TW201116623A - Extracellular polysaccharides with arabinose from Trametes versicolor LH1 and enhancing their production methods - Google Patents

Abstract

The present invention related to the extracellular polysaccharopeptides with arabinose from submerged fermentation with adding trace Scutellaria barbata extracts by a local Trametes versicolor strain LH1 that exhibit significantly enhancing immunomodulatory activity and increasing production.

Description

201116623 VI. Description of the invention: [Technical field to which the invention pertains] The technical field to which the present invention pertains is a medicinal fungus polysaccharide peptide, which is more specific, and relates to an extracellular glycopeptide having an arabinose and a method for promoting the production thereof. Compositions containing the same can be used to modulate immunological activity. [Prior Art] The name of the cloud is Trametes versicolor {syn. Coriolus vaj/co/or), which is parasitic on rot, is a white mycelium, and is distributed throughout the world. It is a widely used medicinal fungus (Wasser, & Weis, 1999). Yunzhi fruiting bodies grow slowly' and are expensive. The Yunzhi polysaccharide peptide produced by liquid culture is known to have immunomodulatory and antitumor biological activities (Cui, & Chisti, 2003). Yunzhi polyacetate can be isolated from different Yunzhi lines such as CM-l〇i (ATCC 20547) or Cov-1 (Kobayashi, Matsunaga, & Fujii, 1993; Cui, &Chisti,.2003; Moradali, Mostafavi, Ghods, & Hediaroude, 2007). The polysaccharides in the polysaccharide peptide have been shown to have immunomodulatory activity in vitro, in vivo and in clinical experiments with 4) and β(ι-3) linkages (Hsieh, Wu, Park, & Wu, 2006). The polysaccharide peptide prepared from the Yunzhi strain Cov-1 strain can increase the division of IL-Ιβ and IL-6 in the immune cells and induce cells of human HL-60 lymphocytes. Apoptosis) (Hsieh, Kunicki, Darzynkiewicz, & Wu, 2002) ° In vivo experiments in animals, macrophages 201116623 isolated from mice fed polysaccharide peptide, containing reactive nitrogen intermediates (RNI), Superoxide anions and Tumor Necrosis Factor-γ » TNF-γ (Liu, Ng, Sze, & Tsui,

1 993). Yunzhi's multi-month can activate giant sputum cells, stimulate the secretion of cytokines TNF-γ, interferon-γ (IFN-γ) and interleukin-1β (Interleukin-1 β, IL-1 β These 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 L. 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, hepatitis and cirrhosis (Wang, & Lu, 2005). Recently, flavonoids and diterpernoids have been isolated from the scutellaria extract (nevonerodines), triterpenic acid, and phytosteryl-pD- Glucose) and other biological activities (Wang, Xu, Yan, & Zhu, 1996; Ducki, Hadfield, Lawrence, Liu, McGown, & Zhang, 1996; Kizu, Imoto, Tomimori, Kikuchi, Kadota, & Tsubono, 1997 Yu, &Lei;2004; Yin, Zhou, & Jie, 2004). Furthermore, Scutellaria barbata extract can inhibit cancer cell proliferation in mouse liver cancer H22 cells and can be used as an adjuvant for chemotherapy (Dai, Liu, Ji, Liu, Kang, Wang, & Dia Dia, 2008). The polysaccharide obtained from Scutellaria barbata L. extract is an important active ingredient of 201116623, which has significant antioxidant activity in animal experiments (Wang Zhiyuan, Dai Ling, & Zhang Kai, 2008) and immune regulation function (Lin Jingming, Liu Huang, & Luo Rongcheng, 2006) The cultivation of general fungal deep fermentation tanks, the growth of mycelium and the production of extracellular polysaccharide peptide are affected by the culture conditions and medium composition. Taking Boletus edulis (wan deiws spp) 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 (Wang, & Lu, 2005). Different strains of Yunzhi strain Wr-74 and ATCC-20545' can be affected by the addition of whey to the medium (Cui, Goh, Archer, & Singh, 2007). The chemical composition and properties of the extracellular polysaccharide peptide are affected by the composition of the medium. For example, Chen, Hsu, Lin, Lai, and Wu (2006) use a medium with different carbon sources to culture, and the resulting extracellular polysaccharide peptide produces different monosaccharides. The composition of its extracellular polysaccharide peptide stimulates macrophages RAW264.7 to produce different amounts of nitric oxide and cytokines. SUMMARY OF THE INVENTION The inventors of the present invention collected different fruit bodies of Yunzhi from the Nantou mountainous area of Taiwan. After isolation and culture, one of the strains of Yunzhi strain LH-1 was found to grow rapidly and the mycelium yield was high. The preservation center identified its scientific name TVameies verhco/or (L.) Lloyd [species identification report, such as attached books], and found that it can produce novel extracellular polysaccharide peptide (ePSP-Cv), and the medium conditions used It has a higher product specific production rate and promotes the immunomodulatory activity of cytokine secretion of 201116623, wherein the cytokine is tumor necrosis factor-a (TNF-a). Adding a small amount of Scutellaria barbata L. (Sc) to the culture medium can significantly increase the ratio of the production rate of the extracellular polysaccharide peptide (ePSP-Cv-SBE) and the immunomodulatory biological activity. The activity comprises promoting macrophage proliferation, promoting the production of nitric oxide in macrophages and promoting the secretion of cytokines; wherein the cytokines are tumor necrosis factor-a (TNF_a), interleukin-ip (IL-lp) and interleukin素_6(IL_6). [Embodiment] Yunzhi (TVflw is a T TT1) is currently deposited in the Bioresource Conservation Center of the Food Industry Development Research Institute, and the registration number is BCRC930112. Scutellaria barbata extract (SBE, ) After adding water to the ratio, the extract of Scutellaria barbata L. will dry the Scutellaria barbata L. 1 (Semabet): 10 (water

The supernatant was concentrated under reduced pressure to a volume of 1 mL, (SBE). Draw the ^·^ state of the culture and the threat. The exopolypeptide peptide is transferred to the above-preserved Yunzhi strain and inoculated into the medium at 25. 〇: At the temperature, in the value of the temperature shock incubator, the oscillation rate (10) is used to oscillate and culture as an inoculum. One of the mediums is formulated as follows: a certain volume of the culture. Ancient 201116623 Ingredients glucose (glucose) 4 protein gland (peptone) 0.3 potassium dihydrogen phosphate (KH2P〇4) 0.15 magnesium sulfate (MgS04 7H20) 0.15 The above medium of Scutellariae Radix Extract (SBE), or the above-mentioned medium supplemented with 0.5% (v/v) Scutellariae Radix Extract, cultured in a 20 liter fermentation tank with 1% of the inoculum 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, and after 24 hours of precipitation, the precipitated polysaccharide peptide was extracellular polysaccharopeptides (ePSP). In the present invention, the isolated Yunzhi extracellular polysaccharide peptide obtained by adding the non-added Scutellaria barbata extract (SBE) in the culture medium is referred to as "the Yunzhi extracellular polysaccharide peptide (ePSP-Cv)" medium, and the addition of Scutellaria barbata L. extract is added. The isolated glutinous exopolysaccharide peptide obtained by culturing the sbe is called "semi-dragon yoghurt extracellular polysaccharide peptide (ePSP-Cv-SBE)". Folding method C biomass measurement The mycelium and supernatant were obtained by centrifuging the fermentation broth at 6000 rpm for 30 minutes. The mycelium was suspended in 10 ml of deionized water, and centrifuged at 6 rpm 201116623 for 30 minutes, three times. The obtained mycelium is freeze-dried and weighed, which is the mycelial body mass. Extracellular polysaccharide peptide assay

The mycelium and the 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, and 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 an exopolysaccharide peptide, and the precipitate is freeze-dried and weighed. Determination of Polysaccharide Peptide (PSP-SB) of Fengzhitang Polysaccharide (PSP-SB) After the Scutellariae Radix Extract (SBE) was precipitated in four times the amount of 95% alcohol for 24 hours, the obtained sediment was separated by centrifugation according to Dubois et al. (1956). The phenol-sulphuric acid method and the AOAC method (1995) analyzed the composition of the polysaccharide and protein, and the preparation was a Scutellaria barbata polysaccharide peptide (PSP-SB), which was freeze-dried and weighed. Statistical Analysis The experimental data in the present invention were analyzed by Statistical Analysis System 6.0 software, analyzed by ANOVA, and Duncan's multiple range test was used to compare the significant differences (p < 0.05). Example 1 Promoting the Extracellular Polysaccharide Peptide (ePSP) of the Yunzhilian Extract (SBE) by the Fengzhilian Extract (SBE) The Yunzhi strain LH1 was inoculated into a 20-liter fermentation tank. There were two kinds of medium 201116623: added and not added ( Control medium) Medium of Scutellaria barbata extract. The extracellular polysaccharide peptide content obtained after 7 days of submerged culture was measured, and the results are shown in Table 1. Compared with the control group, the content of extracellular polysaccharide peptide (ePSP-Cv-SBE) obtained by adding 0.5% of Scutellaria barbata extract (+SBE) was gradually increased with the culture time, and cultured for 7 days. The yield was 1.37 g/L. Compared with the medium without added Scutellaria barbata extract (-SBE), the Yunzhi extracellular polysaccharide peptide (ePSP-Cv) only obtained 0.61 g/L, and the yield increased significantly by 2.2 times. The comparison between the present invention and the literature in the different strains of Yunzhi strain and medium for its extracellular polysaccharide peptide (ePSP), mycelium biomass and product ratio is shown in Table 1. From the point of view of the ratio of product to production, the ratio of the product ratio of the exopolysaccharide peptide (ePSP-Cv) produced by the LH1 strain of Taiwan native Yunzhi LH1 strain and the extracellular polysaccharide peptide of echinacea (ePSP-Cv-SBE) The product specific ratios were 0.26 and 0.38, respectively, which were higher than the product ratio in the prior art. In the past literature, the Yunzhi strain strain INETI was cultured in yeast malt medium and produced only 0.70 g/L extracellular peptide (Tavares, Coelho, Agapito, Coutinho & Xavier, 2005); Yunzhi strain Wr-74 And ATCC-20545, cultured in whey-added medium to produce 1.15 and 1 · 32 g / L extracellular poly-peptide (Cui, Goh, Archer, & Singh, 2007), but the yield is half of the present invention The yield of the extracellular polysaccharide peptide (ePSP-Cv-SBE) of Zhizhiyunzhi was low at 1.66 g/L. 201116623 Exopolymycetes, Ganoderma lucidum, Additives, Glycogen production ratio (g/L), Quality, F rate, Reference LH1 LH1 ΙΝΕΤΙ Wr-74 ATCC20545 J& ***, 0.5% Scutellaria bark extract yeast malt Extract Whey Whey 0.61 1.37 0.70 2.38 3.70 3.8 0.26 0.38 0.18 ί Product ratio generation rate: extracellular polysaccharide/mycelium biomass. The present invention is Tavares et al (2005) Cui et al. (2007) Cui et Al (2007) In the present invention, the addition amount of Scutellaria barbata extract (SBE) in the medium is 0.5% (vΛ〇, that is, a trace amount of Scutellaria barbata extract per ml of the culture solution can produce a significant increase in half-branched The effect of the production of extracellular polysaccharide peptide (ePSp_Cv_SBE) was studied. The general chemical composition of Scutellaria barbata extract was determined by A ◦ AC (1 9 9 5) method, which accounted for 38.10°/carbohydrate and 1627 crude protein. %, crude fat accounted for 6.75%, ash accounted for 10.71 ° /. and crude fiber accounted for 28.17%, indicating that carbohydrate is its main chemical composition. Scutellaria barbata contains scutellarin (apigenin), hibiscus Luteolin, E-1 -(4V-hydroxyphenyl)-bu Tl-en-3-one, protocatechuic acid, acacetin-7-diglucuronide and luteolin-7-diglucuronide and other flavonoids, etc. Microbial culture 201116623 Nutrient components in cell growth and metabolite secretion The process plays an important role. The addition of Scutellaria barbata L. extract is beneficial to the quality of mycelium and the production of ePSP may be related to its components. Paste application 2 Yunzhi extracellular polysaccharide peptide (ePSP-Cv), Fengzhi canopy Yunzhi Chemical characteristics of extracellular polysaccharide peptide (ePSP-Cv-SBE) and polysaccharides of Fengzhilian (PSP-SB) 1. Protein content was analyzed by AOAC method (1995). As shown in Table 2, Yunzhi extracellular polysaccharide peptide ( The protein content of ePSP-Cv) was 13.8 ± 0.3%, and the protein content of the extracellular polysaccharide peptide (ePSP-Cv-SBE) was 15.6 ± 0.3%; Scutellaria barbata polysaccharide peptide (PSP-SB) The protein content was 19.0 ± 0.7%. The protein content of the extracellular polysaccharide peptide (ePSP-Cv-SBE) was 0.13 times that of the Yunzhi extracellular polysaccharide peptide (ePSP-Cv). Polysaccharide peptide (ePSP-Cv), 枸杞云芝 extracellular polysaccharide peptide (ePSP-Cv-SBE) and 枸杞 polysaccharide peptide (PSP-SB) egg Comparison of polysaccharide content of polysaccharide peptide protein (%) Yunzhi extracellular polysaccharide 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 2. Single Cool Composition Analysis The polysaccharide peptide was hydrolyzed with 5 ml of 2 M trifluoroacetic acid (TFA) and allowed to act at 1 ° C for 16 hours. The neutral monosaccharide composition was analyzed by high performance liquid chromatography (HPLC, Jasco PU-2080 PLUS) with SUGAR SP0810 column 11 201116623 (Shodex) (8mm x 3〇〇mm), wherein the tube flow rate was . A 'moving phase is deionized water, the temperature of the column is two. The single domain content of the extracellular auditory form of Yunzhi in the invention is compared with other texts. As shown in Table 3, the composition contains glucose (glue. (4) half Galactose 'mannosese', xyloose (four) squeaking and arabinose. Compared with the literature of Tavares ei αΜ2〇〇5), the composition of monosaccharides in the polysaccharides of Yunxiao No galactose was found, and the present invention: the monosaccharide composition of the extracellular (tetra) peptide (Liu Xin) and the extracellular st peptide (ePSP_Cv_SBE) of the Yunzhi extracellular (tetraploid) produced by the native L. sylvestris LH1 strain in Taiwan Then there are 8.67 mg/g and 21.38 mg/g galactose, respectively, which is obviously the new type of Yunzhi extracellular polysaccharide peptide. Table 1 In the invention, the W content of Yunzhi extracellular township shouted compared with other literatures. Monosaccharide composition (mg/g ratio) Polysaccharide peptide Glucosamine galactose Xylose arabinose References ePSP-Cv*1 82.27 8.67 8.18 0.87 η.ά*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 Invention PSP-LB*6 49.21 17.06 nd 1.33 32.40 Lin et al., 2008

12 201116623 y Yunzhi extracellular polysaccharide peptide obtained from the Chinese herbal extract without extracting *2n.d. The cloud obtained from the addition of the scutellaria extract of the Yunzhi extract of the Scutellaria barbata extract*4 Comparison of the extracellular polysaccharide peptide *5 Scutellaria barbata polysaccharide peptide *6 枸杞 polysaccharide peptide compared to Scutellaria barbata polysaccharide peptide (PSP-SB), Yunzhi extracellular polysaccharide peptide (ePSP-Cv) and Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE), in which the Scutellaria barbata polysaccharide peptide contained more galactose (71.00 mg/g) and mannose (15.45 mg/g), and no xylose and arabinose were detected. However, after 7 days of fermentation, 'arabinose (3.89 mg/g) was detected in the extracellular polysaccharide peptide of Radix Paeoniae Alba, 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. Comparison of the glucose content of the extracellular polysaccharide peptide (ePSP-Cv) and the extracellular polysaccharide peptide (ePSP-Cv-SBE), and the extracellular polysaccharide peptide (ePSP-Cv-SBE) of S. 60.64%) is far lower than the glucose content of Yunzhi extracellular polysaccharide peptide (ePSP-Cv) (82.27%), while the galactose content of echinacea extracellular polysaccharide peptide (ePSP-Cv-SBE) • '21.38% It is much higher than the galactose content (8.67%) of the extracellular polysaccharide peptide (ePSP-Cv) of Yunzhi, and the xylose content (7.43%) of the extracellular polysaccharide peptide (ePSP-Cv-SBE) of S. Yuyunzhi extracellular polysaccharide peptide (ePSP-Cv) xylose content (0.87%), but the mannose content of the exopolysaccharide peptides of the two groups is basically close, ranging from about 6.66-8.18%. . It is shown that the addition of trace amount 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 obvious influence on the chemical composition and monosaccharide content of its extracellular polysaccharide peptide. .

In the literature of Tavares ei <2/. (2 005), two different media were used: culture complete medium and yeast malt extract to cultivate the INETI strain of Yunzhi bacteria. It is shown that the difference in composition of the medium changes the content of mannose and xylose in the extracellular polysaccharide peptide produced therefrom. 3. Fourier Infrared Spectroscopy Analysis of Polysaccharide Peptides The functional groups were detected by Fourier Transform Infrared (FTIR). The samples were dried in a vacuum oven for 24 hours with a KBr powder. After mixing evenly (1:100), The tablets were tested by a Fourier infrared spectrometer with a wavenumber ranging from 500 to 4000 cnT1 and a resolution of 2 cm·1.

Using FTIR to analyze ePSP-Cv and ePSP-Cv-SBE, it was found that there were obvious absorption peaks at 3419, 1641 and 1078 cm_1 (Fig. 1), and there was a peak in the FTIR spectrum of Ng and Chan T. yunzhi PSP at 1078 cm·1. Position, Yang and Zhou pointed out that the FTIR spectra of Yunzhi PSP represent functional groups such as -OH·' -NH2, COC and β-glycosidic at 3400, 1650, 1050 and 893 cm·1. Functional groups such as β-glycosidic are also shown at positions 930, 1038, 1078 and 1161 cm·1. In this experiment, although there are no 893 cnT1 positions in the absorption peaks of the three multi-enzyme peptides, the position of 1078 cm·1 indicates that there is a bond of β(1 -3)glycosidic functional groups. Yang and Zhang report 14 201116623 pointed out that 905-876cm-l>ifr®t±7 female βτΛ 1 ^ 丄1 has absorption peaks of β-D-glucose, and there are three strong absorption peaks in pyronoside in the range of 1100-1010 cm. There are two absorption peaks in furanoside. In this study, there is no obvious m peak in the range of 11〇〇i〇i〇. Mata et al. analyzed the sugar beet experiment with FTIR and indicated that there was a glycic sidic bond at 1146 cm·1. Rau et al. reported that the extracellular polysaccharide peptide of C. veriico/π ATCC 2〇08〇1 contains β(1-3) as the main chain linking glucose, while the Q side chain is linked by β(1-6). Another glucose, indicating that the exopolysaccharide peptide also has a biologically active β(1-3)-bonded structure. A comprehensive analysis of the chemical characteristics of the above-mentioned Yunzhi extracellular polysaccharide peptide (ePSP_Cv), extracellular phage (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide (psp-SB) and the literature In the present invention, the native Japanese genus Yunzhi Lh 1 strain is added with the Scutellaria barbata extract (ePSP-Cv-SBE) to increase the production of extracellular polysaccharide peptide, which also makes its chemical composition protein content. And the composition of the monosaccharide is changed, and the three polysaccharide peptides of PSP_SB, ePSP-Cv and ePSP-Cv-SBE have the 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 content. Children's application of Sanyun glutinous polysaccharide peptide (ePSP-Cv), Scutellaria barbata var. sinensis (ePSP-Cv-SBE, and Fengzhilian polysaccharide peptide (effect on macrophage proliferation 15 201116623) Macrophage RAW 264.7 culture and polysaccharide peptide treatment Mouse macrophage RAW264.7 (ATCC TIB-71) was cultured with 10% fetal bovine serum, 100 units/ml penicillin and 1〇〇g/ml streptomycin. Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco) was cultured in a cell culture chamber containing 5% carbon dioxide at 37 ° C. After 24 hours of culture, the cells were tested at a cell density of 2 χ 105 cells/ml and added to the concentration. Three polysaccharide peptides at 25 0 pg/mL: Yunzhi extracellular polysaccharide peptide (epsP-Cv), φ Scutellaria yunnanensis extracellular polysaccharide peptide (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide peptide (PSP-SB) In the culture of DMEM. II. Effect of polysaccharide peptide on macrophages The cell survival and proliferation rate of mouse macrophage RAW264.7 was analyzed by MTT assay. The MTT assay was used to measure the dehydrogenation of mitochondria in viable cells. Dehydrogenase activity for indirect determination of cell survival and proliferation rate, the method In order to seed the cells in a 96-well plate, the cell density of each well was ΐχ 1〇4 • cells/100 μΐ/well, and the final concentration of the polysaccharide peptides ePSP-Cv and ePSP-Cv- at a concentration of 62.5 pg/ml were added. SBE or PSP-SB solution was cultured in cells for 48 hours, then added with 30 pL of MTT in a C〇2 incubator (37 ° C) for 2 hours. The supernatant was removed and DMSO was added to ELISA reader (Bio- Tek, pQuantTM^j its absorbance at 570 nm. The 〇.d value of the untreated group was the basic control group (100%). The results showed that mega 264.7 was added to 62.5 pg/mL of 16 201116623 ePSP-Cv, The relative cell growth rates of ePSP-Cv-SBE and PSP-SB after 48 hours of culture were 1〇1.3 soil 14_0, 130.3±11.9 and 120.6±17.0%. Statistical analysis showed that these three extracts had no significant effect on macrophage survival. , as shown in Figure 2. f Example: Four-spotted extracellular polysaccharide peptide (ePSP-Cv), Fengzhilian Yunzhi extracellular polysaccharide peptide (ePSP-Cv-SBE) and Fengzhilian polysaccharide peptide (PSP-SB Effects of inducing nitric oxide production on macrophages. Macrophages are seeded in 96-well plates to a cell density of 1 X 104 per well.

Cells. 50 μl of different polysaccharide peptides (final concentration of 62.5 pg/ml) were added to each well, and then 4 μg/ml of 50 μl lipopolysaccharide (LPS) or the same volume of medium dissolved in DMEM was added. 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 by an ELISA reader. One of the nitrogen oxide production yields is measured by a standard curve made of sodium nitrite. The results are shown in Figure 3. When no lpg/ml LPS was added (-LPS), ePSP-Cv was not significantly different from the control group, and the addition of ePSP-Cv-SBE and PSP-SB induced a large number of macrophages. Nitric oxide. When LPS was added (+ 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 NO production. The production of nitric oxide by macrophages is considered to be a defensive work of cells. 17 201116623 Yes, it is common to use LPS to stimulate 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 that activate macrophages and promote macrophages to produce nitric oxide, which is important in many biological immune responses. Chemical delivery of substances. Pang et al. (1998) produced nitric oxide using mouse peritoneal macrophages by the addition of LPS and PSK (a polypeptide derived from the C. strain CM-101 strain). Wang et al (1996) pointed out that C5 7BL/6 mice also induced nitric oxide production after LPS treatment, but extracellular and intracellular production if fed from C. verhco/or strain Cov-1 strain A peptide that is stimulated to produce a higher amount of one of the nitrogen oxides. In this example, when LPS (-LPS) is not added, the scutellaria sylvestris extracellular polysaccharide peptide (ePSP-Cv-SBE) can promote macrophage production of up to 3.20 μΜ of one of the nitrogen oxides, compared with the control group. 0.24 μΜ, or the addition of LPS (+LPS), the average value of the polysaccharide peptide group of 2.5 9 μΜ produced a high amount of nitrogen oxide, indicating that the S. cerevisiae extracellular polysaccharide peptide (ePSP-Cv-SBE) is equivalent. An immunomodulator with potential for application. Example 5: Xueyan extracellular polysaccharide peptide (ePSP-Cv), Scutellaria barbata exopolysaccharide peptide (ePSP-Cv-SBE) and Fengzhilian polysaccharide peptide (PSP-SB) read macrophages to produce cytokines The cells were seeded in 24-well plates, and the cell concentration of each cell was 105 cells/5 00p. 250μ1 polysaccharide peptide and 250μ1 LPS or the same volume of medium were added to make the final volume of each well reach the final concentration of 62.5. Pg/ml polysaccharide peptide. Place the cell supernatant in a cell culture incubator (5% C02, 3 7. Culture medium 48 small 18 201116623), freeze at -80 °C for analysis of cytokine secretion assay. Commercialized cytokine ELISA kit The cytokine concentration of the culture supernatant was determined by using a mouse interleukin ip (m〇use iL ip, e-Bioscience), mouse interleukin-6 (mouse IL_6, e_Bi〇science), mouse tumor Necrosis factor-a (TNF-a) (mouse TNF-α, e-Bioscience) was quantified by commercialization. In the macrophage RAW 264.7 medium, Yunzhi extracellular poly-glycopeptide (ePSP_Cv) was added. Exopolysaccharide peptide (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide (PSP-SB) were studied in the presence or absence of LPS, and the three cytokine secretions were investigated. The effect of macrophage production of TNF-a is shown in Figure 4. 'The results show that when Lps is not added (_Lps), the three polysaccharide peptides can significantly stimulate macrophages to produce 4-5 times of TNF-a relative to the control group. Amount; however, if LPS is added (+Lps), both cells with or without added polysaccharide peptide will significantly increase TNF _a amount, and there is no significant difference between them. The effect of two polysaccharide peptides on the production of IL_ip by macrophages is shown in Figure 5. Compared with the control group, the three polysaccharide peptides can also stimulate the production of IL by macrophages. Ιβ. When LPS is not added (_Lps), the addition of S. cerevisiae extracellular polysaccharide peptide (ePSP-Cv-SBE) and Scutellaria barbata polysaccharide peptide (psp_SB) can promote macrophage production of IL-Ιβ' The extracellular polysaccharide peptide (epSp-CvSBE) of Lianyunzhi has a very significant increase in the biological activity of IL_ip in macrophages; if Lps is added (+ LPS), the extracellular glycopeptide (ePSP-Cv-SBE) ) and Scutellaria barbata 201116623 Peptide (PSP-SB) can also induce iL-1 β in giant sputum cells, but there is no significant difference between them * There is no addition of Scutellaria barbata extract in the medium to produce extracellular The effect of polysaccharide peptide and Scutellaria barbata polysaccharide peptide on the production of cytokine IL-6 produced by macrophage RAW 264.7 in the presence or absence of lipopolysaccharide (LPS) showed that 嗤 264.7 was added to ePSP-Cv-SBE. And PSP-SB, significantly increased IL-6 production, compared with the control group increased by 19 5 and 7. 〇 times. PSP in vivo The study model is widely used in the immunomodulatory activity of megatuber cells or other white blood cells to promote interleukin ability. In this case, ePSP-Cv-SBE can increase macrophages to increase TNF-α, IL-Ιβ and il-6 cells. The amount of hormone secreted. TNF-a and ΙίΛΙβ have a toxic effect on cancer cells and activating sputum lymphocytes. Mtiller and Meineke pointed out that cytokines are related to each other. TNF-a and IL-Ιβ induce IL-6 synthesis, while iL-6 inhibits TNF-a and IL-Ιβ. These three cytokines play an important role in immune regulation. . Extracellular and intracellular polysaccharides obtained by Cui et al. using whey as an additive to Yunzhi medium also have similar immunomodulatory activities. At the same time, the polysaccharide peptide obtained by adding the sputum extract to Yunzhi medium can also increase the activity of IL-Ιβ, IL-6 and TNF-a, indicating that the immunological activity of PSPs induced by macrophages and their different chemical compositions may be Close relationship, that is, the difference in its composition will cause hormones that produce different secretions to RAW264 7 cells.

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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 201116623 [Simple description of the diagram] Figure 1: Analysis of polysaccharide peptide by Fu-Fin Infrared Analyzer (FTIR), in which ePSP-Cv represents Yunzhi extracellular polysaccharide peptide; ePSP-Cv-SBE represents half-branched 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 significant differences (P < 〇 _ 〇 5) 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 addition of LPS; statistical analysis significant differences (P < 〇.〇5) 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 < 〇.〇5) are indicated above the column in English letters. 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 < 〇·〇5) are indicated above the column in English letters. [Main component symbol description] 26 201116623

+ SBE -SBE ePSP-Cv-SBE ePSP-Cv PSP-SB -LPS + LPS Untreated Adding Scutellaria barbata extract without adding Scutellaria barbata extract Scutellaria barbata exopolysaccharide peptide Yunzhi extracellular polysaccharide peptide half branch Lotus polysaccharide peptide without added lipopolysaccharide plus lipopolysaccharide control group TNF-α tumor necrosis factor-a (Tumor Factor-α )

Il-ΐβ interleukin-1β (Interleukin-ΐβ) 11_6 Interleukin-6

Necrosis

27

Claims (1)

201116623 VII. Patent application scope: A kind of extracellular multi-peptide peptide with arabinose, which is produced from the medium of adding annual or perennial herb extracts to the Yunzhi strain. 2. The strain of the Yunzhi extracellular enzyme peptide with arabinose as described in the first paragraph of the patent application is the LH1 strain, which is deposited in the Bioresource Conservation Center of the Food Industry Development Research Institute. The registration number is BCRC. 930112. 3. The extract of the annual or perennial herbaceous plant extract of the genus Ganoderma lucidum, as described in claim 1, is an extract of the genus Astragalus. 4 · The extracellular polyglycolic peptide of Agaricus chinensis with the arabinose as described in the third paragraph of the patent scope of the application of the sho sho. 'The genus Astragalus is a scutellaria (& barbata) ψ ^ 〇5·If applying for a patent The extracellular multi-branched type of the ganoderma lucidum having the arabinose described in the first item is a product obtained by subjecting the Yunzhi strain to a deep culture in a liquid medium. 6. The extracellular peptide of the genus Yunzhi with arabinose as described in claim 5, wherein the liquid medium comprises a carbon source, a nitrogen source and a salt as a nutrient. 7. The liquid medium containing arabinose as described in claim 6 of the patent scope, wherein the liquid medium comprises glucose as a carbon source and peptone is 28 201116623 I source 'potassium dihydrogen phosphate (ΚΗ2Ρ〇4) And magnesium sulfate (MgS04.7H20) is a nutrient component of the salt. 8. The extracellular multi-enzyme of Yunzhi with arabinose as described in claim 7 of the patent application. The liquid medium comprises 10% or less of glucose as a carbon source and 3% or less of a protein gland as a nitrogen source, and 2% or less. Potassium dihydrogen phosphate (KH2P〇4) and 2% or less magnesium sulfate (MgS04.7H20) are the nutrient components of the salt. 9. The ganoderma lucidum extracellular polysaccharide peptide having arabinose as described in claim 4, wherein the liquid medium comprises a scutellaria extract of 5% or less. 10. A method for producing an extracellular polysaccharide peptide of yoghurt having arabinose, comprising: preparing a liquid medium; adding an annual or perennial herb extract to the liquid medium; inoculating the Yunzhi strain in the liquid medium; performing deep culture ; and • Separation of products after deep culture. 11. The method for producing an extracellular peptide of Yunzhi with a gum arabic as described in claim 10, wherein the strain of Yunzhi is Verhco/orLH1, and is deposited in the Biological Resource Conservation Center of the Food Industry Development Research Institute. The login number is BCRC 930112. The method for producing an exopolysaccharide peptide of the arabinose as described in claim 10, wherein the annual or perennial herb extract is an extract of the genus Astragalus. The method of producing the ganoderma lucidum extracellular polysaccharide peptide having arabinose according to the invention of claim 12, wherein the plant of the genus Astragalus is a Scutellaria barbata extract. 14. The method for producing an exopolysaccharide peptide having an arabinose according to claim 10, wherein the liquid medium comprises a carbon source, a nitrogen source and a salt as a nutrient. 15. The method for producing a cyanosugar-free exopolysaccharide peptide having arabinose according to claim 14, wherein the liquid medium comprises glucose as a carbon source, protein gland as a nitrogen source, and potassium dihydrogen phosphate (KH2P〇4). And magnesium sulfate (MgSCV7H2〇) is a salt that is a nutrient component. 16. The method for producing an exopolysaccharide peptide having an arabinose according to claim 15, wherein the liquid medium comprises less than 1% glucose as a carbon source, 3% or less protein, and a gland as a nitrogen source, 2 The following potassium dihydrogen phosphate (KH2P〇4) and 2% or less magnesium sulfate (MgS〇4.7H2〇) are the nutrient components of the salt. 17. The method for producing an exopolysaccharide peptide having an arabinose according to claim 13, wherein the liquid medium comprises a scutellaria extract of 5% or less. 18. A species of yoghurt extracellular polysaccharide peptide having arabinose, comprising a product as described in claim 5, and a composition comprising a diluent, an excipient, a carrier or a formulation. I9·-A kind of yoghurt extracellular polysaccharide peptide composition having arabinose, comprising the product as described in claim 30 201116623, the scope of the patent, and comprising a diluent, a excipient, a carrier or a formulation. combination. 31