TWI379001B - Extracellular polysaccharides with arabinose from trametes versicolor lh1 and enhancing their production methods - Google Patents

Description

1379001 VI. Description of the Invention: [Technical Field of the Invention] The technical field to which the present invention pertains is a medicinal fungus polysaccharide peptide, and more particularly, to a method for promoting the production of a ganoderma lucidum extracellular polysaccharide peptide having arabinose Compositions containing the same can be used to modulate immunological activity. [Prior Art] Yunzhi's scientific name is Trametes versicolor (syn. Coriolus vemco/or) 'Parasitic on rot, white mycelium, growing throughout the world' is a widely used medicinal fungus (Wasser, & Weis, 1999). Yunzhizi has a slow growth and is expensive. The Yunzhi polysaccharide peptide produced by liquid culture is known to have immunomodulatory and antitumor biological activities (Cui, & Chisti, 2003). The Yunzhi polysaccharide peptide can be isolated from different Yunzhi strains such as CM-101 (ATCC 20547) or Cov-1 (Kobayashi, Matsunaga, &

Fujn, 1993, Cui, & Chisti, 2003; Moradali, Mostafavi, Ghods, & Hediaroude, 2007). The polysaccharides in the polysaccharide peptide are bound by α (1-4) and P(l-3), and have been confirmed to have immunomodulatory activity in vitro, in vivo and in clinical experiments (Hsieh, Wu, Park, & Wu , 2〇〇6). The polysaccharide peptide prepared from the strain of Yunzhi strain c〇vi can increase the secretion of interleukin IL-1 and IL-6 and induce apoptosis of human HL_6 lymphoid cancer cells (Hsieh, Kunicki, Darzynkiewicz). , & Wu, 2002) Macrophage fine 3 1379001 cells isolated from rats fed polysaccharide peptide, containing reactive nitrogen intermediates (RNI) and superoxide anions (super〇) Xide anions) and Tumor Necrosis Factor-γ 'TNF-γ (Liu, Ng, Sze, & Tsui, 1993). The polysaccharide peptide of Yunzhi can activate macrophages, stimulate the secretory cytokines TNF-γ, interferon-γ (IFN-γ) and interleukin-1 β (IL-Ιβ). Cytokines are used to fight cell proliferation and cause apoptosis and differentiation. Yunzhi polysaccharide peptide can cause non-specific defense of the immune system and produce anti-tumor effects through the user's own immune system (Cui, & Chisti, 2003; Ng, 1998). Scutellaria barbata (έαΜαία) is a perennial herbal medicine widely distributed in South China and South Korea. It is often used for anti-inflammatory, diuretic and anti-tumor, especially for the treatment of liver cancer' gingivitis and cirrhosis (Wang, & Lu, 2005) Recently, flavonoids (flav〇n〇ids) and diterpernoids of neoclerodane, triterpenic acid, and glutathione have been isolated from Scutellaria barbata extract. (phyt〇Steryl-pD-glUCOSicie) and other biological activities (Wang, Xu, & Zhu, 1996; Ducki, Hadfield, Lawrence, Liu, McGown, & Zhang, 1996; Kizu, Imoto, T〇mimori5 Kikuchi, Kadota &Tsub_,1997;Yu,&Lei,2〇〇4;Yin,Zh〇u,& ...,2〇〇4): and Scutellaria barbata extract can also be found on mouse liver #H22 cells. Inhibits cancer cell proliferation and can be used as an adjuvant for chemotherapy (Dai, Uu,

Wang, &Dia〇, 2_). The polysaccharide obtained from Scutellaria barbata L. extract (IV) is important for the production of 4 Ιό/^ΌΌΙ 'tongue tongue knives, which have significant antioxidant activity in animal experiments (Wang Zhiyuan, Dai Ling, Zhang Kai, 2008) and immune regulation function (Lin Jingming) , Liu Huang, & Luo Rongcheng, 2006) 〇. The culture of the fungus in the deep fermentation tank, the mycelium growth and the production of extracellular polysaccharide peptide and the culture condition and medium composition. Taking Boletus edulis (dwi PP) as an example, the mycelium and extracellular polysaccharide peptides produced by the culture are affected by factors such as temperature stirring speed and initial pH value (Wang, &,, 2〇〇5 ). ® Different strains of Yunzhi strains Wr_74 and ATCC-20545, which can be used to affect the production of mycelium and extracellular polysaccharide peptides by using whey added to the nucleus G〇h, Aixhei·' & Singh, 2007). The chemical composition and properties of the extracellular polysaccharide peptide are affected by i. The effects of nutrient-based ingredients, such as Chen, Hsu, Lin, Lai, and Wu (2006) cultures using different carbon sources, the resulting extracellular polysaccharide peptides produce different monosaccharide compositions, and their extracellular polysaccharide peptides stimulate Macrophage RAW264.7 produces varying amounts of nitric oxide and cytokines. [Inventive content] The inventor of the present invention collected different Yunzhi fruiting bodies from the Nantou mountainous area of Taiwan. After isolation and culture, one of the Yunzhi strains was found to be LH4, which grew rapidly and the mycelium produced 1 was preserved by the Food Industry Development Institute. The center identified its scientific name 7>ame such as vewco/or (L)L1〇yd [species identification report such as attached book]' research found that it can produce novel extracellular polysaccharide peptide (abbreviated as ePSp_Cv), and the medium used Under conditions, the product has a higher ratio of production to immunomodulatory activity that promotes the secretion of fine cytokines, which is tumor necrosis factor alpha (TNF-alpha). Adding a trace amount of Scutellaria barbata extract (SBE) to the culture medium can significantly increase the ratio of the product formation rate and immunomodulatory activity of the Yunzhi extracellular polysaccharide peptide (ePSP-Cv-SBE), which contains immunomodulatory activity. Promote macrophage proliferation, promote the production of nitric oxide in macrophages and promote the secretion of cytokines; the cytokine is tumor necrosis factor-a (TNF-c〇, interleukin _ip (IL-ip) and interleukin素_6(il_6). [Embodiment] Yunzhi versico/or LH1) is currently deposited in the Bioresource Conservation Center of the Food Industry Development Research Institute. The registration number is BCRC930112. Scutellaria barbata extract (SBE) The dried Scutellaria barbata L. is boiled in a ratio of 1 (semi-dragon): 1 〇 (water), and the supernatant is concentrated under reduced pressure to a volume of 1 〇〇 ml to obtain a scutellaria Extract (SBE) Liquid culture of Yuyunzhi and separation of extracellular polysaccharide peptides The above-preserved Yunzhi strains were scraped and inoculated into the medium at a shaking rate of 25 ° C in a constant temperature shaking incubator.丨5 〇卬5 振荡 shaking culture for $ days as an inoculum. One of the mediums is formulated as follows: Medium Formulation 6 1379001 Ingredient Content (%) Glucose 4 Peptone 0.3 Potassium Dihydrogen Phosphate (kh2po4) 0.15 Magnesium Sulfate (MgS04 7H20) 0.15 The above culture medium of lotus extract (SBE), or the above-mentioned medium supplemented with 0.5% (v/v) Scutellaria barbata extract, cultured in a 20 liter fermentation tank at a temperature of 1% by the amount of bacteria (temperature 25 °) C; agitation rate 150 rpm; aeration 1 vvm). The mycelium culture medium after 7 days of culture was centrifuged at 6000 rpm for 30 minutes to obtain mycelium and supernatant. The supernatant was subjected to four times the amount of 95% alcohol' for 24 hours and then centrifuged, and the precipitated polysaccharide peptide was extracellular p〇ly Saccharopeptides (ePSP). In the present invention, the isolated Yunzhi extracellular polysaccharide peptide obtained by culturing the culture medium without the addition of Scutellaria barbata extract (SBE) is called "Yunzhi extracellular polysaccharide peptide (ePSP-Cv)", and the medium is added with the extract of Scutellaria barbata L. The isolated Sesame Extracellular Polysaccharide peptide obtained by the culture of the substance (SBE) is called "Spirulina sinensis extracellular polysaccharide peptide (ePSP-Cv-SBE)". Analytical method Determination of hyphae biomass The mycelium and supernatant were obtained by centrifuging the fermentation broth at 6000 rpm for 3 minutes. The mycelium was suspended in 10 ml of deionized water and centrifuged at 6 rpm 7 137 9001 for 30 minutes, three times. The obtained mycelium is freeze-dried and weighed, which is the mycelial body mass. Exopolysaccharide Peptide Assay Mycelium and supernatant were obtained by centrifuging the fermentation broth at 6000 rpm for 30 minutes. The supernatant was added with four times the amount of 95% alcohol, and after 24 hours of precipitation, it was centrifuged. The precipitate was analyzed according to Dubois et al. (1956) "Phenol-sulphuric acid method" and AOAC method (1995). Containing polysaccharide and protein composition, this preparation is the exopolysaccharide peptide, which is cold in the temple; Scutellaria barbata polysaccharide peptide (PSP-SB) assay The Scutellaria barbata extract (SBE) was precipitated in four times the amount of 95% alcohol for 24 hours, and then the precipitate obtained by centrifugation was separated according to Dubois et al. (1956). The phenol-sulphuric acid method (1995) and the AOAC method (1995) analyzed the composition of the polysaccharide and protein, and the preparation was a scutellaria polysaccharide peptide (PSP-SB). The precipitate was freeze-dried and weighed. Statistical Analysis The experimental data in the present invention were analyzed by Statistical Analysis System 6.0 software, ANOVA was used for mutation analysis, and Duncan, s multiple range test was used to make a significant difference in the mean &<< 〇 〇 5). Example 1 Promoting the exopolysaccharide peptide (epsp) production of Yunzhi extract by using Scutellaria barbata extract (SBE). The strain LH1 was inoculated into a 20-liter fermentation tank, and the medium was 8 1379001. There were two types: addition and no A medium (control group) of Scutellaria barbata extract was added. The results of the extracellular polyheptin content obtained by measuring 7 cells in deep culture are shown in Table--. Compared with the control group, 5% 5% of Scutellaria barbata extract (+sbe) was added. The content of the extracellular polysaccharide peptide (milk p_Cv_sBE) of the saplings of the saplings was increased. The culture time was gradually increased. After 7 days of culture, the yield could reach 137 g/L, compared with the medium without the added Scutellaria barbata extract (_SBE). ), Yunzhi extracellular multi-brewed peptide (ePSP-Cv) only got 0.61 g/L, and the yield increased significantly by 22 times. # 纟Invention and the literature The comparison of the ratio of the extracellular polysaccharide (ePSP), hyphae, body mass and yield & production ratio of different Yunzhi strains and culture medium is shown in Table 1. From the viewpoint of the ratio of product yield, the ratio of the product ratio of the production of the exopolysaccharide peptide (ePSp_Cv) produced by the local Yunzhi strain in Taiwan and the product ratio of the extracellular polysaccharide peptide (ePSP-Cv_SBE) The ratios of 〇26 and 0.38, respectively, are higher than those in the conventional literature. In the past literature, 'Yunzhi strain INETI cultured in yeast malt cultured base can only produce 0.70 extracellular polysaccharide peptide (Tavares, Coelh., Agapit., Coutinho & xavier, 2005); Yunzhi strain Wr_74 and ATCC-20545, cultured in whey-added medium, produces 丨15 and 1.32 g/L exopolysaccharide peptides (Cui, Goh, Archer, & Singh, 2007), but yields are comparable to the half of the present invention. The extracellular polysaccharide peptide of lianyunzhi (epSP_Cv_SBE; ^4 1.66 g/L is low. 9 1379001 Table 1 The different inventions and cultures of the Yunzhi strain and medium for its extracellular polysaccharide peptide (ePSP), mycelium quality and Comparison of product ratio generation rate Yunzhi strain additive extracellular polysaccharide peptide (g/L) Mycelium biomass (§/L) Product ratio generation rate * Reference LH1 No 0.61 2.38 0.26 LH1 0.5% Scutellaria Extract 1.37 3.70 0.38 INETI Yeast Extract of the Invention 0.70 3.8 0.18 Tavares et al. Extract (2005) Wr-74 Whey 1.15 8.9 0.13 Cui et al. (2007) ATCC20545 Whey 1.32 10.6 0.12 Cui et al. (2007) *Product ratio generation rate: exopolysaccharide peptide/mycelium The amount of Scutellaria barbata extract (SBE) added to the medium in the present invention is 0.5% (v/v), that is, a trace amount of Scutellaria barbata extract per ml of the culture solution can produce a significant increase in Scutellaria barbata L. The effect of the production of Yunzhi extracellular polysaccharide peptide (ePSP-Cv-SBE).

The general chemical composition of Scutellaria barbata L. extract was determined by AOAC (1995) method, which accounted for 38.10% of carbohydrates, 16.27% of crude protein, 6.75% of crude fat, 10.71% of ash and 28.17% of crude fiber, showing carbohydrates. Its main chemical composition. Scutellaria barbata contains scutellarin, apigenin, luteolin, E-1-(4V-hydroxyphenyl)-but-1-en-3-one, protocatechuic acid ( Protocatechuic acid), acacetin-7-diglucuronide and luteolin-7-diglucuronide and other yellow compounds. The microbial culture 1379001 nutrient-based ingredients play an important role in the growth of bacteria and the secretion of metabolites. The addition of Scutellaria barbata extract is beneficial to the quality of mycelium and the production of ePSP may be related to its components. Example 2 Chemical properties of Yunzhi extracellular polysaccharide peptide (ePSP-Cv), Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide peptide (PSP-SB) I. Protein content by AOAC Method (1995) analysis. As shown in Table 2, the protein content of the Yunzhi extracellular polysaccharide peptide (ePSP-Cv) was 13.8 ± 0.3%, and the protein content of the echinacea polysaccharide peptide (ePSP-Cv-SBE) was 15.6 ± 0.3%. The protein content of Scutellaria barbata polysaccharide peptide (PSP-SB) was 19.0 ± 0.7%. The protein content of the extracellular polysaccharide peptide (ePSP-Cv-SBE) of S. sinensis was 1.13 times that of the Yunzhi extracellular polysaccharide peptide (ePSP-Cv). Table 2 Comparison of protein content of polysaccharides peptides (ePSP-Cv), extracellular polysaccharide peptides (ePSP-Cv-SBE) and peony polysaccharide peptide (PSP-SB) Exopolysaccharide peptide (ePSP-Cv) 13.8 ±0.3 Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE) 15.6 ±0.3 Scutellaria barbata polysaccharide peptide (PSP-SB) 19.0 ±0.7 II. Analysis of monosaccharide composition 5 ml of 2M trifluoroacetic acid (TFA) hydrolyzed the polysaccharide peptide for 16 hours at 1 °C. The neutral single vinegar component was analyzed by high performance liquid chromatography (HPLC, Jasco PU-2080 PLUS) with SUGAR SP0810 column 1379001 (Sho dex) (8 mm x 3 00 mm), wherein the flow rate of the column was At 0.8 ml/min, the mobile phase was deionized water and the temperature of the column was maintained at 80 °C. In the present invention, the monosaccharide content composition of the Yunzhi extracellular polysaccharide peptide is compared with other literatures, as shown in Table 3, the components include glucose, galactose, mannose, xylose. And arabinose (arabinose). Compared with the literature of Tavares ei αΛ (2005), the presence of galactose in the monosaccharide composition of the Yunzhi extracellular polysaccharide peptide was not found, but the Yunzhi extracellular polysaccharide peptide produced by the native Yunnan Yunzhi LH1 strain in the present invention. The monosaccharide composition of (ePSP-Cv) and S. cerevisiae extracellular polysaccharide peptide (ePSP-Cv-SBE) contained 8.67 mg/g and 21.3 8 mg/g galactose, respectively, which is obviously a new type of cloud. Chitosan polysaccharide peptide. Table 3 Comparison of monosaccharide content of Yunzhi extracellular polysaccharide peptide in the present invention compared with other literatures

Monosaccharide composition (mg/g ratio) polysaccharide peptide glucose galactose mannose xylose arabinose citations ePSP-Cv*1 82.27 8.67 8.18 0.87 nd*2 The present invention ePSP-Cv-SBE*3 60.64 21.38 6.66 7.43 3.89 The present invention ePSP-Cv-LBE*4 80.06 9.29 1.92 8.73 nd Lin et al., 2008 PSP-SB*5 10.78 71.00 15.45 ndnd PSP-LB*6 of the invention 49.21 17.06 nd 1.33 32.40 Lin et al., 2008 12 1379001 No added Chinese medicine The Yunzhi extracellular polysaccharide peptide*2n.d. obtained from the extract did not detect the Yunzhi extracellular polysaccharide peptide obtained from the addition of the scutellaria extract of the Yunzhi extracellular polysaccharide peptide*4 obtained from the extract of Scutellaria barbata D. 5 Scutellaria barbata polysaccharide peptide *6 枸杞 polysaccharide peptide comparison Scutellaria barbata polysaccharide peptide (PSP-SB), Yunzhi extracellular polysaccharide peptide (ePSP-Cv) and Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE ), the Scutellaria barbata polysaccharide peptide contains more galactose (71.00 mg / g) and mannose (1 5.45 mg / g), and no detection of xylose and arabinose. However, after 7 days of fermentation, arabinose (3.89 mg/g) was detected in the extracellular polysaccharide peptide of Scutellaria barbata L., indicating the Scutellaria barbata polysaccharide peptide (SSP-) in Scutellaria barbata extract (SBE). SB) has been used in the fermentation process and converted into different monosaccharides. Comparing the extracellular polysaccharide peptides of Yunzhi (ePSP-Cv) and the extracellular polysaccharide peptides of echinacea (ePSP-Cv-SBE), the glucose content of the scutellaria chinensis (60.64%) is much lower than that of the cloud. The extracellular polysaccharide peptide (ePSP-Cv) has a glucose content (82.27%), while the galactose content (21.38%) of the extracellular polysaccharide peptide (ePSP-Cv-SBE) is much higher than the extracellular polysaccharide of Yunzhi. The peptide (ePSP-Cv) galactose content (8.67%), while the xylose content of the scutellariae extracellular polysaccharide peptide (ePSP-Cv-SBE) (7.43%) is much higher than that of the Yunzhi extracellular polysaccharide peptide (ePSP). - Cv) xylose content (0.87%), but the mannose content of the exopolysaccharide peptides of the two groups is substantially close, ranging from about 6.66 to 8.18%. It is shown that the addition of the traces of Scutellaria barbata L. extract to Yunzhi fermentation medium not only contributes to the substantial increase of the production of extracellular polysaccharide peptide, but also has a significant effect on the chemical composition and monosaccharide content of its extracellular polysaccharide peptide. .

Tavares uses the two different media in the literature of β/· (2005): mushroom complete medium and yeast malt extract medium to culture Yunzhi INETI strain, also showing no medium. The difference in composition changes the amount of mannose and xylose in the extracellular polysaccharide peptide produced. 3. Fourier infrared spectroscopy analysis of polysaccharide peptides The functional groups were detected by Fourier Transf() m Infl_aFed, FTIR. The samples were dried in a vacuum oven for 24 hours after mixing and homogenizing (1:1). 〇〇) Ingot tablets are tested by Fourier infrared spectrometer. The wavenumber range is from 500 to 4000 cm_1, and the resolution is set to 2 cm_i. Using FTIR to analyze ePSP-Cv and ePSP-Cv-SBE, it was found that there were obvious absorption peaks at 3419 '1641 and 1〇78 cm-i positions (Fig. 1). There is a peak in the FTIR spectrum of Ng and Chan T. 1078 cm-i position,

Yang and Zhou pointed out that in the FTIR spectrum of Yunzhi PSP, 3400, 1650, 1050 and 893 cm_丨 represent functional groups such as _0Η, ·ΝΗ2, C_〇_c and p_glyc〇sidic. Functional groups such as β-glycosidic are also shown at positions 930, 1038, 1078 and 1161 cm·1. In the present experiment, although there are no 893 cm·1 positions in the absorption peaks of the three polysaccharide peptides, the position of i〇78 cm·1 indicates that the β(1->3) glycosidic functional group is bonded. Yang and Zhang report 1379001 that there is a absorption peak of p_D-glucose in the position of 905 876 cm-1. In the range of 11〇〇~1010 Cm], there are three strong absorption peaks in Pyranoside, and two absorption peaks in furanoside. The range of 11〇〇_1〇1〇εηΓι also showed significant absorption peaks. Mata et al. analyzed the sugar beet experiment with FTIR and also indicated that there was a gisCosidic bond at 1146 cm. Rau et al. reported that the extracellular polysaccharide peptide of C_ATCC 2〇0801 contains β(1-3) as the main chain linking glucose, while in the Q side chain, p(1-6) is linked to another glucose. The exopolysaccharide peptide also has a biologically active β(1-3) bonded structure. The chemical characteristics analysis of the above-mentioned Yunzhi extracellular polysaccharide peptide (ePSP_Cv), Scutellaria barbata extracellular polyglycolic peptide (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide peptide (PSP-SB) were compared with the literature. In the present invention, the Yunzhi extracellular polysaccharide peptide (ePSP-Cv-SBE) produced by adding the Scutellaria barbata extract to the native L. sylvestris LH1 strain in Taiwan increases the production of extracellular polypeptide, and also makes its chemical composition protein content and The monosaccharide composition is changed, and the three polysaccharide peptides of PSP-SB, ePSP-Cv and ePSP-Cv-SBE have a biologically active structure of β(1 -3) glucan. In comparison with the present invention, the Yunzhi extracellular polysaccharide peptide (ePSP-Cv) and the Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE) have different protein content and monosaccharide composition. Example 3 Effect of Yunzhi Extracellular Polysaccharide Peptide (ePSP-Cv), Scutellariae Radix Glycosides Extracellular Polysaccharide Peptide (ePSP-Cv-SBE) and Scutellaria Barbata Polysaccharide Peptide (pSp_SB) on Macrophage Proliferation 15 1379001 I. Macrophage RAW 264.7 culture and polysaccharide peptide treatment Mouse macrophage RAW264.7 (ATCC TIB-71) was cultured in Dulbecco's Modified Eagle supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 g/ml streptomycin. Medium (DMEM) medium (Gibco) was cultured in a cell culture incubator containing 5% carbon dioxide at 37 ° C. After 24 hours of culture, the cells were tested at a cell density of 2 x 105 cells/ml and added at a concentration of 25 0 respectively. Three polysaccharide peptides of pg/mL: Yunzhi extracellular polysaccharide peptide (ePSP-Cv), Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide peptide (PSP-SB) in DMEM medium in. Second, the polysaccharide peptide on the macrophage

The cell survival and proliferation rate of mouse macrophage RAW264.7 was analyzed by MTT assay. The MTT assay was used to measure the dehydrogenase activity of the viable cells in vitro, which was used to indirectly measure cell survival and proliferation rate. The method is to plant the cells in a 96-well plate, so that the cell density of each well is lxl04 cells/1 ΟΟμΙ/well, and then add a final concentration of 62.5 pg/ml, respectively, peptide ePSP-Cv, ePSP-Cv-SBE or The PSP-SB solution was cultured in the cells for 48 hours, then added with 30 g of MTT in a CO 2 incubator (37 ° C) for 2 hours, the supernatant was removed and DMSO was added to the ELISA reader (Bio-Tek, pQuantTM). The absorbance at 570 nm was measured. The O.D. value of the unmedicated group was the basic control group (100%). The results showed that mega 264.7 was added to 62.5 pg/mL of 16 1379001. ePSP-Cv, ePSP-Cv-SBE and PSP-SB, and the relative cell growth rate after incubation for 48 hours was 1〇1.3±14.〇,13 〇.3±11.9 and 120.6±17·0%, statistical analysis showed that these three extracts had no significant effect on the survival of megatuber cells, as shown in Figure 2. Example 4: Yunzhi extracellular polysaccharide peptide (ePSP_Cv), Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide peptide (PSP-SB) induce nitric oxide production in macrophages The honey is planted in a 96-well plate, so that the cell density per well is 1 X 1 〇 4 Φ cells. 50 μl of different polysaccharide peptides (final concentration of 62.5 pg/ml) were added to each well, and then filtered into 4 μg/ml of 50 μl lipopolysaccharide (LPS) or the same volume of medium in DMEM. After 48 hours of incubation, the content of nitric oxide in the medium was analyzed by Greiss reaction. The supernatant from the cells was removed and added to a 96-well plate, and an equal amount of Greiss reagent (1〇/0 sulfanilamide, 0.1% NED and 5) was added. % H3P〇4) and RNI were reacted at room temperature for 15 minutes, and the absorbance at OD540 nm was read with an ELIS A reader. Yield 9 Nitric oxide production is quantified by a standard curve made of sodium nitrite. • The results are shown in Figure 3. When no pg/ml LPS was added (-LPS), ePSP-Cv was not significantly different from the control group. Adding ePSP-Cv-SBE and PSP-SB induced macrophage production. Nitric oxide. When adding LPS (+LPS), there was no significant difference between the addition of epSP-Cv and ePSP-Cv-SBE and the control group, but the addition of PSP-SB had less production of NO. The production of nitric oxide by megatuber cells is considered to be a cell-based anti-mechanical function. Generally, LPS stimulates cells to produce nitric oxide as a means of detecting immunomodulatory activity. Wasser (2005) pointed out in the study that fungi contain β(1-3)glucan polysaccharides to activate macrophages and promote the production of nitric oxide in giant scorpion cells, which is important for many biological immune responses. Chemical delivery of substances. Pang et al. (1998) used mouse peritoneal macrophages to produce nitric oxide by adding lPS and PSK (a polysaccharide peptide from c. strain CM-101). Wang et al (1996) pointed out that C57BL/6 mice also induced the production of nitric oxide after PS treatment, but if the silver food comes from the extracellular and intracellular polysaccharide peptide produced by C. strain Cov-Ι strain, It is stimulated to produce a higher amount of one of the nitrogen oxides. In the present example, when LPS (-LPS) is not added, the extracellular §1 peptide (ePSP-Cv-SBE) of the scutellaria chinensis can promote the production of up to 3.20 μΜ of one of the nitrogen oxides. In the control group, 0.24 μΜ, or the addition of LPS (+LPS), the average value of the nitrogen oxide produced by the average of 2.59 μΜ of the polysaccharide group was high, indicating that the extracellular polysaccharide peptide (ePSP-Cv-SBE) of S. An immunomodulator with considerable potential for application. Example 5: Analysis of cytokines produced by macrophages induced by Yunzhi extracellular polysaccharide peptide (ePSP-Cv), Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide peptide (PSP_SB) The cells were seeded in 24-well plates, and the concentration of each cell was i〇5 cells/500p. 250μ1 polysaccharide peptide and 250μ1 LPS or the same volume of medium were added respectively, so that the final volume of each well reached lm and the final concentration was 62.5pg/ Ml of polysaccharide peptide. When 48 small prints 9001 were cultured in a cell culture incubator (5% C02, 370C), the cell supernatant was collected and frozen at -8 ° C for assay analysis of cytokine secretion. The cytokine concentration of the culture supernatant was determined by a commercial cytokine ELISA kit. Using mouse interleukin-1β (mouse IL_ip, e-Bi〇science), mouse interleukin-6 (m〇use IL_6, e_Bi〇science), small breast tumor necrosis factor-a (TNF-a) ( Mouse TNF-α, e-Bioscience) commercialized kits to quantify. In the RAW 2 64.7 medium of Jujube cells, Yunzhi extracellular poly-conjugator peptide (ePSP-Cv), Scutellaria barbata exopolysaccharide peptide (ePSp_CvSBE) and Scutellaria barbata polysaccharide peptide (PSP-SB) were added. After treatment with or without the addition of LPS, the above two cytokine secretions were investigated. The effect of three polysaccharide peptides on the production of TNF-a by macrophages is shown in Figure 4. The results show that when the Lps is not added, the three polysaccharide peptides can significantly stimulate the production of macrophages 4-5 times compared with the control group. The amount of TNF_a; however, when Lps was added (+Lps), the cells with or without added polysaccharide peptide significantly increased the amount of D1^17_〇1, and there was no significant difference between them. The effect of IL_ip produced by macrophages is shown in Figure 5. The three multi-branched peptides can also stimulate the production of IL W by giant scorpion cells. When LPS is not added (-LPS), the addition of Scutellaria barbata L. Polysaccharide peptide (ePSP_CvjBE) and Scutellaria barbata (psp_SB) can promote the production of 1L-1P in giant scorpion cells, and the extracellular polysaccharide peptide (ePSP-Cv-SBE) of E. sinensis has a very significant increase in macrophage IL. Biological activity; if Lps is added (LPS) Scutellaria barbata exopolysaccharide peptide (ePSp_Cv_SBE) and Scutellaria barbata 1379001 vinegar (PSP-SB) can also promote you to the beginning. The cells produce IL-Ιβ, but there is no significant difference between them. Is there any addition of Scutellaria barbata extract in the medium? The production of Yunzhi extracellular polysaccharide peptide and Scutellaria barbata multi-st peptide in the presence or absence of lipopolysaccharide (Lps) on the giant scorpion cells. The effect of RAW 264.7 production of cytokines compared to -6 yield, as shown in Figure 6, the results show that the giant cell RAW 264.7 Addition of epsP-Cv-SBE and PSP-SB significantly increased IL-6 production by 19 5 and 7 fold compared with the control group. • PSP promotes interleukin ability by macrophages or other white blood cells in an in vitro study mode. It is widely used in immunomodulatory activity. In this case, ePSP-Cv-SBE can increase the secretion of TNF-ot, IL-Ιβ and IL_6 cytokines by macrophages. TNF-α and IL-Ιβ are poisonous to cancer cells. The effect and activation of T lymphocytes. Miiller and Meineke pointed out that cytokines are related to each other, TNF-ct and IL-Ιβ induce IL_6 synthesis, while iL_6 inhibits TNF-α and IL-1 β 'the three cytokines It plays an important role in immunomodulation. Cui et al. used whey as an additive to Yunzhi medium to obtain similar immunomodulatory activities in extracellular and intracellular, and previously studied the addition of guanidine extract to the cloud. In the medium of 芝The polysaccharide peptide can also increase the activity of IL-Ιβ ' IL-6 and TNF-oc, indicating that the immunological activity of PSPs induced by macrophages may be closely related to its different chemical composition, that is, the difference in its composition will result in RAW264·7 cells produce different secretions of hormones 0 20 1379001 [References] 1. Wang Zhiyuan, Dai Ling, Zhang Kai (Min 97), extraction and purification of polysaccharides from Scutellaria barbata L. and antioxidant activity, Chinese Journal of Biochemical Drugs, 29 (2), 96-103. 2. Wang Feng, Ding Chongyang, Wang Yuhong, Zhang Kechang (Min 94), Effects of Jiuwei f on the deep fermentation of Coprinus comatus, Food Science, 26 (10), 144-146. 3. Zhong Hao ‘Xue Xiaoxia, Yao Qingqiang (Min 97), Research on chemical constituents of Scutellaria barbata, Chinese herbal medicine, 3

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Coriolus versicolor. Immunopharmacology, 26(2), 139-146. 20. Mata, Y. N., Blazquez, M. L., Ballester, A. Gonzalez, F. and Munoz. J. A.

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And immunomodulatory activity. Carbohydrate Polymers, 38(3), 247-253. 28. Spelman, K., Bums, J. J., Nichols, D., Winters, N., Ottersberg, S. and

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30. Tavares, AP, Coelho, MA, Agapito, MS, Coutinho, JA and Xavier, AM (2005) Optimization and modeling of laccase production by Trametes versicolor in a bioreactor using statistical experimental design. Applied Biochemistry and Biotechnology, 134(3) , 233-248. 31. Ueno, S., Yoshikummi, C., Hirosem, F. Omura, Y, Wada, T. and Fujii, T. (1980) Method of producing nitrogen-containing polysaccharides. US Patent 4,202,969. 32 Wang, Η. X., Ng, TB, Liu, WK, Ooi, VE and Chang, ST (1996) Polysaccharide-peptide complexes from the cultured mycelia of the mushroom Coriolus versicolor and their culture medium activate mouse 24 1379001 lymphocytes and macrophages The International Journal of Biochemistry & Cell Biology, 28(5), 601-607. 33. Wang, YX and Lu, ZX (2005) Optimization of processing parameters for the mycelial growth and extracellular polysaccharide production by Boletus spp.ACCC 50328 Process Biochemistry, 40(3-4), 1043-1051. 34. Wang, Y., Xue, X., Xiao, Y., Zhang, F., Xu, Q. and Liang, X. (2008) Purification and preparation of compounds from an extract of Scutellaria barbata D. Don using preparative parallel high performance liquid chromatography. Journal of Separation Science, 31 (10), 1669 - 1676.

35. Wang, ZQ, Xu, FM, Yan, XZ and Zhu, Y. (1996) Scutebarbatine A, a new neoclerodane-type diterpenoid alkaloid from Scutellaria barbata. Chinese Chemical Letters. 7(4), 333-334. Wasser, SP (2005) Reishi or Ling Zhi (Ganoderma lucidum). Encyclop Diet Suppl. 603-622. 37. Wasser, SP and Weis, AL (1999) Therapeutic effects of substances occurring in higher Basidiomycetes mushrooms: a modem perspective. Critical Reviews in Immunology, 19(1), 65-96. 38. Yang, L. and Zhang, LM (2009) Chemical structural and chain conformational characterization of some bioactive polysaccharides isolated from natural sources. Carbohydrate Polymers, 76(3), 349 -361. 39. Yang, QY and Zhou, YF (1993) A protein bound polysaccharide-PSP. In Proceedings of PSP International Symposium (Edited by Yang QY and Kwok, CY). Fundan University Press, Shanghai, China. 22-34 40. Yin, X., Zhou, J. and Jie, C. (2004) Anticancer activity and mechanism of Scutellaria barbata extract on human l Eng cancer cell line A549. Life Sciences, 75(18), 2233-2244. 41. Yu, J. and Lei, J. (2004) Chemical composition and antimicrobial activity of the essential oil of Scutellaria barbata. Phytochemistry, 65(7) ), 881-884. 25 1379001 [Simple diagram of the diagram] Figure 1: Analysis of polysaccharide peptides by FTIR analysis, in which ePSP-Cv represents Yunzhi extracellular polysaccharide peptide; ePSP-Cv-SBE represents half branch Lianyunzhi extracellular polysaccharide peptide; PSP-SB represents Scutellaria barbata polysaccharide peptide. Figure 2: Effect of different polysaccharide peptides on macrophage RAW264.7 proliferation; statistical analysis of significant differences (P < 0.05) marked with English letters above the column. Figure 3: Comparison of the amount of nitric oxide produced by macrophages RAW264.7 induced by different polysaccharide peptides. Black bars indicate no added LPS, gray bars indicate the addition of LPS; statistical analysis significant differences (P < 0.05) are indicated above the column in English letters. Figure 4: Effect of different polysaccharide peptides on the production of cytokine TNF-α by macrophage RAW264.7. Black bars indicate no added LPS, gray bars indicate addition of LPS; statistical analysis significant differences (P < 〇.〇5) are indicated above the column in English letters. Figure 5: Effect of different polysaccharide peptides on the production of cytokines IL-Ιβ by macrophage RAW264.7. Black bars indicate no added LPS, gray bars indicate addition of LPS; statistical analysis significant differences (P < 0.05) are indicated in English letters above the column. Figure 6: Effect of different polysaccharide peptides on the production of cytokines IL-6 by macrophage RAW264.7. Black bars indicate no added LPS, gray bars indicate addition of LPS; statistical analysis significant differences (P < 0.05) are indicated in English letters above the column. [Main component symbol description] ePSP-Cv-SBE Scutellaria barbata extracellular polysaccharide peptide 26 1379001 PSP-SB Scutellaria barbata polysaccharide peptide ePSP-Cv Yunzhi extracellular polysaccharide peptide - LPS no added fat multi-flavor + LPS added fat Polysaccharide TNF-α Tumor Necrosis Factor-α Ι1_1β Interleukin-1β (Interleukin-ΐβ)

11_6 Interleukin-6

27

Claims (1)

j/yu〇i Announcement I • VII. Scope of Application: -I - A method for the preparation of extracellular polysaccharides of Ganoderma lucidum with arabinose, comprising adding Scutellaria barbata extract to the liquid medium, and inoculation with Yunzhi TVameies whco/π LH1 strain In the liquid medium, after the liquid culture is carried out, the obtained polysaccharide peptide is centrifuged. • For the preparation of the polysaccharide peptide described in the first paragraph of the patent application, the strain of the Yunzhi strain is 7>(10)eieiSve^c〇/〇rLH1, deposited in the Food Industry Development Research Institute IX Biological Resource Conservation Center, the registration number is BCRC 930112. 3. The method for preparing a polysaccharide peptide according to the scope of the patent application, the method for preparing the Scutellaria barbata extract is to boil the dried Scutellaria barbata L. with a ratio of 1 (semi-dragon): ίο (water), and then the supernatant is prepared. It was concentrated under reduced pressure to a volume of 1 mL to obtain a extract of Scutellaria barbata. 4. The method for preparing a polysaccharide peptide according to the scope of the patent application is first to carry out liquid culture of Yunzhi, and then centrifuge the culture solution to separate the extracellular polysaccharide peptide. The liquid culture method of Yunzhi is to scrape the Yunzhi strain, inoculate it into a liquid medium, and incubate at a temperature of 25 ° C in a constant temperature shaking incubator at an oscillation rate of l50 rpm for 5 days. The inoculum of the liquid culture medium Μ 1〇% of the 5% (v/v) Scutellaria barbata extract was added, and the culture was carried out in a 20-liter fermentation tank (temperature 25t; search rate BGfpm; aeration amount lvvm). Culture Day P can be taken to Yunzhi culture solution. The mycelium and the supernatant can be obtained from the Yunzhi culture solution at 60 rpm from ~3 knives. The above supernatant is subjected to four times the amount of 95% alcohol, and after centrifugation for 24 hours, it is centrifuged, and the sediment is Yunzhi 28 1379001 extracellular polysaccharide peptide 5. The production of polysaccharide peptide as described in claim 1 of the patent scope The method comprises a liquid medium comprising 4% glucose as a carbon source, 0.3% peptone as a nitrogen source, 〇15 exophosphoric acid dihydrogen (卸2Ρ〇4) and 0.15% magnesium sulfate (MgS〇4.7H2〇) as a salt nutrient. The composition of the object. 6. A polysaccharide peptide obtained by the method of the patent application scope is obtained by culturing a liquid cancer cell of Yunzhi, which contains 4% glucose as a carbon source and 〇·5% (ν/ν) half. TECH lotus extract, 〇3% peptone is the nitrogen source, 〇15% potassium dihydrogen phosphate (ΚΗ2Ρ04) & 〇·15% magnesium sulfate (MgS〇4 7Η2〇) is the nutrient composition of the salt. A polysaccharide peptide composition comprising a polysaccharide peptide as described in claim 6 of the patent application, and a composition comprising a diluent, an excipient or a carrier. 29