马鹿茸血酶解肽免疫活性及抗氧化活性的研究
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摘要
鹿茸血是指加工排血茸时由鲜鹿茸中取出的血液成分。本论文对酶解马鹿茸血制备免疫活性肽的条件进行了优化,并对水解物进行了分离纯化;通过体外、体内试验研究了鹿茸血原血及由木瓜酶水解得到的两种肽对小鼠免疫功能及抗氧化功能的影响。
选用木瓜蛋白酶、酸性蛋白酶、中性蛋白酶在最适条件下对马鹿茸血水解,以水解度(DH)以及水解物对·OH和DPPH·的清除率为指标,确定木瓜蛋白酶作为本次试验水解用酶。确定木瓜蛋白酶水解度最高的水解条件为温度52℃、pH为7.4、酶用量(E/S)为12690 U/g底物、[S]为8%,在该条件下水解得到水解肽Ⅰ水解度为18.1%;清除·OH最高的水解条件温度52℃、pH为7.0、酶用量(E/S)为11269 U/g底物、[S]为10%,该条件下得到的水解肽Ⅱ水解度为12.2%,·OH清除率为51.3%;与原血相比,水解肽Ⅰ和水解肽Ⅱ表现出更高的抗氧化活性。采用HPLC测得马鹿茸血水解物中以相对分子质量小于1000的小肽居多。
对鹿茸血及其水解肽Ⅰ和水解肽Ⅱ进行体外活性检测试验,结果表明水解肽Ⅱ能显著刺激脾淋巴细胞增殖,对·OH和DPPH·有较高的清除作用(P<0.05)。然后,以鹿茸血及其水解肽Ⅰ和水解肽Ⅱ为受试样品,对小鼠进行动物试验。动物试验表明鹿茸血水解肽Ⅱ组能极显著提高小鼠体液免疫和细胞免疫功能(P<0.01),同时,能显著增强小鼠的总抗氧化能力和SOD活性、降低丙二醛含量(P<0.05-0.01),在防止脂质过氧化减轻组织细胞的损伤等具有重要作用。马鹿茸血及其酶解肽抗氧化性与免疫性相关性分析结果显示,具有较强抗氧化活性的肽同时显示出很强的免疫活性,相关系数r=0.87,(P<0.05)。
采用732阳离子交换树脂对马鹿茸血水解肽Ⅱ进行初步分离,得到a、b、c、d四个组分。用大孔吸附树脂对四个组分脱盐处理后,通过抗氧化活性及免疫活性测定,组分a对·OH和DPPH·清除效果较好,并且刺激小鼠脾淋巴细胞增殖效果显著(P<0.05)。对组分a采用Sephadex G-15做进一步分离纯化,得到四个洗脱峰,经抗氧化和脾淋巴细胞增殖活性测定显示,第二和第四个洗脱峰表现出较高的活性(P<0.05)。
The blood of antler has the same chemical component with antler. The optimum enzymatic hydrolysis conditions on preparation of peptides from Cervus elaphus(Chinese Wapiti) antler blood were determined. The immunity and anti-oxidation of the antler blood and its two hydrolysates were studied. Isolation and purification of hydrolysates were studied. Papain, Acid-protease and Neutral-protease were used to hydrolyze antler blood.
According to the degree of hydrolysis (DH) and the·OHand DPPH·clearance of each enzyme, papain was chosed as the experiment enzyme. The optimum conditions of hydrolysate with highest DH was: T52℃, pH 7.4, E/S 9%、[S] 8%, peptideⅠwith DH18.1% was gotten; The optimum conditions of hydrolysis with highest·OHclearance was: T52℃, pH 7.0 , E/S 8, [S]10%, peptideⅡwith DH12.2% and the clearance51.3% was gotten. Compared with antler blood, peptideⅠa ndⅡhad stronger anti-oxidation. HPLC was used to detect the molecular of the two hydrolysates, most of the hydrolysates is small peptides.
The hydrolysates of peptideⅡcan obviously enhance the lymphocyte proliferative response, tested by MTT colorimetry in vitro(P<0.05). It is concluded from the animal experiment that the peptideⅡhas significant effect on humoral and cellular immunity, the result also showed peptideⅡcan obviously enhance anti-oxidative function of mice and prevent cell injury, etc. (P<0.01or P<0.05). And there is significant correlation between anti-oxidation and immunity (r=0.87, P<0.05).
Isolation of peptideⅡwith ion exchange chromatography were studied, four fractions(a, b, c, d) were gotten. Afterwards, macroporous resin was used to desalt the fractions. The immunity and anti-oxidation were detected in vitro, the effect of fraction(a) was obvious(P<0.05). Finally, isolation of the fraction(a) was studied, four fractions were gotten. The activity was detected in vitro, the result showed that the immunity and anti-oxidation of the fraction(2) and fraction(4) was effective(P<0.05).
选用木瓜蛋白酶、酸性蛋白酶、中性蛋白酶在最适条件下对马鹿茸血水解,以水解度(DH)以及水解物对·OH和DPPH·的清除率为指标,确定木瓜蛋白酶作为本次试验水解用酶。确定木瓜蛋白酶水解度最高的水解条件为温度52℃、pH为7.4、酶用量(E/S)为12690 U/g底物、[S]为8%,在该条件下水解得到水解肽Ⅰ水解度为18.1%;清除·OH最高的水解条件温度52℃、pH为7.0、酶用量(E/S)为11269 U/g底物、[S]为10%,该条件下得到的水解肽Ⅱ水解度为12.2%,·OH清除率为51.3%;与原血相比,水解肽Ⅰ和水解肽Ⅱ表现出更高的抗氧化活性。采用HPLC测得马鹿茸血水解物中以相对分子质量小于1000的小肽居多。
对鹿茸血及其水解肽Ⅰ和水解肽Ⅱ进行体外活性检测试验,结果表明水解肽Ⅱ能显著刺激脾淋巴细胞增殖,对·OH和DPPH·有较高的清除作用(P<0.05)。然后,以鹿茸血及其水解肽Ⅰ和水解肽Ⅱ为受试样品,对小鼠进行动物试验。动物试验表明鹿茸血水解肽Ⅱ组能极显著提高小鼠体液免疫和细胞免疫功能(P<0.01),同时,能显著增强小鼠的总抗氧化能力和SOD活性、降低丙二醛含量(P<0.05-0.01),在防止脂质过氧化减轻组织细胞的损伤等具有重要作用。马鹿茸血及其酶解肽抗氧化性与免疫性相关性分析结果显示,具有较强抗氧化活性的肽同时显示出很强的免疫活性,相关系数r=0.87,(P<0.05)。
采用732阳离子交换树脂对马鹿茸血水解肽Ⅱ进行初步分离,得到a、b、c、d四个组分。用大孔吸附树脂对四个组分脱盐处理后,通过抗氧化活性及免疫活性测定,组分a对·OH和DPPH·清除效果较好,并且刺激小鼠脾淋巴细胞增殖效果显著(P<0.05)。对组分a采用Sephadex G-15做进一步分离纯化,得到四个洗脱峰,经抗氧化和脾淋巴细胞增殖活性测定显示,第二和第四个洗脱峰表现出较高的活性(P<0.05)。
The blood of antler has the same chemical component with antler. The optimum enzymatic hydrolysis conditions on preparation of peptides from Cervus elaphus(Chinese Wapiti) antler blood were determined. The immunity and anti-oxidation of the antler blood and its two hydrolysates were studied. Isolation and purification of hydrolysates were studied. Papain, Acid-protease and Neutral-protease were used to hydrolyze antler blood.
According to the degree of hydrolysis (DH) and the·OHand DPPH·clearance of each enzyme, papain was chosed as the experiment enzyme. The optimum conditions of hydrolysate with highest DH was: T52℃, pH 7.4, E/S 9%、[S] 8%, peptideⅠwith DH18.1% was gotten; The optimum conditions of hydrolysis with highest·OHclearance was: T52℃, pH 7.0 , E/S 8, [S]10%, peptideⅡwith DH12.2% and the clearance51.3% was gotten. Compared with antler blood, peptideⅠa ndⅡhad stronger anti-oxidation. HPLC was used to detect the molecular of the two hydrolysates, most of the hydrolysates is small peptides.
The hydrolysates of peptideⅡcan obviously enhance the lymphocyte proliferative response, tested by MTT colorimetry in vitro(P<0.05). It is concluded from the animal experiment that the peptideⅡhas significant effect on humoral and cellular immunity, the result also showed peptideⅡcan obviously enhance anti-oxidative function of mice and prevent cell injury, etc. (P<0.01or P<0.05). And there is significant correlation between anti-oxidation and immunity (r=0.87, P<0.05).
Isolation of peptideⅡwith ion exchange chromatography were studied, four fractions(a, b, c, d) were gotten. Afterwards, macroporous resin was used to desalt the fractions. The immunity and anti-oxidation were detected in vitro, the effect of fraction(a) was obvious(P<0.05). Finally, isolation of the fraction(a) was studied, four fractions were gotten. The activity was detected in vitro, the result showed that the immunity and anti-oxidation of the fraction(2) and fraction(4) was effective(P<0.05).
引文
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