WO2005067950A1 - Extrait de kava destine a reduire des effets secondaires et des effets toxiques, composition et preparation comprenant cet extrait - Google Patents

Extrait de kava destine a reduire des effets secondaires et des effets toxiques, composition et preparation comprenant cet extrait Download PDF

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WO2005067950A1
WO2005067950A1 PCT/CN2004/001558 CN2004001558W WO2005067950A1 WO 2005067950 A1 WO2005067950 A1 WO 2005067950A1 CN 2004001558 W CN2004001558 W CN 2004001558W WO 2005067950 A1 WO2005067950 A1 WO 2005067950A1
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extract
water
kawa
aqueous
content
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Boyang Yu
Jihua Liu
Shengxiang Qiu
Xing Li
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China Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/67—Piperaceae (Pepper family), e.g. Jamaican pepper or kava
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37—Coumarins, e.g. psoralen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes

Definitions

  • the present invention relates to a Kawa extract containing a content of a toxic and side effect reducing ingredient and / or a Kawa extract containing a toxic and side effect component antagonist, a preparation method thereof, and a composition containing the extract. Content, even Kawa extracts with toxic and side effect ingredients removed, and / or medicines, food additives and health foods with toxic side effect ingredient antagonists. Background technique
  • Kapaki Pulaki (Piper methysticum Forster f,, Piperaceae, Kava) is the dried root of the pepper plant (Piper methysticum). With a history of more than 2,000 years in the South Pacific islands, Kawa roots have traditionally been used to make Kawa tea or chew directly, giving people a sense of relaxation and joy. Modern pharmacological studies have shown that the main component of Kawakulides has good anxiolytic, antidepressant and sedative functions and is widely used worldwide. See: MH Pittler, E. Ernst, J. Clin Psychopharmacol 20, 84 (2000).
  • Kawha also contains two yellow pigments: flavokawain A and B (flavokawain A and B)-these two ingredients are generally thought to make long-term Kawha skin yellow.
  • Kawha's traditional manufacturing method is to use coconut milk or water to extract. No obvious toxic and side effects are seen in use. Animal experiments have also confirmed that Kawha's water extract has no liver toxicity. However, due to the low content of kawanolide in water or coconut milk extract, it is not suitable for the needs of modern preparations. Kawa extracts currently used commercially are dissolved in ethanol, acetone or chloroform. The extract is extracted with a chemical agent, and the prepared kawa extract contains more than 30% of kavalactone, which is generally called a "standard extract". The HPLC test showed that the content of Kasugarin B in the Kawa extract extracted according to the standard preparation method of the United States was greater than 2%.
  • the technical problem to be solved by the present invention is to reduce or remove ingredients that have toxic and side effects on the liver in the Kawa extract, to provide a Kawa extract that does not substantially contain a toxic side effect component or significantly reduces the content of the toxic side effect component; or Detoxification substances that can antagonize the effects of hepatotoxicity are added to the extract, which provides a kawaii extract with reduced or no hepatotoxicity, which contains six recognized kawanolide active ingredients: nordroxypiperin and methoxy Gydroxycapsin, dihydrocapsaicin, phytocapsin, dihydrocapsaicin, and capsaicin; the total content of carbavalides (based on the content of the six major carbavalides) is greater than 10%. Up to 11-90%.
  • the present invention also includes research on a method for purifying a Kaw extract, and a composition containing the extract, such as a medicament, a food additive, and a health food.
  • the present invention provides the following technical solutions.
  • a kawaii extract that reduces or eliminates toxic and side effects wherein the content of sage piperin B is 1.0% by weight and / or the content of reduced glutathione 1.0% is added.
  • the content of kavalactone is 10%
  • the content of yellow drizolin B is 0-1.0% based on the total weight of the extract
  • the content is 11-90%
  • the content of sage piperin B can be 0-0.5%.
  • a reduced glutathione which is a substance that antagonizes liver toxicity, is added to the Kawa extract, wherein the content of Kawalide is 10% and the content of reduced glutathione is 1.0% based on the total weight of the extract. .
  • Another aspect of the present invention is to provide a Kawa extract-containing composition containing the aforementioned Kawa extracts and excipients, excipients or carriers for conventional medicinal, food or health products.
  • the composition of the present invention can be in the form of a tablet, granule, solution or suspension, and can also be made into a sustained-release preparation according to methods known in the art.
  • the excipients, excipients or carriers include, but are not limited to, disintegrants such as hydroxypropyl cellulose, sodium carboxymethyl starch, crospovidone, and croscarmel methyl fiber.
  • Sodium such as lactose, microcrystalline cellulose, dextrin, starch, calcium phosphate
  • binders such as pregelatinized starch, povidone, sodium carboxymethyl cellulose, hypromellose
  • lubricants Such as talc, magnesium stearate, micronized silica gel, hydrogenated vegetable oil; wetting agents such as sodium lauryl sulfate, Tween 80; framework materials such as hypromellose, ethyl cellulose, etc.
  • the excipients, excipients, or carriers include, but are not limited to, solubilizers such as Tween 80, Proloni F-68, polyoxyethylene hydrogenated castor oil, etc .; suspending agents such as carboxymethyl Cellulose sodium, povidone, hydroxypropylmethyl cellulose, etc .; Preservatives such as methyl paraben, ethyl, propyl, and butyl esters; pH adjusters such as citric acid and citrate; phosphates, etc. .
  • solubilizers such as Tween 80, Proloni F-68, polyoxyethylene hydrogenated castor oil, etc .
  • suspending agents such as carboxymethyl Cellulose sodium, povidone, hydroxypropylmethyl cellulose, etc .
  • Preservatives such as methyl paraben, ethyl, propyl, and butyl esters
  • pH adjusters such as citric acid and citrate; phosphates, etc. .
  • the crude Kawa extract is added to a suspension of less than 50 times the volume of water to prepare a suspension, and the aqueous phase is extracted with petroleum ether, cyclohexane and / or hexane, and the extracted aqueous phase is concentrated to dryness to reduce yellowing.
  • a reduced glutathione that antagonizes the toxic and side effects of chrysophanol is added to the crude Kawa extract or refined Kawa extract, so that its content in Kawa extract is 10% by weight. At 90%, it is necessary to reduce or eliminate the toxic side effects of the Kawa extract.
  • the aliphatic solvent and / or the alicyclic solvent are petroleum ether, cyclohexane, n-hexane and / or gasoline;
  • the chlorinated lower alkane is dichloroethane, chloroform;
  • Macroporous resin is non-polar or weakly polar macroporous resin;
  • alumina is acidic or neutral alumina;
  • chitosan is the product of deacetylation of chitin; elution of macroporous resin or polyamide chromatography column Liquid ethanol, water methanol, and / or water acetone were used as the eluent;
  • the eluent for silica gel column chromatography was petroleum ether, cyclohexane, and / or ethyl acetate, and then isopropyl alcohol, ethyl acetate, and chloro lower alkanes.
  • alkaline water solution contains inorganic base and / or organic base and water
  • acid water solution contains inorganic acid and / or organic acid and water .
  • the macroporous resin can be selected from HP20, AB-8, XAD-2, D-10K HP21, SP825, SP850, SP70, SP700, SP207, H107;
  • the inorganic base can be sodium carbonate, sodium bicarbonate, potassium carbonate, hydrogen carbonate Potassium and / or disodium hydrogen phosphate
  • the organic base may be citrate, meglumine, sodium pantothenate, L-cysteine, benzamide, nicotinamide, thiourea, ethanolamine and / or urea
  • the inorganic acid is Sulfuric acid and / or hydrochloric acid;
  • the organic acid may be citric acid, acetic acid, oxalic acid, maleic acid, succinic acid, fumaric acid, malic acid, tartaric acid, and / or ascorbic acid.
  • Kawa extract The specific preparation method of Kawa extract is as follows: the crude Kawa extract is put on a silica gel column, and the silica gel column is first eluted with a mixed solvent of petroleum ether or cyclohexane and ethyl acetate in a volume ratio of 10-20: 1, and then Elution with isopropanol, ethyl acetate, chlorinated lower alkanes, methanol or aqueous methanol, ethanol or aqueous ethanol, acetone or aqueous acetone, or a mixture thereof, and recovering the solvent to dryness to reduce the content of chrysin Kawa extract.
  • the specific preparation method of Kawa extract is also: to an aqueous suspension of crude Kawa extract, Add methanol and / or ethanol less than 50 times the volume of the suspension to form a Kawa extract solution, then add chitosan; or add a chitosan solution to the aqueous suspension of the crude Kawa extract; mix well After being left to stand, centrifugation or filtration is used to remove the precipitate, and the supernatant is concentrated and dried to obtain a refined Kawa extract that reduces the content of yellow drizaphanin B.
  • a better method for preparing the Kawa extract is as follows: Add the organic base and / or inorganic base as described above to the aqueous solution or suspension of the crude Kawa extract to make the aqueous solution or water suspension alkaline, and mix After that, the extract is removed with petroleum ether, cyclohexane, and / or hexane to remove chrysophanol B. The obtained aqueous phase is added with an inorganic acid and / or an organic acid to make the solution acidic. After mixing, the mixture is mixed with ethyl acetate and chloroform. And / or dichloroformamidine to extract an acidic solution, and recover the extract to dryness, to obtain a refined Ka-wa extract that reduces the content of chrysanthemum B.
  • the more desirable preparation method of Kawa extract is: Kawa's water, water or non-aqueous ethanol, water or non-aqueous methanol, water or non-aqueous acetone extract, remove the organic solvent to obtain a solution or suspension, or Use a solvent that is less than 10 times the volume of the Kawa extract and miscible with water, such as methanol, ethanol, acetone, etc.
  • Kawa extracts that reduce or eliminate toxic and side effects is to add reduced glutathione to Kawa's crude or refined Kawa extracts, which can antagonize the side effects of chrysophanol.
  • the content of Kaw extract is 1.0% by weight, and the content of glutathione can be up to 90% by weight, so as to reduce or eliminate the side effects of liver toxicity.
  • the technical solution provided by the present invention reduces or removes components that have toxic and side effects on the liver in the Kawa extract, and provides a Kawa extract that does not substantially contain or significantly reduce the content of the toxic side effect components; or Reduced or no toxic side effects are added to the extract or refined extract by reducing glutathione, which provides a reduced or no toxic side effect.
  • It contains the six main active ingredients of kawanolide. : Nordroxycapsaicin, methoxycapryllin, dihydrocapryllin, phytobarrel, dihydrocapryllin, and capryllin; the total content of kawanolide is greater than 10% by weight, but Up to 11-90% by weight.
  • the method for purifying the Kaw extract obtained by the present invention is simple and practical. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 is an HPLC chart of the carbacetone extract (the chromatographic peak indicated by the arrow is yellow drizaphanin B).
  • Figure 2 is an HPLC chart of Kawa extract containing reduced kawaii capsaicin B content after refined with macroporous resin HP20. (The chromatographic peak indicated by the arrow is huangzu capsaicin B).
  • lower alcohol refers to an alcohol containing 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. Specific examples thereof include methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, n-hexanol, n-octanol, and n-decanol, and among them, methanol and ethanol are particularly preferred.
  • lower ketone used in the present invention refers to a ketone containing 3 to 10 carbon atoms, such as acetone, methylbutyl ketone, etc., among which acetone is preferred.
  • chlorinated lower alkane used in the present invention refers to a group in which one or more hydrogen atoms in an alkyl chain are replaced with a chlorine atom, wherein the alkyl chain contains 1 to 10 carbon atoms, preferably 1 to 1 6 carbon atoms, more preferably 1 to 4 carbon atoms, and may be straight or branched.
  • Specific examples of the chlorinated lower alkane include chloroform, carbon tetrachloride, methylene chloride, and the like.
  • the Kawa raw material used in the present invention is a Kawa (Pipe methysticum Forster f., Piperaceae) root powder (100-200 mesh) derived from the Fiji Islands, and was purchased from Kava Kauai (Kava Kauai, PO Box 1202, Kapaa Kauai Hawaii, 96746, USA).
  • silica gel column chromatography sample Take 1 g of the kawan acetone extract prepared as described in Example 1, add 1 ml of ethyl acetate to dissolve, add about 3 g of silica gel to stir the sample, and dry the organic solvent to obtain a silica gel column chromatography sample.
  • the chromatography sample was added to the top of a silica gel column with a column volume of 50 ml, and the silica gel column was eluted with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10: 1.
  • the first yellow color band eluted by chromatography is yellow chrysanthemin B.
  • the absence of chrysophanol B in the subsequent eluate is detected by TLC, and the other components on the column are eluted with methanol.
  • the eluate was concentrated to dryness to obtain a Kawa extract which reduced the content of chrysophanol B.
  • the total content of lactones in the extract was 61.2% by HPLC.
  • Example 5 One gram of the kawan acetone extract prepared as described in Example 1 was taken, dissolved in 1 ml of chloroform, and the tops of the acidic and neutral alumina dry columns with a column volume of 30 ml were added. Acidic and neutral alumina chromatography columns were eluted with 200 ml of chloroform: methanol volume ratio of 2: 1 mixed solvent, and TLC assisted detection. The eluate was concentrated to dryness to obtain a kawaii extract which reduced the content of chrysanthemum B. As determined by HPLC, the total content of kawanolide in the extract was 38.2%, and the content of sage piperin B was 0.3%.
  • Example 5 One gram of the kawan acetone extract prepared as described in Example 1 was taken, dissolved in 1 ml of chloroform, and the tops of the acidic and neutral alumina dry columns with a column volume of 30 ml were added. Acidic and neutral alumina chromatography columns
  • Kaw extract to reduce chrysanthemum B by chitosan treatment Take 1 gram of the kawan acetone extract prepared as described in Example 1, dissolve in 50 ml of 40% ethanol, and add chitosan to this solution so that the concentration of chitosan in the total volume is 1% , Dissolve with sufficient stirring, leave it at room temperature for 60 minutes, remove the precipitate by centrifugation, and concentrate the supernatant obtained by centrifugation to dryness, to obtain a Kawa extract that reduces the content of chrysanthemum B. As determined by HPLC, the total content of kawanolide in the extract was 46%, and the content of sagepinene B was 0.1%.
  • Example 10 Take 1 g of the Kawa water extract prepared as described in Example 1, add 1 ml of ethanol to dissolve, and then add 10 ml of water to make a Kawa extract suspension. Add 50 ml of HP20 macroporous resin chromatography The column was eluted with 200 ml of a 35% ethanol (volume ratio) solution. The eluate was concentrated to dryness and determined by HPLC. The total content of lactone in the extract was 48.2%, and it did not contain the component of chrysin.
  • Example 10 Take 1 g of the Kawa water extract prepared as described in Example 1, add 1 ml of ethanol to dissolve, and then add 10 ml of water to make a Kawa extract suspension. Add 50 ml of HP20 macroporous resin chromatography The column was eluted with 200 ml of a 35% ethanol (volume ratio) solution. The eluate was concentrated to dryness and determined by HPLC. The total content of lactone in the extract was 48.2%, and it did not
  • RPM1640 cell lines L02 medium with 10% fetal calf serum containing culture to a cell concentration of access IX 96 105-well cell culture plate, seeded per well 100 ⁇ 1, after 24 hours of incubation in the carbon dioxide incubator, the medium was discarded Add the medium containing different concentrations of yellow drizolin B, continue to culture for 48 hours, and use the MTT method (see: T. Mossman, J. Immunol. 65, 55 (1983); AP Li et al, Chem. Biol. Interact 121, 117 (1999)) to determine cell viability. The results showed that drunkard capsaicin B had significant cytotoxicity to L02 cells, and its IC 5 (3 was 9.2 ⁇ g / ml.
  • mice Eighteen to twenty grams of male ICR mice were selected and randomly divided into groups of six. Animals were kept in a standardized environment, with free access to food and water. Yellow drunkardin B with 0.5% carboxymethyl cellulose Sodium suspension, mice in the experimental group were orally administered with a dose of 25 mg / kg for 7 days, and the control group was given 0.5% sodium carboxymethyl cellulose in parallel. After 7 days, blood was taken from the mice, and the serum aspartate aminotransferase activity was measured. The results showed that the mice in the experimental group had significantly higher serum aspartate aminotransferase activity than the control group.
  • livers of mice were taken for pathological sectioning, routine staining, and microscopic examination.
  • the results showed that liver cells of all mice in the control group were normal, and liver hepatic ducts infiltrated in all experimental groups of mice (yellow piperine B 25mg / lg).
  • a large number of inflammatory cells, hepatocellular edema, and vacuolation, are consistent with the phenomenon of liver pathological sections in clinical cases of taking liver extracts containing Kawa extract (see: J. G Paul, J. C, Nathan, LH Richard, C. Peter, WA Peter. Med. J. Aust. 178, 442 (2003)) o
  • RPM1640 cell lines L02 medium with 10% fetal calf serum containing culture to a concentration of 1 X 10 5 cells in 96-well cell culture plate access, seeded per well 100 ⁇ 1, after 24 hours of incubation in the carbon dioxide incubator, the medium was discarded Solution, add medium containing 500.00 g / ml reduced glutathione and different concentrations of chrysophanol B; or add 500.00 ug / ml reduced glutathione and different concentrations of kawa acetone extract Medium, continue to culture for 48 hours, using the MTT method (see: T. Mossman, J. iw w "o /. 65, 55 (1983); AP Li et al, Chem. Biol. Interact.

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Description

降低毒副作用的咔哇提取物及含其组合物与制备方法 技术领域
本发明涉及含有降低有毒副作用成分含量的咔哇提取物和 /或加入 毒副作用成分拮抗剂的咔哇提取物、其制备方法和包含有该提取物组合 物， 具体为含有该降低了有毒副作用成分含量、甚至去除了有毒副作用 成分的咔哇提取物和 /或加入有毒副作用成分拮抗剂的药物、 食品添加 剂和保健食品。 背景技术
口卡口圭 (Piper methysticum Forster f, , Piperaceae, Kava) 为胡椒禾斗植 物咔哇 Piper methysticum ) 的干燥根。 在南太平洋岛国具有 2000多年 的使用历史， 传统上咔哇的根被用来制备咔哇茶或直接咀嚼， 使人产生 放松和愉悦的感觉。近代药理学研究表明， 咔哇中的主要成分咔哇内酯 类化合物具有良好的抗焦虑、抗抑郁和镇静功能并在全世界得到广泛的 应用，参见： M. H. Pittler, E. Ernst, J. Clin. Psychopharmacol 20,84(2000)。 咔哇内酯类化合物中已有 6个咔哇吡喃酮经鉴定，其中醉椒素 (kawain)、 二氢醉椒素 （dihydrokawain)、 麻醉椒素 （methysticin)、 二氢麻醉椒素 ( dihydromethysticin ) > 甲氧基醉椒素 （yangonin)、 去甲氧基醉椒素 ( desmethoxyyangonin) 被认为是抗焦虑、 抗抑郁和镇静的活性成分。 咔哇还含有两个黄色色素： 黄醉椒素 A和 B (flavokawain A和 B ) , — 般认为这两种成分使长期食用咔哇者肤色变黄。有药理实验证明黄醉椒 素 A作用于膀胱肿瘤细胞。
咔哇的传统制法是用椰子奶或水提取，在使用中未见明显的毒副作 用， 动物实验也证明咔哇的水提取物没有肝脏毒性。但是由于水或椰子 奶提取的咔哇提取物中的咔哇内酯含量低， 不适合现代制剂的需要。 目 前商业上所使用的咔哇提取物是以乙醇水溶液、丙酮水溶液或氯仿等溶 剂进行提取，制备的咔哇提取物含有 30 %以上的咔哇内酯，一般称为"标 准提取物"。 HPLC检测表明， 按照美国制备标准提取物的方法提取的 咔哇提取物中， 黄醉椒素 B的含量大于 2 %。 自 1999年以后， 大量文 献报道含有该咔哇提取物的药物或保健食品可能引起严重的肝损伤。参 见： Center for Food Safety and Applied Nutrition, Morbidity Mortality Weekly Rep. 51, 1065 (2002)； M. Blumenthal, Herbal. Gram. 47, 64(1999)； A.Denham, M.A.McInt re, J.W itehouse, J. Altern. Complement. Med. 8,237(2002)。 发明内容
本发明要解决的技术问题是降低或去除咔哇提取物中对肝有毒副作 用的成分，提供基本上不含有毒副作用成分或显著降低有毒副作用成分 含量的咔哇提取物； 或者， 在咔哇提取物中加入能拮抗肝毒性作用成分 的解毒物质， 提供降低或没有肝毒性作用的咔哇提取物， 其含有公认的 六种主要咔哇内酯活性成分： 去甲氧基醉椒素、 甲氧基醉椒素、 二氢醉 椒素、 醉椒素、 二氢麻醉椒素和麻醉椒素； 其咔哇内酯的总含量（以六 种主要咔哇内酯含量计）大于 10 %， 可达 11-90 %。 本发明还包括研究 纯化咔哇提取物的方法， 以及含有所述提取物的组合物， 如药物、食品 添加剂和保健食品。
为解决上述技术问题， 本发明提供如下技术方案。
一种降低或去除毒副作用的咔哇提取物， 其中黄醉椒素 B含量按重 量计黄醉椒素 B含量 1.0 %和 /或加入的还原型谷胱甘肽含量 1.0 %。
在本发明的咔哇提取物中，按提取物的总重量计咔哇内酯的含量 10 % , 黄醉椒素 B的含量为 0-1.0 % ; 咔哇提取物中咔哇内酯含量可以 为 11-90 %， 黄醉椒素 B含量可以为 0-0.5 %。或者， 所述咔哇提取物中 加入拮抗肝毒性的物质还原型谷胱甘肽，其中按提取物的总重量计咔哇 内酯的含量 10 %， 而还原型谷胱甘肽的含量 1.0 %。
本发明的另一个方面是提供含咔哇提取物的组合物， 其中含有前述 的咔哇提取物以及常规药用、 食用或保健品用的辅料、 赋形剂或载体。 本发明的组合物可以为片剂、 颗粒剂、 溶液剂或混悬剂的形式， 而 且还可根据本领域已知的方法制成缓释制剂。
对于片剂、 颗粒剂或缓释制剂， 其辅料、 赋形剂或载体包括但不限 于崩解剂如羟丙纤维素， 羧甲基淀粉钠， 交联聚维酮， 交联羧甲基纤维 素钠； 填充剂如乳糖， 微晶纤维素， 糊精， 淀粉， 磷酸钙； 粘合剂如： 预胶化淀粉， 聚维酮， 羧甲基纤维素钠， 羟丙甲纤维素； 润滑剂如滑石 粉， 硬脂酸镁， 微粉硅胶， 氢化植物油； 润湿剂如十二烷基硫酸钠， 吐 温 80; 骨架材料如羟丙甲纤维素， 乙基纤维素等。
对于液体制剂或混悬液， 其辅料、 赋形剂或载体包括但不限于增溶 剂如吐温 80， 普流罗尼 F-68， 聚氧乙烯氢化蓖麻油等； 助悬剂如羧甲 基纤维素钠， 聚维酮，羟丙甲基纤维素等； 防腐剂如尼泊金甲酯、 乙酯、 丙酯和丁酯； pH调节剂如枸櫞酸及枸橡酸盐； 磷酸盐等。
根据本发明的再一个方面， 其提供一种制备咔哇提取物的方法， 其 包括如下步骤：
取咔哇原料， 用水、 含水或不含水的低级醇、 含水或不含水的低级 酮、 含水或不含水的含 4-10个碳原子的酯类、 含水或不含水的氯代低 级垸烃或者这些溶剂的混合物提取，提取液除去溶剂，得粗咔哇提取物; 低级醇可以是甲醇、 乙醇， 低级酮可以是丙酮， 4-10个碳原子的酯类可 以是乙酸乙酯； 用大孔树脂、 聚酰胺、 硅胶、 氧化铝、 壳聚糖常规处理 粗咔哇提取物； 或者， 对碱性水液中的粗咔哇提取物， 用沸点为 30至 100°C的脂族溶剂和 /或脂环族溶剂萃取去除黄醉椒素 B后； 将水相调至 酸性， 用含 4-10个碳原子的酯类和 /或氯代低级垸烃萃取水相， 浓缩萃 取液至干， 得精制咔哇提取物；
或者， 将用沸点为 30至 100°C的脂族溶剂和 /或脂环族溶剂提取了 黄醉椒素 B后的咔哇， 再用含水或不含水的低级醇、含水或不含水的低 级酮、 含水或不含水的含 4-10个碳原子的酯类、 含水或不含水的氯代 低级烷烃或者这些溶剂的混合物提取， 浓缩提取液至干， 得降低黄醉椒 素 B含量的精制咔哇提取物；
或者， 将粗咔哇提取物加入小于 50倍体积的水制备成混悬液， 用 石油醚、 环己烷和 /或己垸萃取水相， 将萃取后的水相浓缩至干， 得降 低黄醉椒素 B含量的精制咔哇提取物；
或者， 向粗咔哇提取物或精制咔哇提取物中加入拮抗黄醉椒素毒副 作用的还原型谷胱甘肽， 使其在咔哇提取物中的含量以重量计 1 0%， 最高可为 90%， 得降低或去除毒副作用的咔哇提取物。
或者， 选择上述方法组合使用。
所述咔哇提取物的制备方法中： 脂族溶剂和 /或脂环族溶剂是石油 醚、 环己垸、 正己烷和 /或汽油； 氯代低级烷烃是二氯乙烷、 三氯甲烷; 大孔树脂为非极性或弱极性大孔树脂； 氧化铝为酸性或中性氧化铝； 壳 聚糖为甲壳素脱乙酰基后的产物；大孔树脂或聚酰胺层析柱的洗脱液选 用含水乙醇、 含水甲醇和 /或含水丙酮； 硅胶柱层析的洗脱液先选石油 醚、 环己烷和 /或乙酸乙酯， 再选异丙醇、 乙酸乙酯、 氯代低级烷烃、 甲醇或含水甲醇、 乙醇或含水乙醇、丙酮或含水丙酮， 或者它们的混合 物洗脱； 碱水液含无机碱和 /或有机碱与水； 酸水液含无机酸和 /或有机 酸与水。其中，大孔树脂可以选用 HP20、 AB-8、 XAD-2、 D-10K HP21、 SP825、 SP850、 SP70、 SP700、 SP207、 H107; 无机碱可以为碳酸钠、 碳酸氢钠、 碳酸钾、 碳酸氢钾和 /或磷酸氢二钠， 有机碱可以为柠檬酸 盐、 葡甲胺、 泛酸钠、 L-半胱氨酸、 苯甲酰胺、 烟酰胺、 硫脲、 乙醇胺 和 /或脲； 无机酸为硫酸和 /或盐酸； 有机酸可以为柠檬酸、 醋酸、 草酸、 马来酸、 琥珀酸、 富马酸、 苹果酸、 酒石酸和 /或抗坏血酸。
咔哇提取物具体的制备方法是： 将粗咔哇提取物上硅胶柱， 先用石 油醚或环己烷与乙酸乙酯的体积比为 10-20: 1的混合溶剂洗脱硅胶柱， 再用异丙醇、 乙酸乙酯、 氯代低级烷烃、 甲醇或含水甲醇、 乙醇或含水 乙醇、 丙酮或含水丙酮， 或者它们的混合物洗脱， 回收溶剂至干得降低 黄醉椒素 B含量的精制咔哇提取物。
咔哇提取物具体的制备方法还有： 向粗咔哇提取物的水混悬液中， 加入小于混悬液 50倍体积的甲醇和 /或乙醇， 形成咔哇提取物溶液后， 加入壳聚糖； 或者向粗咔哇提取物的水混悬液中加入壳聚糖物溶液； 混 匀， 放置后， 离心或过滤去除沉淀物， 将清液浓缩干燥， 得降低黄醉椒 素 B含量的精制咔哇提取物。
咔哇提取物较好的制备方法是： 向粗咔哇提取物的水溶液或水混悬 液中加入如上所述的有机碱和 /或无机碱， 使水溶液或水混悬液呈碱性， 混合后， 用石油醚、环己烷和 /或己烷萃取去除黄醉椒素 B， 获得的水相 加入无机酸和 /或有机酸， 使溶液成酸性， 混匀后， 用乙酸乙酯、 氯仿 和 /或二氯甲垸萃取酸性溶液， 回收萃取液至干， 得降低黄醉椒素 B含 量的精制咔哇提取物。
咔哇提取物更为理想的制备方法是： 将咔哇的水、含水或不含水乙 醇、 含水或不含水甲醇、 含水或不含水丙酮的提取液， 除去有机溶剂得 到溶液或混悬液， 或者用小于咔哇提取物 10倍体积且与水能混溶的溶 剂如甲醇、 乙醇、 丙酮等溶解粗咔哇提取物形成溶液或混悬液， 然后加 入小于所述溶液或混悬液 50倍体积的水制成咔哇提取物的溶液或混悬 液， 上大孔树脂柱， 用含水或不含水的乙醇、 含水或不含水的甲醇、 含 水或不含水的丙酮、 氯代烷烃和 /或甲醇的混和物、 乙酸乙酯和 /或石油 醚的混合物洗脱，洗脱液浓缩至干，得降低黄醉椒素 B含量的精制咔哇 提取物。 ' 降低或去除毒副作用的咔哇提取物的更便捷的制备方法是： 向咔哇 的粗提取物或精制咔哇提取物中加入能拮抗黄醉椒素毒副作用的还原 型谷胱甘肽， 使其在咔哇提取物中的含量 1.0重量％， 谷胱甘肽的含 量最高可达 90重量％， 得降低或去除肝毒副作用的咔哇提取物。
我们的非常偶然地发现， 咔哇提取物中的査尔酮类成分 (flavokawin), 具体地说主要是黄醉椒素 B， 表现出强烈的肝脏毒性作 用， 是咔哇提取物中的主要毒性成分； 还原型谷胱甘肽能拮抗黄醉椒素 的肝毒性作用， 为此， 进行了大量实验研究， 相关的结果可参考以下的 实施例。 本发明 '提供的技术方案降低或去除咔哇提取物中对肝有毒副作用的 成分，提供基本上不含有毒副作用成分或显著降低有毒副作用成分含量 的咔哇提取物； 或者， 在咔哇的粗提取物或精制提取物中加入能拮抗黄 醉椒素肝毒性作用的还原型谷胱甘肽，提供降低或没有毒副作用的咔哇 提取物， 其含有公认的六种主要咔哇内酯活性成分： 去甲氧基醉椒素、 甲氧基醉椒素、 二氢醉椒素、 醉椒素、 二氢麻醉椒素和麻醉椒素； 其咔 哇内酯的总含量大于 10重量％， 可达 11一 90重量％。 本发明研究的纯 化咔哇提取物的方法简便、 实用。 附图说明
图 1是咔哇丙酮提取物 HPLC图 （箭头所指色谱峰为黄醉椒素 B)。 图 2是咔哇丙酮提取物经大孔树脂 HP20精制后， 含有降低黄醉椒 素 B含量的咔哇提取物 HPLC图 （箭头所指色谱峰为黄醉椒素 B)。 具体实施方式
在本发明中， 术语"低级醇"是指包含 1一 10个碳原子的醇， 优选 包含 1一 6个碳原子， 更优选包含 1一 4个碳原子。 其具体的例子包括甲 醇、 乙醇、 正丙醇、 异丙醇、 正丁醇、 叔丁醇、 正己醇、 正辛醇和正癸 醇， 其中尤其优选的是甲醇和乙醇。
本发明所用的术语"低级酮"是指包含 3— 10个碳原子的酮， 例如 丙酮、 甲基丁基酮等， 其中优选是丙酮。
本发明中所用的术语 "氯代低级烷烃"是指烷基链中的一个或者多 个氢原子被氯原子取代的基团， 其中所述烷基链包含 1一 10个碳原子， 优选 1一 6个碳原子，更优选为 1一 4个碳原子，且可以是直链或支链的。 氯代低级烷烃的具体例子包括氯仿、 四氯化碳、 二氯甲烷等。
本发明使用的咔哇原料为来源于斐济群岛的咔哇 ( Piper methysticum Forster f. , Piperaceae)根粉末（100-200目）， 购自美国夏烕 夷 Kava Kauai公司 (Kava Kauai, PO Box 1202， Kapaa Kauai Hawaii, 96746, USA)。
本发明中咔哇提取物中咔哇内酯的含量测定方法见文献 H. S. Alexander , M. Imre， Journal of Chromatography A, 948， 51 (2002)
以下将通过实施例更为详细地描述本发明。但对于本领域技术人员 应理解的是， 这些实施例仅是具体的说明本发明，而绝不是对本发明范 围的限制。 实施例 1
取咔哇原料， 粉碎后， 分别用水、 60%丙酮 （含水 40% )、 丙酮、 95%乙醇提取， 除去溶剂后， 分别得到咔哇水提取物、咔哇 60%丙酮提 取物、咔哇丙酮提取物、咔哇 95 %乙醇提取物， 测定其相应的咔哇内酯 与黄醉椒素 B的含量， 测定结果表明丙酮提取物中黄醉椒素 B的含量 最高。 具体的结果参见下表。 因此， 在以下的制备方法中， 除了咔哇的 水提取物外， 均用咔哇丙酮提取物作为粗咔哇提取物的代表。 不同溶剂提取的咔哇提取物中咔哇内酯的总含量以及黄醉椒素 B 的百 提取溶剂 干提取物中的黄醉椒素 B(%) 干提取物中的咔哇内酯 (％) 水 0.02 4.66
60%丙酮 2.60 47.48
丙酮 3.37 57.00
95 %乙醇 3.23 54.88 实施例 2
以大孔树脂层析制备含有降低的黄醉椒素 B含量的咔哇提取物
取 1克如实施例 1中所述制得的咔哇丙酮提取物，加入 1ml乙醇溶 解，再加入 10ml水制成咔哇提取物悬液，加入柱体积为 50毫升的 HP20 大孔树脂层析柱， 以 200毫升 35 %的乙醇 （体积比） 溶液冼脱， 将洗 脱液浓缩至干， HPLC法测定， 提取物中内酯的总含量为 58.2%， 黄醉 椒素 B的含量为 0.04%。从图 1和图 2的对比可以看出，在经过本发明 的方法处理后， 咔哇提取物中的黄醉椒素 B的含量显著下降。 实施例 3
通过硅胶层析制备降低黄醉椒素 B的提取物
取 1克如实施例 1中所述制得的咔哇丙酮提取物，加入 1ml乙酸乙 酯溶解， 加入约 3克硅胶拌样， 挥干有机溶剂， 获得硅胶柱层析样品。 将层析样品加于柱体积为 50毫升的硅胶柱顶端， 以石油醚与乙酸乙酯 的体积比为 10: 1的混合溶剂洗脱硅胶柱。由于黄醉椒素 B具有黄颜色 并且极性比活性成分咔哇内酯小，层析洗脱的第一个黄颜色的色带即为 黄醉椒素 B。 在层析洗脱第一个黄颜色的色带黄醉椒素 B后， 以 TLC 检测后面的洗脱液中无黄醉椒素 B， 以甲醇洗脱层析柱上的其它成分， 将甲醇洗脱液浓缩至干，获得降低黄醉椒素 B含量的咔哇提取物。 HPLC 测定， 提取物中的内酯总含量为 61.2%， 不含有黄醉椒素 B成分。 实施例 4
通过酸性、 中性氧化铝柱层析制备降低黄醉椒素 B的提取物
取 1克如实施例 1中所述制得的咔哇丙酮提取物，加入 1ml氯仿溶 解， 分别加入柱体积为 30毫升的酸性、 中性氧化铝干柱顶端。 酸性、 中性氧化铝层析柱分别以 200毫升的氯仿： 甲醇体积比为 2: 1混合溶 剂洗脱， TLC辅助检测。 洗脱液浓缩至干， 获得降低黄醉椒素 B含量 的咔哇提取物。 HPLC法测定， 提取物中咔哇内酯的总含量为 38.2%， 黄醉椒素 B的含量为 0.3 %。 实施例 5
通过壳聚糖处理获得降低黄醉椒素 B的咔哇提取物 取 1克如实施例 1中所述制得的咔哇丙酮提取物， 以 50毫升 40% 的乙醇溶解， 向此溶液中加入壳聚糖使壳聚糖的在总体积中的浓度为 1 % , 充分搅拌溶解， 在室温下静置 60分钟， 离心去除沉淀物， 离心所 得清液浓缩至干， 获得降低黄醉椒素 B含量的咔哇提取物。 HPLC法测 定， 提取物中咔哇内酯的总含量为 46%， 黄醉椒素 B的含量为 0.1 %。 实施例 6
通过酸碱处理的降低黄醉椒素 B的提取物的制备方法
取 1克如实施例 1中所述制得的咔哇丙酮提取物，加入 1ml乙醇溶 解， 加入 50毫升水制成咔哇提取物的混悬液， 向咔哇提取物悬液中加 入碳酸钠， 搅拌溶解， 使咔哇提取物悬液的 pH值为 9， 充分混匀， 30 分钟后用 50毫升环己烷萃取三次。 获得的水相以盐酸调节酸碱性， 使 溶液的 pH值为 2， 充分混匀， 静置 2小时后用 50毫升乙酸乙酯萃取三 次，合并萃取的乙酸乙酯并回收溶剂至干，获得降低黄醉椒素 B含量的 咔哇提取物。 HPLC法测定， 提取物中咔哇内酯的总含量为 41 %， 黄醉 椒素 B的含量为 0.05 %。 实施例 7
通过环己烷萃取处理获得降低黄醉椒素 B含量的提取物
取 3克经粉碎的咔哇根， 加入 30毫升 60%的丙酮溶液， 浸泡 30 分钟， 然后再超声提取 60分钟， 离心去除药渣， 去除药渣的上清液减 压回收去除丙酮， 剩下的咔哇提取液以 45毫升的环己烷萃取三次 （每 次 15毫升）， 将经过环己烷萃取后的水相部分浓缩至干， 获得降低黄醉 椒素 B含量的咔哇提取物。 HPLC法测定， 提取物中咔哇内酯的总含量 为 32.5 %， 黄醉椒素 B的含量为 0.2%。 实施例 8
以大孔树脂 AB-8处理制备含有降低的黄醉椒素 B含量的咔哇提取物 取 1克如实施例 1中所述制得的咔哇丙酮提取物，加入 1ml乙醇溶 解， 再加入 4ml水制成咔哇提取物悬液， 加入柱体积为 30毫升的 AB-8 大孔树脂层析柱， 先用 50毫升的水洗脱， 然后用 150毫升体积比为 80 %的乙醇溶液洗脱， 将乙醇洗脱液浓缩至干， HPLC法测定， 提取物中 内酯的总含量为 58.5 %，黄醉椒素 B的含量为 0.05 %。 实施例 9
以大孔树脂层析制备含有降低的黄醉椒素 B含量、 提高咔哇内酯含量 的咔哇提取物
取 1克如实施例 1中所述制得的咔哇水提取物，加入 1ml乙醇溶解， 再加入 10ml水制成咔哇提取物悬液， 加入柱体积为 50毫升的 HP20大 孔树脂层析柱， 以 200毫升 35 %的乙醇（体积比） 溶液洗脱， 将洗脱 液浓缩至干， HPLC法测定， 提取物中内酯的总含量为 48.2 %， 不含黄 醉椒素 B成分。 实施例 10
A: 黄醉椒素 B对正常人肝细胞株 L02的细胞毒性作用
L02细胞株用含有 10%小牛血清的 RPM1640培养基培养， 以 I X 105细胞浓度接入 96孔细胞培养板， 每孔接种 100 μ 1， 于二氧化碳培养 箱培养 24小时后， 弃去培养液， 加入含有不同浓度黄醉椒素 Β的培养 基， 继续培养 48小时， 以 ΜΤΤ法（参见： Τ. Mossman, J. Immunol. 65, 55(1983)； A. P. Li et al, Chem. Biol. Interact. 121， 117(1999))测定细胞 存活率。 结果表明黄醉椒素 B对 L02细胞有显著的细胞毒性， 其 IC5(3 为 9.2 μ g/ml。
B: 黄醉椒素 B对 ICR小鼠的肝脏毒性作用
选用 18-20克雄性 ICR小鼠， 随机分组， 每组 6只。将动物保持在 标准化的环境下， 自由进食和饮水。 黄醉椒素 B以 0.5 %羧甲基纤维素 钠悬浮， 实验组小鼠以 25mg/kg的剂量灌胃给药 7天， 对照组平行给以 0.5 %羧甲基纤维素钠。 7天后小鼠取血， 测定血清谷草转氨酶活性， 结 果显示， 实验组小鼠血清谷草转氨酶活性显著高于对照组。
取小鼠肝脏进行病理切片， 常规染色， 显微镜观擦， 结果显示， 所 有对照组小鼠的肝脏细胞正常，所有实验组小鼠（黄醉椒素 B 25mg/l g) 的肝脏汇管区浸润， 有大量炎症细胞， 肝细胞浊肿， 空泡化， 与文献报 道服用含有咔哇提取物导致肝损伤的临床病例肝脏病理切片现象一致 (参见： J. G Paul, J. C, Nathan, L. H. Richard, C. Peter， W. A. Peter. Med. J. Aust. 178， 442(2003) ) o
C: 还原型谷胱甘肽对黄醉椒素 B和咔哇丙酮提取物干细胞毒性的拮抗 作用
L02细胞株用含有 10 %小牛血清的 RPM1640培养基培养， 以 1 X 105细胞浓度接入 96孔细胞培养板， 每孔接种 100 μ 1， 于二氧化碳培养 箱培养 24小时后， 弃去培养液， 加入含有 500.00 g/ml的还原型谷胱 甘肽和不同浓度黄醉椒素 B的培养基;或者加入含有 500.00 u g/ml的还 原型谷胱甘肽和不同浓度咔哇丙酮提取物的培养基，继续培养 48小时， 以 MTT法 （参见： T. Mossman, J. iw w"o/. 65， 55(1983); A. P. Li et al, Chem. Biol. Interact. 121, 117(1999)) 测定细胞存活率。 结果表明还原型 谷胱甘肽能显著拮抗黄醉椒素 B和咔哇丙酮提取物对正常人肝细胞 L02 细胞毒性。 在含有 500.00 μ g/ml还原型谷胱甘肽 （GSH) 的培养基中， 黄醉椒素 B的 IC5Q大于 200 μ g/ml, 咔哇丙酮提取物的 IC5Q大于 500 μ g/ml, 由此说明还原型谷胱甘肽可以大大降低黄醉椒素 B和咔哇丙酮提 取物的毒性。

Claims

权利要求书
1、 一种降低毒副作用的咔哇 ( Piper methysticum Forster f. , Piperaceae, Kava)提取物， 其中按该提取物的重量计黄醉椒素 B的含 量 1.0 %， 和 /或该提取物含有 1.0 %、最高含量可达 90%的还原型谷 胱甘肽。
2、 如权利要求 1所述的咔哇提取物， 其中按该咔哇提取物的重量 计咔哇内酯的含量 10 %、 优选为 11一 90 %， 黄醉椒素 B的含量为 0 一 1.0 %、 优选为 0— 0.5 %。
3、含咔哇提取物的组合物，其含有如权利要求 1或 2所述的咔哇提 取物以及常规药用、 食用或保健品用的载体。
4、制备如权利要求 1或 2所述的咔哇提取物的方法，其包括如下步 骤：
取咔哇原料， 用水、含水或不含水的低级醇、含水或不含水的低级 酮、 含水或不含水的含 4-10个碳原子的酯类、 含水或不含水的氯代低 级垸烃或者这些溶剂的混合物提取，提取液除去溶剂，得粗咔哇提取物； 用大孔树脂、 聚酰胺、 硅胶、 氧化铝、 壳聚糖处理粗咔哇提取物； 或者，对碱性水液中的粗咔哇提取物，用沸点为 30至 100°C的脂族 溶剂和 /或脂环族溶剂萃取去除黄醉椒素 B后； 将水相调至酸性， 用含 4-10个碳原子的酯类和 /或氯代低级烷烃萃取水相， 浓缩萃取液至干， 得精制咔哇提取物；
或者， 将用沸点为 30至 100Ό的脂族溶剂和 /或脂环族溶剂提取了 黄醉椒素 B后的咔哇，再用含水或不含水的低级醇、含水或不含水的低 级酮、 含水或不含水的含 4-10个碳原子的酯类、 含水或不含水的氯代 低级烷烃或者这些溶剂的混合物提取， 浓缩提取液至干， 得降低黄醉椒 素 B含量的精制咔哇提取物；
或者，将粗咔哇提取物加入小于 100倍体积的水制成咔哇提取物的 溶液或混悬液， 用石油醚、 环己烷和 /或己烷萃取水相， 将萃取后的水 相浓缩至干， 得降低黄醉椒素 B含量的精制咔哇提取物；
或者， 向粗咔哇提取物或精制咔哇提取物中加入能拮抗黄醉椒素毒 副作用的还原型谷胱甘肽， 得降低或去除毒副作用的咔哇提取物，
或者， 选择上述方法组合使用。
5、 如权利要求 4所述的制备方法， 其特征在于：
低级醇是甲醇、 乙醇； 低级酮是丙酮； 4-10个碳原子的酯类是乙酸 乙酯； 脂族溶剂和 /或脂环族溶剂是石油醚、 环己烷、 正己垸和 /或汽油； 氯代低级烷烃是二氯乙烷、三氯甲垸； 大孔树脂为非极性或弱极性大孔 树脂；氧化铝为酸性或中性氧化铝；壳聚糖为甲壳素脱乙酰基后的产物; 大孔树脂或聚酰胺层析柱的洗脱液选用含水乙醇、 含水甲醇和 /或含水 丙酮； 硅胶柱层析的洗脱液先选用石油醚、 环己烷和 /或乙酸乙酯， 再 用异丙醇、 乙酸乙酯、 氯代低级烷烃、 甲醇或含水甲醇、 乙醇或含水乙 醇、 丙酮或含水丙酮， 或者它们的混合物洗脱； 碱水液含无机碱和 /或 有机碱与水； 酸水液含无机酸和 /或有机酸与水。
6、 如权利要求 5所述的制备方法， 其特征在于：
大孔树脂选用 HP20、 AB-8、 XAD-2、 D-10 HP2 SP825、 SP850、 SP70、 SP700、 SP207、 H107; 无机碱为碳酸钠、 碳酸氢钠、 碳酸钾、 碳酸氢钾和 /或磷酸氢二钠， 有机碱为柠檬酸盐、 葡甲胺、 泛酸钠、 L- 半胱氨酸、 苯甲酰胺、 烟酰胺、 硫脲、 乙醇胺和 /或脲； 无机酸为硫酸 和 /或盐酸； 有机酸为柠檬酸、 醋酸、 草酸、 马来酸、 琥珀酸、 富马酸、 苹果酸、 酒石酸和 /或抗坏血酸。
7、 如权利要求 4所述的制备方法， 其包括如下步骤： 将粗咔哇提取物上硅胶柱， 先用石油醚或环己烷与乙酸乙酯的体积 比为 10-20: 1 的混合溶剂洗脱硅胶柱， 再用异丙醇、 乙酸乙酯、 氯代 低级垸烃、 甲醇或含水甲醇、 乙醇或含水乙醇、 丙酮或含水丙酮， 或者 它们的混合物洗脱，回收溶剂至干得降低黄醉椒素 B含量的精制咔哇提 取物。
8、 如权利要求 4所述的制备方法， 其包括如下步骤- 向粗咔哇提取物的水混悬液中加入小于 10倍混悬液体积的甲醇和 / 或乙醇， 形成咔哇提取物溶液后， 加入壳聚糖； 或者向粗咔哇提取物的 水混悬液中加入壳聚糖物溶液；混匀，放置后，离心或过滤去除沉淀物， 将清液浓缩干燥， 得降低黄醉椒素 B含量的精制咔哇提取物。
9、 如权利要求 4所述的制备方法， 其包括如下步骤：
向粗咔哇提取物的水溶液或水混悬液中加入有机碱和 /或无机碱， 使水溶液或水混悬液呈碱性， 混合后， 用石油醚、 环己烷和 /或己烷萃 取去除黄醉椒素 B，获得的水相加入无机酸和 /或有机酸，使溶液成酸性， 混匀后， 用乙酸乙酯、 氯仿和 /或二氯甲垸萃取酸性溶液， 回收萃取液 至干， 得降低黄醉椒素 B含量的精制咔哇提取物。
10、 如权利要求 4所述的制备方法， 其包括如下步骤：
将咔哇的水、 含水或不含水乙醇、 含水或不含水甲醇、 含水或不含 水丙酮的提取液， 除去有机溶剂得到的溶液或混悬液， 或者用小于 10 倍咔哇溶液或混悬液体积的能与水混溶的溶剂如甲醇、 乙醇、 丙酮等溶 解粗咔哇提取物，然后加入小于混悬液 100倍体积的水制成咔哇提取物 的溶液或混悬液， 上大孔树脂柱， 用含水或不含水的乙醇、 含水或不含 水的甲醇、 含水或不含水的丙酮、 氯代垸烃和 /或甲醇的混和物、 乙酸 乙酯和 /或石油醚的混合物洗脱， 洗脱液浓缩至干， 得降低黄醉椒素 B 含量的精制咔哇提取物。
PCT/CN2004/001558 2004-01-13 2004-12-29 Extrait de kava destine a reduire des effets secondaires et des effets toxiques, composition et preparation comprenant cet extrait Ceased WO2005067950A1 (fr)

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