The Effect of Sour Tea (Hibiscus sabdariffa) Plant Extract on Immune System, Plasma Antioxidant Status, Oxidant Balance and Blood Biochemical and Functional Parameters of Old Laying Hens

Document Type : Poultry Nutrition

Authors

• Shahin Sabet sarvestani
• Seyyed Mohammad hosseini
• Seyyed Homayoun Farhangfar

Department of Animal Science, Birjand Faculty of Agriculture, Birjand, Iran

10.22067/ijasr.v12i1.77717

Abstract

Introduction: Stress increases the need for antioxidants, decreases eggs production and weakens immune system of laying hens. Since there is insufficient knowledge about overcoming natural stress conditions with minimal cost and side effects in old laying hens, the medicinal herb contained flavonoid and polyphenolic compounds are concerned. Apart from estrogenic, antibacterial effects and cholesterol reduction property that will result to lower cholesterol deposition in poultry products and where calyx and leaf of Hibiscus sabdariffa are known as potent antioxidant. Therefore, these experiments were designed to investigate the effect of Hibiscus sabdariffa extract as natural antioxidants on the immune system, blood biochemical parameters, plasma antioxidant status and the antioxidant balance of laying hens during molting and post-molting periods, also, laying performance and egg yolk cholesterol in the second phase of laying hen egg production.
Materials and Methods: In these experiments, aqueous-alcoholic extract of calyx and leaf of Hibiscus sabdariffa were prepared and sprayed on feed at levels of 300 and 700 mg/kg. One liter of %96 ethanol/distilled water mixture (30:70) were used to infuse 100 g each of the plant material (calyx or leaf) for 24 h. During the pre-experimental stage, minerals composition and antioxidant properties of Hibiscus sabdariffa were measured. Each of the main experiments, were conducted with 200 laying hens in a completely randomized design. In both experiments, five treatments consisted of control diet (basal diet), basal diet with 300 and 700 mg/kg of leaf extract of Hibiscus sabdariffa and basal diet with 300 and 700 mg/kg Hibiscus sabdariffa calyx extract. In both experiments, the blood cholesterol, total protein, triglyceride, LDL, HDL, glucose and malondialdehyde were evaluated by Spectrophotometer auto analyzer. In the end of each experiment, immune system and plasma antioxidant status for 2 samples from each replicate were determined. The egg production, egg weight, egg mass, feed intake and feed conversion ratio were recorded weekly. The egg malondialdehyde was determined during post molting period. In order to measure the level of yolk cholesterol and triglyceride, in the end of the experimental period, 2 samples were randomly taken from each replicate and after separating the yolks and mixing them, were analyzed by Spectrophotometer auto-analyzer with enzymatic method. The data were statistically analyzed with the general linear model by SAS software. The mean differences between treatments were studied by Tukey's test.
Results and Discussion: Although there was more vitamin C in the leaf compared to calyx, the antioxidant activity including total antioxidants, phenols and anthocyanins in calyx were higher than leaf. In both experiments, plasma antioxidant status of laying hens in a dose-independent manner were improved. During post molt period, attempt to increase oxidative stability of plasma and egg yolk also, improving plasma antioxidant status by dietary supplementation of Hibiscus sabdariffa extract was not significant, however, the Hibiscus sabdariffa calyx at 300 and 700 mg/kg levels, significantly affected plasma MDA and total antioxidant capability during molting period. In both experiments, the relative increase in antibody titers and the ratio of heterophil to lymphocyte were observed by all treatments as compared to the Control treatments. Hibiscus sabdariffa showed to have no significant effect on performance parameters. Probably, the negative effect of polyphenol compounds found in Hibiscus sabdariffa on digestive enzymes decreased lipid, protein and carbohydrates digestibility, thus reduced feed intake, egg weight and total blood protein, insignificantly, blood glucose and cholesterol, significantly. Also, the Hibiscus sabdariffa, especially leaf, decreased the yolk cholesterol and triglyceride. On the other hand, supplementation of Hibiscus sabdariffa at 700 mg/kg, improved production performance following feed conversion ratio compared with Control treatment that's probably due to quercetin and daidzein phytoestrogens found in Hibiscus sabdariffa. It seems that some compounds in Hibiscus sabdariffa, including phytoestrogens and organic acids that has been reduced the negative effect of other Hibiscus sabdariffa components on the performance of laying hens.
Conclusion: It can be concluded that Hibiscus sabdariffa, especially its leaves, which are discarded in most countries, including Iran, have the significant beneficial effects, such as anti-lipid effects, improvement of total plasma antioxidant and oxidative balance of old laying hens, with minimal cost and no significant negative effect on functional and immune parameters. It seems that finding of effective dose of Hibiscus sabdariffa is important to achieve layer hens’ maximum efficacy according to the test conditions and can economically be extended the range of its usage.

Keywords

• Antibody titer
• Malondialdehyde
• Molting
• Sour tea

20.1001.1.20083106.1399.12.1.10.7

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References

1- Abbas, A. K., A. H. Lichtman, and S. Pillai. 2014. Pages 213-493 in Cellular and molecular immunology. Elsevier Health Sciences, Amsterdam.

2- Ademiluyi, A. O., and G. Oboh. 2013. Aqueous extracts of roselle (hibiscus sabdariffa linn.) varieties inhibit α-amylase and α-glucosidase activities in vitro. Journal of Medicinal Food, 16(1):88-93.

3- Adil, S., T. Banday, G. A. Bhat, M. S. Mir, and M. Rehman. 2010. Effect of dietary supplementation of organic acids on performance, intestinal histomorphology, and serum biochemistry of broiler chicken. Veterinary Medicine International, 2010: 1-7.

4- Agoreyo, F., B. Agoreyo, and M. Onuorah. 2008. Effect of aqueous extracts of hibiscus sabdariffa and zingiber officinale on blood cholesterol and glucose levels of rats. African Journal of Biotechnology, 7(21): 3949-3951.

5- Ahmadian-Baghbadorani, N., H. Azhdari-Zarmehri, S. Puzesh, F. S. Mousavi, and F. Rajaei. 2014. Antinociceptive effect of hydroalcoholic extract of green tea in male mice. KAUMS Journal (FEYZ), 17(6):528-536.

6- Al-Batshan, H., S. Scheideler, B. Black, J. Garlich, and K. Anderson. 1994. Duodenal calcium uptake, femur ash, and eggshell quality decline with age and increase following molt. Poultry Science, 73(10):1590-1596.

7- Allan, W., and R. Gough. 1974. A standard haemagglutination inhibition test for newcastle disease. (1). A comparison of macro and micro methods. Veterinary Record, 95(6):120-123.

8- Anwar, H., Z. Rahman, I. Javed, and F. Muhammad. 2012. Effect of protein, probiotic, and symbiotic supplementation on serum biological health markers of molted layers. Poultry Science, 91(10):2606-2613.

9- Bendich, A. 1990. Antioxidant vitamins and their functions in immune responses. Pages 35-55 in Antioxidant nutrients and immune functions. Springer, Boston, MA.

10- Calderon, V. M., and L. S. Jensen. 1990. The requirement for sulfur amino acid by laying hens as influenced by the protein concentration. Poultry Science, 69(6):934-944.

11- Carvajal-Zarrabal, O., S. M. Waliszewski, D. M. Barradas-Dermitz, Z. Orta-Flores, P. M. Hayward-Jones, C. Nolasco-Hipolito, O. Angulo-Guerrero, R. Sanchez-Ricaño, R. M. Infanzon, and P. R. Trujillo. 2005. The consumption of hibiscus sabdariffa dried calyx ethanolic extract reduced lipid profile in rats. Plant Foods for Human Nutrition, 60(4): 153-159.

12- Cason, J., N. Cox, and J. Bailey. 1994. Transmission of salmonella typhimurium during hatching of broiler chicks. Avian Diseases, 583-88.

13- Cetin, E., S. Silici, N. Cetin, and B. Güçlü. 2010. Effects of diets containing different concentrations of propolis on hematological and immunological variables in laying hens. Poultry science, 89(8):1703-1708.

14- Chen, C. C., F. P. Chou, Y. C. Ho, W. L. Lin, C. P. Wang, E. S. Kao, A. C. Huang, and C. J. Wang. 2004. Inhibitory effects of hibiscus sabdariffa l extract on low‐density lipoprotein oxidation and anti‐hyperlipidemia in fructose‐fed and cholesterol‐fed rats. Journal of the Science of Food and Agriculture, 84(15):1989-1996.

15- Cheng, T. K., C. N. Coon, and M. L. Hamre. 1990. Effect of environmental stress on the ascorbic acid requirement of laying hens. Poultry Science, 69(5):774-780.

16- Cherian, G., F. Wolfe, and J. Sim. 1996. Dietary oils with added tocopherols: Effects on egg or tissue tocopherols, fatty acids, and oxidative stability. Poultry Science, 75(3):423-431.

17- Chuah, A. M., Y.-C. Lee, T. Yamaguchi, H. Takamura, L.-J. Yin, and T. Matoba. 2008. Effect of cooking on the antioxidant properties of coloured peppers. Food Chemistry, 111(1):20-28.

18- Davis, G., K. Anderson, and A. Carroll. 2000. The effects of long-term caging and molt of single comb white leghorn hens on heterophil to lymphocyte ratios, corticosterone and thyroid hormones. Poultry Science, 79(4):514-518.

19- de Cordova, C. M. M., and M. M. de Cordova. 2013. A new accurate, simple formula for ldl-cholesterol estimation based on directly measured blood lipids from a large cohort. Annals of Clinical Biochemistry, 50(1):13-19.

20- El-Ghalid, O. 2009. Exogenous estradiol: Blood profile, productive and reproductive performance of female japanese quails at different stages of production. Asian Journal of Poultry Science, 3(1):1-8.

21- Elkin, R. G., Z. Yan, Y. Zhong, S. S. Donkin, K. K. Buhman, J. A. Story, J. J. Turek, R. E. Jr. Porter, M. Anderson, R. Homan, and R. S. Newton. 1999. Select 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors vary in their ability to reduce egg yolk cholesterol levels in laying hens through alteration of hepatic cholesterol biosynthesis and plasma VLDL composition. Journal of Nutrition, 129(5): 1010-1019.

22- Fakaye, T. 2008. Toxicity and immunomodulatory activity of fractions of hibiscus sabdariffa linn (family malvaceae) in animal models. African Journal of Traditional, Complementary and Alternative Medicines, 5(4):394-398.

23- Fiesel, A., D. K. Gessner, E. Most, and K. Eder. 2014. Effects of dietary polyphenol-rich plant products from grape or hop on pro-inflammatory gene expression in the intestine, nutrient digestibility and faecal microbiota of weaned pigs. BMC Veterinary Research, 10(1): 196-206.

24- Gous, R., and T. Morris. 2005. Nutritional interventions in alleviating the effects of high temperatures in broiler production. World's Poultry Science Journal, 61(3):463-475.

25- Gross, W., and H. Siegel. 1983. Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens. Avian Diseases, 972-979.

26- Hansawasdi, C., J. Kawabata, and T. Kasai. 2000. Α-amylase inhibitors from roselle (hibiscus sabdariffa linn.) tea. Bioscience, Biotechnology, and Biochemistry, 64(5):1041-1043.

27- Herranz -Lopez, M., M. Olivares-Vicente, J. Encinar, E. Barrajon-Catalan, A. Segura-Carretero, J. Joven, and V. Micol. 2017. Multi-targeted molecular effects of Hibiscus sabdariffa polyphenols: an opportunity for a global approach to obesity. Nutrients, 9(8): 907-933.

28- Hirunpanich, V., A. Utaipat, N. P. Morales, N. Bunyapraphatsara, H. Sato, A. Herunsalee, and C. Suthisisang. 2005. Antioxidant effects of aqueous extracts from dried calyx of hibiscus sabdariffa linn.(roselle) in vitro using rat low-density lipoprotein (ldl). Biological and Pharmaceutical Bulletin, 28(3):481-484.

29- Hirunpanich, V., A. Utaipat, N. P. Morales, N. Bunyapraphatsara, H. Sato, A. Herunsale, and C. Suthisisang. 2006. Hypocholesterolemic and antioxidant effects of aqueous extracts from the dried calyx of hibiscus sabdariffa l. In hypercholesterolemic rats. Journal of Ethnopharmacology, 103(2):252-260.

30- Karaś, M., A. Jakubczyk, U. Szymanowska, U. Złotek, and E. Zielińska. 2017. Digestion and bioavailability of bioactive phytochemicals. International Journal of Food Science & Technology, 52(2):291-305.

31- Khan, R., S. Naz, Z. Nikousefat, M. Selvaggi, V. Laudadio, and V. Tufarelli. 2012. Effect of ascorbic acid in heat-stressed poultry. World's Poultry Science Journal, 68(3):477-490.

32- Khodadadi, M., and S. Rangbar.2016. Effect of adding green tea powder (Camellia sinensis) to some immune factors and serum proteins in Cyprinus carpio. Iran Veterinary Journal, 12(2): 43-54. (In Persian).

33- Kim, J.-K., H. So, M.-J. Youn, H.-J. Kim, Y. Kim, C. Park, S.-J. Kim, Y.-A. Ha, K.-Y. Chai, and S.-M. Kim. 2007. Hibiscus sabdariffa l. Water extract inhibits the adipocyte differentiation through the pi3-k and mapk pathway. Journal of Ethnopharmacology, 114(2):260-267.

34- Kobayashi, Y., M. Suzuki, H. Satsu, S. Arai, Y. Hara, K. Suzuki, Y. Miyamoto, and M. Shimizu. 2000. Green tea polyphenols inhibit the sodium-dependent glucose transporter of intestinal epithelial cells by a competitive mechanism. Journal of agricultural and food chemistry, 48(11):5618-5623.

35- Leresche, R., U. Seal, and P. Karns. 1974. Review of blood chemistry of moose and other cervidae with emphasis on nutritional assessment. Naturaliste Canadien, 101: 263-290.

36- Lin, H.-H., J.-H. Chen, and C.-J. Wang. 2011. Chemopreventive properties and molecular mechanisms of the bioactive compounds in hibiscus sabdariffa linne. Current Medicinal Chemistry, 18(8):1245-1254.

37- Lubega, A. M., G. S. Bbosa, N. Musisi, J. Erume, and J. Ogwal-Okeng. 2013. Effect of the total crude extracts of Hibiscus sabdariffa on the immune system in the Wistar albino rats. African Journal of Pharmacy and Pharmacology, 7:1942-1949.

38- Lucas, A. M., and P. R. Stettenheim. 1972. Avian Anatomy Integument, Part 1. US Government Printing Office, Washington, DC.

39- Luhman, C. M., B. G. Miller, and D. C. Beitz. 1990. Research note: The effect of feeding lovastatin and colestipol on production and cholesterol content of eggs. Poultry Science, 69(5):852-855.

40- Manivasagam, T. T., K. Dakshayani, S. S. Subash, and R. R. Sivaperumal. 2006. Protective influence of hibiscus sabdariffa, an edible medicinal plant, on tissue lipid peroxidation and antioxidant status in hyperammonemic rats. African Journal of Traditional, Complementary and Alternative Medicines, 3(3):10-21.

41- Mantruad, A., P. Pannangpetch, B. Kongyingyoes, U. Kukongviriyapan, S. Chuanta, S. Nakmareong, and A. Itharat. 2010. Roselle extract and gallic acid improve vascular reactivity of diabetic rats. Srinagarind Medical Journal, 257-261.

42- McFarlane, J. M., and S. E. Curtis. 1989. Multiple concurrent stressors in chicks. 3. Effects on plasma corticosterone and the heterophil: Lymphocyte ratio. Poultry Science, 68(4):522-527.

43- McReynolds, J., K. Genovese, H. He, C. Swaggerty, J. Byrd, S. Ricke, D. Nisbet, and M. Kogut. 2009. Alfalfa as a nutritive modulator in maintaining the innate immune response during the molting process. Journal of Applied Poultry Research, 18(3):410-417.

44- Miller, N. J., C. Rice-Evans, M. J. Davies, V. Gopinathan, and A. Milner. 1993. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clinical Science, 84:407-412.

45- Minka, N., A. Fayomi, and J. Ayo. 2007. Protective influence calyces of hibiscus sabdariffa against heat stress in laying hens during the hot-dry season. Research Journal of Poultry Science, 1(1):7-11.

46- Mohammadi, F., F. Bagherzadeh Kasmani, K. Shojaeian, M. Mehri, and M. A. Karimi Torshizi. 2015. Effect of Hibiscus sabdariffa on performance, immune response, and biochemical parameters in broiler chickens fed normal or aflatoxin contaminated diets. Animal Production, 17(2):301-309. (In Persian).

47- Mohd-Esa, N., F. S. Hern, A. Ismail, and C. L. Yee. 2010. Antioxidant activity in different parts of roselle (hibiscus sabdariffa l.) extracts and potential exploitation of the seeds. Food Chemistry, 122(4):1055-1060.

48- Mungole, A., and A. Chaturvedi. 2011. Hibiscus sabdariffa l a rich source of secondary metabolites. International Journal of Pharmaceutical Sciences Review and Research, 6(1):83-87.

49- Ni, Y., Q. Zhu, Z. Zhou, R. Grossmann, J. Chen, and R. Zhao. 2007. Effect of dietary daidzein on egg production, shell quality, and gene expression of er-α, gh-r, and igf-ir in shell glands of laying hens. Journal of agricultural and food chemistry, 55(17):6997-7001.

50- North, M. O. 1984. Pages 270-292 in Commercial chicken production manual. Third Edition. The Avi Publishing Company, Inc., Dawsonville, Georgia, USA.

51- Ochani, P. C., and P. D’Mello. 2009. Antioxidant and antihyperlipidemic activity of hibiscus sabdariffa linn. Leaves and calyces extracts in rats. Indian Journal of Experimental Biology, 47(4):276-282.

52- Okoko, T., and D. Ere. 2012. Hibiscus sabdariffa extractivities on cadmium—mediated alterations of human u937 cell viability and activation. Asian Pacific Journal of Tropical Medicine, 5(1):33-36.

53- Ologundudu, A., A. Ologundudu, O. Oluba, I. Omotuyi, and F. Obi. 2010. Effect of hibiscus sabdariffa anthocyanins on 2, 4-dinitrophenylhydrazine-induced tissue damage in rabbits. Journal of Toxicology and Environmental Health Sciences, 2(1):1-6.

54- Omotuyi, I., A. Ologundudu, V. Onwubiko, M. Wogu, and F. Obi. 2010. Hibiscus sabdariffa linn. Anthocyanins alter circulating reproductive hormones in rabbits (oryctolagus cuniculus). Journal of Diabetes and Endocrinology, 1(3):36-45.

55- Ortiz, L., C. Centeno, and J. Trevino. 1993. Tannins in faba bean seeds: Effects on the digestion of protein and amino acids in growing chicks. Animal Feed Science and Technology, 41(4):271-278.

56- Perek, M., and F. Sulman. 1945. The basal metabolic rate in molting and laying hens. Endocrinology, 36:240-243.

57- Qureshi, M., and A. Gore. 1997. Vitamin e exposure modulates prostaglandin and thromboxane production by avian cells of the mononuclear phagocytic system. Immunopharmacology and immunotoxicology, 19(4):473-487.

58- Rezaeipoor, R., S. Saeidnia, and M. Kamalinejad. 2000. The effect of plantago ovata on humoral immune responses in experimental animals. Journal of ethnopharmacology, 72(1-2):283-286.

59- Saeed, I. A., L. Ali, A. Jabeen, M. Khasawneh, T. A. Rizvi, and S. S. Ashraf. 2012. Estrogenic activities of ten medicinal herbs from the middle east. Journal of Chromatographic Science, 51(1):33-39.

60- Sahin, K., C. Orhan, M. Tuzcu, S. Ali, N. Sahin, and A. Hayirli. 2010. Epigallocatechin-3-gallate prevents lipid peroxidation and enhances antioxidant defense system via modulating hepatic nuclear transcription factors in heat-stressed quails. Poultry Science, 89(10):2251-2258.

61- Saraswati, T. R., W. Manalu, D. R. Ekastuti, and N. Kusumorini. 2013. Increased egg production of Japanese quail (Cortunix japonica) by improving liver function through turmeric powder supplementation. International Journal of Poultry Science, 12(10): 601-614.

62- SAS Institute. 2015. SAS Users Guide: Statistics. SAS Institute Inc., Cary, NC.

63- Sim, J., W. Kitts, and D. Bragg. 1980. Influence of dietary oil, cholesterol, and soysterols on the fecal neutral and acidic steroid excretion in laying hens. Poultry science, 59(2):325-327.

64- Smith, W. S., C. Green, and M. Jerry. 2003. Determination of ascorbic acid (vitamin C) in commerical tablets by iodometric titration, Department of Chemistry, Stevens Institute of Technology.

65- St-Onge, M.-P., E. R. Farnworth, and P. J. Jones. 2000. Consumption of fermented and nonfermented dairy products: effects on cholesterol concentrations and metabolism. The American Journal of Clinical Nutrition, 71(3):674-681.

66- Sukkhavanit, P., K. Angkanaporn, and S. Kijparkorn. 2011. Effect of roselle (hibiscus sabdariffa linn.) calyx in laying hen diet on egg production performance, egg quality and tbars value in plasma and yolk. The Thai Journal of Veterinary Medicine, 41(3):337.

67- Swain, T. 1965. Page 541 in Analytical methods for flavonoids. Academic Press, London, UK.

68- Tee, P.-L., S. Yusof, S. Mohamed, N. Aimi Umar, and N. Mohamed Mustapha. 2002. Effect of roselle (hibiscus sabdariffa l.) on serum lipids of sprague dawley rats. Nutrition & Food Science, 32(5):190-196.

69- Tiwari, U., B. Rastogi, P. Singh, D. K. Saraf, and S. P. Vyas. 2004. Immunomodulatory effects of aqueous extract of tridax procumbens in experimental animals. Journal of Ethnopharmacology, 92(1):113-119.

70- Turkmen, N., F. Sari, and Y. S. Velioglu. 2005. The effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables. Food Chemistry, 93(4):713-718.

71- Usoh, I., E. Akpan, E. Etim, and E. Farombi. 2005. Antioxidant actions of dried flower extracts of hibiscus sabdariffa l. On sodium arsenite-induced oxidative stress in rats. Pakistan Journal of Nutrition, 4(3):135-141.

72- Vanzo, A., M. Terdoslavich, A. Brandoni, A. M. Torres, U. Vrhovsek, and S. Passamonti. 2008. Uptake of grape anthocyanins into the rat kidney and the involvement of bilitranslocase. Molecular Nutrition & Food Research, 52(10):1106-1116.

73- Wang, J., X. Cao, H. Jiang, Y. Qi, K. L. Chin, and Y. Yue. 2014. Antioxidant activity of leaf extracts from different hibiscus sabdariffa accessions and simultaneous determination five major antioxidant compounds by lc-q-tof-ms. Molecules, 19(12):21226-21238.

74- Wong, P.-K., S. Yusof, H. Ghazali, and Y. Che Man. 2002. Physico-chemical characteristics of roselle (hibiscus sabdariffa l.). Nutrition & Food Science, 32(2):68-73.

75- Xu, X., Y. Hu, W. Xiao, J. a. Huang, X. He, J. Wu, E. P. Ryan, and T. L. Weir. 2012. Effects of fermented camilla sinensis, fuzhuan tea, on egg cholesterol and production performance in laying hens. Agriculture and Food Science, 1:006-010.

76- Yang, M. Y., C. H. Peng, K. C. Chan, Y. S. Yang, C. N. Huang, and C. J. Wang. 2010. The hypolipidemic effect of Hibiscus sabdariffa polyphenols via inhibiting lipogenesis and promoting hepatic lipid clearance. Journal of Agricultural and Food Chemistry, 58(2): 850-859.

77- Yoshioka, T., K. Kawada, T. Shimada, and M. Mori. 1979. Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood. American Journal of Obstetrics and Gynecology, 135(3):372-376.

78- Zhao, R., Y. Zhou, Y. Ni, L. Lu, Z. Tao, W. Chen, and J. Chen. 2005. Effect of daidzein on egg-laying performance in shaoxing duck breeders during different stages of the egg production cycle. British poultry science, 46(2):175-181.

79- Zhao, X., Z. Yang, W. Yang, Y. Wang, S. Jiang, and G. Zhang. 2011. Effects of ginger root (zingiber officinale) on laying performance and antioxidant status of laying hens and on dietary oxidation stability. Poultry science, 90(8):1720-1727.

80- Zhen, J., T. S. Villani, Y. Guo, Y. Qi, K. Chin, M.-H. Pan, C.-T. Ho, J. E. Simon, and Q. Wu. 2016. Phytochemistry, antioxidant capacity, total phenolic content and anti-inflammatory activity of hibiscus sabdariffa leaves. Food Chemistry, 190:673-680.