大豆黄酮和染料木素的RP-HPLC方法的建立及其在禽类体内蛋内含量的研究
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摘要
本文建立了检测大豆黄酮和染料木素的反相高效液相色谱法(RP-HPLC)，并用该法检测了蛋鸡、雏鸡的饲料、血清、肌肉和蛋黄中的大豆黄酮及雏鸡肝脏、空肠食糜中的大豆黄酮、染料木素含量，确立了不同生物样品的预处理方法。初步探讨饲料中大豆黄酮含量与其在蛋鸡体内吸收、转移、分布及在蛋黄中富集的关系，初步探讨蛋鸡血清中大豆黄酮的含量变化与产蛋率的关系，比较了雏鸡的日采食量、增重和食糜、肝脏、肌肉中大豆黄酮和染料木素含量的变化趋势以及初步探讨了饲料中大豆黄酮和染料木素在雏鸡体内释放吸收转移分布的方式。
1．大豆黄酮和染料木素的反相高效液相色谱法(RP-HPLC)的建立
建立RP-HPLC法分离检测大豆黄酮和染料木素的方法。采用的色谱条件为：Waters Spherisorb ODS_2柱(4.6mm i.d.×150mm，5μm)，以甲醇-水(体积比为60:40)为流动相，流速为0.7ml／min，检测波长为254nm，柱温为室温。大豆黄酮在质量浓度为0.05μg／ml-20μg／ml时，其浓度与峰面积呈良好的线性关系，相关系数为0.9984(p＜0.01)。染料木素在质量浓度为0.05μg／ml-10μg／ml时，其浓度与峰面积呈良好的线性关系，相关系数为0.9978(p＜0.01)。大豆黄酮和染料木素的加标回收率分别为91.72％-98.56％和96.80％-98.60％，日内、日间精密度分别为1.30％、5.30％和1.17％、3.84％。样品实测值的重复性范围为2.15％-7.17％。
超声波法提取样品中大豆黄酮和染料木素的方法显著优于有机溶剂热回流法和冷浸法。
2．添喂大豆黄酮对自由采食蛋鸡生产性能的影响及其血清和蛋黄中大豆黄酮含量比较
随机抽取产蛋后期依莎产蛋鸡29只分为2组，对照组饲喂基础日粮(n=13)，大豆黄酮组饲喂基础日粮+6mg／kg大豆黄酮(n=16)。结果显示：日粮中添加大豆黄酮，与对照组相比，产蛋后期蛋鸡摄食量下降5.84％，平均蛋重下降3.03％(p＜0.05)，产蛋率增加8.14％，料蛋比下降2.47％；蛋鸡饲料中大豆黄酮的含量为18.37μg／g。蛋鸡血清中大豆黄酮含量增加93.10％(p＜0.05)，蛋黄中大豆黄酮含量增加68.01％(p＜0.05)。提示，蛋鸡血清中大豆黄酮的含量变化与产蛋率存在正效应关系，日粮中大豆黄酮经蛋鸡吸收、代谢、转移后可在蛋黄内富集。
3．添喂大豆黄酮、染料木素和酶制剂对雏鸡生产性能的影响及其机体中大豆黄酮和染料木素含量
耿志荣 大豆黄酮和染料本素的RI，HPLC方法的建立及其在禽类体内蛋内含量的研究
选取 9日龄罗曼褐蛋公雏鸡 192 ．q，按初重等级随机分为六组：饲喂基础日粮组
＊组）、饲喂基础日粮omg／kg大豆黄酮组＊Da组X饲喂基础日粮历mg／kg大豆黄
6R组的Da组〕饲喂基础5粮十0．05O复合酶制剂十6m叭g大豆黄酮组（Da＊E组）、饲
喂基础日粮十0刀5％复合酶制剂组n组）、饲喂验日粮十6m吵g染料术素组（Ge组）。
试验期持续 2周。与对照组相比，3Da组、6Da组、Da＋E组、E组和 Ge组料重比有
下降的趋势，日增重、生长速率、肝脏重和腿重有增加的趋势，但差异不显著，其中
3Da组第H周肝脏重量增力。12．22％（p＜0＊5），6Da组第一周生长速率增力。11＊1％
（p－0．0543），Da＋E组第H周肝脏重量增力。18．980（p＜0．of），Ge组日增重增力。8．21O
…＜0．l），第H周肝脏重量增加 2256％（p刀刀1）。3Da组食糜和肝脏中大豆黄酮含
量分别增加 197刁 6％（p＜0刀 1）、72．84％（p刃．l＼ 6Da组食糜、肝脏和肌肉中大豆黄
酮含量分别增加288．15％（叶0＊1）、81．48％（p＜0＊5）、155．97％（p＜0刀1）；D＋＋E
组食糜中大豆黄酮和染料术素含量分别增加249．29％（p＜o刀5）、106．76％…＜0刀1），
肝脏中大豆黄酮和染料木素分别增加 127．160（p＜0．of）、107．070（p＜0．of），肌肉中
大豆黄酮增加82刀9％…＜0．05）；E组食糜、肝脏中大豆黄酮和染料本素含量有增加
的趋势，但个体差异较大，肌肉中大豆黄酮增加 373．88％（p＜o01）；Ge组食糜中大
豆黄酮和染料木素含量分别增加315．64％k＜0．01）、门8．13％（p＜0刀1），肝脏中大豆
黄酮和染料本素分别增力。72．84％（p＜0＊5）、48．48％；肌肉中大豆黄酮增加 64．18％
（P＜0．l）。
大豆异黄酮中的染料木素能够提高雏鸡的日增重和饲料转化率，酶制剂在一定程
度上促进雏鸡饲料中大豆黄酮和染料本素的释放和吸收。
1.The development of HPLC for daidzein and genistein
A reverse-phase high performance liquid chromatographic method was established for the determination of daidzein and genistein. Waters Spherisorb ODS2 column (4.6mm i.d.× 150mm, 5μm) was used with a mixture of methanol-water (60:40,v/v) as the mobile phase and a detector set at 254nm. The calibration curve was linear(r=0.9984,p<0.01) in the range of 0.05μg/ml-20μg/ml of dadizein and was linear(r=0.9978,p<0.01) in the range of 0.05μg/ml-10μg/ml of genistein. The recoveries of dadidzein and genistein were 91.72%-98.56% and 96.80%-98.60% respectively with the intra-day and inter-day RSDs of 1.30%, 5.30% and 1.17%, 3.84%, respectively.
2.Effect of daidzein preparation supplemented to basal diets on the performance and daidzein concentration in diet% sera and yolks of laying hens fed ad libitum
29 forty-four-week-old laying hens were randomized into 2 groups: basal diet(n=13), basal diet+6mg/kg daidzein(n=16). The blood samples were collected in the 4th day. Compared with control, the feed intake was decreased and laying performance was improved; daidzein concentrations in sera and yolks of laying hens increased by 93.10%(p<0.05) and 68.01%(p<0.05) respectively. Daidzein concentration in feed stuff of laying hens was 18.37μg/g. It indicated that daidzein from feed stuff can be enriched in the yolk.
3.Effect of daidzein, genistein and enzyme preparations supplemented to basal diets on the performance and daidzein, genistein concentration in diet, chyme, liver and muscle of chicks
192 nine-day-old layer chicks were randomized into 6 groups: basal diet (C group), basal diet plus 3mg/kg daidzein(3Da group), basal diet plus 6mg/kg daidzein(6Da group), basal diet plus 0.05% enzyme preparation+6mg/kg daidzein (Da+E group), basal diet plus 0.05% enzyme preparation(E group) , basal diet plus 6mg/kg genistcin(Ge group). Compared with control, the experiment groups increased chicks body weight gain per day, the feed conversion rate, growth rate and weights of livers and legs. 3Da group increased weight of livers of the second week by 12.22%(p<0.05), 6Da group increased growth rates of the first
week by 11.01%(p=0.0543), Da+E group increased weight of livers of the second week by 18.98%(p<0.01), Ge group respectively increased weight gain per day and weight of livers of the second week by 8.21%(p<0.1) and 22.56%(p<0.01). 3Da group respectively increased daidzein concentrations in the chyme and liver by 197.16%(p<0.01), 72.84%(p<0.1); 6Da group respectively increased daidzein concentrations in the chyme, liver and muscle by 288.15%(p<0.01),81.48%(p<0.01), 155.97%(p<0.01); Da+E group respectively increased daidzein concentrations in the chyme, liver and muscle by 249.29%(p<0.01), 127.16%(p<0.01), 82.09%(p<0.05), increased genistein concentrations in the chyme and liver by 106.76%(p<0.01),107.07%(p<0.01); E group increased daidzein concentration in muscle by373.88%(p<0.01) , daidzein and genistein concentrations in the chyme and liver had the tendency of increase, however had difference individually; Ge group respectively increased daidzein concentrations in the chyme, liver and muscle by315.64%(p<0.01
),72.84%(p<0.05),64.18%(p<0.1),increased genistein concentrations in the chyme and liver by 138.13%(p<0.01),48.48%.
In conclusion, daidzein, genistein and enzyme preparation increased chicks performances and daidzein and genistein concentration in the chyme, liver and muscle of chicks. To a certain degree, enzyme preparation can speed daidzein and genistein released from diets and improve daidzein and genistein's absorptivity.
1．大豆黄酮和染料木素的反相高效液相色谱法(RP-HPLC)的建立
建立RP-HPLC法分离检测大豆黄酮和染料木素的方法。采用的色谱条件为：Waters Spherisorb ODS_2柱(4.6mm i.d.×150mm，5μm)，以甲醇-水(体积比为60:40)为流动相，流速为0.7ml／min，检测波长为254nm，柱温为室温。大豆黄酮在质量浓度为0.05μg／ml-20μg／ml时，其浓度与峰面积呈良好的线性关系，相关系数为0.9984(p＜0.01)。染料木素在质量浓度为0.05μg／ml-10μg／ml时，其浓度与峰面积呈良好的线性关系，相关系数为0.9978(p＜0.01)。大豆黄酮和染料木素的加标回收率分别为91.72％-98.56％和96.80％-98.60％，日内、日间精密度分别为1.30％、5.30％和1.17％、3.84％。样品实测值的重复性范围为2.15％-7.17％。
超声波法提取样品中大豆黄酮和染料木素的方法显著优于有机溶剂热回流法和冷浸法。
2．添喂大豆黄酮对自由采食蛋鸡生产性能的影响及其血清和蛋黄中大豆黄酮含量比较
随机抽取产蛋后期依莎产蛋鸡29只分为2组，对照组饲喂基础日粮(n=13)，大豆黄酮组饲喂基础日粮+6mg／kg大豆黄酮(n=16)。结果显示：日粮中添加大豆黄酮，与对照组相比，产蛋后期蛋鸡摄食量下降5.84％，平均蛋重下降3.03％(p＜0.05)，产蛋率增加8.14％，料蛋比下降2.47％；蛋鸡饲料中大豆黄酮的含量为18.37μg／g。蛋鸡血清中大豆黄酮含量增加93.10％(p＜0.05)，蛋黄中大豆黄酮含量增加68.01％(p＜0.05)。提示，蛋鸡血清中大豆黄酮的含量变化与产蛋率存在正效应关系，日粮中大豆黄酮经蛋鸡吸收、代谢、转移后可在蛋黄内富集。
3．添喂大豆黄酮、染料木素和酶制剂对雏鸡生产性能的影响及其机体中大豆黄酮和染料木素含量
耿志荣 大豆黄酮和染料本素的RI，HPLC方法的建立及其在禽类体内蛋内含量的研究
选取 9日龄罗曼褐蛋公雏鸡 192 ．q，按初重等级随机分为六组：饲喂基础日粮组
＊组）、饲喂基础日粮omg／kg大豆黄酮组＊Da组X饲喂基础日粮历mg／kg大豆黄
6R组的Da组〕饲喂基础5粮十0．05O复合酶制剂十6m叭g大豆黄酮组（Da＊E组）、饲
喂基础日粮十0刀5％复合酶制剂组n组）、饲喂验日粮十6m吵g染料术素组（Ge组）。
试验期持续 2周。与对照组相比，3Da组、6Da组、Da＋E组、E组和 Ge组料重比有
下降的趋势，日增重、生长速率、肝脏重和腿重有增加的趋势，但差异不显著，其中
3Da组第H周肝脏重量增力。12．22％（p＜0＊5），6Da组第一周生长速率增力。11＊1％
（p－0．0543），Da＋E组第H周肝脏重量增力。18．980（p＜0．of），Ge组日增重增力。8．21O
…＜0．l），第H周肝脏重量增加 2256％（p刀刀1）。3Da组食糜和肝脏中大豆黄酮含
量分别增加 197刁 6％（p＜0刀 1）、72．84％（p刃．l＼ 6Da组食糜、肝脏和肌肉中大豆黄
酮含量分别增加288．15％（叶0＊1）、81．48％（p＜0＊5）、155．97％（p＜0刀1）；D＋＋E
组食糜中大豆黄酮和染料术素含量分别增加249．29％（p＜o刀5）、106．76％…＜0刀1），
肝脏中大豆黄酮和染料木素分别增加 127．160（p＜0．of）、107．070（p＜0．of），肌肉中
大豆黄酮增加82刀9％…＜0．05）；E组食糜、肝脏中大豆黄酮和染料本素含量有增加
的趋势，但个体差异较大，肌肉中大豆黄酮增加 373．88％（p＜o01）；Ge组食糜中大
豆黄酮和染料木素含量分别增加315．64％k＜0．01）、门8．13％（p＜0刀1），肝脏中大豆
黄酮和染料本素分别增力。72．84％（p＜0＊5）、48．48％；肌肉中大豆黄酮增加 64．18％
（P＜0．l）。
大豆异黄酮中的染料木素能够提高雏鸡的日增重和饲料转化率，酶制剂在一定程
度上促进雏鸡饲料中大豆黄酮和染料本素的释放和吸收。
1.The development of HPLC for daidzein and genistein
A reverse-phase high performance liquid chromatographic method was established for the determination of daidzein and genistein. Waters Spherisorb ODS2 column (4.6mm i.d.× 150mm, 5μm) was used with a mixture of methanol-water (60:40,v/v) as the mobile phase and a detector set at 254nm. The calibration curve was linear(r=0.9984,p<0.01) in the range of 0.05μg/ml-20μg/ml of dadizein and was linear(r=0.9978,p<0.01) in the range of 0.05μg/ml-10μg/ml of genistein. The recoveries of dadidzein and genistein were 91.72%-98.56% and 96.80%-98.60% respectively with the intra-day and inter-day RSDs of 1.30%, 5.30% and 1.17%, 3.84%, respectively.
2.Effect of daidzein preparation supplemented to basal diets on the performance and daidzein concentration in diet% sera and yolks of laying hens fed ad libitum
29 forty-four-week-old laying hens were randomized into 2 groups: basal diet(n=13), basal diet+6mg/kg daidzein(n=16). The blood samples were collected in the 4th day. Compared with control, the feed intake was decreased and laying performance was improved; daidzein concentrations in sera and yolks of laying hens increased by 93.10%(p<0.05) and 68.01%(p<0.05) respectively. Daidzein concentration in feed stuff of laying hens was 18.37μg/g. It indicated that daidzein from feed stuff can be enriched in the yolk.
3.Effect of daidzein, genistein and enzyme preparations supplemented to basal diets on the performance and daidzein, genistein concentration in diet, chyme, liver and muscle of chicks
192 nine-day-old layer chicks were randomized into 6 groups: basal diet (C group), basal diet plus 3mg/kg daidzein(3Da group), basal diet plus 6mg/kg daidzein(6Da group), basal diet plus 0.05% enzyme preparation+6mg/kg daidzein (Da+E group), basal diet plus 0.05% enzyme preparation(E group) , basal diet plus 6mg/kg genistcin(Ge group). Compared with control, the experiment groups increased chicks body weight gain per day, the feed conversion rate, growth rate and weights of livers and legs. 3Da group increased weight of livers of the second week by 12.22%(p<0.05), 6Da group increased growth rates of the first
week by 11.01%(p=0.0543), Da+E group increased weight of livers of the second week by 18.98%(p<0.01), Ge group respectively increased weight gain per day and weight of livers of the second week by 8.21%(p<0.1) and 22.56%(p<0.01). 3Da group respectively increased daidzein concentrations in the chyme and liver by 197.16%(p<0.01), 72.84%(p<0.1); 6Da group respectively increased daidzein concentrations in the chyme, liver and muscle by 288.15%(p<0.01),81.48%(p<0.01), 155.97%(p<0.01); Da+E group respectively increased daidzein concentrations in the chyme, liver and muscle by 249.29%(p<0.01), 127.16%(p<0.01), 82.09%(p<0.05), increased genistein concentrations in the chyme and liver by 106.76%(p<0.01),107.07%(p<0.01); E group increased daidzein concentration in muscle by373.88%(p<0.01) , daidzein and genistein concentrations in the chyme and liver had the tendency of increase, however had difference individually; Ge group respectively increased daidzein concentrations in the chyme, liver and muscle by315.64%(p<0.01
),72.84%(p<0.05),64.18%(p<0.1),increased genistein concentrations in the chyme and liver by 138.13%(p<0.01),48.48%.
In conclusion, daidzein, genistein and enzyme preparation increased chicks performances and daidzein and genistein concentration in the chyme, liver and muscle of chicks. To a certain degree, enzyme preparation can speed daidzein and genistein released from diets and improve daidzein and genistein's absorptivity.
引文
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