WO2009124420A1 - 促进营养素被吸收的医药组合物及茯苓萃取物 - Google Patents

促进营养素被吸收的医药组合物及茯苓萃取物 Download PDF

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WO2009124420A1
WO2009124420A1 PCT/CN2008/000749 CN2008000749W WO2009124420A1 WO 2009124420 A1 WO2009124420 A1 WO 2009124420A1 CN 2008000749 W CN2008000749 W CN 2008000749W WO 2009124420 A1 WO2009124420 A1 WO 2009124420A1
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use according
absorption
extract
nutrients
medicament
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PCT/CN2008/000749
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English (en)
French (fr)
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林汉钦
张自忠
张温良
宋一洋
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杏辉天力（杭州）药业有限公司
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Priority to MYPI2010004439A priority Critical patent/MY159260A/en
Priority to CA2716948A priority patent/CA2716948C/en
Priority to JP2011503323A priority patent/JP5593305B2/ja
Priority to PCT/CN2008/000749 priority patent/WO2009124420A1/zh
Priority to AU2008354673A priority patent/AU2008354673B2/en
Priority to EP08733950A priority patent/EP2272520A4/en
Priority to KR1020107022686A priority patent/KR101488332B1/ko
Priority to CN2008801277851A priority patent/CN101959518B/zh
Publication of WO2009124420A1 publication Critical patent/WO2009124420A1/zh

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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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/076—Poria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A23L33/11—Plant sterols or derivatives thereof, e.g. phytosterols
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • composition and sputum extract for promoting nutrient absorption are provided.
  • the present invention relates to a novel use of a lanthanide compound for promoting the absorption of nutrients, and more particularly to a pharmaceutical composition containing a lanthanide compound as an effective ingredient for promoting nutrient absorption.
  • the applicant's invention patent application No. 921 13393 discloses a pharmaceutical composition for enhancing human immunity, which contains lanostane A class of compounds as an active ingredient.
  • the invention also provides a cockroach extract which enhances human immunity, comprising 5-60% by weight of a lanthanane compound and substantially free of a ring-opening serotonin-like compound.
  • the extract is a metabolite or a fermented product or a hyphae extracted from the Polyacobacter (Pow cocos (Schw) Wolf).
  • intestinal absorption cells are well absorbed by nutrients.
  • These nutrients can be used by human cells, which is the basis of a healthy body.
  • glucose is converted into high-energy substances (ATP) by cells, and human cells use ATP to perform tissue and organ functions such as heart beat, nerve conduction, and skeletal muscle movement. Therefore, substances or methods that promote the absorption of nutrients are not only the subject of concern to researchers in the relevant field but also to all. Summary of the invention
  • a primary object of the present invention is to provide a novel use of a lanthanide compound as an effective ingredient to promote absorption of nutrients.
  • Another object of the invention is to use a lanthanide compound as a food or beverage additive to promote absorption of nutrients.
  • Still another object of the present invention is to provide a pharmaceutical composition containing a lanthanane compound as an active ingredient for promoting absorption of nutrients.
  • pharmaceutical composition as used herein, in addition to its literal meaning, further includes a nutritional supplement composition, the nutritional supplement composition In addition to nutrients, it contains a lanthanane compound as an active ingredient for promoting nutrient absorption.
  • the invention provides a pharmaceutical composition for promoting absorption of nutrients into a mammal (for example, a human), comprising a lanosterane having the following formula (I) or an pharmaceutically acceptable amount thereof, which comprises an effective amount of nutrient absorption as an active ingredient.
  • Salt
  • R 3 is H or OH
  • R 5 is H or OH
  • R 6 is CH 3 or CH 2 OH.
  • the present invention also provides a use of the lanosterane having the above formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient for the preparation of a nutrient-enhancing drug or pharmaceutical composition for absorption by a mammal.
  • the lanostane (I) has the following chemical formula:
  • the composition of the present invention contains 0.1-20% by weight.
  • composition of the invention is orally administered.
  • the present invention uses a guanidine extract as the source of the lanthanane (1), the bismuth extract comprising from 1 to 60 weight percent. Wool decane having the above formula (I) and substantially free of open loop lanolin.
  • the cockroach extract is prepared by a method comprising the steps of: a) extracting a metabolite of sputum, a fermentation product of sputum or a hyphae of sputum by using water, sterol, ethanol or a mixed solvent thereof; ;
  • step c) introducing the concentrate obtained in step a) into a silica gel column;
  • the solvent used in the extraction of step a) is 95% alcohol.
  • step a) comprises extracting the metabolite of the fungus, the fermentation product of the fungus or the mycelium hyphae by boiling water; adding a base to the aqueous extract to a pH of 9-1; separating the alkaline aqueous solution Adding an acid to the alkaline aqueous solution to a pH of 4-7 to produce a precipitate; separating the precipitate; extracting the precipitate with alcohol and separating the extract liquid.
  • the concentrate of step b) is further extracted with a volume ratio of 1:1 of a 95% v/v sterol aqueous solution and a two-phase solvent of n-hexane; separating the sterol layer; and concentrating the sterol layer
  • the concentrate obtained was used as a feed to the silica column of step c).
  • the low polarity eluent of step d) is a mixed solvent of dichlorosilane and decyl alcohol in a volume ratio of 96.5:3.5.
  • the cockroach extract comprises from 5 to 35 parts by weight. /. Wool decane (1).
  • the composition of the present invention further comprises a nutrient such as glucose, amino acid, vitamins or a combination thereof.
  • a nutrient such as glucose, amino acid, vitamins or a combination thereof.
  • the present invention can be applied to all the following conditions by using the lanthanane of the formula (I) or a pharmaceutically acceptable salt thereof, or the above-mentioned cerium extract as an active ingredient for promoting nutrient absorption: (1) improvement of nutritional status of the elderly, The aging relationship also causes the digestive system to decline, which often causes the absorption function to deteriorate, and the nutritional status is insufficient. Taking the active ingredient obviously contributes to the improvement of nutritional status. (2) The body of a thin child is improved. The child has some physical problems during the growth process. The body is thin and can't be improved due to the increased supply of nutrients. Taking the active ingredient obviously helps the body to be thin and improved.
  • Surgical or cancer patients cause weakness and urgent need to supplement nutrients (including amino acids, glucose, vitamins) to improve, such as taking this active ingredient obviously helps patients to quickly return to health.
  • the active ingredient of the present invention having an immunopotentiating function (killing virus), together with its nutrient absorption ability, can synergistically guide water to enter the human body with nutrient absorption (change osmotic pressure), thereby preventing diarrhea. Save children's lives.
  • the active ingredient of the present invention may be added to milk powder, beverages, food for nutritional purposes, or may be used as a medicine, such as a tablet, a capsule, a granule, a liquid, an injection, etc. Medical purposes.
  • a medicine such as a tablet, a capsule, a granule, a liquid, an injection, etc. Medical purposes.
  • Figures 1 to 3 show the effects of the lanostane compounds K2, ⁇ 3 and ⁇ 4 of the present invention on the absorption of glucose by intestinal cells (Caco-2), respectively.
  • Figures 4 to 7 show the effects of the lanostane compounds K l, ⁇ 2, ⁇ 3 and ⁇ 4 of the present invention on the absorption of arginine by intestinal cells (Caco-2), respectively.
  • Figures 8 to 10 show the effects of the lanostane compounds K1, ⁇ 3 and ⁇ 4 of the present invention on the absorption of tryptophan cells (Caco-2) by trypsin.
  • Fig. 1 to Fig. 14 show the effects of the lanostane compounds K1, ⁇ 2, ⁇ 3 and ⁇ 4 of the present invention on the absorption of folic acid by intestinal cells (Caco-2), respectively. Best way to implement the invention
  • the transport of protein in the intestinal lumen in combination with sodium ions causes a structural change in the transported protein, thereby opening the glucose-binding portion.
  • the combination of glucose and sodium ions transports proteins causing a change in the conformation of this transported protein, allowing glucose and sodium ions to enter the cell (inside the cell).
  • Sodium ions leave the cells first, causing instability of glucose and transport proteins.
  • the glucose also leaves and enters the cell, and the protein that removes both glucose and sodium ions returns to the original direction of the intestine.
  • the protein is transported by another type, which is a facilitated diffusion protein that causes glucose to leave the intestinal cells and enter the blood vessel in a concentration gradient.
  • Caco2 cells are a cell line derived from human colorectal cancer. The most characteristic feature of this cell line is the rapid differentiation of a polar monolayer into a human-like intestinal membrane. 0 Generally, two to three cells are cultured. After a week, Caco2 cells differentiate into highly hydrolyzed brush borders and tight junctions between cells, making them a monolayer that blocks and controls the passage of substances. The monolayer of Caco2 cells has a resistance value similar to that of the intestine, which is about 300 ⁇ cm 2 . In addition, transport proteins with various nutrients have been confirmed on Caco2 cell monolayer membranes, including transport proteins such as amino acids, glucose, and vitamins.
  • monolayers formed by Caco2 cells are often used to study the effects or tests of drugs or nutrients penetrating intestinal cells, and the test results are used to illustrate the absorption of drugs or nutrients by human-like intestinal cells. It is accepted by those skilled in the relevant fields [Hidalgo IJ, al" Gastroenterology, 1989; 96: 736-749.; Artursson P., J Pharm Sci 1990; 79: 476-482.].
  • the strong Chinese herbal extract and/or the strong Chinese herbal purified component have such enhanced nutrient absorption.
  • the inventors of the present application are using Caco2 cells In the study of typical sturdy Chinese medicine (ginseng, radix astragalus), the atypical and strong Chinese medicine, ⁇ was also studied. It was unexpectedly found that the ⁇ extract and the lanthanane in it had the ability to enhance the ability of nutrients to pass through Caco2 cells.
  • the present invention discloses a cockroach extract which can promote the absorption of nutrients into a mammal (for example, a human), and the preparation method thereof is the same as the method disclosed in the aforementioned Taiwan Patent Publication No. 200425900, which comprises extracting hydrazine by a conventional extraction method.
  • the crude extract is separated into a small polar lanostane (by dichloromethane: methanol (96:4) as an eluent) and a highly polar ring-opened lanane (secolanostane) by chromatography.
  • Example 1 The present invention will be further described in detail below with reference to the embodiments, but without limiting the invention.
  • Example 1
  • the methylene chloride/sterol mixture was used as an eluent (eluent), and the mixture was eluted in a ratio of 96:4, 90:10, and 0:100, and the mixture was washed with a thin layer of silicon.
  • the PCM fraction was further subjected to a silica gel column using dichloromethane: methanol (96.5:3.5) as an eluent. Chromatography (same as the above silica gel column), further separation of purified lanostane-like components Kl (K1-1 and Kl-2), K2 (K2- 1 and K2-2), K3, K4, K4a , K4b, K5, K6a and K6b.
  • Chromatography (same as the above silica gel column), further separation of purified lanostane-like components Kl (K1-1 and Kl-2), K2 (K2- 1 and K2-2), K3, K4, K4a , K4b, K5, K6a and K6b.
  • the yields of the lanosterane compounds K l to K6b separated from the PCM fraction are shown in the following table.
  • the PCM portion contains about 15% by weight of lanosterane compounds K 1 to K6b.
  • the lanosterane compounds K 1 , K2, K3 and K4 prepared in Example 1 of the present invention, and the PCM extract were evaluated for their effects of promoting nutrient absorption by the following methods.
  • the lanthanide compound or PCM extract of the present embodiment promotes absorption of nutrients such as glucose, amino acids, vitamins, etc. by Caco2 cells
  • Caco2 cells are seeded in polycarbonate S ranswell® inserts (Polycarbonate Membrane Transwell® inserts) (No. 34 14, Corning Incorporated, NY, USA), the cell culture medium was changed every 2 to 3 days, and after 14 to 21 days, Caco2 cells formed a monolayer film.
  • the trans-epithelial electrical resistance (TEER) formed on the upper and lower sides was measured using Millicell®-ERS (Millipore EVOM-6; World Precision Instrument, Sarasota, FL, USA).
  • the Caco2 monolayer membrane was cultured for two days in a cell culture medium containing different concentrations of the test substance, wherein the serum was charcoal-dextran-treated fetal bovine serum (CD-FBS). After culturing for two days, it was washed once with PBS, and changed to a buffer containing no specific nutrient (such as a glucose-free buffer when glucose absorption was measured), and then the buffer was changed to contain a predetermined concentration. This particular nutrient and radioactively calibrated fresh buffer of nutrients to track the rate at which the nutrient molecules pass through the Caco2 monolayer.
  • CD-FBS charcoal-dextran-treated fetal bovine serum
  • Radioactively calibrated nutrients such as [ l 4 C]-D-glucose or [ l4 C]-D-2-deoxyglucose, [ 3 H]-L-arginine (arginine), [ 3 H]-L-color Tryptophan, [ 3 H]-folic acid.
  • Simultaneous determination of carbon-14 or hydrogen-3 labeled -D-xylool is used to determine the integrity of Caco-2 cell monolayers [Ref. Literature: Artursson, P" J Pharm Sci, 1990, 79: 476-482; Ferraris RP, et al., Am J Physiol, 1993, 264: G285-G293.
  • Human Caco-2 cells are planted in transwell and differentiated to form a complete monolayer. Before the measurement, the cell monolayer membrane was treated with the analyte for two days, followed by a glucose-free buffer (composition of 80 mM NaCl, 100 mM mannitol, 20 mM Tris-HCl, pH 7.4, 3 mM K 2 HP0).
  • a glucose-free buffer composition of 80 mM NaCl, 100 mM mannitol, 20 mM Tris-HCl, pH 7.4, 3 mM K 2 HP0.
  • this experiment simultaneously determined that the carbon-14-labeled -D-xylool was used to determine the integrity of the Caco-2 cell monolayer; whereas the carbon-14-calibrated-L-glucose was used to determine Non-specific background values for absorption by the glucose transport protein pathway.
  • the value of the radiation intensity is converted to represent the concentration of glucose molecules passing through the Caco-2 cell monolayer to the lower layer at that time point.
  • the lower glucose concentration at each time point is plotted against the time point, and a linear analysis is performed to obtain a straight line whose slope represents the average rate of glucose molecules passing through the monolayer of Caco-2 cells under the treatment of the analyte at this concentration. .
  • the control in the experiment refers to data that is not processed by the analyte.
  • the main references for the above assays are as follows: Kimura T. et al., J. Pharm. Pharmacol., 2001, 54, 213-219. Analytical determination of amino acid uptake
  • the absorption of amino acids such as arginine and tryptophan is carried out in a cell culture medium containing different concentrations of the test substance in a Caco2 monolayer. After two days of culture, it is washed once with PBS and replaced with a buffer containing no such amino acid (
  • the components of arginine were: 137 mM NaCl, 10 mM Hepes pH 7.4, 0.3 mM NaH 2 P0 4 , 0.3 mM K 2 HP0 4 , 5.4 mM C1, 2.8 mM CaCl 2 , 1 mM MgS0 4 , 10 mM glucose;
  • the buffer composition for tryptophan uptake was: 137 mM choline chloride, 10 mM Hepes pH 7.4, 0.6 mM KH 2 P0 4 , 5.4 mM KC1, 2.8 mM CaCl 2 , 1 mM MgS0 4 , 10 mM glucose).
  • the lower amino acid concentration at each time point is plotted against the time point, and a linear analysis is performed to obtain a straight line whose slope represents the average rate of amino acid molecules passing through the Caco-2 cell monolayer film at the concentration of the analyte.
  • the control in the experiment refers to the data obtained without being treated with the analyte.
  • the main references for the above determination methods are as follows: Pan M., et al., Am J Physiol. Gastrointest Liver Physiol 1995, 268: G578-G585.
  • Human Caco-2 cells were seeded in 10-cm plates and assayed after about two weeks of differentiation. Before the assay, the cells were first treated with various concentrations of the analyte for two days, followed by Folate transport incubation buffer (pH 6.0): Hank's Balanced Salt Solution (HBSS), supplemented with 0.14 g L CaCl. 2 , 0.1 g/L MgCl 2 , and 0.1 g/L MgS0 4 ) After incubation for 1 hour, it was then replaced with a final concentration of 5 ⁇ folic acid containing 2 Ci/mL of hydrogen-3 calibrated folic acid (3,5 , 7,9- 3 H-folic acid, 25 mCi/mmol, ARC, St.
  • HBSS Hank's Balanced Salt Solution
  • the cells were washed with PBS at specific time points, then the cells were lysed by 0.2 mL of 0.2 N NaOH, and the cells were concentrated by scraping.
  • the quantitative supernatant cell solution was taken to determine the protein concentration, and the supernatant cell solution 20 was taken to determine the concentration at which folic acid molecules were absorbed into and accumulated in Caco2 cells.
  • the calculated value represents the concentration of folic acid molecules contained in the cell fluid of equal protein amount in Caco-2 cells at that time point.
  • the intracellular folate concentration at each time point was plotted against time points and a linear analysis was performed to determine the average rate at which folate molecules were absorbed into Caco-2 cells at this concentration of analyte.
  • the control in the experiment refers to the data obtained without being treated with the analyte.
  • the above determination methods are mainly referred to in the literature: Dudeja PK., et al., Am J Physiol Gastrointest Liver Physi, 2001, 281 ( 1 ): G54 - G60.
  • PCM1 0.033 g/cc
  • PCM2 0.0033 g/cc
  • intestinal cells promote the absorption of these two amino acids under low doses of lanostere compounds (1 ⁇ - 0.001 ⁇ ) and the absorption is linear, as shown in Figures 4-10. This linear relationship indicates that these lanthanane compounds increase the ability of the two amino acids to be absorbed by affecting or increasing the transport protein.
  • Table 3 Effects of lanthanane compounds Kl, K2, K3 and K4 on the absorption of arginine nutrients by intestinal cells (Caco-2)
  • K2 did not have a significant effect on the uptake of tryptophan by intestinal cells (Caco-2).
  • Control group 0.0759 ⁇ 0.0169 100.00 -
  • the precipitate was separated and washed with 40 LH 2 0, then the precipitate was separated by a centrifuge, and spray dried with 8 L of water to obtain about 380 g of a powder.
  • the powder was extracted three times with 4 L of alcohol, and the extract was combined and concentrated to obtain 238.9 g of an alcohol extract, which was separated by HPLC.
  • the extract was obtained as a main component K2 185.93 mg, K320.34 mg, K4 per gram of the extract. 15.82 mg and a small amount of K 14.52 mg, ie the extract contains about 226.07 mg of lanostanes per gram.
  • Example 4 Preparation of capsules
  • a capsule containing the PCM component obtained in Example 1 was prepared according to the following composition: The PCM and sodium aluminosilicate were respectively sieved through a #80 mesh sieve, the potato starch was sieved through a #60 mesh sieve, magnesium stearate was sieved through a #40 mesh sieve, and placed in a mixer to be stirred. Evenly, and then filled into vacant capsules, each capsule contains about 1.68 mg (0.42 wt%) of active ingredient Kl-K6.

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Description

促进营养素被吸收的医药组合物及茯苓萃取物 技术领域
本发明是有关一种使用羊毛 烷类化合物来促进营养素被吸收的新 用途， 尤其有关一种含有羊毛 烷类化合物作为促进营养素被吸收的有效 成分的医药组合物。 背景技术
本申请申请人在台湾发明专利申请案第 921 13393 号 （公开号 200425900 , 2004年 12月 1 日公开）揭示了一种用以提升人体免疫力的医 药组合物， 此组合物含羊毛 烷（lanostane ) 类化合物作为有效成分。 该 发明也提供一种提升人体免疫力的茯苓萃取物， 包含 5-60重量％的羊毛甾 烷类化合物且实质上不含开环羊毛甾烷 （ secolanostane ) 类化合物。 该萃 取物是萃取自多孔菌科植物茯苓菌 （Porea cocos (Schw)Wolf ) 的代谢产物 或发酵产物或茯苓菌菌丝。
一般来说， 要使人体更健康、 有活力， 对营养素的吸收是非常重要 的。 如肠冒道细胞对营养素的吸收状况良好， 这些营养素能被人体细胞利 用， 就是健康身体的基础。 例如葡萄糖被细胞转化为高能物质（ATP ), 人 体细胞利用 ATP, 可执行组织器官功能， 如心脏跳动、 神经传导、 骨骼肌 的运动。 因此促进营养素被吸收的物质或方法， 不仅是相关领域研究者、 也是所有人所关切的课题。 发明内容
本发明的一主要目的是提供一种使用羊毛 烷类化合物作为有效成 分来促进营养素被吸收的新用途。
本发明的另一目的是将羊毛 烷类化合物作为食品或饮料添加物来 促进营养素被吸收。
本发明的又一个目的是提供一种含有羊毛 烷类化合物作为促进营 养素被吸收的有效成分的医药组合物。 本发明所指的 "医药组合物" 一词 除了其字面意义外， 进一步包括营养补充品组合物， 该营养补充品组合物 除了营养素外还包含作为促进营养素吸收的有效成分的羊毛 烷类化合 物。 本发明所提供的一种促进营养素为一哺乳类动物 （例如人类） 吸收 的医药组合物， 包含一促进营养素吸收有效量作为有效成分的具下列化学 式 (I)的羊毛甾烷或其医药可接受的盐：
Figure imgf000004_0001
其中 1 ,为 1^或 CH3 ; R2为 OCOCH3, =0或 OH； R3为 H或 OH； R4为 -C(=CH2)-C(CH3)2Ra, 其中 Ra为 H或 OH， 或 -CH=C(CH3)-Rb， 其中 Rb为 CH3 或 CH2OH; R5为 H或 OH; 及 R6为 CH3或 CH2OH。
本发明也提供一种使用具上述化学式 (I)的羊毛甾烷或其医药可接受 的盐作为活性成分在制备促进营养素被一哺乳类动物吸收药物或医药组 合物中的用途。
较佳的， 该羊毛甾烷 (I)具有具下列化学式：
Figure imgf000004_0002
Figure imgf000005_0001
Figure imgf000005_0002
Figure imgf000005_0003
较佳的， 本发明的组合物其含有 0.1-20重量 °/。的羊毛 烷 (I)或其医药 可接受的盐。
- 较佳的， 本发明的组合物为口服的。
较佳的， 本发明使用一茯苓萃取物作为该羊毛 烷(1)的来源， 该茯 苓萃取物包含 1-60 重量 °/。的具前述式 (I)的羊毛甾烷且实质上不含开环羊 毛甾 。 较佳的， 该茯苓萃取物是由包含下列步骤的方法所制备： a) 以一水， 曱醇， 乙醇或它们的混合溶剂萃取茯苓菌的代谢物， 茯苓菌的 发酵产物或茯苓菌菌丝；
b) 浓缩步骤 a)萃取所荻得的液体；
c) 将步骤 a)所荻得的浓縮物导入一硅胶管柱；
d) 以一低极性洗脱剂（eluent)洗脱该硅胶管柱， 及收集所产生的洗出液
(eluate); 及
e) 浓缩步骤 d)的洗出液。
较佳的，从步骤 e)所荻得的洗出液的浓缩物以硅胶薄层层析法分析具 有一层析值 (Rf)≥0.1 , 其中以紫外光灯及碘作检侧， 展开液为二氯曱烷： 曱醇 = 96:4。
较佳的， 步骤 a)的萃取所使用的溶剂为 95%酒精。
较佳的， 步骤 a)包含以沸水萃取茯苓菌的代谢物， 茯苓菌的发酵产物 或茯苓菌菌丝； 加入一碱至该萃取水溶液至其 pH值 9- 1 1 ; 分离出该碱性水 溶液； 加入一酸至该碱性水溶液至其 pH值 4-7， 以产生沉淀物； 分离出该 沉淀物； 再以酒精萃取该沉淀物， 并分离出萃取液体。
较佳的， 步骤 b)的浓缩物被进一步以一体积比为 1： 1的 95% v/v曱醇水 溶液及正己烷的两相溶剂萃取； 分离出曱醇层； 及浓缩该曱醇层， 所获得 的浓缩物被用作为步骤 c)的硅胶管柱的进料。
较佳的， 步骤 d)的低极性洗脱剂为体积比为 96.5 :3.5的二氯曱烷及曱 醇的混合溶剂。 '
较佳的， 该茯苓萃取物包含 5-35重量。 /。的羊毛甾烷 (1)。
较佳的， 本发明的组合物进一步包含一营养素， 例如葡萄糖、 氨基 酸、 维生素或其组合。
本发明以具式 (I)的羊毛 烷或其医药可接受的盐， 或前述茯苓萃取 物作为促进营养素吸收的有效成分可应用在下列所有状况： （1)老年人营养 状况改善， 老年人因老化关系其消化系统也随之衰退， 常造成吸收功能也 变差， 营养状况因而不足， 服用该有效成分显然有助于营养状况改善。 （2) 瘦弱小孩身体改善， 小孩在成长过程中有些因体质问题， 身体瘦弱无法因 营养素增加供应而改善， 服用该有效成分显然有助于身体瘦弱改善。 （3) 工作压力大的人或经常熬夜赶工作的人， 此类人因压力引起的身心症而造 成胃肠功能受到影响， 吸收不良为病症之一， 进而衍生出疲劳、 精神不振. 的后遗症， 故此类人可借助服用该有效成分来改善。 （4)运动量大的运动员 或工作量大的工人 /上班族常需补充营养素， 因工作量大或耗体力大的人， 相对应的是能量 （ATP ) 消耗量也大， 常需补充能量而能量最主要是由葡 萄糖而来， 因此加速补充葡萄糖有利于能量制造， 因此对于寻求佳绩的运 动员及工作表现工人 /上班族， 服用该有效成分是有效补充葡萄糖的方法。 (5)手术或癌症病人（接受化疗或放射疗法）造成身体虛弱， 急需要补充营 养素 （含氨基酸、 葡萄糖、 维他命） 来改善， 如搭配服用该有效成分显然 有助于病人快速恢复健康。 （6)病毒性腹泻， 小孩在冬春时期， 常会发生不 同病毒感染引起的感冒症状及腹泻 （病毒之一， 如轮状病毒）， 严重者会 因腹泻造成水、 电解质及营养素流失而死亡， 因此已知具有免疫增强功能 (杀灭病毒） 的本发明的有效成分， 再加上其具有营养素吸收能力， 会因 协同作用引导水分随营养素吸收 （改变渗透压） 而进入人体， 因此阻止腹 泻而拯救孩童生命。 综合上述 6项应用， 本发明的有效成分可以被添加在 奶粉、 饮料、 食品作为营养目的， 也可作成医药品， 如锭剂、 胶嚢、 颗粒 剂、 液剂、 注射剂等形式出现， 用于医疗目的。 附图的简要说明
图 1至图 3分别显示本发明羊毛甾烷化合物 K2、 Κ3及 Κ4对葡萄糖 被肠细胞 （Caco-2 ) 吸收的影响。
图 4至图 7分别显示本发明羊毛甾烷化合物 K l、 Κ2、 Κ3及 Κ4对精 氨酸被肠细胞 （Caco-2 ) 吸收的影响。
图 8至图 10分别显示本发明羊毛甾烷化合物 Kl、 Κ3及 Κ4对色氨 酸被肠细胞 （Caco-2 ) 吸收的影响。
图 1 1 至图 14分别显示本发明羊毛甾烷化合物 Kl、 Κ2、 Κ3 及 Κ4 对叶酸被肠细胞 （Caco-2 ) 吸收的影响。 实施本发明的最佳方式
由现代的生物化学知道人类从饮食中获得的营养素有非常多的种 类， 其中能被人体利用吸收而产生能量或细胞基础代谢所必须的营养素主 要有葡萄糖 （ glucose )、 象基酸 ( amino acid ) 和维他命 ( vitamin ) 等类。 然而这些营养素并不能自由进出人体的组织或细胞， 而是受到严密的机转 所调控。 此种情形与一般我们服食化学物质 （如药物） 被人体吸收情形不 同， 化学物质则是利用浓度差异性以扩散方式进出人体。 但是营养素在细 胞间的出入则不同， 必须由特定的输送蛋白质或是细胞膜上的特别的通道
( channel ) 来负责。 以下利用人体肠道对葡萄糖吸收来说明。 首先， 肠腔 内的输送蛋白质与钠离子结合使输送蛋白质产生结构上的改变， 因而打开 葡萄糖结合部份。 葡萄糖与钠离子共同结合输送蛋白质引起此输送蛋白质 的构型改变， 而让葡萄糖与钠离子能进入细胞内 （面向细胞内 ）。 钠离子 先离开进入细胞内， 进而引起葡萄糖与输送蛋白质的不稳定。 最后， 葡萄 糖也离开而进入细胞内， 而脱去葡萄糖与钠离子两者的输送蛋白质也重新 回到原来面对肠腔的方向。 当细胞内的葡萄糖累积至一定量时， 便借助另 一类输送蛋白质， 此类蛋白质是属于促进扩散 （ facilitated diffusion ) 蛋白 质， 使葡萄糖利用浓度梯度的方式离开肠细胞进入血管中。
Caco2细胞为一种由人体大肠癌衍生的细胞株，此细胞株最大的特色 是能自 主性地快速分化成类似人类肠道的具有极性的细胞单层膜 ( monolayer )0 一般培养两到三星期后， Caco2 细胞会分化形成具有高度 水解能力的絨毛状边缘 ( brush border ) 以及细胞间的紧密连结 ( tight junctions ), 成为能阻隔及控制物质通透的细胞单层膜。 Caco2细胞的单层 膜具有与肠道相似的电阻值， 其约 300 Ω cm2左右。 除此之外， 在 Caco2 细胞单层膜上也被证实具有各种营养素的输送蛋白质， 包含如氨基酸、 葡 萄糖及维他命等的运输蛋白质。 因此， Caco2 细胞形成的单层膜常被用于 研究药物或营养素穿透肠道细胞的实猃或测试， 而其测试结果用以说明人 -类肠道细胞对于药物或营养素的吸收作用， 也为熟悉相关领域者所接受 [Hidalgo IJ, al" Gastroenterology, 1989;96:736-749.; Artursson P., J Pharm Sci 1990;79:476-482.]。
由于中国传统医学对长得瘦小的小孩， 或身体因老化变得衰弱老人， 或因生病或因疾病（其中之一如癌症）而开刀， 身体变成虛弱的病人， 均会 采用强壮性中药来改善或治疗身体， 使他们变回健康的身体， 但是强壮性 中药如何改善身体朝向健康状态？ 有可能是经由强壮中药本身即为营养 素的提供者， 亦有可能是增强营养素吸收， 也有可能是透过上述二种情形 来增强人体的营养状态。 如果经由增强营养素吸收机制， 则那些有效成分 能够影响运输蛋白， 至今也未有人在此方面提出科学上的证据。 故利用上 述 Caco2 细胞吸收营养素的实验即可证明强壮性中药萃取物及 /或强壮性 中药纯化成分具有此种增进营养素吸收。 本申请发明人在利用 Caco2细胞 筛选典型强状性中药（人参、 黄芪）研究时， 同时对非典型的强壮性中药， 茯苓也进行研究， 结果意外发现茯苓萃取物及其中的羊毛 烷具备了增强 营养素通过 Caco2细胞的能力事实。
本发明所揭示的可促进营养素为一哺乳类动物 （例如人类） 吸收的茯 苓萃取物， 其制备方法与前述的台湾发明专利公开号 200425900所揭示的 方法相同， 包括利用传统萃取法萃取茯苓得到一粗萃取物，再经由层析法， 分成极性小的羊毛 烷（ lanostane )类部位（以二氯甲烷： 甲醇 (96:4)为洗 脱剂） 和极性大的开环羊毛 烷 （ secolanostane ) 类部位 （以二氯曱烷： 甲醇 (90: 10或 0: 100)为洗脱剂）， 其中， 利用硅胶薄层层析法， 显示出羊毛 甾烷（ lanostane )类部位的所在位置， 即展开溶媒为二氯曱烷-曱醇 (96:4) 时， 层析值 (1 0为≥0.1； 至于开环羊毛甾烷 （secolanostane ) 类成分， 则层 析值小于 0.1。 用硅胶管柱层析法可进一步分离该羊毛甾烷类部位， 其中 洗脱剂使用二氯曱烷： 曱醇 (97:3至 95:5),分离出数种羊毛 烷（ lanostane ) 类化合物。
下面结合实施例对本发明做进一步详细的描述， 但不以此限制本发 明。 实施例 1
以云南产茯苓 30公斤， 磨成粉后， 利用 120 L酒精 （浓度 95% ) 萃 取 24 小时， 及过滤分离。 再重复前述萃取及固液分离三次。 合并滤液， 并将滤液浓缩后得干燥萃取物 265.2克。 再利用一两相萃取剂（己烷： 95% 曱醇 = 1 : 1 ) 对该干燥萃取物进行分配萃取。 取出曱醇层并加予浓缩后得 到千燥固体 246.9克。 利用硅胶管柱层析对该千燥固体进行分离， 该硅胶 管柱填充有该干燥固体重量 10-40倍的硅胶，购自 Merck公司， Silica gel 60, 70-230 目。 以二氯甲烷 /曱醇混合液作为洗脱剂 （eluent ), 依序以 96:4、 90: 10、 0: 100比例的混合液进行洗脱， 洗出液（duate ) 以硅 薄层层析法 ( Thin Layer Chromatography )(紫外光灯及碘作检测，展开液为二氯甲烷： 曱醇 = 96:4 ) 检测成分， 将相同成分合并。
以二氯曱烷-曱醇 (96:4)混合液进行硅胶管柱层析， 可得到属 PCM部 份 78克， PCM部份依上述硅胶薄层层析法可明显看到 6个迹点。 以二氯 曱烷： 曱醇 (90: 10)及 (0: 100)洗脱液层析合并可得到 PCW部份 168克。
PCM部份进一步以二氯甲烷： 甲醇 (96.5:3.5)作为洗脱剂进行硅胶管柱 层析（同上述硅胶管柱）， 进一步分离可得纯化的羊毛 烷（lanostane )类 成分 Kl (K1-1及 Kl -2), K2 (K2- 1及 K2-2), K3, K4, K4a, K4b, K5, K6a及 K6b。 详细分离步骤及鉴定分析数据请参见台湾发明专利公开号 200425900。
上述的 K1至 K6b化合物， 其结构如下：
Figure imgf000010_0001
Figure imgf000011_0001
从 PCM部份分离出来羊毛甾烷化合物 K l至 K6b的产量如下表所示。 PCM部份含有约 1 5重量％的羊毛甾烷化合物 K 1至 K6b。
Figure imgf000011_0002
实施例 2
本发明的实施例 1所制备的羊毛甾烷化合物 K 1 , K2, K3及 K4 ,及该 PCM萃取物依下列方法来评估其促进营养素吸收的功效。
人类 Caco-2细胞的培养及营养素的吸收测试
在本实施例的羊毛 烷化合物或 PCM萃取物促进 Caco2细胞对营养 物如： 葡萄糖、 氨基酸、 维生素等的吸收测试中， Caco2细胞被种在聚碳 酸 S旨 ranswell®插入件 (Polycarbonate Membrane Transwell® inserts) (No. 34 14, Corning Incorporated, NY, USA)上，每隔 2~3天换一次细胞培养液，待 14~21天后， Caco2细胞会形成单层膜。 利用 Millicell®-ERS (Millipore EVOM-6; World Precision Instrument, Sarasota, FL, USA)测其上下两侧形成 的电阻值 (trans-epithelial electrical resistance, TEER) , 当 TEER达到 300~450 a cm2 , 并且 Caco2细胞呈现出分化的边缘绒毛状（brush border )后， 即可 用以进行营养物吸收的测试。 Caco2单层膜以含不同浓度的待测物的细胞 培养液培养两天， 其中血清均为活性炭-葡聚糖（charcoal-dextran )处理过 的胎牛血清 (CD-FBS)。 培养两天后， 用 PBS洗涤一次， 改换以不含特定营 养物的緩沖液 （如测定葡萄糖吸收时， 以不含葡萄糖的緩沖液） 培养 1小 时， 接着将该緩沖液更换为含有一预定浓度的该特定营养物及放射线标定 的营养物的新鲜緩沖液， 以追踪该营养物分子穿过 Caco2单层膜的速率。 放射线标定的营养物例如 [l 4C]-D-葡萄糖或 [l4C]-D-2-脱氧葡萄糖， [3H]-L- 精氨酸 (arginine), [3H]-L-色氨酸 (tryptophan), [3H]-叶酸 (folic acid). 同时测 定碳 - 14或氢 -3标定的 -D-木醇糖是用以确定 Caco-2细胞单层膜的完整性 [参考文献: Artursson, P" J Pharm Sci, 1990, 79: 476-482; Ferraris RP, et al., Am J Physiol, 1993, 264: G285-G293. ]。
葡萄糖吸收的分析测定
人类 Caco-2细胞植于 transwell , 待分化形成完整的单层膜。 测定前， 细胞单层膜先以待测物处理两天， 再以不含葡萄糖的緩沖液（组成成分为 80 mM NaCl, 100 mM mannitol, 20 mM Tris-HCl, pH 7.4, 3 mM K2HP04, 1 mM CaCl2, 1 mg/ml BSA )培养 1小时后， 然后将膜上层的緩冲液更换为含 有终浓度为 10 mM葡萄糖， 其中含有 2 μ /mL的碳 - 14标定的 D-葡萄糖 或 D-脱氧葡萄糖 (60 mCi/mmol, American Radiolabeled Chemicals, St. Louis MO, USA)的新鲜緩沖液， 以追踪葡萄糖分子穿过 Caco2单层膜的速率。 在特定时间点自下层緩沖液取出 10 测定其放射强度， 其数值经换算为 葡萄糖浓度，代表该时间点下层緩沖液葡萄糖分子的浓度。除了测定 TEER 值外，此实验同时测定碳 14标定的 -D-木醇糖是用以确定 Caco-2细胞单层 膜的完整性； 而碳 14标定的 -L-葡萄糖则用以测定不经葡萄糖运输蛋白途 径吸收的非专一性背景值。 放射强度数值经换算， 代表该时间点通过 Caco-2细胞单层膜到下层的葡萄糖分子浓度。 就各时间点的下层葡萄糖浓 度对时间点作图， 并以数值分析方法得出一直线， 其斜率代表在该浓度的 待测物处理下， 葡萄糖分子通过 Caco-2 细胞单层膜的平均速率。 实验中 的对照组 (control)是指未以待测物处理所得的数据。 以上测定方法主要参 考文献如下： Kimura T. et al., J. Pharm. Pharmacol., 2001 , 54, 213-219. 氨基酸吸收的分析测定
氨基酸如精氨酸及色氨酸的吸收， 是以 Caco2 单层膜以含不同浓度 的待测物的细胞培养液， 培养两天后， 用 PBS洗涤一次， 改换以不含该氨 基酸的緩沖液（测精氨酸的成分为： 137 mM NaCl, 10 mM Hepes pH 7.4, 0.3 mMNaH2P04, 0.3 mM K2HP04, 5.4 mM C1, 2.8 mM CaCl2, 1 mM MgS04, 10 mM glucose; 而测色氨酸吸收时的緩沖液组成为： 137 mM 氯化胆碱 (choline chloride), 10 mM Hepes pH 7.4, 0.6 mM KH2P04， 5.4 mM KC1, 2.8 mM CaCl2, 1 mM MgS04, 10 mM葡萄糖）， 培养 1 小时后， 利用氢 -3标定 的氨基酸 (L-3H-amino acid)来观察 Caco2单层膜在不同浓度的待测物作用 下对氨基酸的吸收是否有所影响。 除了测定 TEER值外， 此实验同时测定 氢 -3标定的 -D-木醇糖是用以确定 Caco-2细胞单层膜的完整性。 放射强度 数值经换算， 代表该时间点通过 Caco-2 细胞单层膜到下层的氨基酸分子 浓度。 就各时间点的下层氨基酸浓度对时间点作图， 并以数值分析方法得 出一直线， 其斜率代表在该浓度的待测物处理下， 氨基酸分子通过 Caco-2 细胞单层膜的平均速率。 实验中的对照组 （control ) 是指未以待测物处理 下所得的数据。 以上测定方法主要参考文献如下： Pan M., et al., Am J Physiol. Gastrointest Liver Physiol 1995, 268: G578-G585.
叶酸吸收的分析测定
将人类 Caco-2细胞植于 10-cm培养盘， 待约二星期之后分化完成后即 进行测定。 测定前， 细胞先以各种浓度的待测物处理两天， 再以不含叶酸 的緩沖液 (Folate transport incubation buffer (pH6.0): Hank's Balanced Salt Solution (HBSS), supplemented with 0.14 g L CaCl2, 0.1 g/L MgCl2, and 0.1 g/L MgS04)培养 1小时后， 然后更换为含有终浓度为 5 μΜ叶酸， 其中含有 2 Ci/mL的氢 -3标定的叶酸 （3，5,7,9-3H-folic acid, 25 mCi/mmol, ARC, St. Louis, MO, USA) 的新鲜緩沖液。 在特定时间点将细胞以 PBS洗过， 然后 以 0.2 mL的 0.2 N NaOH使细胞溶解打破， 刮下集中离心。 取出定量上清细 胞液用以测定蛋白质浓度， 并取上清细胞液 20 测定叶酸分子吸收进入 并累积于 Caco2细胞内的浓度。 计算得数值代表该时间点 Caco-2细胞中相 等蛋白质量的细胞液内所含有的叶酸分子浓度。 就各时间点的细胞内的叶 酸浓度对时间点作图， 并以数值分析方法得出一直线， 其斜率代表在该浓 度待测物处理下叶酸分子被吸收进入 Caco-2细胞的平均速率。 实验中的对 照 (control)是指未以待测物处理下所得的数据。 以上测定方法主要参考文 献^口下： Dudeja PK., et al., Am J Physiol Gastrointest Liver Physi , 2001 , 281 ( 1 ): G54 - G60.
结果
( 1 ) 实施例 1 的 PCM萃取物对促进 2-脱氧葡萄糖被肠细胞 (Caco-2) 吸收的影响被示于表一。表一显示该 PCM萃取物在低剂量下（0.0033 g/cc) 具有显著的促进 2-脱氧葡萄糖被肠细胞 (Caco-2)吸收的功效。
I I 表一： PCM萃取物对 2-脱氧葡萄糖 (2-deoxyglucose)被肠细胞 (Caco-2)吸收 的影响
Figure imgf000014_0001
*实施例 1 的 PCM萃取物被调整成羊毛甾烷化合物浓度为 0.033 g/cc (PCM1 )及 0.0033 g/cc (PCM2)
(2) 实施例 1所制备的羊毛甾烷化合物 K1 , K2，K3及 K4对葡萄糖营 养素被肠细胞 (Caco-2)吸收的影响被示于表二及图 1至 3。如表二所显示羊 毛甾烷化合物 K2, 3及 K4在低剂量下（1 μΜ - 0.001 μΜ)对葡萄糖营养素 被肠细胞吸收有促进作用。 如图 1至图 3所显示， Κ2-Κ4能促进吸收葡萄 糖而且吸收呈线性关系， 此种线性关系表示茯苓成分透过影响或增加运输 蛋白而使葡萄糖被吸收能力增力。。 K1虽无作用但其为 Κ2的前药（prodrug), 易于在肠中或血中被转化成 k2而成为有效物质。
表二： 羊毛甾烷化合物对葡萄糖被肠细胞 (Caco -2)吸收的影响 化合物 1 输送速率 2 百分比
影响
(μΜ) (nmol/min) (%)
对照组 3.0420 ± 0.0605 100.00 ―
0.001 3.9220 ± 0.0388 128.93 个
7 0.01 4.1350 ± 0.0688 135.93 个
0.1 3.0860 ± 0.1 104 101.45 -
1.0 2.9690 ± 0.0974 97.60 -
0.001 2.6170 ± 0.1982 86.03 -
V 1 0.01 3.5970 ± 0.1285 1 18.24 个
0.1 3.4030 ± 0.1794 1 1 1.87 个
1.0 3.3490 ± 0.1940 1 10.09 个
0.001 3.7320 ± 0.1447 122.68 个
0.01 4.1730士 0.0989 137.18 γ 个
0.1 4.7450 ± 0.1745 155.98 个
1.0 3.9740 ± 0.2231 130.64 个
1.结果是以平均值土 SD (n=3)表示
(3) 实施例 1 所制备的羊毛甾烷化合物 K 1 , K2, K3及 K4对氨基酸 (精氨酸及色氨酸） 营养素被肠细胞 （Caco-2 ) 吸收的影响被示于表三及 表四，及图 4至 10。如表三所示， K l , K2, K3及 K4在低剂量下（1 μηι - 0.001 μΜ)下对精氨酸被肠细胞 （ Caco-2 ) 吸收有促进作用。 如表四所示 Kl， K3 及 K4低剂量下（1 μπι - 0.001 μΜ)下对色氨酸被肠细胞 ( Caco-2 )吸收有促 进作用。 而有意义的是肠细胞在低剂量羊毛甾烷化合物下（1 μΐΏ - 0.001 μΜ) 下能促进此二种氨基酸的吸收而且吸收是呈线性关系， 如图 4至 10所示。 此线性关系表示这些羊毛 烷化合物透过影响或增加运输蛋白使此二种 氨基酸被吸收能力增加。 表三：羊毛 烷化合物 Kl, K2, K3及 K4对精氨酸营养素被肠细胞 (Caco-2) 吸收的影响
输送速率 1 百分比 t
化合物（μΜ) 影响
(nmol/min) (%)
对照组 6.1390 ± 0.6935 100.00 -
0.001 8.5490 ±0.6102 139.26 个
K1 0.1 7.1640 ± 0.3526 113.37 个
1.0 7.8920 ± 0.5695 128.56 个
0.001 8.4400 ± 2.3890 137.48 个
Κ2 0.1 8.7740 ±2.7020 142.92 个
1.0 8.5550 ± 1.3090 139.35 个
0.001 7.6100士 0.3255 123.96 个
Κ3 0.1 9.0900 ± 0.9357 148.07 个
1.0 6.9050 ±0.0814 112.48 个
0.001 9.7980 ± 0.0843 159.60 个
Κ4 0.1 7.6420 ± 1.3290 124.48 个
1.0 6.8730 ± 1.0980 111.96 个
1.结果是以平均值土 SD (n
表四：羊毛甾烷化合物 Kl， K2, K3及 K4对色氨酸营养素被肠细胞 (Caco-2) 吸收的影响
输送速率 2 百分比
化合物 1 (μΜ) 影响
(nmol/min) (%)
对照组 17.780 ± 0.501 100.00 -
0.001 23.550土 1.304 132.45 个
K1 0.01 24.160士 1.063 135.88 个
1.0 21.390 ±0.886 120.30 个
0.001 21.520士 1.298 121.03 个
Κ3 0.01 24.220 ±2.257 136.22 个
1.0 21.610±2.419 121.54 个
0.01 15.720 ±2.575 88.41
Κ4 0.1 27.390 ± 1.818 154.05 个
1.0 27.200 ± 1.370 152.98 个
1. K2对色氨酸被肠细胞 （Caco-2) 吸收未产生显著的影响。
2.结果是以平均值士 SD (n=3 )表示
(4) 实施例 1所制备的羊毛甾烷化合物 K1,K2,K3及 K4对维他命叶 酸营养素被肠细胞 （Caco-2) 吸收的影响被示于表五及图 11至 14。 如表 五所示， K1,K2, K3及 K4在低剂量下（0.1 μΜ - 0.001 μΜ)对叶酸被肠细胞 (Caco-2)吸收有促进作用。 而有意义的是肠细胞在低剂量羊毛 烷化合物 下（1 μπι- 0.001 μΜ)能促进叶酸的吸收而且吸收是呈线性关系， 如图 11至 14所示。此种线性关系表示这些羊毛 烷化合物是透过影响或增加运输蛋 白而使叶酸吸收能力增加。 表五： 羊毛甾烷化合物 K1, 2, K3及 K4对叶酸营养素被肠细胞（Caco-2) 吸收的影响
输送速率 1 百分比
化合物（μΜ) 影响
(nmol/mg/min) (%)
对照组 0.0759 士 0.0169 100.00 -
1 ^ 1 0.001 0.1105 士 0.0157 145.59 个
IV 1
0.1 0.1042 ± 0.0153 137.29 个 tf 0.001 0.1094 土 0.0194 144.14 个
0.1 0.1083 士 0.0280 142.69 个
1^ 0.001 0.0864 土 0.0157 113.83 个
0.1 0.0852 ± 0.0174 112.25 个
士
τ ' A 0.001 0.0665 0.0126 87.62 -
0.1 0.0852 土 0.0174 112.25 个
1.结果是以平均值士 SD (n=3)表示 实施例 3
茯苓药材 100公斤以 800公斤水煮沸 3小时后， 静置冷却至 50°C, 以 5NNaOH调节溶液至 pH 11， 再搅拌溶液 3小时。 接着用离心机分离液 体和固体， 固体用 800公斤水加入， 同上述方法， 用 NaOH调至 ρΗ 11、 搅拌和离心机分离， 去掉固体。 合并两次液体， 在 50°C将液体真空浓缩至 100公斤溶液， 再加入 3NHC1至 pH6.5, 产生沉淀物。 分离出该沉淀物， 再以 40 L H20清洗， 接着用离心机分离出沉淀物， 加入 8 L水喷雾干燥 (spray dry), 得到约 380 g粉末。 再以 4 L酒精萃取该粉末三次， 合并萃 取液并浓缩可得 238.9克酒精萃取物， 再经过 HPLC分离该萃取物， 每克 该萃取物可得主成分为 K2 185.93mg, K320.34 mg, K4 15.82 mg及少量 成分 K14.52mg, 即萃取物每克约含 226.07 mg羊毛 烷 （ lanostanes )。 实施例 4:胶嚢制备
依下列组成制备含有实施例 1所制得的 PCM成份的胶嚢：
Figure imgf000019_0001
将 PCM与硅铝酸钠分别以 # 80 目（mesh)筛网过筛， 马铃薯淀粉以 # 60 目筛网过筛， 硬脂酸镁以 # 40 目筛网过筛后， 置入混合机搅拌均匀， 接着填充入壹号空胶囊，每颗胶嚢含有约 1.68 mg (0.42 wt%)的有效成份有 效成份 Kl -K6。

Claims

权 利 要 求
1 . 一种使用具下列化学式 (I)的羊毛甾烷或其医药可接受的盐作为活 性成分在制备促进营养素被一哺乳类动物吸收药物中的用途：
Figure imgf000020_0001
(I)
式中 R1为H或 CH3 ; R2为 OCOCH3， =0或 OH ; R3为 H或 OH ; R4为 -C(=CH2)-C(CH3)2Ra, 其中 Ra为 H或 OH, 或 -CH=C(CH3)-Rb, 其中 为(： 或 CH2OH; R5为 H或 OH; 及 1^为（： 或 CH2OH。
2. 如权利要求 1的用途， 其中该羊毛甾烷 (I)具有具下列化学式：
Figure imgf000020_0002
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更正页（细则第 91条)
Figure imgf000021_0001
3. 如权利要求 1的用途，其含有 (U-20重量。 /0的羊毛甾烷 (I)或其医药可 接受的盐。
4. 如权利要求 1的用途， 其中该药物为口服的。
5. 如权利要求 1的用途， 其中该哺乳类动物为人类。
6.如权利要求 1的用途， 其中包含使用一茯苓萃取物作为该活性成分， 该茯苓萃取物包含 1-60重量％的具权利要求 1的羊毛甾烷 (I)且实质上不含 开环羊毛甾烷。
19
更正页（细则第 91条)
7. 如权利要求 6的用途， 该茯苓萃取物是由包含下列步骤的方法所制 备：
a)以一水、 甲醇、 乙醇或它们的混合溶剂萃取茯苓菌的代谢物， 茯苓 菌的发酵产物或茯苓菌菌丝；
b)浓缩步骤 a)萃取所获得的液体；
c)将步骤 a)所获得的浓缩物导入一硅胶管柱；
d)以一低极性洗脱剂（duent )洗脱该硅胶管柱， 及收集所产生的洗出 液 ( eluent ); 及
e)浓缩步骤 d)的洗出液。
8. 如权利要求 7的用途， 其中从步骤 e)所获得的洗出液的浓缩物以硅 胶薄层层析法分析具有一层析值 (Rf) > 0.1 , 其中以紫外光灯及碘作检测， 展开液为二氯甲烷： 曱醇 = 96:4。
9. 如权利要求 7的用途， 其中步骤 a)的萃取所使用的溶剂为 95%酒精。
10.如权利要求 7的用途， 其中步骤 a)包含以沸水萃取茯苓菌的代谢物， 茯苓菌的发酵产物或茯苓菌菌丝； 加入一碱至该萃取水溶液至其 pH值 9-11； 分离出该碱性水溶液； 加入一酸至该碱性水溶液至其 pH值 4-6, 以产 生沉淀物； 分离出该沉淀物； 再以酒精萃取该沉淀物， 并分离出萃取液体。
11.如权利要求 9或 10的用途， 其中步骤 b)的浓缩物被进一步以一体积 比为 1 : 1的 95% v/v曱醇水溶液及正己烷的两相溶剂萃取； 分离出甲醇层； 及浓缩该甲醇层， 所获得的浓缩物被用作为步骤 c)的硅胶管柱的进料。
12.如权利要求 7的用途， 其中步骤 d)的低极性洗脱剂为体积比为 96.5:3.5的二氯曱烷及曱醇的混合溶剂。
13.如权利要求 6的用途， 其中该茯苓萃取物包含 5-35重量。 /。的羊毛甾 烷 (1)。
14.如权利要求 6的用途， 其中该羊毛甾烷 (I)具有具下列化学式：
Figure imgf000022_0001
20
更正页（细则第 91条)
Figure imgf000023_0001
Figure imgf000023_0002
更正页（细则第 91条)
Figure imgf000024_0001
15.如权利要求 1或 6的用途， 其中该药物进一步包含一营养素。
16.如权利要求 14的用途， 其中该营养素为葡萄糖、 氨基酸、 维生素或 其组合。
1 7.如权利要求 15的用途， 其中该药物被用于老年人营养状况改善。
18.如权利要求 1 5的用途， 其中该药物被用于瘦弱小孩身体改善。
19.如权利要求 15的用途，其中该药物被用于促进营养素被工作压力大 的人或经常熬夜赶工作的人所吸收。
20.如权利要求 15的用途，其中该药物被用于促进营养素被运动量大之 运动员或工作量大的人所吸收。
21 .如权利要求 1 5的用途，其中该药物被用于手术后的病人或接受化疗 后的癌症病人。
22.如权利要求 1 5的用途， 其中该药物被用于患有病毒性腹泻的病人。
22
更正页（细则第 91条)
PCT/CN2008/000749 2008-04-11 2008-04-11 促进营养素被吸收的医药组合物及茯苓萃取物 WO2009124420A1 (zh)

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CN101959518B (zh) 2013-04-10
KR101488332B1 (ko) 2015-02-02
CA2716948C (en) 2012-09-25
CA2716948A1 (en) 2009-10-15
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