TWI299665B - A method for preparing the extracts from taiwanofungus camphoratus with a capacity for inhibiting the activity of matrix metalloproteinases - Google Patents

Description

1299665 IX. Description of the Invention: [Technical Field] The present invention relates to an extract of Antrodia camphorata which can inhibit the activity of matrix metalloproteinase and its preparation green, and the extract of Antrodia camphorata is composed of; The extracellular polysaccharide obtained by the deep culture of the mycelium of Antrodia camphorata is isolated, and in particular, the activity of the matrix metalloproteinase can be inhibited. [Prior Art] Antrodia camphorata (η/w dirty) / (10) g secreted C (10) plus) Also known as Antrodia camphorata or Burdock mushroom, it is a unique species in Taiwan that grows only in Burdock trees belonging to Taiwan's conservation tree species. According to the literature, it is known that the active constituents of Antrodia camphorata are multi-body (p〇lysacchari (tetra) and triterpenoids.) At this stage, Antrodia camphorata extract has been widely used in immune regulation, improving human hematopoietic function, and improving human body movement ability. Anti-fatigue, treatment of cancer or tumor diseases, and medical treatments such as hepatitis B. /σ In addition, the ethanol extract of mycelium of Antrodia camphorata is a scavenger of superoxide anion and hydrogen-oxygen free radical, which has antioxidant function. Moreover, the polysaccharides of the A. camphorata fruit body and the mycelium can restore liver damage caused by the four gasified carbon.

Only the polysaccharide extracts of Ganoderma lucidum used in the above research are obtained by a wide range of alcohol precipitation, and the alcohol concentration is mainly 75 〇~95 〇%; that is, the above studies are mainly based on total polysaccharides. Experimental extract. L [Abstract] The anthraquinone extract capable of inhibiting the activity of matrix metalloproteinase and the preparation method thereof are the extracellular polysaccharides obtained by the deep culture of the mycelium of the isolates of Antrodia camphorata, using different alcohol concentration ranges, The extracellular polysaccharide of Antrodia camphorata was precipitated, and ten polysaccharides of the same molecular weight range were not isolated. The protein electrophoresis activity analysis (gelatin_|3ased zymography) of gelatin as a substrate metalloproteinase was carried out, and it was found that the polysaccharide separated by 5〇·〇~66·7 % alcohol concentration range and 66.7 ~75.0% alcohol concentration range of precipitated polysaccharides, the molecular weight distribution range of 1000~30000, can effectively inhibit the activity of matrix metalloproteinase_2 (MMP_2). Therefore, the present invention utilizes an alcohol concentration range of 50 〇 to 75 〇% ("50.0 to 66.7% alcohol concentration range plus Γ66/7 to 75 〇% alcohol concentration range"). The molecular weight distribution ranges from 1000 to 30000, and is effective for inhibiting the activity of matrix metalloproteinase J. [Embodiment] The present invention can inhibit the matrix metalloproteinase activity of the Antrodia camphorata extract and the preparation method thereof, and the different alcohol concentration ranges used in the stroke (as shown in Table 1), including one different alcohol concentration range (0~) 10.0%, 10.0~20.0%, 2〇.〇~33.3 %, 33.3~50 〇% ^50.0-66.7 % >66.7^75.0 %>75.0^80.0 % ^80.0^85.7 %>85.7^90.0 %, 90·0~95.0%) 'And this step type alcohol concentration (alcohol concentration from low to high concentration) / child; wide and the separation method of the exopolysaccharide body from the deep layer culture of the scorpion, the dream is different The extraction method of the extracellular polysaccharides from the deep-culture of 10 fracti〇ns mycelium is as follows: θ σ (1) The ratio of multi-hear solution to 100% alcohol is 9 :丨(Alcohol concentration: 1%) Place overnight at -20 °C, centrifuge (centrifugation at 12 〇〇〇φηη for 2 〇 minutes), and precipitate the polysaccharide. (2) The ratio of polysaccharide solution to 100% alcohol is 4:1 (alcohol concentration is 2〇〇〇/〇), and the remaining mixed solution of the previous item is added with 1% alcohol in proportion, and it is placed at _2〇 Overnight 'centrifugation (centrifugation 212 minutes by 12__), sink the multi-enzyme. 1299665 (3) The ratio of multi-enzyme solution to 100% alcohol is 2: U alcohol concentration is 33 3 %), and the remaining mixed solution of the upper-term is added in proportion to the alcohol in the proportion, and centrifuged at the overnight (centrifugation at 12_ _ 2〇) Minutes), Shen Dang is more daring. (4) The ratio of multi-hearing solution to excitation-% alcohol is i: 1 (alcohol concentration is 5〇.〇/〇) 'The remaining mixed solution of the above item is added to Na% alcohol according to the ratio, placed in overnight, and centrifuged. 12_ _ Centrifugation 2 〇 minutes), Shen Dian multiple body. (5) The ratio of multi-body solution to 1 () 〇% alcohol is: 2 (alcohol concentration is (10) / 〇) 'The remaining mixed solution of the above item is added with %% alcohol in proportion, and it is centrifuged overnight (centrifuged at 12000 rpm) 2 minutes), Shen Dian listens to the body. (6) The ratio of multi-body solution to excitation-% alcohol is 丨: 3 (alcohol concentration is 〇%), and the remaining mixed solution of the upper-term is added _% alcohol in proportion, and it is placed overnight at 2 离心, centrifuged (to 12_—centrifugation for 2 minutes) to precipitate the polysaccharide. (7) The ratio of multi-brewed solution to 100% alcohol is i: 4 (alcohol concentration is 〇〇/〇) 'The remaining mixed solution of the previous one is added with 1% alcohol in proportion, and it is placed overnight at _2〇t Centrifuge (centrifugation at 12,000 rpm for 2 minutes) to precipitate the polysaccharide. (8) The ratio of multi-enzyme solution to loo% alcohol is: 6 (alcohol concentration is 85.7 %), and the remaining mixed solution of the previous one is added with 1% alcohol in proportion, and placed overnight at _2〇t, centrifuged ( The polysaccharide was precipitated by centrifugation at 12,000 rpm for 2 minutes. (9) The ratio of polyacetate solution to 100% alcohol is i: 9 (alcohol concentration is %), the remaining mixed solution of the previous one is added with 1% alcohol in proportion, and veight is centrifuged at _2 (Centrifugation at 12,000 rpm for 2 minutes), the polysaccharide was precipitated. (1〇) The ratio of polysaccharide solution to 1〇〇% alcohol is 1:19 (alcohol concentration is 95〇%), and the remaining mixed solution of the previous item is added with 1% alcohol in proportion, and it is _2〇 The overnight was centrifuged (centrifuged at 12,000 rpm for 2 minutes) to precipitate the polysaccharide. 1299665

Fractions The concentration range of alcohol used by the extracellular polysaccharides of A. sinensis 〇~10.0% 10·0 ～20.0%

20.0 to 33.3% 33.3 to 50.0% 50.0^66.7% 66·7 to 75.0%

Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8 Fraction 9 Fraction 10 75.0 ~80.0% 80.0^85.7% 85.7^90.0% 90·0 ～95.0%

Using a different concentration of 赖 四 Ρ Ρ Ρ 四 樟 樟 樟 樟 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞 胞The retention time and molecular weight distribution range obtained (see Table 2). The 3T3 mouse fibroblast culture medium [containing matrix metalloproteinases (MMPs)] was subcultured for 48 hours, and the 1299665 cell culture medium was treated with 0. 5 mg/ml of 1〇fractions. After reacting for 48 or 48 hours, gelatin-based zymography with gelatin as a substrate metalloproteinase was performed, and 10 fractions of different molecular weight ranges of Antrodia camphorata extracellular polysaccharides were extracted. Whether it inhibits the enzyme activity of MMPs (MMP-9 and MMP-2); the "cell culture solution" used in the above experiments is a cell culture medium in which 3T3 cells have been cultured for 48 hours, and the "cell culture medium" is first separated from 3Τ3 cells. Use only "cell culture fluid" for the experiment.

. Each fraction of anthraquinone exopolysaccharide

Fractions ................................................. .................................................. .................................................. .................................................. .................................................. ........................................... Residence time (min) Molecular weight Distribution (Da)

37.69 > 950,000 39.87 450,000~950,000 43.69 120,000~450,000 46.61 30,000~120,000 47.81 3,000~30,000 54.22 1,000~3,000 58.87 <1000 59.29 <1000 <1000

Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8

Fraction 9 59.29 <1000

Fraction 10 57.41 By the enzyme activity analysis of gelatin-based zymography, it was proved that fracti〇n 1299665 5 (precipitated by the precipitation of 5〇·〇~66.7 % alcohol concentration range) was fractionated (with 66.7~) The anthocyanin extracellular polysaccharide obtained by precipitation separation in a 75.0% alcohol concentration range can effectively inhibit the activity of matrix metalloproteinase-2. Therefore, the present invention utilizes a mixture of fraction 5 and fraction 6 of anthraquinone exopolysaccharide, that is, a concentration range of 5〇.〇~75.0% ("50.0~66.7% alcohol concentration range plus 66.7~75.0% alcohol concentration range" The precipitated and isolated anthraquinone exopolysaccharide has the best effect on inhibiting the activity of matrix metalloproteinase-2 [see the first figure]; wherein, the molecular weight of the extracellular polysaccharide of fractionation 5 and fraction 樟The distribution range is between 1〇〇〇 and 30000 (as shown in Table 2). The invention relates to an extract of Antrodia camphorata which can inhibit the activity of matrix metalloproteinase and a preparation method thereof. The extraction method of the cytoplasmic domain of the culturing of the lion riding layer is carried out by the above-mentioned borrowing and the concentration of the concentrating position, and the separation method is detailed as follows: After the deep liquid culture of 樟 _ _ body liquid _ float liquid centrifuged _ ah, divided into solid _ silk body and clarified culture liquid, and then the clarified culture liquid and % alcohol in proportion to 丨: 丨 mixed (: 酉 essence concentration For %), on ·2〇. (: Place Gvemight, centrifuge (by centrifugation for 12 minutes for 12 minutes). 'Shenfang separates the clarified liquid (containing 5G G% alcohol concentration) after centrifugation with a larger molecular weight (molecular weight greater than the extracellular mass of the rod Add Na% alcohol to adjust the alcohol concentration to 75.Q%, place it in _2() (4) and coffee, centrifuge (12000, centrifuge for 20 minutes), sink; the molecular weight range is between 1〇〇〇~30000 The extracellular carcass of Antrodia camphorata. The extracellular polysaccharide of Ganoderma lucidum isolated by this step has the ability to significantly inhibit the activity of MMP_2 [please refer to Figures 5 and 6 of the first figure]. The method for preparing the protease activity and the preparation method comprises the following steps: solid state culture, liquid mycelial liquid deep culture and separation of 1299665 Antrodia camphorata extract, and the preparation steps are as follows: (1) Solid state culture of Antrodia camphorata isolates : Inoculum mycelium is inoculated on a circular flat plate, the composition of the medium is malt extract (malt extract) 2 0 /., ° sugar) 2%, bacto peptone (protein gland) 0 · 1 〇 /〇, bact〇月月==Portuguese placed at a constant temperature of 26 °C The medium is allowed to stand for about 20 to 30 days; the mycelium of the 曰/〇(2) cup is in liquid deep culture: the round mycelium is scraped, and the mycelium is inoculated into the conical flask, and the starch-free component is used. Cultivate cockroaches

Injury includes glucose (glucose) 2%, malt extract (malt extract) bacto peptone (peptone) i 〇 / 〇, MgS 〇 4 (sulfate town) 〇〇 5%, potassium dihydrogen hydride) 0. 05 % , K2HP〇1 2 (dihydrogen potassium dibasic) 〇·05 %, the oscillating speed is rpm, temperature 28, ρΗ 5·5 (before inoculation), in a rotary oscillating incubator

11 1 5 liter fermentation tank mycelium liquid deep culture of Antrodia camphorata isolate · Inject the liquid deep culture of the dilute flask into the fermentation tank. The composition of the medium is the same as above, and the stirring speed is 120 rpm and the temperature is 28 t. Ventilation volume 丨vvm, culture ^ 5~7 days; " 2 separation of Antrodia camphorata extract (molecular weight range from looo~30000): after deep culture of Antrodia camphorata mycelium The suspension was centrifuged at 8000 rpm, and separated into a solid mycelium and a clarified medium, and the clarified medium was mixed with 100% alcohol in a ratio of 丨: 丨 (alcohol concentration: 5〇〇%), at _2 〇C placed overnight 'centrifugation (centrifugation at 12000 rpm for 20 minutes), and precipitated a large molecular weight (molecular weight greater than 30,000) of anthraquinone exopolysaccharide, and the centrifuged clear liquid (containing 50.0% alcohol concentration) was added proportionally. 1〇〇% alcohol adjust the alcohol concentration to 75·0'. Place overnight on -20, centrifuge (1299665 hearts at 12000 rpm for 2 〇 minutes), and separate the 分子量 from molecular weight range of 1000~3〇〇〇〇. Exopolysaccharide . In summary, the present invention can inhibit the matrix metalloproteinase activity of the extract of the extract of the genus, the preparation method thereof, the extract of the material extract __ concentration separation of the extracellular polysaccharide obtained by the deep culture of the scorpion scorpion filament, obviously Effectively inhibit the activity of matrix metalloproteinase (MMP-2), the extracellular polysaccharide fraction is between 1000 and 30000, and the riding concentration range of H # 5〇w reversal age is (S) 12 1299665. Brief Description] The first picture: for the use of fractions 1 ~ 5 disk frjw; a [ /, extractions 6 ~ 1 〇 樟 菌 培养 培养 ^ ^ ^ ^ ^ ^ ^ m m m m m m m m m m m m m m The activity of matrix metalloproteinase in cell culture medium was analyzed, and it was found that mound acti〇n 5 (lane and fracti〇n 6 (lane 6) can significantly inhibit the activity of matrix metalloproteinase. [Key element symbol description]

C represents that no extracellular polysaccharide of Antrodia camphorata was added to the 3T3 cell culture medium and left for 〇 hours. N represents that no extracellular polysaccharide of Antrodia camphorata was added to the 3T3 cell culture medium and left for 48 hours. ΜΜΡ-9 stands for matrix metalloproteinase-9. MMP-2 stands for matrix metalloproteinase-2. 92 kDa represents the molecular weight of MMP-9. 72 kDa represents the molecular weight of MMP-2.

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Claims (1)

Ι29966%Η;?9 1 - :..π上一>; X. Patent scope · · An extract of Zhizhi that inhibits the activity of matrix metalloproteinases, and the deep-seated Ganoderma lucidum After the culture, it is a special sucrose body of A. sinensis, and the molecular weight ranges from 1000 to 30000. The sexual patents are as described in Item 1 of the "Suppressed __ White Enzyme 2 Stroke", in which the 'Antrodia extract is derived from the deep cultivation of the mycelium of the Antrodia camphorata isolate, and the whole suspension. The nature of the "^ can be used in the second paragraph of the second paragraph of the "can inhibit the base # metalloproteinase live sound: Lin t" 'the towel, the extract of Antrodia camphorata fine from the separation of the secret silk body 曰 deep 曰 β, after the income Clarified medium in the entire suspension. 4. As described in the third paragraph of the patent application, the "stroke inhibiting matrix metalloproteinase^ can be inhibited", wherein the extract of Antrodia camphorata is derived from the mycelial culture of the mycelium of the offspring. The extracellular polysaccharide contained in the clarified culture solution in the entire suspension. 5. The "Anthraquinone extract which inhibits matrix metalloproteinase activity" as described in the fourth paragraph of the patent application, wherein the extract of Antrodia camphorata is derived from the mycelium of Antrodia camphorata isolates. The extracellular polysaccharide contained in the clarified culture solution in the floating liquid is obtained by extraction. 6. A method for preparing an anthraquinone extract capable of inhibiting the activity of a base metalloproteinase as described in the above item, wherein the preparation steps are as follows: (1) solid state culture of a stalk isolate: anthrax The silk body was inoculated on a circular flat culture plate. The medium consisted of malt extract (malt extract) 2%, curry (glucose) 2%, bacto peptone (protein gland) 〇 1%, _ 〇 ( (slow fat) 2 %, placed in a 26 C temperature incubator for about 2 to 3 days of standing culture; (S) 14 1299665 (2) Mycelium liquid deep culture of Antrodia camphorata isolates · Scrap a circular plane The silkworm body is inoculated into a three-layer bottle towel, and the medium containing no thief is used. The formula includes glUC〇Se (glucose) 2〇/〇, malt extract (malt extract) 2%, bacto _0ne (protein gland)丨%, MgS〇4 (sulfate town) (10) 5%, called noisy (dish acid-yttrium potassium) (10) 5%, Κ2ΗΡ〇4 (dipotassium hydrogen phosphate) (10) 5%, oscillation speed is milk rpm / dish 28 C, pH 5 · 5 (Before inoculation), culture in a rotary vibrating incubator for about one week; ' (3) 5 liter fermentation tank mycelium liquid deep culture of Antrodia camphorata isolate : Injecting the liquid deep scales of the Erlenmeyer flask into the scales _, the composition of the culture medium is the same as above, the stirring speed is 120 rpm, the temperature is 28 ° C, the aeration amount is 1 vvm, and the culture is about 5 to 7 days; (4) Antrodia camphorata Separation of extracts (exopolysaccharide of Antrodia camphorata with molecular weight ranging from 1〇〇〇3 to 3〇〇(8)): The suspension after liquid deep culture of Antrodia camphorata was centrifuged at 8000 rpm in a centrifuge to separate into solids. Mycelium and clarified medium, then mix the clarified medium with 100% alcohol in a ratio of 1: ( (alcohol concentration: 50%), place overnight at _2 〇C, centrifuge (centrifuge at 12000 rpm) Minutes), the extracellular polysaccharides with larger molecular weight (molecular weight greater than 30,000) were separated by precipitation, and the clarified liquid (containing 50. 0% alcohol concentration) after centrifugation was added to 1QQ% alcohol in proportion to adjust the alcohol concentration to 75.0%. The overnight was placed at 20 ° C, centrifuged (centrifuged at 12,000 rpm for 20 minutes), and an extracellular body of Antrodia camphorata having a molecular weight ranging from 1000 to 3 Å was separated by precipitation. 15