红三叶草异黄酮在奶牛瘤胃中的代谢

doi:10.16431/j.cnki.1671-7236.2024.10.010

摘要/Abstract

摘要： 【目的】本试验旨在研究红三叶草异黄酮在瘤胃内的稳定性及其代谢规律。【方法】体内试验：选择3头安装有瘤胃瘘管的荷斯坦奶牛，于晨饲前在瘤胃内投入2 g红三叶草提取物(异黄酮含量为22.91%)，分别在0、0.5、1、2、4、8、12、14 h采集瘤胃液，利用HPLC法分析异黄酮含量。体外瘤胃发酵试验：设置3个处理组，分别为添加0.1 g/120 mL(高剂量组)、0.01 g/120 mL(低剂量组)和不添加红三叶草提取物组(对照组)，每组均为5个重复，在39 ℃条件下发酵，分别在0、0.5、1、2、4、8、12、14 h采集瘤胃液，利用HPLC法分析异黄酮含量。【结果】在体内试验中，刺芒柄花素和鹰嘴豆芽素A分别于14和8 h完成代谢。2 h后，瘤胃内出现代谢产物S-雌马酚。鹰嘴豆芽素A和刺芒柄花素的半衰期分别为1.02和2.25 h，大豆苷元和染料木素的半衰期分别为3.38和0.77 h。在体外试验中，高剂量组和低剂量组发酵液中4种异黄酮的半衰期均不同：高剂量组中，鹰嘴豆芽素A、刺芒柄花素、大豆苷元和染料木素的半衰期分别为2.45、0.70、12.62和5.74 h。低剂量组中，这4种异黄酮的半衰期分别为2.82、0.87、13.11和3.45 h。【结论】红三叶草异黄酮在奶牛瘤胃中不稳定，会被微生物分解代谢，主要异黄酮刺芒柄花素和鹰嘴豆芽素A半衰期不超过4 h，推测这两种异黄酮代谢终产物分别为S-雌马酚和对乙基苯酚。

关键词: 瘤胃; 红三叶草异黄酮; 代谢; 半衰期

Abstract: 【Objective】 The purpose of this experiment was to study the stability and metabolism of red clover isoflavones in rumen. 【Methods】 In in vivo experiment，3 fistulated Holstein cows were selected and fed 2 g of red clover extract (isoflavone content 22.91%) into the rumen before morning feeding. Rumen fluid was collected at 0，0.5，1，2，4，8，12 and 14 h，respectively，and HPLC was used to analyze the isoflavone contents. In in vitro rumen fermentation，3 treatment groups were set up，0.1 g/120 mL (high-dose group)，0.01 g/120 mL (low-dose group)，and no red clover extract group (control group)，with 5 replicates in each group，and fermented at 39 ℃. Rumen fluid was collected at 0，0.5，1，2，4，8，12 and 14 h，respectively. HPLC was used to analyze the contents of isoflavones. 【Results】 In in vivo experiments，biochanin A and formononetin were metabolized at 14 and 8 h,respectively. After 2 h，the metabolite S-equol was present in the rumen. The half-lives of biochanin A and formononetin were 1.02 and 2.25 h，respectively，while those of daidzein and genistein were 3.38 and 0.77 h，respectively.In in vitro experiment，the half-lives of the four isoflavones in the high-dose group and the low-dose group were different. In the high-dose group，the half-lives of biochanin A，formononetin，daidzein and genistein were 2.45，0.70，12.62 and 5.74 h，respectively. In the low-dose group，the half-lives of biochanin A，formononetin，daidzein and genistein were 2.82，0.87，13.11 and 3.45 h，respectively. 【Conclusion】 Red clover isoflavones were unstable in the rumen of dairy cows and could be catabolized by microorganisms. The half-lives of the main isoflavones，biochanin A and formononetin were all less than 4 h. It was speculated that the metabolic end products of the two isoflavones were S-equol and ethylphenol，respectively.

Key words: rumen; red clover isoflavones; metabolism; half-life

中图分类号:

S816.7

卜莹, 郑楠, 王典, 王加启, 赵圣国. 红三叶草异黄酮在奶牛瘤胃中的代谢[J]. 中国畜牧兽医, 2024, 51(10): 4292-4300.

BU Ying, ZHENG Nan, WANG Dian, WANGJiaqi, ZHAO Shengguo. The Metabolism of Red Clover Isoflavones in the Rumen of Dairy Cows[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4292-4300.

参考文献

[1] KURZER M S，XU X. Dietary phytoestrogens[J]. Annual Review of Nutrition,1997,17:353-381.
[2] SALONIEMI H，WAHALA K，NYKANEN-KUEKI P，et al. Phytoestrogen content and estrogenic effect of legume fodder[J]. Proceedings of the Society for Experimental Biology & Medicine,1995,208(1):13.
[3] KIM M R，KIM H J，YU S H，et al. Combination of red clover and hops extract improved menopause symptoms in an ovariectomized rat model[J]. Evidence-based Complementary and Alternative Medicine,2020,2020(1): 7941391.
[4] ADLER S A，PURUP S，HANSEN-MOLLER J，et al. Phytoestrogens and their metabolites in bulk-tank milk: Effects of farm management and season[J]. PLoS One,2015,10(5):e0127187.
[5] KENNY A M，MANGANO K M，ABOURIZK R H，et al. Soy proteins and isoflavones affect bone mineral density in older women: A randomized controlled trial[J]. American Journal of Clinical Nutrition,2009,90(1):234-42.
[6] JEMINIWA B O，KNIGHT R M，BRADEN T D，et al. Regulation of the neuroendocrine axis in male rats by soy-based diets is independent of age and due specifically to isoflavone action[J]. Biology of Reproduction,2020,103(4):892-906.
[7] HARLOW B E，FLYTHE M D，KAGAN I A，et al. Biochanin A (an isoflavone produced by red clover) promotes weight gain of steers grazed in mixed grass pastures and fed dried-distillers’grains[J]. Crop Science,2016,57(1)：506-514.
[8] HALMEMIES-BEAUCHET-FILLEAU A，VANHATALO A，TOIVONEN V，et al. Effect of replacing grass silage with red clover silage on nutrient digestion，nitrogen metabolism，and milk fat composition in lactating cows fed diets containing a 60∶40 forage-to-concentrate ratio[J]. Journal of Dairy Science,2014,97(6):3761-3776.
[9] WEINERT-NELSON J R，ELY D G，FLYTHE M D，et al. Red clover supplementation modifies rumen fermentation and promotes feed efficiency in ram lambs[J]. Journal of Animal Science,2023,101:skad036.
[10] XU Q，LI Y，DU W，et al. Effect of dietary biochanin A on lactation performance，antioxidant capacity，rumen fermentation and rumen microbiome of dairy goat[J]. Frontiers in Microbiology,2023,14:1101849.
[11] LIU S，ZHANG Z，HAILEMARIAM S，et al. Biochanin A inhibits ruminal nitrogen-metabolizing bacteria and alleviates the decomposition of amino acids and urea in vitro[J]. Animals,2020,10(3):368.
[12] NILSSON A，HILL J L，DAVIDS H L. An in vitro study of formononetin and biochanin A metabolism in rumen fluid from sheep[J]. Biochimica et Biophysica Acta,1967,148(1):92-98.
[13] BARNES S. The biochemistry，chemistry and physiology of the isoflavones in soybeans and their food products[J]. Lymphatic Research and Biology,2010,8(1):89-98.
[14] LIANG F，CAO W，HUANG Y，et al. Isoflavone biochanin A，a novel nuclear factor erythroid 2-related factor 2(Nrf2)-antioxidant response element activator，protects against oxidative damage in HepG2 cells[J]. Biofactors,2019,45(4):563-574.
[15] DADÁKOVÁ K，TRNKOVÁ A，KAPAROVSKÁ J，et al. In vitro metabolism of red clover isoflavones in rumen fluid[J]. Journal of Animal Physiology and Animal Nutrition,2020,104(6):1647-1654.
[16] DAY A J，DUPONT M S，RIDLEY S，et al. Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity[J]. FEBS Letters,1998,436(1):71-75.
[17] AKIN D E，BORNEMAN W S. Role of rumen fungi in fiber degradation[J]. Journal of Dairy Science,1999,73(10):3023-3032.
[18] DICKINSON J M，SMITH G R，RANDEL R D，et al. In vitro metabolism of formononetin and biochanin A in bovine rumen fluid[J]. Journal of Animal Science,1988,66(8):1969-1973.
[19] KASPAROVSKA J，PECINKOVA M，DADAKOVA K，et al. Effects of isoflavone-enriched feed on the rumen microbiota in dairy cows[J]. PLoS One,2016,11(4):e0154642.
[20] LAMPE J W，KARR S C，HUTCHINS A M，et al. Urinary equol excretion with a soy challenge: Influence of habitual diet[J]. Proceedings of the Society for Experimental Biology and Medicine,1998,217(3):335-339.
[21] ROWLAND I R，WISEMAN H，SANDERS T A，et al. Interindividual variation in metabolism of soy isoflavones and lignans: Influence of habitual diet on equol production by the gut microflora[J]. Nutrition and Cancer,2000,36(1):27-32.
[22] PETIT H V，CORTES C，DA SILVA D，et al. The interaction of monensin and flaxseed hulls on ruminal and milk concentration of the mammalian lignan enterolactone in late-lactating dairy cows[J]. The Journal of Dairy Research,2009,76(4):475-482.
[23] LIANG H，XU L，ZHAO X，et al. Effect of daidzein on fermentation parameters and bacterial community of finishing Xianan cattle[J]. Italian Journal of Animal Science,2018,17(4):950-958.
[24] LUCA S V，MACOVEI I，BUJOR A，et al. Bioactivity of dietary polyphenols: The role of metabolites[J]. Critical Reviews in Food Science and Nutrition,2020,60(4):626-659.
[25] TRNKOVA A，SANCOVA K，ZAPLETALOVA M，et al. Determination of in vitro isoflavone degradation in rumen fluid[J]. Journal of Dairy Science,2018,101(6):5134-5144.
[26] DENG Y，PANG Y，GUO Y，et al. Host-guest inclusion systems of daidzein with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD): Preparation，binding behaviors and water solubility[J]. Journal of Molecular Structure,2016,1118:307-315.
[27] TAJIMA K，AMINOV R I，NAGAMINE T，et al. Diet-dependent shifts in the bacterial population of the rumen revealed with Real-time PCR[J]. Applied and Environmental Microbiology,2001,67(6):2766-2774.
[28] MELCHIOR E A，SMITH J K，SCHNEIDER L G，et al. Effects of red clover isoflavones on tall fescue seed fermentation and microbial populations in vitro[J]. PLoS One,2018,13(10):e0201866.
[29] LUNDH T J，PETTERSSON H，KIESSLING K H. Liquid chromatographic determination of the estrogens daidzein，formononetin，coumestrol，and equol in bovine blood plasma and urine[J]. Journal - Association of Official Analytical Chemists,1988,71(5):938-941.
[30] WOCLAWEK-POTOCKA I，PISKULA M K，BAH M，et al. Concentrations of isoflavones and their metabolites in the blood of pregnant and non-pregnant heifers fed soy bean[J]. The Journal of Reproduction and Development,2008,54(5):358-363.
[31] KELEMEN K，PALDI A，TINNEBERG H，et al. Early recognition of pregnancy by the maternal immune system[J]. American Journal of Reproductive Immunology,1998,39(6):351-355.
[32] LIPOVAC M，PFITSCHER A，HOBIGER S，et al. Red clover isoflavone metabolite bioavailability is decreased after fructooligosaccharide supplementation[J]. Fitoterapia,2015,105:93-101.