TWI500628B - Preparation and Application of New Glycoprotein (Anchao) in Myrica - Google Patents

Description

Preparation method of Astragalus mycelium new glycoprotein (anilose) and its use

The invention relates to a method for preparing a glycoprotein and a use thereof, in particular to a preparation method and a use thereof for preparing a novel glycoprotein (amplein) from the mycelium of Antrodia camphorata.

Antrodia cinnamomea (AC, also known as Antrodia camphorate, Taiwanofimgus camphoratus and Ganoderma comphoratum) is a new Taiwanese native basidiomycete (basidiomycete) originally grown only in the hollow trunk of the decaying eucalyptus ( Cinnamomum kanehirai Hay), but because of excessive forest The development, limited number of eucalyptus and the slow growth rate of eucalyptus have made the use of submerged liquid culture systems for the mass production of burdock to become an emerging technology.

Many bioactive ingredients have been found in Antrodia camphorata, including triterpenoids, polysaccharides, benzenoids, benzoquinone derivatives, succinic and maleene. Maleic acid derivatives. On the fruiting body of Antrodia camphorata, it has also been confirmed that it is rich in various biological activities of triterpenoids, including biological immune response, fatigue recovery, liver protection, anti-oxidation, anti-inflammatory and anti-cancer. The nature. However, the most abundant and biologically active product obtained by culturing AC Mycelia with a liquid culture system is a polysaccharide; while polysaccharides are widely considered to be potential immunomodulatory products. .

Polysaccharides have various biological activities, such as: with inflammation regulators The role of (inflammatory mediator), alleviating allergic asthma, anti-angiogenic effects, regulating gene expression caused by LPS, and inhibiting tumorigenesis; other literature also pointed to mycelium Neutral polysaccharides may have a good liver protection effect. In Japan, a similar product, protein-bound polysaccharide K (PSK, Krestin), has been licensed for chemotherapeutic therapy since 1989 to prolong gastric cancer and colorectal (colorectal). Cancer) and survival of patients with small-cell lung carcinoma. However, the records on the chemical structure and biological function of glycoptotein are still lacking; and considering the importance of protein-bound polysaccharides and their outstanding potential, the biological activity for improving health is derived from the mycelium of Antrodia camphorata. The potential value of the glycoprotein is worth further research and application.

The object of the present invention is to provide a method for preparing a mycelium mycelium new glycoprotein (ampule), wherein the method comprises the steps of: (1) extracting the mycelium of Antrodia camphorata with hot water to remove water-soluble substances; (2) the residue remaining after the hot water extraction is extracted with a base to obtain an alkali-soluble product; (3) the alkali-soluble product is obtained by isoelectric precipitation to obtain an alkali-soluble crude polysaccharide; The alkali-soluble crude polysaccharide is purified by colloidal chromatography using Sepharose CL-6B resin, and finally a polysaccharide is obtained, which is an ampoule.

The ampule has a molecular weight of 440 kDa; wherein the ampule contains 71% protein and 14.1% sugar; wherein the protein contains 18.49% leucine and 13.76% valine. Wherein the saccharide contains 53% xylose and 21% glucose.

A second object of the present invention is to provide a pharmaceutical composition comprising ampoules and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, and the like.

The ampoules are used to inhibit inflammation; and the inhibition of inflammation inhibits inflammation by inhibiting the production of nitric oxide.

The ampoules have the effect of protecting the liver; the effect of protecting the liver is inhibiting serum glutamate transaminase (ALT, GPT), aspartate aminotransferase (aspartate aminotransferase) , AST, GOT), interleukin-6 (IL-6), malondialdehyde (MDA) or nitric oxide, and improve superoxide dismutase (SOD), The amount of catalase (CAT) or glutathione peroxidase (GPX).

Fig. 1 The mycelium of Antrodia camphorata was purified by colloidal chromatography on Sepharose CL-6B (3.0×82 cm) resin to obtain polysaccharide AC-2 (polysaccharides AC-2); the fragments were collected (5.0 mL per fragment); The sugar (490 nm) and protein (280 nm) contents were separately analyzed; the vertical dotted line is the fraction of the fragments collected from the preparation of ampoules.

Figure 2: The polysaccharide antrodan isolated by high performance size-exclusion chromatography, detected by 280 nm ultraviolet light and evaporative light scattering detectors (ELSD), and predicted Molecular weight; the molecular weight of pullulans (788, 404, 212, 112, 47.3, 22.8, 11.8, and 5.9 kDa) is used as a reference value to correspond to the retention time to obtain a regression equation: log Da=-0.466 x+10.009, R ² =0.9937; where X is the retention time of the target polymer; the samples used are antrodan, biobran, and yeast beta-glucan (yeast) --glucan).

Figure 3 shows the total ion chromatograms of gas chromatography-mass spectrometry (GC/MS analysis); (a) standard monosaccharides, and (b) obtained from hydrolyzed ampoules Monosaccharide; Ara: arabinose, Rha: rhamnose, Fuc: fucose, Xyl: xylose, IS: internal standard arabitol, Man : mannose, Gal: galactose, Glc: glucose.

Figure 4 Comparison of Fourier transform infrared spectroscopy (FTIR spectra); (a) ampoules (b) rice bran, and (c) yeast beta-glucan; two milligrams of completely dried sample and 300 mg completely dried The KBr is mixed and pressed into a disk; the full infrared spectrum (400 to 4000 cm ^-1 ) is recorded by a FTIR spectrophotometer; rice bran is a reference for a commercially available polysaccharide sugar, and the yeast β- Glucan is a beta-glucan (β-glucan) reference.

Figure 5 Hydrogen nuclear magnetic resonance spectroscopy ( ¹ H NMR spectra) (a) ampoules, and (b) a standard Catalan polysaccharide (curdlan) β-D- (1 → 3, 1 → 6) bonded oligosaccharide Sugar (β-D-(1→3,1→6)-linked glucan).

Figure 6 (a) Effect of ampoules and rice bran, and (b) lipopolysaccharide (LPS) on the survival rate of mouse macrophage cell line (RAW 264.7 cell); (a) cells with ampoules or Rice bran was cultured for 24 hours and cultured with 0.05~5μg/mL lipopolysaccharide for 24, 48 and 72 hours respectively; cell proliferation was evaluated by MTT assay; the value obtained was the average of three independent experiments (mean ± mean standard error (SEM); survival rate as a percentage of survival compared to the control group; * p < 0.05 vs. control group; ** p <0.01; *** p < 0.001.

Figure 7: Lipopolysaccharide on (a) mouse macrophage cell line (RAW 264.7 cell) nitric oxide production, treatment with ampoules and rice bran polysaccharides (b) 24 hours, (c) 48 hours and (d) The effect of treatment for 72 hours; the cells were treated with different concentrations of lipopolysaccharide for 24, 48 and 72 hours respectively; the nitrite content in the culture solution was analyzed by Griess reaction assay; the obtained values were three independent experiments. Mean ± mean standard error (SEM); * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control group.

Figure 8: Effect of ampoules on the increase of serum aspartate transaminase (GOT) induced by LPS; LPS: LPS 5 mg/kg; Antrodan: ampoules 40 mg/kg; Antrodan (L): low dose Ampoule (40mg/kg); Antrodan (H): high dose ampoules (80mg/kg); a, b: different letters represent significant differences (p<0.05); values are average (mean) ± standard deviation (SD) to represent (n = 6).

Figure 9: Effect of ampoules on the increase of serum glutamate pyruvate transaminase (GPT) induced by LPS; LPS: LPS 5 mg/kg; Antrodan: ampoules 40 mg/kg; Antrodan (L): Low-dose ampoules (40 mg/kg); Antrodan (H): high-dose ampoules (80 mg/kg); a, b: different letters represent significant differences (p<0.05); values are averages ( Mean) ± standard deviation (SD) to represent (n = 6).

Figure 10: Effect of ampoules on the increase of serum interleukin-6 (IL-6) induced by LPS; LPS: LPS 5 mg/kg; Antrodan: ampoules 40 mg/kg; Antrodan (L): low dose Ampoule (40mg/kg); Antrodan (H): high dose ampoules (80mg/kg); a, b: different letters represent significant differences (p<0.05); values are average (mean) ± standard deviation (SD) to represent (n = 6).

Figure 11 Effect of ampoules on the increase in the amount of nitric oxide (NO) induced by LPS; LPS: LPS 5 Mg/kg; Antrodan: ampoules 40 mg/kg; Antrodan (L): low dose ampoules (40 mg/kg); Antrodan (H): high dose ampoules (80 mg/kg); a, b, c , bc: Different letters represent significant differences (p < 0.05); values are expressed as mean ± standard deviation (SD) (n = 6).

Figure 12: Effect of guanose on LPS-induced liver antioxidant capacity (superoxide dismutase, SOD); LPS: LPS 5 mg/kg; Antrodan: ampoules 40 mg/kg; Antrodan (L): low dose Ampoule (40mg/kg); Antrodan (H): high dose of ampoules (80mg/kg); a, b, c, d, cd: different letters represent significant differences (p <0.05); It is expressed by mean ± mean standard error (SEM) (n = 6).

Figure 13: Effect of guanose on LPS-induced liver antioxidant capacity (catalase, CAT); LPS: LPS 5 mg/kg; Antrodan: ampoules 40 mg/kg; Antrodan (L): low dose Caramel (40mg/kg); Antrodan (H): high dose of ampoules (80mg/kg); a, b, c, d, cd: different letters represent significant differences (p <0.05); The mean (mean) ± mean standard error (SEM) is expressed (n = 6).

Figure 14: Effect of ampoules on LPS-induced liver antioxidant capacity (glutathione peroxidase, GPX); LPS: LPS 5 mg/kg; Antrodan: ampoules 40 mg/kg; Antrodan (L): Low-dose ampoules (40 mg/kg); Antrodan (H): high-dose ampoules (80 mg/kg); a, b, c, d, cd: different letters represent significant differences (p<0.05); Values are expressed as mean ± mean standard error (SEM) (n = 6).

Figure 15: Effect of ampoules on LPS-induced liver antioxidant capacity (malondialdehyde, MDA); LPS: LPS 5 mg/kg; Antrodan: ampoules 40 mg/kg; Antrodan (L): low dose ampoule Sugar (40mg/kg); Antrodan (H): high dose ampoules (80mg/kg); a, b, c, d, cd: different The letters represent significant differences (p < 0.05); the values are expressed as mean ± mean standard error (SEM) (n = 6).

Figure 16 Hematoxylin-Eosin staining (HE staining) of liver tissue; A: control group; B: LPS of 5 mg/kg by intraperitoneal injection (ip injection), severe inflammatory cell infiltration can be found (inflammatory cell infiltration), infiltration and vacuolar degeneration of apparently diffuse cells; C: administration of 40 mg/kg of antrodan; D: administration of 40 mg/kg of antrodan by gastric tube feeding followed by LPS It can be found that there is neutrophil infiltration and necrosis in the convergent area of liver parenchyma; E: 80 mg/kg of antrodan is administered by gastric tube feeding, and then LPS is given. No pathological changes were found, except for a small number of neutrophils found in the sinusoid; this result showed that 40 mg/kg of Antrodan has a liver-protecting effect against inflammatory reactions caused by LPS; magnification All are 200 times.

Figure 17 Western blotting method for cytoplasmic NFkB and iNOS; A: control group; B: LPS: 5 mg/kg LPS; C: 40 mg/kg Antrodan; D: Antrodan (L) (low dose ampoules 40 mg/kg) +LPS; E: Antrodan (H) (high dose ampoules 80 mg/kg) + LPS; a, b, c, ab: different letters represent significant differences (p < 0.05).

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Embodiment 1 1.1 Separation and preparation of polysaccharides from Antrodia camphorata

The freeze-dried mycelium (Institute of Food Industry Development, registration number BCRC 35398) is provided by the Grape King Biotechnology Center of Zhongli City, and the voucher specimens of the freeze-dried mycelium are Hosted in the Bioproducts Laboratory of the Department of Biotechnology, Hong Guang University. Mycelium production and polysaccharide separation are described in Chen, CC et al J. Agric. Food Chem. 2007, 58, 5007-5012. Briefly, the defatted powdery mycelium (1 kg) obtained by supercritical fluid extraction was refluxed at 90 ° C with 20 L of double-distilled water (DDW). 3 times, and continued to stir at 400 rpm for 2 hr to remove water-soluble substances - including sugars and amino acids. After the mixture formed in the above step was filtered, the residue was vacuum dried, and then extracted three times with a hot alkaline solution (pH 9.0, 1:10, w/v) at 80 ° C for 2 hours. The resulting extract was filtered off with suction after cooling; the filtrate was adjusted to pH 4.0 with 1 N HCl; then concentrated and lyophilized to give base-soluble extracts; the extract was The alkali-soluble polysaccharide was precipitated by dissolving it in 10 times its volume of twice-distilled water, and then adding 3 volumes of 95% ethanol, and then collecting the polysaccharide and lyophilizing it (AC-2).

1.2 Producing ampoules by gel permeation chromatographic separation

To pulverize AC-2, 10 mL of 0.05 M NaOH was added to AC-2 (100 mg) and heated to 50 ° C while stirring to facilitate dissolution; then the solution was centrifuged at 13,000 xg for 5 minutes to remove Insoluble residue; the supernatant containing ampoules was first applied to a Sephadex G-100 column (2.5 x 100 cm) in a 0.05 M NaOH solution (containing 0.02% of NaN ₃ ) at a flow rate of 0.5 mL/min (flow Rate) to wash (elute). The flow wash was collected as a fraction collector (ISCO Retriever 500, Lincoln, NE, USA), 6 mL per tube, and 100 tubes were collected. The flow washings of the 16th to 40th tubes were added together and concentrated; the obtained concentrate was added to a Sepharose CL-6B column (column, 3.0 x 82 cm) (the column was twice pH 11.0) Distilled water to equilibrate), then flow wash with the same solution at a flow rate of 0.5 mL/min to separate the polysaccharide; flow wash to a liquid separator (SF-2120, Advantec MFS, Inc., Dublin, CA, USA) ) Collected at a speed of 1 tube (1 tube/10 min) for 10 minutes, and collected 100 tubes in total. The total sugar content in the flow wash was determined by OD 490 nm and simultaneously detected at OD 280 nm. The main fraction of OD 280 nm, including the 29th to 43th tubes, was combined; the combined solution was freeze-dried to obtain the antrodan of the present invention. The ampoules were analyzed by a series of physicochemical analysis methods, and compared with ampoules using Biobran polysaccharide and yeast β-glucan as reference compounds.

Preparation of 1.3 蕈Biobran polysaccharide

Distilled water was added to rice bran powder (Biobran powder, Daiwa Pharmaceutical Co. Ltd., Tokyo, Japan) at a ratio of 1:10 (w/v); the mixture was refluxed at 80 ° C and continued at 400 rpm. Stirring; the extraction process lasted for 2 hours, then the solution was cooled at room temperature and centrifuged at 6000 xg for 10 minutes. Then, the supernatant obtained after centrifugation is filtered with Toyo No. 1 filter paper; the filtrate is retained; the extraction process is repeated 3 times, and then each filter is combined. together. In order to precipitate the polysaccharide, 3 times of 95% ethanol was added to the combined filtrate, and the solution was statically placed at 4 ° C overnight, and then centrifuged at 6000 x g for 10 minutes; finally, the precipitate was freeze-dried to obtain rice bran polysaccharide. (Biobran polysaccharide).

Example 2 Physical and chemical properties of ampoules 2.1 Structural analysis

The molecular weight of ampoules is detected by high performance size-exclusion chromatography (HP SEC) using PolySep-GFC-P (75 x 7.8 mm) and PolySep-GPC-P 4000 (300 x 7.8 mm, Phenomenex, Torrance, CA. USA) tandem columns mounted with a pre-column PolySep-GFC-P ( l x id = 75 mm x 7.8 mm) and combined At 280 nm ultraviolet light and evaporative light scattering detectors (ELSD; SEDERE, SA, Alfortville, Cedex, France). The evaporative light scattering detector system operates with the following settings: a 50 ° C drift tube, a 5 ° C increase, a 2.3 bar nitrogen gas pressure atomizer (nebulizer nitrogen gas pressure); mobile phase (mobile phase The deionized water was used at a flow rate of 0.8 ml/min. The temperature of the column oven is 40 ° C; the reference polymer used is Pullulan, and the pullulan is a series of different molecular weights: 788, 404, 212, 1 12, 47.3, 22.8, 11.8, 5.9 kDa . The molecular weight of ampoules is predicted by the following regression equation (R ² =0.9937).

Log Da=-0.466 x+10.009, R ² =0.9937

x is the retention time of the target polymer

The infrared spectrum is derived from a Fourier transform infrared spectrometer (FTIR 8400s Shimadzu). After the ampoules sample was completely dried, it was thoroughly mixed with infrared grade potassium bromide (IR grade KBr) at a ratio of 1:100 to prepare a tablet, which was then fixed in a sample chamber. Fourier transform infrared spectrum (FTIR spectra) at 4cm ^-1 resolution scan in Intercropping 4000-400cm ^-1; scan each sample 10 times to ensure accuracy. Hydrogen nuclear magnetic resonance spectroscopy ( ¹ H NMR spectrum) was obtained from Varian MR 400 MHz; the sample was dissolved in deuterated dimethyl sulfoxide (DMSO-d ⁶ ) at a final concentration of 20 mg/0.6 mL.

2.2 Quantification of total sugar content

According to Masuko et al., the total sugar content is quantified by the phenol-H ₂ SO ₄ method. Briefly, 250 μL of 5% phenol was added to 250 μL and uniformly mixed; 1 mL of 12 M H ₂ SO ₄ was added to the above mixture, and vigorously shaken to promote the reaction; the reaction mixture was cooled at room temperature. The OD 490 nm was used; glucose was used as a reference compound to establish a calibration curve, and the total glucose content was predicted accordingly.

2.3 Quantification of total protein content

The total protein content was quantified by Bradford protein binding assay. At 795μL of two 200 μL of protein assay dye Coomassie Brilliant Blue G-250 was added to the sub-distilled water; 5 μL of ampoulose was added to the above mixture, and after mixing uniformly, the reaction was allowed to stand for 5 minutes; OD 595 nm was used; and bovine serum albumin was used. (BSA) is a reference compound to establish a calibration curve and to predict total protein content.

2.4 Detection of monosaccharide components

200 μL of 0.5 N methanolic HCl was added to 1 mg of ampoules; the mixture was then placed at 80 ° C for 14-16 hours, then 500 μL of MeOH, 10 μL of pyridine and 50 μL of acetic anhydride were added. The mixture was shaken vigorously and centrifuged. The mixture in the reaction was then allowed to stand for 15 minutes; to avoid oxidation, immediately after drying with nitrogen, 200 μL of Sylon HTP kit (HMDS: TMCS: pyridine = 3:1:9) was added; after thorough shaking, the mixture was Centrifuge and let stand for 15-30 minutes. The mixture was blown with nitrogen until the solution was centrifuged to 100 μL; n-hexane was added to the mixture, shaken and centrifuged for 5 minutes; the supernatant was transferred to a new tube; the above-mentioned n-hexane extraction step was repeated 3 After the second time, the supernatant obtained each time (3 times in total) was added together, and then dried with nitrogen. The residue was stored at -20 ° C for use as needed. Gas chromatography-mass spectrometry (GC/MS analysis) was performed by dissolving the residue in methanol, taking 1 μL of sample each time from an Agilent 6890 gas chromatograph (GC) with a HP-5MS capillary column ( Agilent 5973A MSD mass spectrometer (EI mode, 70 eV, Agilent Technologies, Santa Clara, CA, USA) was analyzed for l = 30 m, id = 0.25 mm, Agilent Technologies. The column temperature is planned to be 60 ° C for 1 min, then heated to 150 ° C at a rate of 25 ° C / min, then heated from 150 ° C to 200 ° C at a rate of 5 ° C / min, and finally from 200 ° C at a rate of 10 ° C / min Heat to 300 °C. The temperature of the GC injector and the GC-MSD interface were 250 ° C and 265 ° C, respectively. The temperature of the injection port and the detector is set to 250 ° C / 265 ° C. Helium gas was used for the carrier gas at a flow rate of 1 ml/min.

Standard monosaccharides are also treated in the same manner as described above, and the amount of each monosaccharide is compared with the peak of its corresponding standard monosaccharide to estimate its amount.

Analysis of 2.5 amino acid components

The method of Sobolevsky et al. is modified in the present invention to perform analysis of an amino acid; the advantage of this method is that it can successfully detect glutamine and asparagine; In a word, powdered ampoules (2 mg) were placed in a microreactor (microreactor 1 mL, Supelco, Mellefonte, PA, USA); heated at 60 ° C and dried with nitrogen blowing; 6 N, 1 mL of HCl was added, and Hydrolysis was carried out at 100 ° C for 24 hours; the reaction vessel was cooled to room temperature and devaporated with nitrogen at 40 ° C; 0.2 methanol was added and the evaporation was repeated 3 times until the water was completely removed. To the residue, 100 μL of norleucine (0.4 μl of pyridine in 100 μL of ortho-amine) was added as an internal reference standard; the mixture was stirred to promote dissolution; 100 μL was contained in 1% TCMS derivatization reagent-MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide) is dissolved in 100 μL of pyridine, and 80 μL of acetonitrile is added, and then uniformly mixed by stirring; the derivation reaction ) was carried out at 120 ° C for 30 minutes. 1 μL of the sample was analyzed by gas chromatography-mass spectrometry (GC/MS analysis). Each 0.3 mg standard amino acid sample was also treated in a similar manner; the gas chromatography/mass spectrometry used was an HP-5MS capillary column ( l × id = 30 m × 0.25 mm, film thickness) ) = 0.25 μm; Angilent Technologies). The inlet temperature was maintained at 280 °C. The carrier gas used was helium gas at a flow rate of 1.0 ml/min. The temperature of the GC and MS interfaces was maintained at 290 ° C; the elution temperature was set to increase from 40 ° C to 280 ° C at 10 ° C / min and then at 280 ° C for 10 minutes. The split rate is set at 40:1 and measured at 70eV electron impact (EI); and a complete scan state is used to ensure complete capture of all derivative fragments; the scope of the scan It is 40-700 m/z ; finally, MSD ChemStation software (Agilent technologies, Agilent Technologies) is used for data collection and analysis.

2.6 Determination of uronic acid content

The method of Galambos was used to analyze the content of uronic acid in antrodan, Biobran and yeast z-glucan. Briefly, after adding 1 mL of 2M methanolic HCl to 2 mg antrodan, the mixture was allowed to stand at 80 ° C for 16 hours; 1 mL of 2 M TFA was added to the mixture, and reacted at 100 ° C for 2 hours. 50 μL of hydrolyzate was measured each time and 200 μL of 25 mM sodium tetraborate (dissolved in H ₂ SO ₄ ) was added; after heating at 100 ° C for 10 minutes, the mixture was allowed to stand at room temperature for 15 minutes; 50 μL of 0.125% w/w carbazole (absolute alcohol) was added to the mixture and heated at 100 ° C for 10 minutes; the mixture was allowed to stand at room temperature for 15 minutes; finally read at 550 nm wavelength Its optical density (OD). Standard D-galacturonic acid was also treated in the same manner to establish a calibration curve; then the amount of uronic acid was calculated.

2.7 Quantification of dextran (glucan)

(1) Total glucan

Total glucan was analyzed according to the manufacturer's manual using the mushroom and yeast beta-glucan kit (Megazyme, Ireland). Briefly, 100 mg of powdered antrodan was placed in a spiral test tube and 1.5 mL of HCl (37% v/v) was added, then uniformly mixed and the mixture was heated at 30 ° C for 45 min. Stir once every 15 minutes; add 10 ml of water to the reaction mixture and mix well; after removing the lid, the mixture was heated at 100 ° C for 5 minutes; cover was capped and then heated for 2 hours; Cool at room temperature; add 10 mL of 2N KOH solution, and add 200 mM sodium acetate (pH 5.0). Make the final volume 100 ml; shake the solution gently to obtain a homogeneous solution, then centrifuge at 1500 × g 10 Minutes to remove contaminants; transfer the supernatant obtained after centrifugation to 2 spiral tubes, 0.1 ml each; add 0.1 mL of exo-1,3-β-glucanase to each tube ( 20 U/mL) and β-glucosidase (4 U/mL, dissolved in 200 mM sodium acetate, pH 5.0); uniformly mixed and placed at 40 ° C for 20 minutes; added 3 mL glucose oxidase containing 0.65 U / mL peroxidae (GOPOD) (glucose oxidase, 12 U/mL), and uniformly mixed; the enzyme reaction mixture was placed at 4 The reaction was carried out at 0 ° C for 20 minutes; the mixture was cooled at room temperature, and finally the optical density (OD) was measured at a wavelength of 510 nm.

(2) α -Glucan (α-Glucans)

100 mg of powdered Antrodan was transferred into a spiral tube; 2 mL of 2 M KOH was added, and stirring was continued on ice for 20 minutes to uniformly mix; after adding 8 mL of sodium acetate (1.2 M, pH 3.8) , quickly add amyloglucosidase (1630 U / mL) + invertase (500 U / mL), and mix evenly; the enzyme solution was reacted at 40 ° C for 30 minutes while stirring frequently; the reaction mixture was centrifuged at 1500 × g for 10 minutes; Add 200 mM sodium acetate solution (pH 5.0) to make a volume of 10.3 mL; transfer the supernatant into 2 spiral tubes, each 0.1 ml each; then add 0.1 mL of sodium acetate solution (200 mM, pH 5.0). And 3 mL of GPPOD reagent, and uniformly mixed; the mixed solution was reacted at 40 ° C for 20 minutes, and allowed to stand for cooling; finally, the optical density (OD) was measured at a wavelength of 510 nm.

The total glucan, α-glucan, and β-glucan content were calculated using the following formula.

Total dextran (% w/w) = △ E × (F / W) × 90

α-Glucan (%w/w))=ΔE×(F/W)×9.27 (note the final volume was 10.3mL), and β-glucan=total glucan-α-glucan

Where ΔE is OD _{sample -} OD _blank .

F=100×(W _ag )/OD _ag

W _ag : the weight of the standard D-glucose used

OD _ag: optical density (OD) of standard D-glucose

W: is the sample weight

2.8 Characteristics of Antrodan

The polysaccharide-AC-2 obtained by the alkali extraction and acid precipitation method is further purified in the present invention by a Sepharose CL-6B column. Then, it was washed with an equal degree of secondary distillation (pH=11.0, DDW) and a rate of 0.5 mL/min; Figure 1 is a complete view of the flow washing; the 29th and 43rd washing fragments of the washed-washed section were combined. Dialysis was carried out with distilled water; the resulting concentrated solution (ie, antrodan) was then analyzed by high performance size exclusion chromatography (HP-SEC). As shown in Fig. 2A, from the absorption mode of UV and evaporative light-scattering detection (ELSD), the isolated antrodan is highly purified and has an average molecular weight of 442 kDa; Regression analysis was performed with reference to pullulans, and the regression equation was: log Da=-0.466X+10.009, R ² =0.9937

Where X is the retention time of the target polymer

However, HP-SEC analysis showed that the Biobran sample prepared by the present invention has various components, at least three main components (Fig. 2B); arabinoxylan (arabinoxylan) which is confirmed as a main component It has a xylose in its main chain and an arabinose polymer in its side chain. Similarly, the yeast beta-glucan detected by HPLC-UV and HPLC-ELSD is a complex mixture of polysaccharides. However, the results show that the non-peptide chemical portion of the yeast β-glucan has no absorbance at a wavelength of UV 280 nm (Fig. 2C). --1,3-glucan (β-1,3-glucan), polymannose (mannan) and chitin The β-1,6-glucosidic cross-linking between chitins was analyzed as a component of yeast β-glucan.

In the present invention, the original crude extracted polysaccharide AC-2 fraction (10.38%) can be purified to obtain 9.19% of antrodan; the antrodan has a molecular weight of 442 kD and contains 152.6±0.0 mg/g of uronic acid (uronic). Acid); its total glucan content was 15.65%, and the content of β-glucan (14.20%) was significantly higher than that of α-glucan (1.45%) (Table 1). In contrast, the yeast β-glucan contained 47.17% total glucan, and the contents of α-glucan and β-glucan were 1.7% and 45.47%, respectively (Table 1).

Antrodan's polysaccharide component consists mainly of four monosaccharides, namely glucose (38.2% 0, xylose (33.7%), mannose (16.6%, mannose) and trehalose (8.4%, fucose) (see Figure 2 and Table 2), it is obvious that it has the compositional characteristics of xyloglucan. The peptide part of antrodan contains 19 kinds of amino acids, mainly essential amino acids (up to 53.66%), of which the main five are leucine (17.62%, leucine), proline (13.21%, valine), isoleucine (10.53%, isoleucine), phenylalanine (9.85%, phenylalanine) and alanine (9.12%, alanine) (see Table 3) The newly isolated antrodan complex biomolecule has a bond between carbohydrate and protein, which can be a different glycopeptide bond arranged by an amino acid and a sugar; the exclusive GlcNAc-β-Asn N-linked glycosylation and O-linked glycosylation (N- and O-glycosylation) which occur with GalNAc-α-Ser/Thr linkages are the most well-known protein glycation modifications. Comprehensive data show that N-acetylglucosamine (GlcNAc) and asparagine (Asn) β-glycosylamine linkage will be the main bond Because glucose (38.2%) and aspartic acid (2.23%, asparagine) are more linked than galactose (1.80%) and serine (3.61%, serine) or threonine (0.85%, threonine) The number is high (see Table 2 and Table 3). Figure 4 shows the infrared (IR) absorption spectrum of antrodan. For comparison, Figure 4 and Figure 3C show the rice bran polysaccharide and yeast. Fourier transform infrared spectroscopy (FTIR spectra) of bacteria β-glucan; biobran (commercial name BioBran/MGN-3) is an effective choice for treating patients with hepatic metastasis; Composed of denatured hemicellulose, which is hydrolyzed by rice boiled by multiple carbohydrate hydrolyzing enzymes; absorption peak of antrodan (absorption peak) ) is: 3316 cm ^-1 , broad, s, ν _OH , (intermolecular hydrogen bond and amide A band NH stretching, 3300 H-bonded) (Fig. 3 A); 2924.2 cm ^-1 , s, and 2890 cm ^-1 , s, ν _CH2 ; 1735.5 cm ^-1 , s, ν _{C = O} (nonconjugated, perhaps uronic acid); 164 7.5cm ^-1 , s, ν _C=O (amide I band, sec-amide, -CONH ₂ ); 1547.8cm ^-1 , s, δ _NH , or ν _CN (amide II band, sec-amide, -CONH ₂ ,couple CN stretching and NH bending);1385.9cm ^-1 ,s,δ _CH3 ;1250cm ^-1 ,s, δ _CN ;1230-1000cm ^-1 (the sec-cyclic alcohols of β-pyranoside);1108.5-1041.6cm ^{- 1} , s, v _CO (Note: 1275-800cm ^-1 represents ν _{C-0-C in} monosaccharides such as glucose or galactose; or endocyclic torsion in furanose ring The three absorption peaks appearing between the changes of angles) are 875.5 cm ^-1 , m, (β-glycosidic linkage) and 800.4 cm ^-1 , s, ν _C-0-C , m, (monosaccharide units) ,monosaccharide units). Multiple absorption peaks 800-640 cm ^-1 , s, out of plane δ _NH . Based on the above results, these characteristics and infrared absorption spectra all indicate that antrodan is a typical glucoxylan-protein complex. Other studies have pointed out that some polysaccharide-protein complexes from mushrooms can stimulate the non-specific immune system and exert anti-tumor activity by stimulating the host's defense mechanism. Among them, a similar structure of a xyloglucan-protein complex prepared from Hericium erinaceus (Bull.: Fr.) Pers. was also found to have related biological activity.

In order to determine the α- or β-configuration of glucose in the protein-bound polysaccharide in the purified antrodan glycoprotein, the ¹ H-NMR signal of antrodan (Fig. 5A) is a signal with the curlan (the fifth). Figure B) Comparison; Cattlenan is composed of β-D-(1→3,1→6) bonded glucose residues (β-D-(1→3,1→6)-linked glucose residues) Polysaccharide body. The distribution of the linkages in the sugar, the ¹ H-NMR signal at 4.55 ppm belongs to the (1→3)-β-D-glucan anomeric signals; 5.14 ppm belongs to (1→4)-α- D-glucan; both proved to be the α-and β-glucans complex in the polysaccharide position of antrodan (Fig. 5A). In addition, the unique olimeric signal (4.4~5.5ppm) and methyl (1.2ppm) signals again illustrate the chemical structure of polysaccharides in antrodan. The structure of the polysaccharide and the glycoprotein can be further distinguished. The present invention finds that no signal dispersion is observed at about 8.5 ppm or less (the region is a characteristic region of the backbone amides); The Catalan polysaccharide has no signal at 0.8 ppm (the strong methyl peak) (see Figure 5B); however, the 8.5 ppm signal and the strong methyl peak 0.8 ppm clearly indicate the protein in the antrodan Random-coil conformation feature.

Embodiment 3 3.1 Culture of mouse macrophage cell line (RAW 264.7 Cell)

Add 10% fetal bovine serum (FBS), 1% penicillin (10,000 units/mL), streptomycin (10 mg/mL) to DMEM medium (Dulbecco's Modified Eagle's Medium, HyClone, Logan, UT, USA). And 2 mM L-glutamine; RAW 264.7 cell was lightly washed twice with 3 ml of 0.01 M phosphate buffered saline (PBS), and added 1 mL of 0.25% (1×) trypsin-EDTA; Finally, the cells were cultured in a 5% CO ₂ , 37 ° C incubator.

3.2 Effects of Antrodan and Biobran on Cell Viability

The cell survival rate was examined by the method disclosed by Mosmann et al. (1983) and the like; RAW 264.7 cells were cultured in a 96-well plate, and the number of cells per well was 6000 cells (6000 cells). /well). The cells were cultured for 24, 48, and 72 hours at different antrodan or biobran concentrations on the next day; then colorimetric MTT (3-(4,5-dimethylthioazol-2-yl)-5-(3) -carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetr azolium])test) to determine cell viability. Briefly, after adding 0.5 mg/mL of MTT to the medium, the reaction was allowed to stand for 2 hours; the medium was centrifuged at 12000× g , and the supernatant was removed; 1 ml of DMSO was added to dissolve the formazan crystals; Finally, the optical density (OD) was read by an ELISA reader at a wavelength of 570 nm.

3.3 Effect of LPS on cell viability

The 6000 cells / mL in 96-well culture plate, and the cells were cultured in DMEM culture medium; As shown, a final concentration of between 0.0-5.0μg / mL of LPS; then continued for 72 hours; and added to LPS The latter 24, 48 and 72 hours were analyzed by MTT colorimetry.

3.4 Effects of lipopolysaccharide (LPS) and antrodan on nitric oxide production (NO production) in cell lines

The method of Green et al. (1982) was used and slightly improved. Briefly, taking the determination of NO in serum as an example, 100 μL of VCl ₃ solution (0.8% in 1N HCl) was transferred to a 96-well culture dish; 100 μL of serum (Serum) was added; 100 μL of Griess reagent (Griess reagent) was added. Wherein the Griess reagent is prepared by mixing 1:1 v/v 1% sulfanilamide dissolved in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride dissolved in water. The mixture was then allowed to stand for 15 minutes and avoid direct sunlight; then the optical density (OD) was read by ELISA reader at a wavelength of 540 nm; a calibration curve was established with NaNO ₂ , and the calibration curve was used to predict nitrous acid. Nitrite and nitrate content.

The production of nitric oxide (NO) in the cell line was carried out by culturing the RAW264.7 cell line at a density of 6000 cells/mL in a 96-well culture dish containing DMEM medium. 24 hours; as shown, LPS was added at a final concentration of 0.0-5.0 μg/mL; culture was continued for 72 hours; and at 24, 38 and 72 hours, 100 μL of the medium was analyzed in the same manner as mentioned above. The production of NO.

3.5 statistical analysis

The obtained data were processed by One-Way Analysis of Variance (ANOVA) method; and analyzed by Tukey's test or least significant difference test (LSD). Difference; when p < 0.05, there was a significant difference between the groups.

Embodiment 4 4.1 Effects of Antrodan and LPS on cell viability

After treatment, mouse macrophages (RAW 264.7 cells) were treated with anrodan for 24 hours, and the number of cells was counted. Figure 5A shows that the survival rate of RAW 264.7 cells is not affected by antrodan, even at a high dose of 400 μg/mL, the inhibition rate is still less than 20%. Obviously, the purification step of polysaccharides AC-2 improved the cytotoxicity of the purified antrodan compared to previous studies; At 500 μg/mL, the apparent inhibition of cell viability was >20% (Fig. 6A). 72 hours, when, in a manner of LPS apparently dose responsive to inhibition of cell survival, and its EC ₅₀ of approximately 0.08μg / mL (FIG. 6 B).

Embodiment 5 5.1 Antrodan inhibits the production of NO in RAW264.7cells induced by LPS in vitro

In vitro, LPS induces NO production in RAW264.7 cells in a dose-and time-responsive manner (Figure 7A); NO production in the control group is 4.70 ± 1.12 μM; At a concentration of 5 μg/mL LPS, the NO production reached 72.11±7.08 μM after 72 hours, an increase of 7.3 times ( p < 0.001) (Fig. 7A); Antrodan proved to be moderately and effectively inhibited by The NO production induced by LPS (Fig. 7B, C and D) Antrodan's effectiveness in scavenging NO production may be due to its high content of uronic acid (Table 1). Previous studies have shown that even small differences in molecular weight, solubility, and higher order structures (such as branching and branching) between beta β (1→3) or β(1→6) glucans, proteins and lipids attached to the stem and formation Higher order aggregates can lead to large differences in innate immune activity.

In animal experiments, the specific backbone 1→3 linear β-glycosidic chain of β-glucan cannot be digested after oral administration. Most of the β-glucan enters the proximal small intestine, while others capture it by macrophages; they are then internalized and fragmented in the cell, and finally transported by macrophages to the bone marrow and the endothelial network. Endothelial reticular. The small β -glucan fragment is finally released by macrophages and absorbed by other immune cells, which in turn leads to various immune responses. In addition, other literatures have indicated that after oral administration of β -glucan, β -glucans, such as scleroglucan and laminarin, can bind to intestinal epithelial cells and Gut-associated lymphoid tissue cells are, and are internalized by, and produce an amount detectable in plasma. However, no bioactivity report related to antrodan has been published so far.

Conclusion: In short, the unique glycoprotein antrodan prepared by AC mycelia is an excellent candidate for further development of functional foods, especially since they can be standardized for fermentation and Separate the program to produce. And our experimental results also prove that by using a standardized alkali extraction step and GPC purification step, an antirodan with anti-inflammatory potential activity can be repeatedly obtained.

Example 6 Animal experiment

The invention used was Sprague-Dawley rats born from the BioLasCo Animal Centre in Taiwan for 6 weeks and weighing 265-287 g; the rats were randomly divided into 5 groups of 6 each; Control, LPS control, antrodan control, LPS+ low-dose antrodan (40mg/kg) control group (Antrodan L+LPS) and LPS+ high-dose antrodan (80mg/kg) control Group (Antrodan H+LPS); rats were housed in cages, 2-3 per cage; rats were given Antrodan by gastric tube feeding; placebo group was fed only basic food; LPS control group The physiological saline solution was used instead of Antrodan; the rats were free to obtain food and water; the whole experiment lasted for 7 days; the rats' body weight was recorded on the first and last days of the experiment; One day, 5 mg/kg of LPS (dissolved in physiological saline) was induced by intraperitoneal injection (ip injection) to induce acute hepatic injury; then the rats were anesthetized with carbon dioxide and hepatic portal vein (hepatic portal vein) Extract blood, and finally store the blood at 4 ° C; The heart, kidneys, and liver were excised and washed twice with PBS; then the water was removed with a soft tissue, weighed and quickly stored at -80 ° C in liquid nitrogen for later use; and the collected blood was The serum was centrifuged at 3000 xg for 15 minutes for subsequent biochemical analysis; the biochemical analysis included analysis of GOT, GPT, IL-6, MDA, SOD, CAT, GPX, NF _k B, and iNOS.

6.1 Changes in body weight and liver weight and LW (liver weight) / BW (body weight) ratio

After 7 days of feeding, there was no significant change in body weight between the groups (Table 4); there was a significant increase in the net weight of the liver in the antrodan L+LPS group, but the net weight of the liver in the antrodan H+LPS group was The antrodan control group is quite (Table 4). However, in LPS-treated rats, the LW (liver weight)/BW (weight) ratio appeared to be significantly reduced; the treatment of antrodan or antrodan+LPS (including high and low dose antrodan) had no effect on the LW/BW ratio. (Table 4).

6.2 Antrodan inhibits GOT, GPT, IL-6 and MDA induced by LPS in vivo Increase in quantity

The increase in LPS height of GOT, GPT and IL-6 in serum was 168±45 U/L, 160±76 U/L and 6.8±2.0 ng/mL, respectively (Fig. 8 to Fig. 10); 40 mg/kg The Antrodan dose was the most effective dose to slow these adverse effects (Figures 8 to 10, p < 0.05); however, in the higher doses of the antrodan treatment group, it was found to be less dose dependent. (Fig. 10, p < 0.05).

6.3 Effects of LPS and Antrodan on liver superoxide dismutase (SOD)

After 24 hours of treatment with LPS, the amount of liver SOD was inhibited to 0.56 ± 0.17 U / mg protein; the amount of liver SOD was only 0.74 ± 0.06 U / mg protein in rats given antrodan; therefore LPS reduced the amount of SOD approximately 2.8 times (p < 0.05), and antrodan was 2.1 times (Fig. 12); low doses of antrodan did not improve the amount of SOD, but high doses of antrodan were significantly more efficient at restoring SOD.

6.4 Effects of LPS and Antrodan on liver catalase (hepatic catalase)

After 24 hours of treatment with LPS, LPS reduced the activity of catalase by 3.4-fold compared with the control group (46.17±0.24 vs. 158.26±14.29 U/mg protein); even antrodan alone inhibited the amount of catalase to 68.54±15.74 U. /mg protein, which means a 2.3-fold decrease (p<0.05); it is worth noting that the antrodan L+LPS and antrodan H+LPS groups did not show good results, and the activity of catalase remained at 61.55±4.68, respectively. And 46.23 ± 7.18 U / mg protein (Figure 13).

6.5 Antrodan slows down inhibition of liver glutathione peroxidase (hepatic GPX) induced by LPS in vivo

The activity of GPX in all groups was 5.76±0.26 in the control group, 4.83±0.4 in the LPS control group, 5.15±0.03 in the antrodan control group, 6.13±0.72 in the anrodan L+LPS, and 5.64±0.15 U/mg protein in the antrodan H+LPS; LPS inhibits GPX; while low doses of antrodan (40 mg/mL) are effective in restoring inhibition of GPX induced by LPS, with higher doses of antrodan (Figure 14).

6.6 Antrodan inhibits the increase of liver malondialdehyde (MDA) induced by LPS in vivo

Compared with the control group's MDA amount of 92.11±0.70 nmole/mg protein, LPS significantly increased the amount of MDA to 127.16±4.10 nmole/mg protein, meaning that the amount of MDA increased by 1.4 times (p<0.05) (15th Figure). Surprisingly, antrodan alone can further increase the amount of MDA by 1.6 times to 150.14±8.64 nmole/mg protein; in contrast, in the antrodan L+LPS and antrodan H+LPS treatment groups, MDA can be significantly The values returned to 107.48 ± 14.37 and 107.95 ± 19.84 nmole / mg protein, respectively; and this also seems to mean that low doses of antrodan (40 mg / kg) are sufficient to effectively alleviate the adverse reactions caused by LPS.

6.7 Antrodan inhibits the increase of plasma nitric oxide (NO) induced by LPS in vivo

The LPS height increased the concentration of nitric oxide in plasma to 55.5 ± 6.0 mM; while the low dose of Antrodan (40 mg / kg) moderately alleviated this adverse reaction (Fig. 11 (p < 0.05).

6.8 Hematoxylin-Eosin staining (HE staining) of liver tissue

Histopathological examination revealed no morphological changes in both the control group and the Antrodan group (Fig. 16A and Fig. 16C); in contrast, LPS impaired liver tissue and increased neutrophilies and lymphocytes. (lymphocytes), which cause hepatic necrosis and cell lysis, which release a large number of nuclei and cause degenerative death of the hepatocytes (Fig. 16B, where the arrow points). It is known by HE staining that anrodan is promoting hepatocyte proliferation (Fig. 16C); and the dose-responsiveness of antrodan seems to be advantageous for alleviating these cytotoxicities. At low doses, the rats treated with antrodan have white blood cells (leucocytes) around the blood vessels, and leucocytes infiltration can be found in the liver cells (Fig. 16D); however, they are treated with LPS alone. This phenomenon has been significantly improved compared to the mouse. Interestingly, high doses of antrodan aggravate LPS-induced liver damage; inflammation with neutrophils is common (Figure 16 E, where the arrow points)

6.9 Western point ink method for detecting cytoplasmic NF _k B and iNOS

LPS highly increased iNOS in the cytoplasm and significantly reduced NF _k B activity (Fig. 18); the expression of iNOS and NF _k B treated with Antrodan alone was comparable to that of the control group; and Antrodan L+LPS and Antrodan H The +LPS treatment group was able to moderately inhibit the increase in the expression level of iNOS induced by LPS; surprisingly, the efficacy of high dose Antrodan was small in the lower dose Antrodan (Fig. 18).

The above-mentioned embodiments and the drawings are only the preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, that is, the equivalent variations and modifications of the scope of the present invention should be covered by the present invention. Within the scope.

Claims (11)

A method for preparing an anthocyanin myosin (antrodan), wherein the method comprises the steps of: (1) extracting an anthrax mycelium (BCRC 35398) with hot water to remove a water-soluble polysaccharide; (2) extracting the residue after the hot water extraction with alkali to obtain an alkali extract; (3) obtaining the alkali-soluble crude polysaccharide by isoelectric precipitation of the alkali extract; (4) The alkali-soluble crude polysaccharide is purified by colloidal chromatography using Sepharose CL-6B resin, and finally a polysaccharide is obtained, which is an ampoule. The method of claim 1, wherein the ampoules have a molecular weight of 440 kDa. The method of claim 1, wherein the ampoules contain 71% protein and 14.1% sugar. The method of claim 3, wherein the protein comprises 18.49% leucine and 13.76% valine. The method of claim 3, wherein the saccharide comprises 53% xylose and 21% glucose. A medicinal composition comprising ampoules according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant. The pharmaceutical composition according to claim 6, wherein the ampoules are used to inhibit inflammation. The pharmaceutical composition according to claim 6, wherein the anti-inflammatory system inhibits inflammation by inhibiting the production of nitric oxide. The pharmaceutical composition according to claim 6, wherein the ampoules have liver protection effect fruit. The pharmaceutical composition according to claim 9, wherein the liver-protecting effect is inhibition of serum glutamate transaminase (ALT), aspartate transaminase (aspartate) Amount of aminotransferase, AST), interleukin-6 (IL-6), malondialdehyde (MDA) or nitric oxide. The pharmaceutical composition according to claim 9, wherein the effect of protecting the liver is to improve superoxide dismutase (SOD), catalase (CAT) or glutathione. The amount of oxidase (glutathione peroxidase, GPX).