南五味子多糖提取及其对雏鸡免疫活性的研究
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摘要
南五味子多糖(Kadsura polysaccharides,KPS)是从五味子科(Schisandraceae)南五味子属(Kadsura)植物中提取的多糖类化合物。国内外对KPS的提取、化学组成及其药理活性研究报道较少,尤其是关于大叶南五味子(Kadsura marmorata)的相关研究报道更少。因此,本研究以大叶南五味子的果实为原料,进行了南五味子粗多糖的提取工艺、粗多糖免疫活性的检测、分离纯化及化学组成、免疫活性评价的相关研究,筛选出了KPS中具有明显免疫活性的多糖组分KPSⅢ,并进行了临床应用试验研究。
为获得试验用KPS,本研究首先优化并确定了南五味子水溶性多糖的最佳提取条件。依次用石油醚、乙酸乙酯和95%乙醇对大叶南五味子干燥果实粉末进行预处理,再以料液比为1:10～1:30,水提温度为60℃～100℃,水提时间为1 h～5 h进行单因素试验,确定水提取多糖最优水平。根据单因素实验结果,将料液比、水提温度、水提时间作为考察因素,多糖提取率为响应值,中心组合设计和响应面分析法优化确定KPS最佳提取条件为提取时间2.4 h、料液比1:28(m/V)、提取温度为95℃,提取率达到3.50 %,蒽酮-硫酸比色法检测糖含量为76.01 %。
本研究接着初步评价了自制KPS粗品对雏鸡免疫活性的影响。分别给7日龄雏鸡皮下注射KPS粗品,检测外周血淋巴细胞转化率、CD4+与CD8+淋巴细胞比值、ND-HI抗体效价、血细胞总数、脾脏、胸腺、法氏囊的脏器指数等免疫指标。结果显示,皮下注射KPS粗品高剂量组(20mg/Kg体重),于免疫后7 d,T淋巴细胞转化率明显高于APS组、CTX组、BCG组(P<0.01);免疫后第21 d,CD4+/CD8+比值明显高于CTX组、BCG组(P<0.01),胸腺、法氏囊脏器指数显著高于APS组(P<0.05),极显著高于BCG组、CTX组(P<0.01);于28日龄时,ND-HI抗体效价极显著高于APS组、CTX组、BCG组(P<0.01);还能促进外周血细胞总数的增多。由此表明,KPS粗品对雏鸡细胞免疫和体液免疫应答存在一定的促进作用。
为了获得具有免疫活性的KPS纯品,本研究先用TCA对KPS粗品脱蛋白、活性炭脱色纯化,再用20%~80 %乙醇分级沉淀,得到KPSI、KPSⅡ、KPSⅢ、KPSⅣ、KPSV五个级分,其中KPSⅢ最多,占52.31 %,糖含量高达95.13 %。各级分经SephadexG25柱梯度洗脱,洗脱曲线显示,KPSⅡ、KPSⅢ为单一组分,KPSI、KPSⅣ、KPSV含有不同分子量的糖组分。选取各级分中含量最多的组分,经PC和GC检测其单糖组成,结果显示,KPSI-1由Rib、Xyl及GalA组成(摩尔比为1.13:1.04:0.26);KPSⅡ由Fru、Glc及未知糖组成(摩尔比为0.26:0.10:0.11);KPSⅢ由Ara、Glc及GalA组成(摩尔比为0.22:0.24:0.09);KPSⅣ-6由Fru、Glc、GalA及未知糖组成(摩尔比为0.24:0.09:0.06:0.04);KPSV-1由Rha、Glc及GalA组成(摩尔比为0.33:0.15:0.10)。
其次,本研究经体外免疫活性测定试验,分别检测了KPSI-1、KPSⅡ、KPSⅢ、KPSⅣ-6、KPSV-1五个组分对雏鸡T淋巴细胞、B淋巴细胞、腹腔巨噬细胞增殖能力的影响,对T淋巴细胞分泌IL-2、IFN-γ及PMφ分泌TNF-α、NO的影响。结果显示,KPSⅡ、KPSⅢ均能直接和协同刺激源明显地促进淋巴细胞增殖,拮抗CTX对淋巴细胞的抑制作用;KPSⅢ、KPSⅣ-6均能显著地促进腹腔巨噬细胞增殖,拮抗CTX对巨噬细胞的抑制作用;KPSⅢ、KPSⅣ-6显著升高T淋巴细胞分泌IL-2、IFN-γ的含量及巨噬细胞分泌TNF-α、NO的含量。
基于以上结果,本研究选取KPSIII组分进一步研究其体内免疫活性及临床应用价值。以增强疫苗抗体效价、免疫器官脏器指数、促进动物生长和抗病作为检测指标进行了动物临床试验。结果显示,将KPSⅢ与传染性法氏囊D78株疫苗混合后饮水免疫可显著提高IBDV-AGP抗体效价;将KPSⅢ与新城疫Losata株疫苗混合后皮下注射免疫亦可显著提高ND-HI抗体效价,且能显著上调雏鸡脾脏、胸腺及法氏囊免疫器官的脏器指数。KPSⅢ还能够促进雏鸡生长,提高饲料报酬,减少疾病的发生,提高雏鸡的存活率。皮下注射0.8 mL 2×108 cfu/mL O2型大肠杆菌成功建立了雏鸡大肠杆菌病病理模型,在KPSⅢ的预防试验中发现,皮下注射20 mg/Kg体重的KPSⅢ能有效预防O2型大肠杆菌病的发生,使发病率极显著低于感染对照组(P<0.01),保护率达到75 %以上;在治疗试验中,死亡率控制在27.5 %,有效率达到82.0 %,与阴性对照组有极显著性差异(P<0.01)。
综上所述,本项研究为南五味子多糖的进一步研发及在兽医临床的应用提供了宝贵的实验技术依据。
Kadsura polysaccharides(KPS)is a type of polysaccharides compound extracted from Schisandraceae Kadsura plants. Up to date, few studies or reports focus on the extraction, chemical composition and pharmacological activities of KPS, especially reports on Kadsura marmorata was rarely. In this study, we developed an extraction method to extract crude Kadsura polysaccharides from Kadsura marmorata fruits, tested its immunity activity, separated and purified the crude polysaccharides, analysed the chemical composition of single component from KPS and evaluated its the immunity activities. An active component, KPSⅢ, was purified and selected to investigate the clinical application.
To extract KPS, Kadsura marmorata fruits were treated with petroleum ether, ethyl acetate and 95% ethanol successively, and followed by extraction with water. In order to optimize extraction conditions, single factor tests, material liquid ratio (1:10~1:30), extraction temperature (60℃~100℃), extraction time (1h~5h), were performed. Center combination design(CCD) and response surface analysis (RSA) was applied to select the optimum combination for these extraction conditions. Based on the analysis from single factor tests and RSA experiments, the optimum extraction material liquid proportion was 1:28, the extraction temperature was 95℃and the extraction time was 2.4 hours. The extraction rate reached peak, 3.50 %, under the optimum extraction conditions and the sugar purity was 76.01 % measured by anthrone-vitriol colorimetric assay.
Following extraction, the KPS was tested for immunomodulatory effects on chicken in vivo. Seven-days old chicken were administrated with KPS by subcutaneous injection. A series of parameters closely related to immune system were measured, such as transformation of lymphocyte (LTT), ratio of CD4+ T cells and CD8+ cells, antibody titer of Newcastle disease blood clots inhibition (ND-HI), total peripheral blood cell numbers and viscera index of spleen, thymus and bursa. The results showed that LTT from KPS high dose group (20 mg/Kg weight) was significantly higher than from astragalus polysaccharides group (APS), cytoxan group (CTX) and black control group (BCG) (P<0.01) at 7 days post immunization. The ratio of CD4+ T cells and CD8+ cells in KPS immunized group was significantly higher than CTX and BCG (P<0.01) at 21 days post immunization. The KPS immunization made viscera indexes of thymus and bursa higher than APS (P<0.05) and significantly higher than CTX and BCG (P<0.01) at 21 days post immunization. The KPS caused ND-HI antibody titer significantly higher than APS, CTX and BCG (P<0.01) at 28 days post immunization. Also, the KPS immunization increased total peripheral blood cell number while the other group did not. These data indicated that KPS could up-regulate immune responses on chicken.
To obtain pure immunomodulatory KPS, the crude Kadsura polysaccharide was subjected to a series of purification processes, such as excluding proteins with trichloroacetic acid (TCA), discoloring with active carbon and classifying by 20%~80% alcohol. Then the water soluble KPS was classified into five components by different concentration of alcohol, named as KPSI, KPSⅡ, KPSⅡ, KPSⅡ, KPSV respectively. Among which, KPSⅡwas the biggest proportion, 52.31 %, and its sugar purity reached the highest, namely 95.13 %. These five components were then subjected to gradient elution with Sephadex G-25 column respectively. The gradient elution curves showed that KPSⅡ, KPSⅡwere composed by single component, while KPSI, KPSⅡand KPSVwere mixed with polysaccharides with different molecular weight. To analyze the monosugar composition, all these five components were subjected to Gas chromatography (GC) and paper chromatography (PC) analysis. GC and PC analysis results demonstrated that KPSI-1 was heteropolysaccharide consisting of Rib, Xyl and GalA in the molar ratios of 1.13: 1.04 : 0.26, KPSⅡconsisted of Fru, Glc, and a unknow sugar in the molar ratio of 0.26: 0.10: 0.11, KPSⅡconsisted of Ara, Glc, and GalA in the molar ratios of 0.22 : 0.24 : 0.09, KPSⅡ-6 consisted of Fru, Glc, GalA and a unknow sugar in the molar ratio of 0.24 : 0.09: 0.06: 0.04 and KPSV-1 consisted of Rha, Glc and GalA in the molar ratios of 0.33 : 0.15 : 0.10.
Next, these five components were applied to immunize chickens to evaluate their immunomodulatory effects in vitro. To do so, T and B lymphocyte proliferations, PMφin vitro and IL-2, IFN-γ, TNF-αand NO secretion levels were measured. The results demonstrated that KPSII and KPSⅢsignificantly stimulated lymphocytes proliferation, KPSⅢand KPSIV-6 induced PMφproliferation efficiently, as well as secretion of IL-2, IFN-γ, TNF-αand NO. Taken together, the KPSIII was selected for further clinical application research.
Finally, we applied the KPSⅢto do some preliminary clinical trials on chicken to figure out its commercial value. The results showed that KPSⅢsignificantly enhanced HI antibody titer of ND Losata vaccination and AGP antibody titer of IBD D78 vaccination. Additionally, KPSⅢenhances viscera index of spleen, thymus and bursa as well. KPSⅢalso improved the growth of chicken, raised feed rewards, reduced the happening of diseases and increased the survival rate of chicken. In the preventive trial, subcutaneous injection of KPSⅢat 20 mg/Kg body weight prevented the occurrence of O2 type E. coli disease effectively. The protective rate reached 75%, which was significantly higher than that of infected control group (P <0.01). In the treatment trial, mortality rate from KPS injection group was under 27.5 % which was significantly lower than that of infected control group (P <0.01) and efficient reached 82.0 %, which was significantly higher than that of infected control group (P <0.01).
In conclusion, this study laid a solid ground for the further development and veterinary clinical application of kadsura polysaccharide.
为获得试验用KPS,本研究首先优化并确定了南五味子水溶性多糖的最佳提取条件。依次用石油醚、乙酸乙酯和95%乙醇对大叶南五味子干燥果实粉末进行预处理,再以料液比为1:10～1:30,水提温度为60℃～100℃,水提时间为1 h～5 h进行单因素试验,确定水提取多糖最优水平。根据单因素实验结果,将料液比、水提温度、水提时间作为考察因素,多糖提取率为响应值,中心组合设计和响应面分析法优化确定KPS最佳提取条件为提取时间2.4 h、料液比1:28(m/V)、提取温度为95℃,提取率达到3.50 %,蒽酮-硫酸比色法检测糖含量为76.01 %。
本研究接着初步评价了自制KPS粗品对雏鸡免疫活性的影响。分别给7日龄雏鸡皮下注射KPS粗品,检测外周血淋巴细胞转化率、CD4+与CD8+淋巴细胞比值、ND-HI抗体效价、血细胞总数、脾脏、胸腺、法氏囊的脏器指数等免疫指标。结果显示,皮下注射KPS粗品高剂量组(20mg/Kg体重),于免疫后7 d,T淋巴细胞转化率明显高于APS组、CTX组、BCG组(P<0.01);免疫后第21 d,CD4+/CD8+比值明显高于CTX组、BCG组(P<0.01),胸腺、法氏囊脏器指数显著高于APS组(P<0.05),极显著高于BCG组、CTX组(P<0.01);于28日龄时,ND-HI抗体效价极显著高于APS组、CTX组、BCG组(P<0.01);还能促进外周血细胞总数的增多。由此表明,KPS粗品对雏鸡细胞免疫和体液免疫应答存在一定的促进作用。
为了获得具有免疫活性的KPS纯品,本研究先用TCA对KPS粗品脱蛋白、活性炭脱色纯化,再用20%~80 %乙醇分级沉淀,得到KPSI、KPSⅡ、KPSⅢ、KPSⅣ、KPSV五个级分,其中KPSⅢ最多,占52.31 %,糖含量高达95.13 %。各级分经SephadexG25柱梯度洗脱,洗脱曲线显示,KPSⅡ、KPSⅢ为单一组分,KPSI、KPSⅣ、KPSV含有不同分子量的糖组分。选取各级分中含量最多的组分,经PC和GC检测其单糖组成,结果显示,KPSI-1由Rib、Xyl及GalA组成(摩尔比为1.13:1.04:0.26);KPSⅡ由Fru、Glc及未知糖组成(摩尔比为0.26:0.10:0.11);KPSⅢ由Ara、Glc及GalA组成(摩尔比为0.22:0.24:0.09);KPSⅣ-6由Fru、Glc、GalA及未知糖组成(摩尔比为0.24:0.09:0.06:0.04);KPSV-1由Rha、Glc及GalA组成(摩尔比为0.33:0.15:0.10)。
其次,本研究经体外免疫活性测定试验,分别检测了KPSI-1、KPSⅡ、KPSⅢ、KPSⅣ-6、KPSV-1五个组分对雏鸡T淋巴细胞、B淋巴细胞、腹腔巨噬细胞增殖能力的影响,对T淋巴细胞分泌IL-2、IFN-γ及PMφ分泌TNF-α、NO的影响。结果显示,KPSⅡ、KPSⅢ均能直接和协同刺激源明显地促进淋巴细胞增殖,拮抗CTX对淋巴细胞的抑制作用;KPSⅢ、KPSⅣ-6均能显著地促进腹腔巨噬细胞增殖,拮抗CTX对巨噬细胞的抑制作用;KPSⅢ、KPSⅣ-6显著升高T淋巴细胞分泌IL-2、IFN-γ的含量及巨噬细胞分泌TNF-α、NO的含量。
基于以上结果,本研究选取KPSIII组分进一步研究其体内免疫活性及临床应用价值。以增强疫苗抗体效价、免疫器官脏器指数、促进动物生长和抗病作为检测指标进行了动物临床试验。结果显示,将KPSⅢ与传染性法氏囊D78株疫苗混合后饮水免疫可显著提高IBDV-AGP抗体效价;将KPSⅢ与新城疫Losata株疫苗混合后皮下注射免疫亦可显著提高ND-HI抗体效价,且能显著上调雏鸡脾脏、胸腺及法氏囊免疫器官的脏器指数。KPSⅢ还能够促进雏鸡生长,提高饲料报酬,减少疾病的发生,提高雏鸡的存活率。皮下注射0.8 mL 2×108 cfu/mL O2型大肠杆菌成功建立了雏鸡大肠杆菌病病理模型,在KPSⅢ的预防试验中发现,皮下注射20 mg/Kg体重的KPSⅢ能有效预防O2型大肠杆菌病的发生,使发病率极显著低于感染对照组(P<0.01),保护率达到75 %以上;在治疗试验中,死亡率控制在27.5 %,有效率达到82.0 %,与阴性对照组有极显著性差异(P<0.01)。
综上所述,本项研究为南五味子多糖的进一步研发及在兽医临床的应用提供了宝贵的实验技术依据。
Kadsura polysaccharides(KPS)is a type of polysaccharides compound extracted from Schisandraceae Kadsura plants. Up to date, few studies or reports focus on the extraction, chemical composition and pharmacological activities of KPS, especially reports on Kadsura marmorata was rarely. In this study, we developed an extraction method to extract crude Kadsura polysaccharides from Kadsura marmorata fruits, tested its immunity activity, separated and purified the crude polysaccharides, analysed the chemical composition of single component from KPS and evaluated its the immunity activities. An active component, KPSⅢ, was purified and selected to investigate the clinical application.
To extract KPS, Kadsura marmorata fruits were treated with petroleum ether, ethyl acetate and 95% ethanol successively, and followed by extraction with water. In order to optimize extraction conditions, single factor tests, material liquid ratio (1:10~1:30), extraction temperature (60℃~100℃), extraction time (1h~5h), were performed. Center combination design(CCD) and response surface analysis (RSA) was applied to select the optimum combination for these extraction conditions. Based on the analysis from single factor tests and RSA experiments, the optimum extraction material liquid proportion was 1:28, the extraction temperature was 95℃and the extraction time was 2.4 hours. The extraction rate reached peak, 3.50 %, under the optimum extraction conditions and the sugar purity was 76.01 % measured by anthrone-vitriol colorimetric assay.
Following extraction, the KPS was tested for immunomodulatory effects on chicken in vivo. Seven-days old chicken were administrated with KPS by subcutaneous injection. A series of parameters closely related to immune system were measured, such as transformation of lymphocyte (LTT), ratio of CD4+ T cells and CD8+ cells, antibody titer of Newcastle disease blood clots inhibition (ND-HI), total peripheral blood cell numbers and viscera index of spleen, thymus and bursa. The results showed that LTT from KPS high dose group (20 mg/Kg weight) was significantly higher than from astragalus polysaccharides group (APS), cytoxan group (CTX) and black control group (BCG) (P<0.01) at 7 days post immunization. The ratio of CD4+ T cells and CD8+ cells in KPS immunized group was significantly higher than CTX and BCG (P<0.01) at 21 days post immunization. The KPS immunization made viscera indexes of thymus and bursa higher than APS (P<0.05) and significantly higher than CTX and BCG (P<0.01) at 21 days post immunization. The KPS caused ND-HI antibody titer significantly higher than APS, CTX and BCG (P<0.01) at 28 days post immunization. Also, the KPS immunization increased total peripheral blood cell number while the other group did not. These data indicated that KPS could up-regulate immune responses on chicken.
To obtain pure immunomodulatory KPS, the crude Kadsura polysaccharide was subjected to a series of purification processes, such as excluding proteins with trichloroacetic acid (TCA), discoloring with active carbon and classifying by 20%~80% alcohol. Then the water soluble KPS was classified into five components by different concentration of alcohol, named as KPSI, KPSⅡ, KPSⅡ, KPSⅡ, KPSV respectively. Among which, KPSⅡwas the biggest proportion, 52.31 %, and its sugar purity reached the highest, namely 95.13 %. These five components were then subjected to gradient elution with Sephadex G-25 column respectively. The gradient elution curves showed that KPSⅡ, KPSⅡwere composed by single component, while KPSI, KPSⅡand KPSVwere mixed with polysaccharides with different molecular weight. To analyze the monosugar composition, all these five components were subjected to Gas chromatography (GC) and paper chromatography (PC) analysis. GC and PC analysis results demonstrated that KPSI-1 was heteropolysaccharide consisting of Rib, Xyl and GalA in the molar ratios of 1.13: 1.04 : 0.26, KPSⅡconsisted of Fru, Glc, and a unknow sugar in the molar ratio of 0.26: 0.10: 0.11, KPSⅡconsisted of Ara, Glc, and GalA in the molar ratios of 0.22 : 0.24 : 0.09, KPSⅡ-6 consisted of Fru, Glc, GalA and a unknow sugar in the molar ratio of 0.24 : 0.09: 0.06: 0.04 and KPSV-1 consisted of Rha, Glc and GalA in the molar ratios of 0.33 : 0.15 : 0.10.
Next, these five components were applied to immunize chickens to evaluate their immunomodulatory effects in vitro. To do so, T and B lymphocyte proliferations, PMφin vitro and IL-2, IFN-γ, TNF-αand NO secretion levels were measured. The results demonstrated that KPSII and KPSⅢsignificantly stimulated lymphocytes proliferation, KPSⅢand KPSIV-6 induced PMφproliferation efficiently, as well as secretion of IL-2, IFN-γ, TNF-αand NO. Taken together, the KPSIII was selected for further clinical application research.
Finally, we applied the KPSⅢto do some preliminary clinical trials on chicken to figure out its commercial value. The results showed that KPSⅢsignificantly enhanced HI antibody titer of ND Losata vaccination and AGP antibody titer of IBD D78 vaccination. Additionally, KPSⅢenhances viscera index of spleen, thymus and bursa as well. KPSⅢalso improved the growth of chicken, raised feed rewards, reduced the happening of diseases and increased the survival rate of chicken. In the preventive trial, subcutaneous injection of KPSⅢat 20 mg/Kg body weight prevented the occurrence of O2 type E. coli disease effectively. The protective rate reached 75%, which was significantly higher than that of infected control group (P <0.01). In the treatment trial, mortality rate from KPS injection group was under 27.5 % which was significantly lower than that of infected control group (P <0.01) and efficient reached 82.0 %, which was significantly higher than that of infected control group (P <0.01).
In conclusion, this study laid a solid ground for the further development and veterinary clinical application of kadsura polysaccharide.
引文
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