TWI379001B - Extracellular polysaccharides with arabinose from trametes versicolor lh1 and enhancing their production methods - Google Patents
Extracellular polysaccharides with arabinose from trametes versicolor lh1 and enhancing their production methods Download PDFInfo
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1379001 六、發明說明: 【發明所屬之技術領域】 本發明所屬之技術領域為藥用菌類多醣肽,更特定而 言,係關於具有阿拉伯糖之雲芝胞外多醣肽及促進其生產之 方法與含彼之組合物’可用以調節免疫活性。 【先前技術】 雲芝學名為 Trametes versicolor (syn. Coriolus vemco/or) ’寄生於腐木,為白色菌絲體,生長分佈於全世 界’是一種廣泛使用之藥用真菌(Wasser,& Weis,1999)。雲 芝子貫體生長缓慢,且價格昂貴。以液態培養方式所生產之 雲芝多醣肽已知具有免疫調節與抗腫瘤之生物活性(Cui,& Chisti, 2003)。雲芝多醣肽可從不同雲芝品系如 CM-101(ATCC 20547)或 Cov-l 分離(Kobayashi,Matsunaga,&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)。多醣肽中之多醣體以α (1—4)及 P(l—3)之鍵結,於體外、體内及臨床實驗上已證實其具免疫 調節活性(Hsieh,Wu,Park,& Wu,2〇〇6)。從雲芝菌株 c〇v i 品系製備所得之多醣肽可增加免疫細胞介白素IL_ip及 IL-6之分泌量,且誘發人類HL_6〇淋巴癌細胞之細胞凋亡 (apoptosis) (Hsieh, Kunicki, Darzynkiewicz, & Wu, 2002) 〇 動物體内實驗中,從餵食多醣肽之老鼠分離出之巨噬細 3 1379001 胞,含有活性氮中間體(reactive nitrogen intermediates, RNI)、超氧化物陰離子(super〇xide anions)及腫瘤壞死因子 -7(Tumor Necrosis Factor-γ ' TNF-γ) (Liu, Ng, Sze, & Tsui, 1993)。雲芝的多醣肽可以活化巨噬細胞、刺激分泌細胞激素 TNF-γ、干擾素-γ ( interferon-γ,IFN-γ )及介白素-1β (Interleukin-1 β, IL-Ιβ),這些細胞激素係用以對抗細胞增生 及引起細胞凋亡及分化。雲芝多醣肽可引起免疫系統非專一 性防禦及透過服用者本身免疫系統進而產生抗腫瘤作用(Cui, & Chisti,2003; Ng,1998)。 半枝蓮(έαΜαία )為一種多年生中草藥,廣 泛分佈於南中國及韓國,常被用於抗發炎、利尿與抗腫瘤, 尤其是肝癌' 旰炎及肝硬化之治療(Wang,& Lu, 2005),最近 從半枝蓮萃取物中分離出類黃酮(flav〇n〇ids )及二莊類 (diterpernoids )的新克羅烷(neoclerodane )、三萜酸 (triterpenic acid ) 及谷留醇葡萄甙 (phyt〇Steryl-p-D-glUCOSicie )等具有生物活性(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):且 在小鼠肝# H22細胞上也發現半枝蓮萃取物可抑制癌細胞 增生,且可做為化學治療的輔助劑(Dai,Uu,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_)。半枝蓮萃取物中所得多醣㈣重要生 4 Ιό/^ΌΌΙ '物舌丨生成刀,在動物試驗中明顯具抗氧化活性(王志遠,戴玲, 張凱,2008)及免疫調節功能(林敬明,劉煌,&羅榮城, 2006) 〇 . 般真菌深層發酵槽培養,其菌絲體生長及胞外多醣肽 生產又培養條件環境及培養基組成影響。以牛肝菌(价/dwi PP )為例,進行培養所產生之菌絲體及胞外多醣肽受如溫 度攪拌速度及起始pH值等因子影響(Wang,&以,2〇〇5)。 ®不同雲芝菌株品系Wr_74和ATCC-20545,利用乳清添加於 坨養基中可影響其菌絲體及胞外多醣肽之產量G〇h, Aixhei·’ & Singh,2007)。胞外多醣肽之化學組成和特性會受 到i。養基成分之影響,例如Chen,Hsu,Lin,Lai,and Wu (2006)利用不同碳源之培養基’培養乃, 所得之胞外多醣肽會產生不同單醣組成,其胞外多醣肽會刺 激巨噬細胞RAW264.7產生不同量之一氧化氮及細胞激素。 【發明内容】 本案發明人自台灣南投山區採集不同雲芝子實體,經分 離培養後發現其中一雲芝菌株品系LH4,生長快速且菌絲體 產1尚’經食品工業發展研究所生物資源保存中心鑑定其學 名為7>ame如vewco/or (L)L1〇yd【菌種鑑定報告如附送書 件】’研究發現能產生新穎胞外多醣肽(簡稱ePSp_Cv),且在 所採用之培養基條件下具有較高之產物比生成率與促進細 5 1379001 胞激素分泌之免疫調節活性,其中該細胞激素為腫瘤壞死因 子·α ( TNF-α)。於培養基中添加微量半枝蓮( )萃取液(簡稱SBE ),更可顯著提高雲芝胞外多醣 肽(簡稱ePSP-Cv-SBE)產物比生成率及免疫調節生物活性, 該免疫調節活性包含促進巨噬細胞增生、促進巨噬細胞一氧 化氮產生及促進細胞激素分泌量;其中該細胞激素為腫瘤壞 死因子-a(TNF-c〇、介白素_ip(IL-ip)及介白素_6(il_6)。 【實施方式】 雲芝versico/or LH1) 目前寄存於財團法人食品工業發展研究所生物資源保 存中心,寄存編號為BCRC930112。 半枝蓮萃取液(SBE) 將乾燥半枝蓮以1(半枝蓮):1〇(水)比例加水煮沸後, 將上清液減壓濃縮到體積為1〇〇毫升,得到半枝蓮萃取液 (SBE) 〇 雲芝液態培養及胞外多醣肽分離製備 刮取上述保存之雲芝菌種,將其接種於培養基,在25〇c 服度下,於恆溫震盪培養箱中以振盪速率丨5〇卬爪振盪培養$ 天做為種菌。 其中該培養基之一實施配方如下: 培養基配方 6 1379001 成分 含量(%) 葡萄糖(glucose) 4 蛋白腺(peptone) 0.3 磷酸二氫鉀(kh2po4) 0.15 硫酸鎂(MgS04 7H20) 0.15 分別以無添加半技蓮萃取液(SBE)之上述培養基,或添 加0.5% (v/v)半枝蓮萃取液之上述培養基,以1〇%種菌接 • 種量,於20公升發酵槽中進行培養(溫度25 °C ;攪拌速率 150 rpm ;通氣量1 vvm)。將培養7天後之醱酵培養液,以 6000 rpm離心30分鐘,可得菌絲體及上清液。將上清液經 四倍量的95 %酒精’澱析24小時後再經離心,沉澱之多醣 肽即為胞外多醣肽(extracellular p〇lySaccharopeptides, ePSP)。本發明中將培養基中無添加半枝蓮萃取物(SBE)培養 所得分離之雲芝胞外多醣肽稱為「雲芝胞外多醣肽 ® (ePSP-Cv)」,培養基中添加半枝蓮萃取物(SBE)培養所得分離 之雲芝胞外多醣肽稱為「半枝蓮雲芝胞外多醣肽 (ePSP-Cv-SBE)」。 分析方法 菌絲敢生質量(biomass)測定 將醱酵培養液以6000 rpm離心3〇分鐘,可得菌絲體及 上清液。將菌絲體加入10 ml去離子水懸浮清洗,以6〇〇〇rpm 7 1379001 離心30分鐘,重複三次。所得菌絲體冷凍乾燥後秤重,即 為菌絲體生質量。 胞外多醣肽測定 將醱酵培養液以6000 rpm離心30分鐘,可得菌絲體及 上清液。上清液加入四倍量的95%酒精,澱析24小時後再 經離心’沉澱物依據Dubois等人(1956 )「酚硫酸法 (phenol-sulphuric acid method)」及 AOAC 方法(1995)分析其 中含多醣及蛋白質組成,此一製備物為即為胞外多醣肽,將 此沉殿物冷;東乾燥後秤重。 半枝蓮多醣肽(PSP-SB)測定 將半枝蓮萃取液(SBE)以四倍量95%酒精沉澱24小時 後’離心分離所得之沉澱物,依據Dubois等人(1956)「盼 硫酸法(phenol-sulphuric acid method)」及 AOAC 方法(1995) 分析其中含多醣及蛋白質組成,此一製備物為半枝蓮多醣肽 (PSP-SB ) ’將此沉澱物冷凍乾燥後秤重。 统計分析 本發明中之實驗數據以Statistical Analysis System 6.0 軟體進行分析,以ANOVA作變異分析,並以Duncan,s multiple range test做平均值顯著性差異之比較& < 〇 〇5)。 實施例一以半枝蓮萃取液(SBE)促進雲芝胞外多醣肽(epsp) 生產 雲芝菌株LH1接種於20公升發酵槽中,其中之培養基 8 1379001 .有兩種:分別為添加及無添加(對照組)半枝蓮萃取液之培 養基。測ϊ深層培養7錢所得之胞外多聽肽含量結果如 表-所示。與對照組比較,經添加〇 5%的半枝蓮萃取液(+sbe) 。養所得之半枝蓮雲芝胞外多醣肽(奶p_Cv_sBE)含量隨者 .培養時間逐漸增加,經7天培養後產量可達到137g/L,相 對於無添加半枝蓮萃取液之培養基(_SBE),雲芝胞外多酿肽 (ePSP-Cv)只得到0.61 g/L,產量顯著增加2 2倍。 # 纟發明與文獻中不同雲芝品系與培養基對其胞外多醣 狀(ePSP)、菌絲、體生質量及產&比生成率之比較如表一所 示。就產物比生成率之觀點,台灣本土雲芝品系所產生 之雲芝胞外多醣肽(ePSp_Cv)之產物比生成率及半枝蓮雲芝 胞外多醣肽(ePSP-Cv_SBE)之產物比生成率分別為〇 26及 0.38,皆較習知文獻中產物比生成率為高。 過去文獻中’雲芝菌株品系INETI培養於酵母麥芽培養 參基中只能產生0·70 胞外多醣肽(Tavares,Coelh。,Agapit。, Coutinho & xavier, 2005);雲芝菌株品系 Wr_74 及 ATCC-20545,培養於添加乳清之培養基中能產生丨1 5和 1.3 2 g/L 胞外多醣肽(Cui,Goh,Archer,& Singh,2007),但產 量均較本發明之半枝蓮雲芝胞外多醣肽(epSP_Cv_SBE;^4 1.66 g/L 為低。 9 1379001 表一本發明與文獻中不同雲芝品系與培養基對其胞外多醣肽(ePSP)、 菌絲體生質量及產物比生成率之比較 雲芝品系 添加物 胞外多 醣肽 (g/L) 菌絲 體生 質量 (§/L) 產物比 生成率 * 參考文獻 LH1 無 0.61 2.38 0.26 本發明 LH1 0.5%半枝 蓮萃取液 1.37 3.70 0.38 本發明 INETI 酵母麥芽萃 0.70 3.8 0.18 Tavares et al. 取物 (2005) Wr-74 乳清 1.15 8.9 0.13 Cui etal. (2007) ATCC20545 乳清 1.32 10.6 0.12 Cui etal. (2007) *產物比生成率:胞外多醣肽/菌絲體生質量 本發明中半枝蓮萃取液(SBE)於培養基中之添加量為 0.5% (v/v),亦即每毫升培養液中含有極微量半枝蓮萃取物 即可產生顯著增加半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE)產 量之效果。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).
利用AOAC (1995)方法檢測半枝蓮萃取物之一般化學組 成,其碳水化合物佔38.10%、粗蛋白佔16.27%、粗脂肪佔 6.75%、灰分佔10.71 %及粗纖維佔28.17%,顯示碳水化合物 為其主要化學组成。半枝蓮中含有黃芩素苷(scutellarin)、芹 菜 素(apigenin)、木 犀草素(luteolin)、 E-1-(4V-hydroxyphenyl)-but-1-en-3-one 、原 兒茶酸 (protocatechuic acid) 、 acacetin-7-diglucuronide 及 luteolin-7-diglucuronide及其他黃酌類化合物等。微生物培 1379001 養基之成分在菌體生長及代謝物分泌過程扮演了重要角 色,半枝蓮萃取液之添加有益於菌絲生質量及ePSP生產可 能與其所含成分有關。 實施例二雲芝胞外多醣肽(ePSP-Cv)、半枝蓮雲芝胞外多醣 肽(ePSP-Cv-SBE)及半枝蓮多醣肽(PSP-SB)化 學特性 一、蛋白質含量 以AOAC方法(1995)分析之。 如表二所示,雲芝胞外多醣肽(ePSP-Cv)之蛋白質含量為 13.8 士 0.3%,半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE)之蛋白 質含量為15.6 ± 0.3%;半枝蓮多醣肽(PSP-SB)之蛋白質 含量為19.0 ± 0.7%。半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE) 之蛋白質含量為雲芝胞外多醣肽(ePSP-Cv)之1.13倍。 表二雲芝胞外多醣肽(ePSP-Cv)、枸杞雲芝胞外多醣肽(ePSP-Cv-SBE) 及枸杞多醣肽(PSP-SB)蛋白質含量比較 多醣肽 蛋白含量(%) 雲芝胞外多醣肽(ePSP-Cv) 13.8 ±0.3 半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE) 15.6 ±0.3 半枝蓮多醣肽(PSP-SB) 19.0 ±0.7 二、單醣組成分析 以 5 毫升之 2M 三氟醋酸(trifluoroacetic acid, TFA )水 解多醣肽,於1 〇〇°C作用16小時。再以高效能液相層析儀 (HPLC,Jasco PU-2080 PLUS ),以 SUGAR SP0810 管柱 1379001 (S ho dex) (8 mm x 3 00mm)分析中性單醋成分,其中該管柱之 流速為0.8ml/min,移動相為去離子水,管柱之溫度維持於 80oC。 本發明中雲芝胞外多醣肽之單醣含量組成與其他文獻 之比較,如表三所示,成分包含葡萄糖(glucose)、半乳糖 (galactose)、甘露糖(mannose)、木糖(xylose)及阿拉伯糖 (arabinose)。與Tavares ei αΛ (2005)之文獻比較,其雲芝胞 外多醣肽中單糖組成中未發現有半乳糖之存在,而本發明中 台灣本土雲芝LH1品系所產生之雲芝胞外多醣肽(ePSP-Cv) 及半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE)之單糖組成中則分 別含有8.67 mg/g及2 1.3 8 mg/g之半乳糖,顯然為一新型雲 芝胞外多醣肽。 表三本發明中雲芝胞外多醣肽之單醣含量組成與其他文獻之比較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
單糖組成(mg/g ratio) 多醣肽 葡萄糖 半乳糖 甘露糖 木糖 阿拉伯糖 引用文獻 ePSP-Cv*1 82.27 8.67 8.18 0.87 n.d.*2 本發明 ePSP-Cv-SBE*3 60.64 21.38 6.66 7.43 3.89 本發明 ePSP-Cv-LBE*4 80.06 9.29 1.92 8.73 n.d. Lin et al., 2008 PSP-SB*5 10.78 71.00 15.45 n.d. n.d. 本發明 PSP-LB*6 49.21 17.06 n.d. 1.33 32.40 Lin et al., 2008 12 1379001 無添加中藥萃取液所得之雲芝胞外多醣肽 *2n.d.未偵測到 *3添加半枝蓮萃取液所得之雲芝胞外多醣肽 *4添加枸杞萃取液所得之雲芝胞外多醣肽 *5半枝蓮多醣肽 *6枸杞多醣肽 比較半枝蓮多醣肽(PSP-SB )、雲芝胞外多醣肽(ePSP-Cv) 及半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE),其中半枝蓮多醣 肽含有較多的半乳糖(71.00 mg/g)及甘露糖(1 5.45 mg/g),且 無檢測出木糖及阿拉伯糖。然而,經過7天發酵培養後,於 雲芝半枝蓮胞外多醣肽中檢測到阿拉伯糖(3.89 mg/g),表示 半枝蓮萃取液(SBE)中之半枝蓮多醣肽(PSP-SB),在發酵過程 中已被使用且轉化成不同之單醣。 比較雲芝胞外多醣肽(ePSP-Cv)及半枝蓮雲芝胞外多醣 肽(ePSP-Cv-SBE),半枝蓮雲芝胞夕卜多之 葡萄糖含量(60.64%)遠低於雲芝胞外多醣肽(ePSP-Cv)葡萄 糖含量(82.27%),而半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE) 之半乳糖含量(21.38%)遠高於雲芝胞外多醣肽(ePSP-Cv)半 乳糖含量(8.67%),同時半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE) 之木糖含量(7.43%)遠高於雲芝胞外多醣肽(ePSP-Cv)木糖 含量(0.87%),但該兩組之胞外多醣肽所含之甘露糖含量,基 本上是接近的,介於約為6.66-8.18%。顯示微量半枝蓮萃取 液添加於雲芝醱酵培養基中,不僅有助於其胞外多醣肽產量 之大幅提升,對於其胞外多醣肽之化學組成及單糖含量亦有 13 1379001 明顯之影響。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以β/· (2005)之文獻中,利用兩種不同培養基: 菇完全培養基(mushroom complete medium)及酵母麥芽萃取 物(yeast malt extract)培養基培養雲芝菌INETI品系,亦顯 不培養基之組成差異會改變其所產生之胞外多醣肽中甘露 糖與木糖之含量。 三、多醣肽之傅氏紅外線光譜分析 以傅氏紅外線光譜儀試驗(Fourier Transf()m Infl_aFed, FTIR)檢測其官能基,將樣品與KBr粉末於真空烘箱乾燥24 小時’混合均勻後(1:1 〇〇)打成錠片’以傅立葉紅外線光譜儀 測試’其波數範圍由500〜4000 cm_1,解析度設定2 cm_i之 圖譜。 利用 FTIR 分析 ePSP-Cv 和 ePSP-Cv-SBE 發現在 3419 ' 1641及1〇78 cm-i位置有明顯之吸收波峰(如圖一),Ng及 Chan提雲芝PSP的FTIR圖譜中有一波峰在1078 cm-i位置,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及Zhou指出雲芝PSP的FTIR圖譜中在3400、 1650、 1050 及 893 cm_丨各代表 _0Η、·ΝΗ2、C_〇_c 及 p_glyc〇sidic 等官能基鍵結。在930、1038、1078及1161 cm·1位置亦表 現出有β-glycosidic等官能基鍵結。本實驗中三種多醣肽之 吸收波峰中雖無893 cm·1位置,但有i〇78 cm·1位置可說明 具有β(1->3) glycosidic官能基之鍵結。Yang及zhang報告 1379001 中才曰出905 876 cm-1位置中有p_D-glucose之吸收波峰,在 11〇〇~1010 Cm】範圍Pyranoside有三個強烈吸收峰, furanoside有二個吸收峰,本研究於11〇〇_1〇1〇 εηΓι範圍亦 顯示有明顯吸收峰出現。Mata等分析甜菜實驗以FTIR之資 料亦說明1146 cm】位置上有giyCosidic鍵結存在。Rau等 研究報導中指出C_ ATCC 2〇0801之胞外多醣肽中 含有以β(1—3)為主鏈連結葡萄糖,而在Q側鏈以p(1—6) 連結另一葡萄糖’說明胞外多醣肽同樣具有生物活性之 β( 1—3)鍵結之結構。 综合上述雲芝胞外多醣肽(ePSP_Cv)、半枝蓮雲芝胞外多 醋肽(ePSP-Cv-SBE)及半枝蓮多醣肽(PSP-SB)化學特性分 析與文獻之比較討論,顯示本發明中台灣本土雲芝LH1品系 添加半枝蓮萃取液所產生之雲芝胞外多醣肽 (ePSP-Cv-SBE),使其胞外多餹肽產量增加,也使其化學組 成蛋白質含量及單醣組成分改變,而PSP- SB、ePSP-Cv和 ePSP-Cv-SBE這三種多醣肽均具有β(1 —3)葡聚醣之生物活 性結構。比較本發明中雲芝胞外多醣肽(ePSP-Cv)及半枝蓮雲 芝胞外多醣肽(ePSP-Cv-SBE),兩者在蛋白質含量及單糖組 成含量亦有所不同。 實施例三雲芝胞外多醣肽(ePSP-Cv)、半枝蓮雲芝胞外多醣 肽(ePSP-Cv-SBE)及半枝蓮多醣肽(pSp_SB) 對巨噬細胞增生之影響 15 1379001 一、巨噬細胞RAW 264.7培養及多醣肽處理 將老鼠巨噬細胞RAW264.7 (ATCC TIB-71)培養於添加 10%胎牛血清、100 units/ml青黴素及100 g/ml鏈黴素之 Dulbecco's Modified Eagle Medium (DMEM)培養基(Gibco 公司)中, 置於含有5%二氧化碳、37°C的細胞培養箱中培養,培養24 小時後,以2x 105 cells/ml細胞密度進行試驗,分別加入濃 度為25 0 pg/mL的三種多醣肽:雲芝胞外多醣肽(ePSP-Cv)、 半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE)及半枝蓮多醣肽 (PSP-SB)於DMEM培養基中。 二、多醣肽對巨噬細胞之影牢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
以MTT測試法分析老鼠巨噬細胞RAW264.7之細胞存活 及增生率,MTT測試法是測量存活細胞之粒線體内去氫酵素 (dehydrogenase)活性,用以間接測定細胞存活及增生率,該 方法為將細胞種於96孔盤中,使每孔之細胞密度為lxl04 cells/1 ΟΟμΙ/well,再分別添加最終濃度為62.5pg/ml之多· 肽ePSP-Cv、ePSP-Cv-SBE或PSP-SB溶液於細胞中,培養 48小時後,再加入30 gL MTT於C02培養箱(3 7°C)中培養2 小時,去除上清液再加入DMSO,以ELISA reader (Bio-Tek, pQuantTM)測其570 nm之吸光值。未加藥物組之O.D值為基 本對照組(100%)。 結果顯示巨嗤細胞RAW 264.7添加入62.5 pg/mL的 16 1379001 . ePSP-Cv、ePSP-Cv-SBE 和 PSP-SB,經 48 小時培養後相對 細胞生長率為 1〇1.3±14.〇、13〇.3±11.9 及 120.6±17·0%,統計 分析顯示此三種萃取物對巨嗟細胞存活沒有顯著影響,如圖 二所示。 實施例四雲芝胞外多醣肽(ePSP_Cv)、半枝蓮雲芝胞外多醣 肽(ePSP-Cv-SBE)及半枝蓮多醣肽(PSP-SB) 對誘導巨噬細胞產生一氧化氮之影蜜 將巨噬細胞種於96孔盤中,使每孔之細胞密度為1 X 1 〇4 Φ cells。每孔中分別加入50μ1不同之多醣肽(最終濃度為 62.5pg/ml )後,再加入過濾後溶於DMEM之4pg/ml之50μ1 脂多糖(LPS)或同體積之培養基。48小時培養後,培養基中 一氧化氮之含量係以Greiss反應分析之,取出細胞之上清液 加入 96孔盤中,再加入等量的 Greiss 試劑(1〇/0 sulfanilamide、0.1% NED 及 5% H3P〇4)和 RNI,於室溫反 應15分鐘,以ELIS A reader讀OD540 nm的吸光值。產生 9 之一氧化氮產量以硝酸納(sodium nitrite)製作標準曲線來對 照定量。 • 結果如圖三所示,於無添加1 pg/ml LPS時(-LPS), ePSP-Cv與對照組無顯著差異’而添加ePSP-Cv-SBE及 PSP-SB可誘導巨噬細胞產生大量的一氧化氮。添加LPS時 (+LPS) ’添加epSP-Cv及ePSP-Cv-SBE與對照組間並無明顯 差異,但添加PSP-SB卻有較少NO的產量。 巨嗤細胞產生一氧化氮被認為是細胞的—種防货功 17 1379001 能,一般係以LPS刺激細胞產生一氧化氮,作為一種檢測免 疫調節活性之方式。Wasser (2005)於研究中指出真菌含有 β( 1 —3 )葡聚醣多醣體可活化巨噬細胞,並促使巨嗟細胞產生 一氧化氮,而一氧化氮於許多生物免疫反應令是很重要化學 傳遞物質。Pang等(1998)利用老鼠腹腔巨噬細胞經添加lPS 及PSK(為一種來自c. 菌株CM-101品系之多醣 肽)後,可產生一氧化氮。Wang等(1996)研究中指出經[PS 處理後C57BL/6老鼠也會誘導一氧化氮之產生,但若银食來 自C. 菌株Cov-Ι品系所產生之胞外及胞内多醣 肽’會被刺激產生更高量之一氧化氮。 於本實施例中,未添加LPS(-LPS)時,半枝蓮雲芝胞外 多§1肽(ePSP-Cv-SBE)即可促使巨嗔細胞產生高達3.20 μΜ 之一氧化氮量’比對照組0.24 μΜ,或添加LPS(+LPS)時多 餹肽組之平均值2.59 μΜ所產生之一氧化氮量為高,顯示半 枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE)為一相當具有應用潛力 之免疫調節劑。 實施例五雲芝胞外多醣肽(ePSP-Cv)、半枝蓮雲芝胞外多醣 肽(ePSP-Cv-SBE)及半枝蓮多醣肽(PSP_SB) 誘導巨噬細胞產生細胞激素之分析 將細胞種於24孔盤中,使每孔細胞濃度為i〇5 cells/500p卜分別加入250μ1多醣肽及250μ1 LPS或同體積之 培養基,使每孔的最終體積達到lm卜最終濃度為62.5pg/ml 之多醣肽。放入細胞培養箱(5% C02,370C)中培養48小 印9001 時,收集細胞上清液,冷凍於-8〇°C以便進行細胞激素分泌 的測定分析。以商業化細胞激素ELISA套組測定培養上清液 的細胞激素濃度。採用小鼠介白素_1β (mouse IL_ip, e-Bi〇science)、小鼠介白素 _6 (m〇use IL_6, e_Bi〇science),小 乳腫瘤壞死因子-a(TNF-a) (mouse TNF-α,e-Bioscience)商業 化套組來定量之。 於巨嗤細胞RAW 2 64.7培養基中,分別添加雲芝胞外多 聰肽(ePSP-Cv)、半枝蓮雲芝胞外多醣肽(ePSp_CvSBE)及半 枝蓮多醣肽(PSP-SB),於有/無添加LPS之處理後,探討前 述二種細胞激素分泌量。三種多醣肽對於巨噬細胞產生 TNF-a之影響如圖四所示,結果顯示未添加Lps時卜^”, 该三種多醣肽相對於對照組,均能顯著刺激巨噬細胞產生 4-5倍之TNF_a量;但若添加Lps時(+Lps),無論是有/無添 加多醣肽之細胞,均會顯著增加丁1^17_〇1量,且彼此間並無顯 著差異。 三種多醣肽對於巨噬細胞產生IL_ip之影響如圖五所 不’與m比較’該三種多酿肽亦均能刺激巨嗟細胞產生 IL W。於未添加LPS時(-LPS),添加半枝蓮雲芝胞外多醣肽 (ePSP_CvjBE)及半枝蓮多膽肽(psp_SB)可促使巨嗟細胞 產生1L-1P,其中半枝蓮雲芝胞外多醣肽(ePSP-Cv-SBE)具有 非常顯著提高巨噬細胞IL,之生物活性;若添加Lps時 (LPS)半枝蓮雲芝胞外多醣肽(ePSp_Cv_SBE)及半枝蓮多 1379001 醋狀(PSP-SB)亦可促你么始方 . 』炖便巨噬細胞產生IL-Ιβ,但彼此間並 無顯著差異。 有無添加半枝蓮萃取液於培養基中所產生雲芝胞外多 醣肽及半枝蓮多st肽於有無添加脂多醣(Lps)對巨嗟細胞 . RAW 264.7產生細胞激素比-6產量之影響如圖六所示,結果 顯示巨么細胞RAW 264.7添加epsP-Cv-SBE和PSP-SB,明 顯增加IL-6產量,與對照組比較增加19 5及7 〇倍。 • PSP於體外研究模式以巨噬細胞或其他白血球促進白細 胞介素能力之探討廣泛使用於免疫調節活性上。在本案中 ePSP-Cv-SBE可使巨噬細胞增加TNF-ot、IL-Ιβ及IL_6細胞 激素之分泌量。TNF-α及IL-Ιβ對癌細胞有毒殺之效果及活 化T淋巴細胞之作用。Miiller及Meineke指出細胞激素相互 間關係,TNF-ct及IL-Ιβ會誘導IL_6合成,而iL_6又會抑 制TNF-α及IL-1 β ’此三種細胞激素在免疫調節上扮演重要 • 角色。Cui等人利用乳清作為雲芝培養基之添加物所得到之 胞外及胞内多亦有類似的免疫調節活性。同時先前研究添 • 加枸杞萃取液於雲芝培養基中所得之多醣肽也可增加 IL-Ιβ ' IL-6及TNF-oc之活性,顯示PSPs誘導巨噬細胞所產 生免疫活性與其不同之化學組成可能有密切關係,亦即其組 成分之差異會造成對RAW264·7細胞產生不同分泌量之激 素0 20 1379001 【參考文獻】 1.王志遠、戴玲、張凱(民97),半枝蓮多醣的提取純化及抗氧化活性研 究, 中國生化藥物雜誌,29(2),96-103。 2. 王峰、丁重陽、王玉紅、章克昌(民94),九味f藥對雞腿蘑深層發酵 的影響,食品科學,26 ( 10),144-146。 3. 仲浩’薛曉霞,姚慶强(民97),半枝蓮化學成分的研究,中草藥,3The 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. 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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. andAnd immunomodulatory activity. Carbohydrate Polymers, 38(3), 247-253. 28. Spelman, K., Bums, J. J., Nichols, D., Winters, N., Ottersberg, S. and
Tenborg, M. (2006) Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. Alternative MedicineTenborg, M. (2006) Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. Alternative Medicine
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30. Tavares, A. P., Coelho, M. A., Agapito, M. S., Coutinho, J. A. and Xavier, A. M. (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. U.S. Patent 4,202,969. 32. Wang, Η. X., Ng, T. B., Liu, W. K., Ooi, V. E. and Chang, S. T. (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, Y. X. and Lu, Z. X. (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 - 1676.30. 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, Z. Q., Xu, F. M., Yan, X. Z. and Zhu, Y. (1996) Scutebarbatine A, a new neoclerodane-type diterpenoid alkaloid from Scutellaria barbata. Chinese Chemical Letters. 7(4), 333-334. 36. Wasser, S. P. (2005) Reishi or Ling Zhi (Ganoderma lucidum). Encyclop Diet Suppl. 603-622. 37. Wasser, S. P. and Weis, A. L. (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, L. M. (2009) Chemical structural and chain conformational characterization of some bioactive polysaccharides isolated from natural sources. Carbohydrate Polymers, 76(3), 349-361. 39. Yang, Q. Y. and Zhou, Y. F. (1993) A protein bound polysaccharide-PSP. In Proceedings of PSP International Symposium (Edited by Yang Q. Y. and Kwok, C. Y.). 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 lung 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 【圖式簡單說明】 圖一:俜式紅外線分析儀(FTIR)分析多醣肽圖譜,其中 ePSP-Cv表示雲芝胞外多醣肽;ePSP-Cv-SBE表示半枝蓮雲 芝胞外多醣肽;PSP-SB表示半枝蓮多醣肽。 圖二:不同多醣肽對巨噬細胞RAW264.7增生之影響; 統計分析顯著性差異(P < 0.05)以英文字母標示於立柱上方。 圖三:不同多醣肽誘導巨噬細胞RAW264.7產生一氧化 氮量之比較。黑柱表示無添加LPS,灰柱表示添加LPS ;統 計分析顯著性差異(P < 0.05)以英文字母標示於立柱上方。 圖四:不同多醣肽誘導巨噬細胞RAW264.7產生細胞激 素TNF-α之影響。黑柱表示無添加LPS,灰柱表示添加LPS ; 統計分析顯著性差異(P < 〇.〇5)以英文字母標示於立柱上方。 圖五:不同多醣肽誘導巨噬細胞RAW264.7產生細胞激 素IL-Ιβ之影響。黑柱表示無添加LPS,灰柱表示添加LPS ; 統計分析顯著性差異(P < 0.05)以英文字母標示於立柱上方。 圖六:不同多醣肽誘導巨噬細胞RAW264.7產生細胞激 素IL-6之影響。黑柱表示無添加LPS,灰柱表示添加LPS ; 統計分析顯著性差異(P < 0.05)以英文字母標示於立柱上方。 【主要元件符號說明】 ePSP-Cv-SBE半枝蓮雲芝胞外多醣肽 26 1379001 PSP-SB半枝蓮多醣肽 ePSP-Cv雲芝胞外多醣肽 —LPS未添加脂多酿 + LPS添加脂多醣 TNF-α 腫瘤壞死因子·α ( Tumor Necrosis Factor-α ) Ι1_1β 介白素-1β ( Interleukin-ΐβ)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 介白素-6 ( Interleukin-6)11_6 Interleukin-6
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