TW201028178A - Ganoderma extract with high amounts of ganoderic/lucidenic acids and method for producing the same - Google Patents

Abstract

Disclosed herein is a Ganoderma extract having at least 50% (wt%) of Ganoderic acids/Lucidenic acids, and a method for producing the same.

Description

201028178 - VI. Description of the invention: [Technical field of the invention] The present invention generally relates to a method for preparing a dried ganoderma lucidum powder, in particular, a method for preparing a dried ganoderma lucidum powder bundle having a high amount of ganoderic acid/anthuric acid. Prior art ❹ medicinal fungi are widely used as health foods, among which Ganoderma lucidum is a large gamma-like ganoderma lucidum, which refers to both Ganoderma lucidum and Ganoderma lucidum. According to taxonomy, UGanoderma lucidum contains only vestibules. {G_derma and other agaric caps are reddish brown Ganoderma lucidum. As for Zizhi, it is represented by G.Slnense, and its umbrella cover is purple-black or dark brown. Because Lingzhi contains a variety of active ingredients, it is often used in South East Asia as a medicine for Ganoderma lucidum. These active ingredients include ganoderma triterpenoids, polysaccharide proteins, nucleic acids, polypeptides, and phytosterol compounds. Ganoderma lucidum triterpenoids are the most important active ingredients of Ganoderma lucidum, including Ganoderma lucidum A, B, C, D, E, F, G and strontium, as well as other Ganoderma lucidum, Ganoderma aldehyde and lanosterane compounds. Take the class. A number of literatures have been published to date to prove that these active ingredients have anti-tumor and immunity (see U.S. Patent No. 4, No. 51,314, No. 6,613,754, No. 1) and antioxidant effects, and therefore various extracts from Ganoderma lucidum fruit bodies have also been proposed. The most common way to extract these active ingredients is to extract the harvested, fresh or dried Ganoderma lucidum raw material using a polar solvent such as water. For example, the Chinese Patent Application No. 96116559 6 discloses a method for preparing an active ganoderma lucidum polysaccharide and a high amount of ganoderic acid, which comprises crushing, water extracting, filtering and concentrating the ganoderma lucidum raw material to prepare a desired 201028178 f. According to the specification of the patent specification, the extraction rate of the method disclosed therein is about 40%, and the pharmacological activity of the Ganoderma lucidum polysaccharide extracted by the special method is improved by about 3〇〇~5〇 compared with the conventional preparation method. 〇%, but it does not mention the efficiency of this method for improving the content of Ganoderma lucidum, the most important active ingredient of Ganoderma lucidum. Therefore, there is a need for an improved method for preparing a dried ganoderma lucidum powder which not only achieves a high extraction rate but also preserves the most important active ingredient in the raw material of the ganoderma lucidum (ie, fruit body) - a ganoderic acid/anthoic acid compound to dry Ganoderma lucidum powder contains at least 50% by weight of ganoderic acid/erythric acid. SUMMARY OF THE INVENTION The present invention provides a dried ganoderma lucidum powder having a high amount of Ganoderma lucidum triterpenoid active ingredients (including ganoderic acid/erythric acid), and a preparation method thereof. The method of the present invention is characterized by comprising the steps of: (a) extracting a crude extract of a ganoderma lucidum fruit body with a first polar solution; (b) in a fast liquid chromatography chromatography column at a concentration of 20%~80°/. a second polar solution (volume ° /.) to extract the ganoderic acid / gibberellic acid compound in the extract obtained in the step (4); and (c) subjecting the extract obtained in the step (b) to freeze-drying, The dried ganoderma powder "In one embodiment, the first and second polar solutions comprise ethanol. The crude extract of Ganoderma lucidum fruit body which can be used in the method of the present invention is preferably prepared by a method comprising the following steps: (1) fresh ganoderma lucidum which will not be dried for more than 4 to 8 hours after harvesting The body or the Ganoderma lucidum fruit body which has been dried and pulverized after harvesting is immersed in a polar solution for about 2 to 16 hours; (2) the temperature of the polar solution containing the fresh Ganoderma lucidum fruit body is raised to not more than about 1 〇 〇t, and stirring for about 1 to 4 hours; and (3) subjecting the liquid obtained in the step (2) to concentration under reduced pressure and cooling and drying. In a preferred embodiment, the ganoderma lucidum comprises a combination of Ganodder tsugae and genus Ganoderma lucidum, which is not more than 4 to 8 hours after harvesting, and is not dried. Fresh pine yew or Ganoderma lucidum is preferred. In one example, the polar solution used in this method step comprises ethanol. In another example, the extraction rate of the process is about 2 〇 0 / 〇 (% by weight) and the resulting crude extract of Ganoderma lucidum fruit body contains about 2 to 4% of a Ganoderma lucidum compound. The method step (b) of the present invention further comprises the following treatment: (bi) at a concentration of between 20 and 40. /. (% by volume) of the ethanol solution is subjected to a first flushing; and (b2) is at a concentration of 50 to 70 Å/. The (vol%) ethanol solution was subjected to a second flush. In one example, the fast liquid chromatography column comprises a C-18 reverse phase chromatography column. In a preferred embodiment, the extraction rate of the method of the present invention is about 1 〇 to 3 〇%, and the resulting dried $zhi powder contains at least about 5% by weight of a ganoderic acid/anthoic acid compound comprising at least 6 ganoderic acid. / 芝ic acid compounds (including GA, GB, GC, GN, GD2, and GE2) or at least 9 ganoderic acid/anthodic acid compounds containing ganoderic acid A (Ga), ganoderic acid A Derivatives, Ganoderma D (GD) and combinations thereof, and derivatives of Ganoderma acid a include gb, GC, GD, GE, GC5, GC6 and GG. Another object of the present invention is to provide a dried ganoderma lucidum powder prepared by the method and having a high amount of a ganoderic acid/anthoic acid compound. In one example, the dried ganoderma lucidum powder prepared according to the method contains at least about 50% by weight of a ganoderic acid/anthoic acid compound. 201028178 [Embodiment] Extraction of high dry extracts According to the present invention, there is provided a method for recognizing an active compound from a medicinal fungus, and a method for preparing a Ganoderma lucidum three-type active ganoderma lucidum powder by the method. In the present invention, a method for preparing a dry invention having a high content (i.e., a ganoderma acid/anthoic acid compound) is provided, which is characterized in that: • (a) extracting a crude extract powder of a ganoderma lucidum fruit body with a first polar solution. (b) in a fast liquid chromatography column, 'Λ, a second polarity solution between ～70% (% by volume) in the morning to extract the extract from step (a) The ganoderma acid/acanthoic acid compound; and (c) the freeze-dried extract of the extract obtained in the step (b) to obtain the dried ganoderma lucidum powder. The Ganoderma lucidum fruiting body φ crude extract powder which can be used in the method of the present invention can be produced in any conventional manner. In a preferred embodiment, the Ganoderma lucidum fruit body crude extract powder is prepared in the following manner: First, the Ganoderma lucidum fruit body is subjected to a rough stroke with a polar solvent. The Ganoderma lucidum fruiting body referred to herein includes the fruiting body of fresh Ganoderma lucidum, especially fresh Ganoderma lucidum fruiting bodies which are not more than 4 hours after harvesting, preferably no more than 5, 6, 7 or 8 hours and have not been dried; Or a Ganoderma lucidum fruiting body that has been harvested, dried, and comminuted in a suitable manner (including 'mechanical grinding, cutting, vacuum pulverization, etc.). The ganoderma lucidum includes Gawderwa or Gawoi/erwifl /wcz'dww or a combination thereof. The crude extraction step is generally carried out in two stages: first, at room temperature, the above-mentioned Ganoderma lucidum fruit body is completely Soak in a polar solvent at least about 2, 3, 4, 5, 6, 7, 8, 9, 201028178 1 liquid 0: two up to 13 not 1 2 15 or 16 hours; then, raise this soak ΐ = 7 The temperature of the second f?, preferably not more than 9 〇 n ^ oc or 50 c, is preferably about 50 ° C, and the mixing of the soaking liquid is at least about 1, 2, 3 ^. 3 or 4 hours, optimally at least two: examples of the dibasic solvent include, but are not limited to, water and alcohols such as ethanol, propanol, isopropanol, n-butanol, iso-butanol, etc. in the extraction step After that, the liquid (ie, the extract) can be collected at any appropriate ^ ^ ^ ^ ^

And dry. The condensation and drying can be carried out in any conventional manner. In the example, the extract is concentrated under a reduced pressure environment, such as a pressure environment, and the amount of solids 3 in the extract is up to 20 /. ^, that is, stop this concentration process. Next, the concentrate is dried, and J is cooled at 30 C to -5 0 C; According to the method of the present invention, a crude extract powder of Ganoderma lucidum fruit body can be obtained, wherein the Ganoderma lucidum triterpenoid content containing ganoderic acid/erythric acid is between about 2% and 4%. In the practice of the method of the present invention, firstly, an appropriate amount of the crude extract powder of Ganoderma lucidum fruit body prepared in the above manner is mixed with a first pole steep/liquid mixture in a container, and the powder weight: polarity is mixed. The solution volume is mixed in a ratio of about 1:10 to 1.30. For example, an i kilogram of Ganoderma lucidum fruiting body crude extract powder can be mixed with a first polar solution of about 1 liter liter to 3 liters liter. The first polar solution suitable for use in this step includes, but is not limited to, water and alcohols such as ethanol, propanol, isopropanol, n-butanol, iso-butyrate, and the like. In the example, this first polar solution is 5% by volume of ethanol. Next, the present compound is at a temperature of not more than 1 〇〇〇c, preferably not more than 9 〇e > c, 8 〇 C, 70 ° C, 60 ° C or 50 ° C, preferably about 50 °. Stir at a temperature of C for at least about 30, 40, 50, 60, 70, 80 or 90 minutes, preferably at least 7 201028178. About 6 minutes. Thereafter, the residue was removed by filtration or centrifugation, and the liquid was collected and the filtrate was concentrated until it was gelatinous. This concentration step can be performed in any conventional manner. Next, an appropriate amount of the first polar solution was successively added to the gel, and the gel was dissolved back until it was completely dissolved. Next, each of the active components of the ganoderma lucidum in the solution was separated by rapid liquid chromatography (flas c〇lumn chromatography). In operation, the remelted colloidal solution is first loaded into a fast liquid chromatography column; then, at a flow rate of about 80 to 180 ml/min (preferably about 8 Torr, 9 Torr, ❹ 100, 110, 120, 130, 140, 150, 160, 170 or 18 〇 ml / min, more preferably about 100, 110, 120, 130, 140 or 150 ml / min ;), to a concentration of 20% ~ 70% (preferably 20%, 30%, 35%, 40%, 450/., 50%, 55% '60% ' 65%, 7〇%, 75% or 8〇%, more preferably about 35%, 4%, 45%, 50%, 55%, 6%, 65% or 7% of the second polar solution is rinsed and the resulting extract is collected. In one example, the fast liquid chromatography column is a C 18 reverse phase chromatography column. In another example, the stripping treatment can be carried out in two stages. First, at a flow rate of about 1 〇〇 ml/min, about 2 〇 4 4 〇 β /. The first extraction is carried out in an ethanol solution of (products/(>); then, the second extraction is carried out in an ethanol solution having a concentration of 50 to 70% by volume. The extracts are collected together and concentrated under reduced pressure and lyophilized to obtain the desired dry powder of ganoderic acid in a yield of about 1 〇 to 3 〇%. This concentration and drying can be carried out in any conventional manner. In one example, the extract is concentrated under a reduced pressure environment, for example, under a pressure of about 〇·〇〇rr, and the solid content in the extract is up to 2%, ie The concentration treatment is stopped. Next, the concentrate is dried, for example, lyophilized at _3 〇 * > C 〜 5 〇〇 C. According to the method of the present invention, dried ganoderma lucidum powder can be obtained which contains ganoderic acid 8 201028178 • / The content of the triterpenoids of Ganoderma lucidum is at least about 50% by weight. Therefore, the present invention also provides a dry Ganoderma lucidum powder prepared according to the method of the present invention, and wherein the total amount of cinnamic acid/Ganoderma lucidum is The content is up to at least about 50% by weight. In a preferred embodiment The dried ganoderma lucidum powder obtained contains at least 5% by weight of 9 kinds of ganoderic acid compounds, and the 9 ganoderic acid 7 zhizhi acid compounds comprise ganoderic acid A (GA), a derivative of ganoderic acid A, and ganoderma lucidum Acid D (GD) and combinations thereof, and derivatives of Ganoderma A include GB, GC, GD, GE, GC5, GC6 and GG. In another preferred embodiment, the dried Ganoderma lucidum powder is contained At least 51% by weight of the erythroic acid compound, including GA, gb, GC, GN, GD2, and GE2. Hereinafter, preferred embodiments of the present invention will be described by way of examples. Example 1 Preparation of Ganoderma lucidum Fruiting Body Crude extract powder - 板 黧 黧 :: extract 狢 狢 参 参 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子In the extraction tank, add an appropriate amount of 95% alcohol to the extraction tank until the ganoderma lucidum fruit body is completely immersed in the alcohol solution, soak it at room temperature for about 16 hours. Heat the mixture to raise the temperature of the solution to About 5G ° C ' and extract at a rate of 30 rpm for 3 hours. The extract is pumped Drain into a vacuum concentration tank, concentrate at 65 〇, 45 lt 匸 under reduced pressure and recover alcohol. When the solid content is 2〇%~25%, the concentration is stopped. The concentrate is taken out at _30°. C~-5〇V freezes to a solid at low temperature. After high temperature~oi t called freeze-drying for 48 hours, it can obtain 45 kg of yellow-brown powder with a yield of 45%. 9 201028178 According to high performance liquid chromatography method ( HPLC) (J. Chin. Chem. Soc. 1999 46: 47-51) analysis shows that the total triterpenoid content of the product is 20%, and according to the conventional method (J_Food Drug Anal· 2003 1 1:195-200) The measurement includes a total of nine kinds of ganoderic acid in a group consisting of a derivative of Ganoderma lucidum A, a derivative of Ganoderma lucidum A (including GB, GC, GD, GE, GC5, GC6, and GG), Ganoderma lucidum D, and combinations thereof. The content of these nine kinds of Ganoderma lucidum was found to be about 2%. The results of the analysis of the method are shown in the high performance liquid chromatography (HPLC) chart of Figure 1. The above nine kinds of Ganoderma lucidum acid structure are as follows

R! Rj R3 R4 GA 0 α-Η,β-ΟΗ α-ΟΗ,β-Η Η GB α-Η,β-ΟΗ α-Η,β-ΟΗ Ο Η

GC α-Η,β-ΟΗ α-Η,β-ΟΗ α-ΟΗ,β-Η GD GD Ο α-Η,β-ΟΗ Η Η GE Ο Ο Η Η GC5 Ο α-ΟΗ,β-Η Ο ΟΗ GC6 α-Η,β-ΟΗ Ο ΟΗ GG GG α-Η,β-ΟΗ α-ΟΗ,β-Η Η ΟΗ

GA: Ganoderic acid A

Ganoderenic acid D

Structures of nine ganoderic acids 1.2 Ganoderma lucidum fruit body (YK-02) fe extract powder East in addition to the Ganoderma lucidum fruit body (YK-02) (strain name: YK-02, 10 201028178 / wcWwm) to replace the pine sage In addition to the strain name: YK-01, iwgae, the crude extract of the Ganoderma lucidum fruit body (YK-02) was prepared in accordance with the method described in the above Example i^. The HPLC chromatogram of the obtained crude extract is shown in Fig. 3, which contains six kinds of erythric acid compounds (including GA, GB, GC, GN, GD2 and GE2) in an amount of about 4%, and its chemical structure is shown in the following figure:

LA = Lucidenic acid A Example 2 Preparation of dried Ganoderma lucidum powder _ The crude extract of Ganoderma lucidum fruit body (YK-01) as in Example 1.1 In a container, 100 g of the crude extract of Ganoderma lucidum fruit body prepared according to the method of Example 1 was mixed with 1 liter of ethanol (concentration: 50% by volume), and stirred at 50 ° C for about 60 minutes. Filtration (or centrifugation), removal of the solids in the filtrate was concentrated under reduced pressure to form a gel. The solution was reconstituted by adding 500 ml of ethanol to the colloidal solution, and then the solution was loaded into a fast liquid chromatography column (ie, a C-18 reverse phase chromatography column). The solution was eluted with a 35% ethanol solution at a flow rate of about 100 ml/min, followed by 11 201028178. The extraction was continued with a 70% ethanol solution at a flow rate of about 150 ml/min, and the two rinses were collected. Liquid, about 3 liters. After the collected extract was concentrated and lyophilized, about 15.6 g of a pale yellow powder was obtained. After HPLC analysis (shown in Figure 2), it was found that the polar signal of the front end of the crude extract of the Ganoderma lucidum fruit extract of Example 1.1 (ie, Figure 1) had disappeared, leaving most of the Ganoderma lucidum. There are 9 kinds of acid compounds, and the structure thereof is as shown in the above Example 1.1 and the total content is as high as 50.2%. H is the dried Ganoderma lucidum powder prepared from the extract of the Ganoderma lucidum fruit body (YK-02) group of Example 1.2, and the crude extract of Ganoderma lucidum (YK-02) of Example 1.2 is used as the raw material. Dry Ganoderma lucidum powder was prepared in substantially the manner described in Example 2.1. Approximately 3 liters of the extract was also obtained after the implementation of the rapid liquid chromatography chromatography, and the collected extract was concentrated and lyophilized to obtain about 13.6 g of a pale yellow powder. After HPLC analysis (shown in Figure 4), it was found that the polar signal of the front end of the crude extract of the genus Ganoderma lucidum which was originally present in Example 1.2 (i.e., Figure 3) had disappeared, leaving most of the A total of 6 phthalic acid compounds, the structure of which is as shown in the above Example 1.2, and the total content is as high as 5 1 · 2 %. Although the present invention has been disclosed above in a preferred embodiment, it is not intended to limit the present invention. The invention is to be understood as being limited by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS 12 201028178 ^ Fig. 1 is an HPLC spectrum of a crude extract of Ganoderma lucidum fruit body according to Example I of the present invention; Fig. 2 is a raw material of the crude extract of Ganoderma lucidum fruit body of Example I1. HPLC spectrum of dried Ganoderma lucidum powder prepared according to the method described in Example 2.1 of the present invention; FIG. 3 is an HPLC spectrum of the crude extract of Ganoderma lucidum fruit body according to Example 1 of the present invention; and FIG. For the crude extract of the Ganoderma lucidum fruit body of Example 1, 2, the HPLC spectrum of the dried Ganoderma lucidum powder prepared according to Example 2.2 (4) of the present invention. [Explanation of main component symbols]

Claims (1)

201028178 VII. Patent application scope. ι_ A method for preparing dried ganoderma lucidum powder, comprising: (4) extracting a crude extract powder of a ganoderma lucidum fruit body with a -polar solution; (b) in a fast liquid chromatography column Extracting the ganoderic acid/acanthoic acid compound in the extract obtained in the step (a) with a second polar solution having a concentration between 20% and 7% by volume (v); and (c) stepping The dried extract of the hybrid (b) is freeze-dried to obtain the dried ganoderma lucidum powder. 2. The method according to claim 1, wherein the crude extract of the Ganoderma lucidum fruit body is prepared by a method comprising the following steps: (1) no more than 4 to 8 hours after harvesting and not dried. The fresh ganoderma lucidum + entity or the dried and pulverized Ganoderma lucidum fruit body after soaking in the "polar solution" liquid for about 2 to 16 hours; (7) raising the temperature of the polar solution containing the Ganoderma lucidum fruit body to not exceed About 100 ° C, and stirring for about 4 hours; and (3) The liquid obtained in the step (2) is subjected to vacuum concentration and freeze-drying treatment. 3. The method of claim 2, wherein the fresh ganoderma lucidum comprises Ganoderma lucidum U sei ae, Ganoderma lucidum (Gaw 〇 β β 岣 岣 岣 。 。 。 4. 4. 4. 4. 4. 4. 4. 4. 4. 4. 4. 4. 4. The method of the present invention, wherein the polar solution comprises ethanol. The method of claim 2, wherein the method for preparing the crude material of the ganoderma lucidum fruit body has an extraction rate of about 2% and is prepared. The crude extract of the genus Ganoderma lucidum has about 2 to 4% of a ganoderic acid/anthoic acid compound. The method of claim 1, wherein the first polar solution or the second polarity The solution comprises ethanol. wherein step (b) comprises 7. The method as described in claim 6 ❹ the following steps: (bl) at a concentration of the second extraction; and (b2) at a concentration between the second extraction 20~40% (volume./〇) of the ethanol solution is subjected to a first 50~70% (% by volume) ethanol solution for a second 'where the fast liquid phase' wherein the method is extracted 8 as claimed The method of the first aspect of the present invention comprises a chromatography column comprising C-18 The colorimetric chromatography column is as described in the first aspect of the patent application. The method of the method of claim 1 wherein the drying method comprises at least about 50%. (% by weight) of ganoderic acid/anthoic acid compound 15 201028178 GC, GN, GD2 and GE2. 12. The method of claim 10, wherein the ganoderic acid/anthoic acid compound comprises at least 9 ganoderic acid compounds Wherein the ganoderic acid compound comprises Ganoderma lucidum A, a derivative of Ganoderma lucidum A, Ganoderma lucidum D, and a combination thereof, wherein the derivatives of Ganoderma lucidum A include GB, GC, GD, GE, GC5, GC6 and GG 〇-13 A dry Ganoderma lucidum powder containing at least about 50% by weight of a ganoderic acid/anthoic acid compound prepared by the method of claim 1 of the patent application. The dried ganoderma lucidum powder containing at least about 50% by weight of a ganoderic acid/anthoic acid compound, wherein the ganoderic acid/anthoic acid compound comprises at least 6 phthalic acid compounds, including GA, GB, GC, GN, GD2 and GE2. 15. The dry ganoderma lucidum powder containing at least about 50% by weight of a ganoderic acid/anthoic acid compound according to claim 13 wherein the ganoderic acid/anthoic acid compound comprises at least 9 ganoderic acid compounds, wherein the ganoderic acid The compound comprises Ganoderma A, a derivative of Ganoderma A, Ganoderma D, and a combination thereof, wherein the derivatives of Ganoderma A include GB, GC, GD, GE, GC5, GC6 and GG.