine alkaloid-removed ephedra extract as the active ingredient can be provided.

[0015] Based on the above, the ephedrine alkaloid-removed ephedra extract of the present invention is expected to be used as a safer anti-cancer / anti-metastatic agent, pain reliever, and anti-influenza virus agent. Furthermore, the ephedrine alkaloid-removed ephedra extract is a new type of cancer treatment that can simultaneously treat cancer and cancer pain.

[0016] Since ephedra is widely used as a component herbal medicine, replacing the ephedra in herbal medicines containing ephedra with ephedrine alkaloid-free ephedra extract can provide a new herbal preparation that is safer.

[0017] For example, when using Ma Huang Tang as an anti-cancer and anti-metastatic drug, it is possible to add new indications and effects while expanding its applicability to patients who were previously susceptible to Ma Huang Tang.

[0018] Furthermore, it can be applied, for example, to the treatment of joint pain in the elderly. Joint pain in the elderly is one of the causes of a decline in quality of life (QOL), and much of it is due to osteoarthritis. It occurs. Such joint pain accounts for about 10% of the causes of needing support or care in the elderly. It is the leading cause (Matsui, Y., National Center for Geriatrics & Gerontology, 42, 1-4, 2013). Treatment for joint pain mainly involves nonsteroidal anti-inflammatory drugs (NSAIDs). However, due to gastrointestinal side effects, long-term use is difficult, and there is a need for analgesics that can replace NSAIDs. Recently, a pharmaceutical company developed an antibody drug with analgesic effects and conducted clinical trials, but the trials were discontinued because osteoarthritis worsened in some patients (Yamaguchi, A, Drug). (Delivery System, 26(5), 457-460, 2011). This exacerbation of symptoms is thought to be caused by a sudden increase in the patient's activity due to the strong analgesic effect. On the other hand, herbal medicines containing ephedra (such as Kakkonto, Makyoyokukanto, Yuepijiajutsuto, and Yokuirento) are effective in treating joint pain, and their analgesic effect is gradual, so they are less likely to cause a sudden increase in the patient's activity and do not worsen osteoarthritis. However, due to the side effects of ephedra (palpitations, increased blood pressure, insomnia, urinary disorders, etc.), caution is required when administering ephedra-containing herbal medicines to the elderly. By using herbal preparations in which ephedra in these herbal medicines is replaced with ephedrine alkaloid-free ephedra extract, joint pain in the elderly can be safely controlled without worsening osteoarthritis, improving their quality of life. It can contribute to good.

[0019] Furthermore, while clinical studies have reported that Ma Huang Tang, used in the early stages of influenza, has a similar level of efficacy in treating influenza as the Western drug oseltamivir (Nabeshima, S., et al., J. Trad. Med., 27, 148-156, 2010; Kimoto Hiroshi, Therapeutics, 40, 385-388, 2006), its administration to the elderly and patients susceptible to ephedra should be avoided due to side effects from ephedra. However, using Ma Huang Tang with ephedrine alkaloid-removed ephedra extract makes it possible to administer it to the elderly and patients susceptible to ephedra. In addition, while Ma Huang Tang has been a difficult herbal medicine to administer long-term due to the side effects of ephedra, using Ma Huang Tang with ephedrine alkaloid-removed ephedra extract makes it possible to administer it prophylactically to people at high risk of influenza infection for long-term prevention.

[0020] This invention reveals that ephedrine alkaloid-free ephedra extract possesses the anti-cancer, anti-metastatic, pain-relieving, and anti-influenza virus properties of ephedra extract, and that it can be used as a highly safe pharmaceutical product with selectively removed ephedrine alkaloids, which are the cause of side effects. This invention is an extremely important fundamental technology for providing ephedrine alkaloid-free ephedra extract as a pharmaceutical product. The clinical application of this invention is expected. [Brief explanation of the drawing]

[0021] [Figure 1] The HPLC charts for ephedra extract before and after ion-exchange chromatography purification and ephedrine alkaloid-removed ephedra extract, as described in Example 4, are shown. [Figure 2] The fingerprints of the ephedra extract and the ephedrine alkaloid-removed ephedra extract before and after ion-exchange chromatography purification, as described in Example 5, are shown. [Figure 3] The LC-MS charts of ephedra extract before and after ion-exchange chromatography purification and ephedrine alkaloid-removed ephedra extract, as described in Example 6, are shown. [Figure 4] The MET kinase inhibitory activity of ephedra extract and ephedrine alkaloid-removed ephedra extract, as described in Example 7, is demonstrated. [Figure 5] The inhibitory effect of ephedra extract and ephedrine alkaloid-free ephedra extract on the proliferation of human non-small cell lung cancer H1975 cells, as described in Example 8, is demonstrated. [Figure 6] The pain-suppressing effects of ephedra extract and ephedrine alkaloid-removed ephedra extract, as described in Example 9, are demonstrated. [Figure 7A] The influenza virus infection-suppressing effects of ephedra extract and ephedrine alkaloid-removed ephedra extract, as described in Example 10, are demonstrated. [Figure 7B] The influenza virus infection-suppressing effects of ephedra extract and ephedrine alkaloid-removed ephedra extract, as described in Example 10, are demonstrated. [Modes for carrying out the invention]

[0022] Ephedra extract The ephedra used in this invention is the above-ground stem of the Ephedra sinica Stapf, Ephedra intermedia Schrenk et CA Meyer, or Ephedra equisetina Bunge (Ephedraceae) plants of the Ephedraaceae family. Yes, it is available. It can be used fresh, dried, or as processed above-ground stems.

[0023] The extraction step of ephedra in the manufacturing method of the present invention can be carried out according to any known method. As the extraction solvent, water or warm water, hot water, alcoholic solvents, and other organic solvents such as acetone can be used. Examples of alcoholic solvents include methanol, ethanol, propanol, isopropanol, butanol, and isobutanol. These solvents may be used individually or in combination.

[0024] The amount of extraction solvent is preferably 2-100 parts by weight relative 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, or supercritical fluid extraction.

[0025] The extracted liquid obtained, or the filtrate obtained by filtering the extract, or the concentrate obtained by concentrating the filtrate, or the dried product obtained by drying the concentrate, may be subjected to the next purification step by cation exchange chromatography, but it is preferable to roughly separate the components beforehand. For example, fine solid components can be easily removed by filtering with a suitable filter or by centrifugation, which prevents problems such as column clogging when performing the next chromatography.

[0026] In Example 1, described later, the ephedrine alkaloid content in the ephedra extract obtained in this way was 4.74% (3.19% ephedrine and 1.55% pseudoephedrine).

[0027] Ephedrine alkaloid-free ephedra extract The ephedrine alkaloid-free ephedrine extract obtained through the above process can be further processed by removing ephedrine using cation exchange chromatography and then concentrated and dried to obtain ephedrine alkaloid-free ephedrine extract. Ephedrine alkaloid-free ephedrine extract is EFM (ephedrine It is sometimes abbreviated as "alkaloids-free Mao extract."

[0028] To determine a suitable packing material for ion exchange chromatography for the production of ephedrine alkaloid-removed ephedra extract according to the present invention, we conducted studies. After treating 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 included 13 types of cation exchange resins, 1 type of amphoteric ion exchange resin, and an anion exchange resin. There are a total of 22 types, including 8 types of exchange resins. As a result, it is suitable for removing ephedrine alkaloids. It was found that the ion-effect resin was a cation exchange resin. Therefore, using 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 1060H The ephedrine alkaloid content in the extract after processing with ephedra extract was quantified by HPLC. The results are shown in the table below.

[0029] [Table 1]

[0030] Based on the above results, the cation exchange column suitable for the production of ephedrine alkaloid-removed ephedra extract is as follows: For the production of EFM with an ephedrine alkaloid content of 0.23% or less, the column packing material should be selected from the following: weakly acidic cation exchange resin WK20, strong acidic cation exchange resins SK104, SK110, SK1B, UBK530, PK216, IR120B, FPC3500, and 1060H. It is possible to select from the following for EFM production with an ephedrine alkaloid content of 0.023% or less: weakly acidic cation exchange resin WK20, and strongly acidic cation exchange resins SK104, SK110, SK1B, UBK530, PK216, IR120B, and 1060H as column packing materials. Furthermore, for EFM production with an ephedrine alkaloid content of 0.05 ppm or less, strongly acidic cation exchange resin PK216 can be selected as the column packing material, and from Examples 2 and 3, Strong acid cation exchange resins SK1B and IR120B can also be selected.

[0031] The specific method for performing the chromatography shall be one of the methods well known to those skilled in the art.

[0032] Drying can be carried out by any method, such as vacuum drying, freeze-drying, or spray drying. Excipients such as dextrin may be added if necessary.

[0033] The ephedrine alkaloid-free ephedra extract of the present invention is an ephedrine alkaloid-free ephedra extract obtained by removing ephedrine alkaloids from ephedra extract, and contains ephedrine alkaloids (total of ephedrine and pseudoephedrine) in an amount of 0.23%% or less. Preferably, it contains ephedrine alkaloids in an amount of 0.023%% or less, and even more preferably in an amount of 0.05 ppm (detection limit) or less.

[0034] The Japanese Pharmacopoeia stipulates that ephedra contains 0.7% or more total alkaloids (ephedrine and pseudoephedrine) in its dried crude drug form. This amount is calculated to be approximately 2.3% to 3.5% or more when converted to ephedra extract. Therefore, the ephedrine alkaloids in the present invention... Lloyd's Ephedra Extract is clearly distinct from the Ephedra Extract currently in use.

[0035] The conversion of the total alkaloid content in the ephedra extract mentioned above can be determined from non-patent literature (Ichiro Narikawa, "The Claims of Kampo - Modern Science and Kampo Preparations," pp. 162-163, Kenyuukan Co., Ltd., published in 1991) and Example 1. When ephedra extract is prepared from ephedra according to the Japanese Pharmacopoeia used in the production of EFM, almost all of the ephedrine alkaloids contained in ephedra are transferred to the ephedra extract. However, since the yield of ephedra extract obtained from ephedra is 20% to 30%, the total alkaloids contained in the ephedra extract are concentrated 3.3 to 5 times. Therefore, the total alkaloid content of ephedra extract is 2.3% to 3.5% or more, which is 3.3 to 5 times the 0.7% or more total alkaloid content of ephedra specified in the Pharmacopoeia. This will be Lloyd's default value.

[0036] The alkaloid content of this EFM was determined from the following non-patent literature: Preparation of a placebo for herbal medicine. In this case, since 10% of the active ingredient is mixed in (Katsutoshi Terazawa, Toshiaki Kita, eds., "EBM Kampo," p. 8, March 20, 2003, Ishiyaku Publishers, Inc.), if the content is less than one-tenth, the pharmacological effect will not appear. The possibility is considered extremely low. Since ephedra extract contains 2.3% to 3.5% or more of ephedrine alkaloids, reducing it to 0.23% to 0.35% or less (one-tenth of that amount) is considered to be a value that makes the possibility of ephedrine alkaloid side effects extremely low. In this invention, the dealkaloidized ephedra extract (residual alkaloid content 0.33% to 0.5%) described in Non-Patent Literature 2 is used. To clearly distinguish it from (), it was set to 0.23% or less. To further increase certainty, it was set to 0.023% or less. This was specified. More preferably, the concentration is 0.05 ppm or less (below the detection limit), as shown in Example 1.

[0037] Ephedrine alkaloid-free ephedra extract-containing herbal preparation Since ephedra is widely used as a component herb in traditional Chinese medicine, replacing the ephedra in herbal medicines containing ephedra with ephedra extract from which ephedrine alkaloids have been removed can provide a safer herbal preparation that eliminates side effects caused by ephedrine alkaloids. For example, Ma Xing Yu Gan Tang, which is used to treat joint pain, is composed of ephedra, apricot kernel, coix seed, and licorice. By adding ephedra extract from which ephedrine alkaloids have been removed to extracts prepared from the other component herbs, namely apricot kernel, coix seed, and licorice, it is possible to create Ma Xing Yu Gan Tang with fewer side effects.

[0038] Furthermore, since Ma Huang Tang, which is used in the early stages of influenza, is composed of ephedra, cinnamon bark, apricot kernel, and licorice, it is possible to create a Ma Huang Tang with fewer side effects by adding ephedrine alkaloid-free ephedra extract to an extract prepared from cinnamon bark, apricot kernel, and licorice.

[0039] Pharmaceutical composition The present invention further provides a composition comprising ephedrine alkaloid-decontaminated ephedra extract, which can be used as an anticancer / antimetastatic agent, an analgesic, or an anti-influenza virus agent, and may be in the form of a food, nutritional supplement, or pharmaceutical.

[0040] The ephedrine alkaloid-free ephedra extract used in the composition is preferably one having an ephedrine alkaloid content of 0.23% or less or 0.023% or less. More preferably... The important thing is that the ephedrialkaloid content is 0.05 ppm (detection limit) or less.

[0041] The method of administration of the composition of the present invention when it is a pharmaceutical composition is not particularly limited, but an orally administrative dosage form is preferred. The pharmaceutical composition of the present invention can be in various dosage forms.

[0042] For example, for oral administration, the formulation can be, but is not limited to, tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, alcoholic preparations, syrups, extracts, or elixirs. Furthermore, various pharmaceutically acceptable carriers can be added to the formulation.

[0043] For example, it may, but is not limited to, contain excipients, binders, disintegrants, lubricants, flavoring agents, colorants, sweeteners, flavor modifiers, solubilizers, suspending agents, emulsifiers, coating agents, vitamin C, and antioxidants.

[0044] The dosage of the pharmaceutical composition of the present invention can be used in the range of 100 mg to 3 g per day for adults, when converted to ephedrine alkaloid-free ephedra extract. Based on the general dosage of ephedra extract, the estimated daily dose for adults can be estimated to be 740 mg to 770 mg, but since the side effects caused by ephedrine alkaloids have been eliminated, it is possible to increase the dosage.

[0045] Of course, the dosage can be individually adjusted according to the recipient's age, weight, symptoms, route of administration, duration of administration, treatment progress, etc.

[0046] The daily dose can also be administered in several divided doses. Additionally, other pain relievers or anti-pain medications may be used. It can also be administered in combination with cancer drugs.

[0047] The composition of the present invention can also be in the form of food or nutritional supplements. For example, by incorporating ephedrine alkaloid-free ephedra extract as a raw material, it can be made into noodles, bread, candy, jelly, cookies, soup, or health drinks.

[0048] In addition to ephedrine alkaloid-free ephedra extract, these foods and nutritional supplements may also contain inorganic components such as iron and calcium, various vitamins, dietary fiber such as oligosaccharides and chitosan, protein such as soybean extract, lipids such as lecithin, and sugars such as sucrose and lactose.

[0049] Anti-cancer and anti-metastatic drugs The types of cancers in which the composition of the present invention can be used as an anticancer / anti-metastatic agent are mainly METs. It is a type of cancer that manifests as MET. METs are found in various cancers such as gastric cancer, colorectal cancer, lung cancer, ovarian cancer, breast cancer, osteosarcoma, and brain tumors. Because it is expressed on the surface of cancer cells, it can be used to treat these cancers. The HGF-c-Met signaling pathway promotes cancer cell proliferation, motility, cell dispersion, survival, and angiogenesis. This plays a key role in the proliferation, invasion, and metastasis of cancer cells. Therefore, the composition of the present invention is also effective as a drug to prevent cancer recurrence and metastasis.

[0050] Pain relievers Diseases in which the composition of the present invention can be used as a pain suppressant include muscle pain, joint pain, lower back pain, rheumatism, gout, and cancer pain.

[0051] Antiviral drugs for influenza The composition of the present invention is effective in preventing influenza virus infection and treating the initial stages of infection. [Examples]

[0052] The present invention will now be described in detail with reference to the following examples, but the present invention is not limited thereto.

[0053] Example 1: Preparation of ephedra extract. The dried raw material of ephedra is ground in a mixer, and 50g of the ground material is mixed with 500mL of water. The mixture was extracted at 95°C for 1 hour while stirring. After solid-liquid separation, the extract was centrifuged at 3000 rpm for 10 minutes. The resulting supernatant was concentrated under reduced pressure at 60°C and dried under reduced pressure overnight at 60°C to obtain 9.6 g of ephedra extract.

[0054] Example 2: Preparation of ephedrine alkaloid-removed ephedra extract using ion exchange resin SK1B Made. The dried raw material of ephedra is ground in a mixer, and 50g of the ground material is mixed with 500mL of water. The mixture was extracted at 95°C for 1 hour while stirring. After solid-liquid separation, the extract was centrifuged at 3000 rpm for 10 minutes, and the resulting supernatant was passed through 25 mL of strong acid type cation exchange resin SK1B (manufactured by Mitsubishi Chemical). The permeate was adjusted to pH=5.2 with 5% NaHCO3, concentrated under reduced pressure at 60°C, and dried under reduced pressure overnight at 60°C to obtain 6.3 g of ephedrine alkaloid-removed ephedra extract.

[0055] Example 3: Ephedrine alkaloid removal from ephedra extract using ion exchange resin IR120B Created. The dried raw material of ephedra is ground in a mixer, and 50g of the ground material is mixed with 500mL of water. Extraction was carried out at 95°C for 1 hour while stirring. Solid-liquid separation was performed, and the extract was centrifuged at 3000 rpm for 10 minutes. The obtained supernatant was then mixed with 25 mL of strong acid type cation exchange resin IR120B (O The solution was passed through a (Lugano) filter. The permeable solution was adjusted to pH=5.2 with 5% NaHCO3, concentrated under reduced pressure at 60°C, and dried overnight under reduced pressure at 60°C to obtain 6.3 g of ephedrine alkaloid-removed ephedra extract.

[0056] Example 4: Comparison of ephedrine content in ephedra extract (Example 1) and ephedrine alkaloid-removed ephedra extracts (Examples 2 and 3) by high-performance liquid chromatography (HPLC). Figure 1 shows the HPLC charts for ephedra extract (Example 1) and ephedrine alkaloid-free ephedra extract (Example 2). Analysis conditions Column: SHISEIDO AG120 4.6×150mm 5μ Mobile phase: Sodium lauryl sulfate solution (1→128) / acetonitrile / phosphate mixture ( 640:360:1) Temperature: 45℃ Detection: Ultraviolet absorbance photometer (measurement wavelength: 210 nm) Flow rate: 0.6 mL / min (adjust so that ephedrine is present around 14 minutes) Sample: Ephedra extract, or dried ephedrine alkaloid-free ephedra extract. 10 mg was taken, 1 mL of methanol was added and mixed, and 20 μL of each was injected. Standard solutions: 20 μL each of ephedrine standard solution (1.0 mg / 10 mL of 50% methanol) and pseudoephedrine standard solution (1.0 mg / 10 mL of 50% methanol) were injected.

[0057] The results obtained are shown in Table 1 below. These results demonstrate that ephedrine alkaloids (ephedrine and pseudoephedrine) can be removed from ephedra extract to below the detection limit (0.05 ppm) by column chromatography using the strong acid cation exchange resin SK1B or IR120B.

[0058] [Table 2]

[0059] Example 5: Comparison of the compositional components of ephedra extract (Example 1) and ephedrine alkaloid-removed ephedra extract (Example 2) by three-dimensional high-performance liquid chromatography (3D-HPLC). Analysis conditions Column: TSK-GEL 80TS 4.6×250mm 5μ Mobile phase: (A) 0.05M ammonium acetate (pH 3.6) (B) Acetonitrile 0 minutes: B liquid 10% → 60 minutes: B liquid 100% Temperature: 40℃ Detection: Photodiode array (PDA) Flow rate: 1.0mL / min Sample: Ephedra extract, or dried ephedrine alkaloid-free ephedra extract. 10 mg was taken, 1 mL of methanol was added and mixed, and 20 μL of each was injected. Standard solution: Ephedrine standard solution (1.0 mg / 10 mL methanol) and pseudoephedrine Each sample was injected with 20 μL of fedrine standard solution (1.0 mg / 10 mL methanol).

[0060] The 3D-HPLC fingerprint is shown in Figure 2.

[0061] 3D-HPLC analysis of the compositional components of ephedra extract and ephedra extract with ephedrine alkaloids removed revealed that the ephedrine alkaloid peak disappeared in the ephedra extract with ephedrine alkaloids removed, but the patterns of other components were almost the same as those of the ephedra extract.

[0062] Example 6: Ephedra extract (Example 1) and ephedrine alkaloid-removed ephedra by LC / MS Compositional analysis and comparative study of the extract (Example 2) Analysis conditions 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℃ 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℃; sheath gas flow rate, 50; auxiliary gas flow rate, 25; scan range, m / z 150-2000; mass resolution, 30,000 full width. Flow rate: 0.2 mL / min Sample: Injection volume, 1 μL at 5 mg / mL

[0063] The LC / MS chart is shown in Figure 3.

[0064] LC / MS comparison of the compositional components of ephedra extract and ephedrine alkaloid-removed ephedra extract. As a result, it was confirmed that the peaks of l-ephedrine, pseudoephedrine, methylephedrine, and norephedrine disappeared from the ephedrine alkaloid-free ephedra extract. Figure 3).

[0065] Example 7: Ephedra extract (Example 1) and ephedrine alkaloid-removed ephedra extract (fruit) MET kinase inhibitory effect in Example 2) MET kinase activity is determined by the recombinant MET kinase domain using the Poly E4Y1 peptide as a substrate. The amount of ADP produced in conjunction with ATP consumption was used as an indicator. A mixture of a reaction buffer containing a recombinant MET kinase domain, Poly E4Y1 peptide, and ATP was mixed with ephedra extract or ephedrine alkali. The effluvium extract (2 lots) is prepared to contain 1 μg / mL, 5 μg / mL, and 10 μg / mL respectively. It was added and incubated at room temperature for 1 hour. After stopping the reaction, the amount of ADP was measured using ADP-Glo ​​reagent. The dependent luminol emission intensity was measured, and relative MET kinase activity was investigated.

[0066] As a result, ephedrine alkaloid-removed ephedra extract was comparable to METquina in ephedra extract. It was revealed that the enzyme activity was inhibited. Figure 4 shows the MET kinase inhibition curves for ephedra extract (Example 1) and ephedra extract with ephedrine alkaloids removed (Example 2).

[0067] Example 8: Inhibitory effect of ephedra extract (Example 1) and ephedrine alkaloid-free ephedra extract (Example 2) on the proliferation of human lung cancer-derived H1975 cells. H1975 cells were purchased from the United States Bioresource Bank (ATCC). H1975 cells were cultured in 10% FCS-RPMI culture medium. Suspend in soil, 2 × 10⁶ per well of a 96-well culture plate. 3 Cells were plated at 100 μL / cell. After overnight incubation, 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. The control was given 10% FCS-RPMI medium. After 72 hours of incubation, 10 μL of Cell counting kit-8 was added to each well. After culturing for 4 hours, the absorbance (450 nm) of each well was measured using a plate reader. Four wells were measured for each extract concentration, and the relative cell number was calculated from the average value (Figure 5).

[0068] As a result, it was revealed that ephedrine alkaloid-free ephedra extract suppresses the proliferation of human lung cancer-derived H1975 cells, similar to ephedra extract.

[0069] Example 9: Inhibitory effect of ephedra extract (Example 1) and ephedrine alkaloid-free ephedra extract (Example 2) on pain in animal studies. Forty-eight four-week-old male ICR mice were used as experimental animals. Weight was measured on the first day of the experiment, and each The participants were divided into a control group, two groups of ephedra extract, and two groups of ephedrine alkaloid-free ephedra extract (daily doses of 350 and 700 mg / kg, respectively) to ensure that the group was nearly identical. Yellow extract and ephedrine alkaloid-removed ephedra extract were combined with injection-grade distilled water and suspended at 37°C for 30 minutes with stirring. The suspension was homogenized by vortexing immediately before oral administration and then administered via tube. On days 1 and 2, the single dose was 175 mg / kJ. The dosage was 375 mg / kg, administered twice a day (9:00 and 17:00). On the third day, the afternoon... A single dose of 350 mg / kg or 700 mg / kg was administered before or during the period.

[0070] Six hours after the final dose on the morning of the third day, administer 20 μL of 2.5% formalin solution subcutaneously to the sole of the foot. The substance was administered. 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 analyzed in two phases: Phase 1, which occurred from immediately after administration to 10 minutes later, and Phase 2, which occurred from 15 minutes to 30 minutes later. The duration of licking or biting the treated foot was measured at regular intervals. Statistical analysis was performed using Dunnett's tests to determine the duration of pain behaviors in the control group and the group administered the test substance, with a significance level of 5%.

[0071] As a result, when ephedra extract was administered at 700 mg / kg, it significantly suppressed pain-related behaviors in phase 2. It was conquered. Ephedrine alkaloid-free ephedra extract was administered in both doses of 350 mg / kg and 700 mg / kg. The dosage significantly suppressed pain-related behaviors in phase 2 in a concentration-dependent manner, and the pain-suppressing effect was higher than that of ephedra extract before ephedrine removal (Figure 6). Based on these results, ephedrine alkaloids It has been revealed that the ephedra extract used for decongestion has a higher pain-suppressing effect than ephedra extract.

[0072] Example 10: Inhibitory effect of ephedra extract (Example 1) and ephedrine alkaloid-free ephedra extract (Example 2) on influenza virus infection. The assay utilized the fact that MDCK cells are lysed by influenza virus infection, and the remaining cells were detected by staining them with a dye.

[0073] Ephedra extract and ephedrine alkaloid-removed ephedra extract were each prepared as a 200 μg / mL solution in 10% FBS-MEM, and then a 10-step 2x dilution series was prepared to use as sample solutions. A solution containing influenza virus (A / WSN / 33 (H1N1) strain) was diluted to 1000 TCID in 10% FBS-MEM. 50 Diluted to 1 / mL to prepare influenza virus solution. MDCK cells were suspended in 10% FBS-MEM and 3 x 10⁶ cells were added to each well of a 96-well culture plate. 4 Seeds were seeded at a rate of 1 cell / 100 μL. After 24 hours of incubation, the culture supernatant was removed, 100 μL of sample solution was added to each well, and then 100 μL of influenza virus solution or 10% FBS-MEM was added. After 72 hours of incubation, the plates were stained with crystal violet, and the absorbance at 560 nm of each well was measured using a microplate reader.

[0074] As a result, in the series to which 10% FBS-MEM was added, no decrease in cell number was observed in any of the extracts within the sample concentration range of 1.56 to 25 μg / mL, confirming that cytotoxicity was not observed in this concentration range (Figure 7B). On the other hand, in the series to which influenza virus solution was added, concentration-dependent cell lysis was observed in all extracts within this sample concentration range (Figure 7A). An approximate formula for the 4-parameter logistic curve was obtained, and From the parameters of IC 50 The calculated values ​​showed that ephedra extract contained 8.6 μg / mL, and ephedrine extract contained 8.6 μg / mL. The ephedrine concentration in the lucaloid-free ephedra extract was 8.3 μg / mL. Based on these results, ephedrine alcohol It was revealed that the caloid-removed ephedra extract inhibited influenza virus infection of MDCK cells to a similar degree as the ephedra extract.

Claims

1. A method for producing an ephedra extract from which ephedrine alkaloids have been removed, comprising passing a hot water extract of ephedra and / or an aqueous solution of the hot water extract through an acidic cation exchange resin and collecting the fraction that passes through without being adsorbed to remove ephedrine and pseudoephedrine.

2. The method for producing ephedra extract according to claim 1, wherein ephedrine alkaloids refer to the sum of ephedrine and pseudoephedrine, and ephedrine alkaloids are removed until the ephedrine alkaloid content is 0.05 ppm or less.

3. The manufacturing method according to claim 1 or 2, characterized by using a strong acid type cation exchange resin.

4. The manufacturing method according to claim 3, using a strong acid type cation exchange ion resin SK104, SK110, SK1B, UBK530, UBK12, PK216, IR120B, or 1060H.

5. The manufacturing method according to claim 3, wherein a strong acid type cation exchange ion resin PK216, SK1B, or IR120B is used.

6. An ephedrine alkaloid-removed ephedrine extract obtained by the method described in any one of claims 1 to 5, wherein ephedrine alkaloids have been removed from the ephedrine extract.

7. A traditional Chinese medicine preparation comprising ephedrine alkaloid-removed ephedra extract as described in claim 6.

8. A food or nutritional supplement containing ephedrine alkaloid-free ephedra extract as described in claim 6.

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