Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 8: 5059 - 5068

DOI: https://doi.org/10.12418/CJAN2023.469

RESEARCH PAPER

In Vitro Antioxidant Activity of Mulberry Leaf Extract and Its Effects on Antioxidant Capacity of Yellow Feather Broilers

  • WANG Yinling ,
  • XUE Yuyang ,
  • SUN Haobin ,
  • NAN Shanshan ,
  • ZHANG Wenju , * ,
  • NIE Cunxi , *
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  • College of Animal Science and Technology, Shihezi University, Shihezi 832000, China
*ZHANG Wenju, professor, E-mail: ;
NIE Cunxi, associate professor, E-mail:

Received date: 2023-02-17

  Online published: 2023-08-10

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Abstract

This experiment was conducted to study the in vitro antioxidant activity of mulberry leaf extract and its effects on serum and liver antioxidant indexes and liver antioxidant gene mRNA relative expression levels of yellow feather broilers, and to investigate the comprehensive antioxidant capacity of mulberry leaf extract. The scavenging ability and reducing ability of 1,1-diphenyl-2-picrylhydrazine (DPPH) radical, 2,2'-diazobis (3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS) radical and hydroxyl radical were analyzed to evaluate the in vitro antioxidant activity of mulberry leaf extract. A total of 120 healthy 42-day-old male medium speed yellow feather broilers with similar body weight were randomly divided into 4 groups with 6 replicates per group and 5 broilers per replicate. Broilers in the control group (CON group) were fed a basal diet, and others in experimental groups were fed basal diets supplemented with 0.05%, 0.10% and 0.20% mulberry leaf extract, respectively. The trial period was 42 days. The results showed as follows: 1) the mulberry leaf extract had scavenging effects on hydroxyl radical, ABTS radical and DPPH radical when the concentration was 0.2 to 1.5 mg/mL, and the reducing ability, hydroxyl radical clearance rate and ABTS radical clearance rate of mulberry leaf extract were significantly increased with the mulberry leaf extract concentration increased (P<0.05). 2) Compared with the CON group, the serum glutathione peroxidase (GPx) activity and total antioxidant capacity (T-AOC) of 0.05%, 0.10% and 0.20% groups were significantly increased (P<0.05), and the serum reactive oxygen species (ROS) content was significantly decreased (P<0.05); the serum catalase (CAT) activity of 0.20% group was significantly increased (P<0.05). 3) Compared with the CON group, the liver GPx activity of 0.05%, 0.10% and 0.20% groups was significantly increased (P<0.05), and the contents of malondialdehyde (MDA) and ROS in liver were significantly decreased (P<0.05); the liver T-AOC of 0.20% group was significantly increased (P<0.05), and the liver CAT activity of 0.10% and 0.20% groups was significantly increased (P<0.05). 4) Compared with the CON group, the liver nuclear factor E2 correlation factor 2 (Nrf2) mRNA relative expression level of 0.10% group was significantly increased (P<0.05), the liver superoxide dismutase 1 (SOD1) mRNA relative expression level of 0.20% group was significantly increased (P<0.05), the liver CAT mRNA relative expression level of 0.10% and 0.20% groups was significantly increased (P<0.05), and the liver GPx mRNA relative expression level of 0.05%, 0.10% and 0.20% groups was significantly increased (P<0.05). In conclusion, the mulberry leaf extract has good in vitro antioxidant activity, dietary mulberry leaf extract can significantly improve the antioxidant capacity of yellow feather broilers, and the appropriate addition of mulberry leaf extract is 0.10% to 0.20%.

Key words： mulberry leaf extract; yellow feather broilers; in vitro antioxidant activity; antioxidant capacity

Cite this article

WANG Yinling , XUE Yuyang , SUN Haobin , NAN Shanshan , ZHANG Wenju , NIE Cunxi . In Vitro Antioxidant Activity of Mulberry Leaf Extract and Its Effects on Antioxidant Capacity of Yellow Feather Broilers[J]. Chinese Journal of Animal Nutrition, 2023 , 35(8) : 5059 -5068 . DOI: 10.12418/CJAN2023.469

桑属植物具有抗炎、抗菌、抗癌、降血糖、降血脂和抗氧化作用[1]。以桑叶为原料,采用适当的溶剂或提取工艺,将桑叶中活性物质浓缩聚集到一起的产物即为桑叶提取物,目前主要的提取工艺有微波提取法[2]、超声提取法[3]、热水提取法和酶辅助提取法等[4]。桑叶提取物物中活性成分具有抗氧化、抗炎、抗菌、抗癌、抗糖尿病、神经保护、心脏保护、肝保护、抗高血压、抗凋亡、抗病毒、抗动脉硬化和抗抑郁作用[5]。在“饲料禁抗”的大背景下,桑叶提取物对促进畜禽业的健康可持续发展提供了新思路[6]。在我国,桑叶每亩(1亩≈666.67 m2)年产量可达2 136 kg[7],开发桑叶资源具有不可估量的经济效益。不同的加工方法会改变桑叶提取物中某些化合物的含量,进而影响桑叶提取物的生物活性[8]。据报道,桑叶提取物具有良好的抗氧化活性。体外抗氧化活性可作为评定待测物质抗氧化活性行之有效的方法,具有简单快速、成本低的优点。例如,有研究者利用体外抗氧化活性分析丙酮、乙醇和甲醇提取到的桑叶提取物的抗氧化活性,发现丙酮提取到的桑叶提取物具有更高的抗氧化活性[9]。待测物质对各种自由基的清除能力是体外抗氧化活性分析中常用的一种方法,例如对羟自由基、2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)自由基和1,1-二苯基-2-苦基肼(DPPH)自由基等清除能力的研究。有研究者比较不同溶剂提取的桑叶提取物对羟自由基清除能力时发现,无水乙醇桑叶提取物清除自由基能力达到43.7%[10]。桑叶黄酮浓度为0.125 mg/mL时,对ABTS自由基清除率达到100%;桑叶黄酮浓度在0~0.1 mg/mL时对DPPH自由基清除率呈剂量依赖性增加,当桑叶黄酮浓度高于0.1 mg/mL时趋于稳定,其半抑制浓度(IC50)为0.045 2 mg/mL,说明桑叶黄酮具有良好的自由基清除活性[11]。
氧化应激会影响畜禽的健康状况,给养殖业带来经济损失。因此,提高畜禽的抗氧化能力对养殖业至关重要。添加外源性的抗氧化剂是机体对抗氧化应激以及提高抗氧化能力的有效措施[12]。据报道,桑叶提取物具有提高动物抗氧化能力、缓解氧化应激损伤的作用。饲粮中添加桑叶提取物可显著提高蛋鸡血清超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GPx)活性[6],还可以提高断奶仔猪机体GPx和过氧化氢酶(CAT)活性[13]。2,2-偶氮二(2-甲基丙基咪)二盐酸盐(AAPH)诱导绵羊红细胞氧化损伤,导致SOD、CAT和GPx活性发生显著变化,添加桑叶黄酮后,SOD、CAT和GPx活性与阴性对照组无显著差异,并且抑制了细胞中丙二醛(MDA)的积累,从而有效缓解AAPH对细胞膜的损伤[11]。桑叶提取物也可显著减少脂多糖(LPS)刺激的小鼠单核巨噬细胞白血病细胞(RAW 264.7细胞)中活性氧(ROS)的产生[14]。
本试验从桑叶提取物体外抗氧化活性及其对黄羽肉鸡抗氧化能力的影响2个方面进行研究,评价桑叶提取物作为抗氧化添加剂的潜力。首先通过桑叶提取物的还原能力对羟自由基、ABTS自由基和DPPH自由基清除活性的研究,为其抗氧化能力做一个初步判断;随后以黄羽肉鸡为研究对象,测定桑叶提取物对黄羽肉鸡血清抗氧化指标、肝脏抗氧化指标和肝脏抗氧化基因mRNA相对表达量的影响,以期为桑叶提取物在肉鸡抗氧化方面的应用提供理论基础和生产指导。

1 材料与方法

1.1 试验材料

桑叶提取物购自西安某生物科技有限公司,桑叶提取物中总糖含量≥24%,总黄酮含量≥20%,总酚含量≥12%。DPPH自由基、ABTS自由基和二甲基亚砜(DMSO)均购自北京索莱宝科技有限公司,过硫酸钾、铁氰化钾、三氯化铁、30%过氧化氢、水杨酸、七水硫酸亚铁、磷酸二氢钠、磷酸氢二钠均购自天津市富宇精细化工有限公司。

1.2 试验设计

1.2.1 桑叶提取物体外抗氧化活性的测定

取一定量的桑叶提取物,用DMSO配制成0.2、0.4、0.6、0.8、1.0和1.5 mg/mL桑叶提取物DMSO溶液。桑叶提取物对DPPH自由基清除能力和还原能力的测定参照Lin等[15]的方法,对ABTS自由基清除能力的测定参照Lee等[16]的方法,对羟自由基清除能力的测定参照Feng等[17]的方法。

1.2.2 试验动物分组设计与饲养管理

饲养试验选用体重相近、健康状况良好的42日龄雄性中速型黄羽肉鸡120羽,随机分为4组,每组6个重复,每个重复5羽鸡。对照组(CON组)饲喂基础饲粮,试验组分别在基础饲粮的基础上添加0.05%、0.10%和0.20%的桑叶提取物。试验期共42 d。采用干粉料饲粮类型,营养水平参考国家农业行业标准《黄羽肉鸡营养需要量》(NY/T 3645—2020),按中期(42~60日龄)和后期(61~84日龄)2个阶段进行配制,基础饲粮组成及营养水平见表1。
表1 基础饲粮组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of basal diets (DM basis) %

项目
Items		 含量Content
42~60日龄
42 to 60
days of age		 61~84日龄
61 to 84
days of age
原料Ingredients
玉米Com 62.00 65.00
豆粕Soybean meal 28.00 28.00
植物油Vegetable oil 1.00 1.00
葵花籽粕Sunflower seed meal 4.00 1.00
预混料Premix1) 5.00 5.00
合计Total 100.00 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 11.95 12.10
粗蛋白质CP 17.00 16.33
钙Ca 0.12 0.11
总磷TP 0.38 0.36
蛋氨酸Met 0.31 0.30
赖氨酸Lys 0.94 0.92

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets:CaCO3 11.34 g,CaHPO4 9.8 g,NaCl 2.94 g,赖氨酸 Lys 0.234 g,蛋氨酸 Met 1.791 g,VA 12 000 IU,VD3 2 500 IU,VE 35 IU,VK3 2.5 mg,VB12 15 μg,VB6 20.02 mg,烟酸 nicotinic acid 45 mg,泛酸 pantothenic acid 25 mg,叶酸 folic acid 1.2 mg,硫胺素 thiamine 2.5 mg,核黄素 riboflavin 6.6 mg,生物素 biotin 0.2 mg,胆碱 choline 500 mg,Cu 8.0 mg,Fe 80 mg,Zn 80 mg,Mn 100 mg,I 0.80 mg,Se 0.20 mg。

2)粗蛋白质为测定值,其余营养水平均为计算值。CP was a measured value, while the other nutrient levels were calculated values.

肉鸡饲养试验在石河子大学动物科技学院试验站进行,采用3层笼养方式,每天分别于早、晚定时饲喂。鸡只自由采食,充足饮水,15 lx的光照强度,16 h光照,室温控制在24 ℃左右。每天观察鸡只的采食、排便和健康状况,按正常免疫程序进行免疫接种。

1.3 样品采集及指标测定

1.3.1 血清抗氧化指标

饲养结束后,每个重复随机选取1羽鸡(每组6羽),共24羽体况健康的鸡,用采血管进行翅下静脉采血,静置2 h,3 000 r/min,离心10 min,分离血清,于-20 ℃保存。测定血清总抗氧化能力(T-AOC)和CAT、SOD、GPx活性及MDA、ROS含量。CAT、T-AOC和MDA试剂盒购自南京建成生物试剂有限公司,SOD、GPx和ROS剂盒购自北京盒子生工科技有限公司,样品处理和测定按照相关试剂盒说明书进行操作,用全波长酶标仪(美国Thermo公司)测定。

1.3.2 肝脏抗氧化指标

饲养结束后,每个重复随机选取1羽鸡(每组6羽),共24羽体况健康的鸡,屠宰后取肝脏,用磷酸盐缓冲液(PBS)冲洗干净后,剪切成小块,放入2 mL冻存管中,立即放入液氮中,随后从液氮中取出,于-80 ℃保存。测定肝脏T-AOC和SOD、CAT、GPx活性及MDA、ROS含量。试剂盒均购自北京盒子生工科技有限公司。将肝脏组织与预冷的生理盐水以1∶9(g∶mL)混合,随后用组织研磨机(上海净信实业发展有限公司)匀浆,制成10%肝脏组织匀浆液,离心后取上清液,按照试剂盒说明书进行操作,用全波长酶标仪(美国Thermo公司)测定。

1.3.3 肝脏抗氧化基因mRNA相对表达量

采用实时荧光定量PCR(qRT-PCR)法检测肝脏抗氧化基因mRNA相对表达量。使用Trizol法按试剂盒说明书提取肝脏总RNA,使用超微量分光光度计(美国Thermo公司)检测RNA的浓度和纯度,使用电泳仪(北京市六一仪器厂)检测RNA的完整性,于-80 ℃保存。将提取到的RNA用反转录试剂盒按说明书反转录为cDNA,于-20 ℃保存,用于后续qRT-PCR。用荧光定量仪(瑞士Roche公司)测定,qRT-PCR反应体系为5 μL模板(cDNA),上、下游引物各0.4 μL(共0.8 μL),10 μL的2×TransStart®Green qPCR SuperMix,4.2 μL无酶水,总体积为20 μL。反应条件为94 ℃ 30 s→94 ℃ 5 s→60 ℃ 30 s(45个循环)。试剂盒均购自北京全式金生物技术股份有限公司。以β-肌动蛋白(β-actin)为内参基因,2-ΔΔCt法计算各基因mRNA相对表达量。引物合成由北京睿博兴科生物科技有限公司合成,引物序列见表2。
表2 引物序列

Table 2 Primer sequences

基因
Genes		 登录号
Accession No.		 引物序列
Primer sequences (5'—3')		 大小
Size/bp		 参考文献
Reference
核因子E2相关因子2
Nrf2		 XM_046921130.1 F:GGGACGGTGACACAGGAACAAC
R:GCTCTCCACAGCGGGAAATCAG		 97 [18]
超氧化物歧化酶1
SOD1		 NM_205064.1 F:TTGTCTGATGGAGATCATGGCTTC
R:TGCTTGCCTTCAGGATTAAAGTGA		 98 [19]
过氧化氢酶
CAT		 NM_001031215.2 F:GGTTCGGTGGGGTTGTCTTT
R:CACCAGTGGTCAAGGCATCT		 213 [20]
谷胱甘肽过氧化物酶
GPx		 NM_001163245.2 F:CAAAGTTGCGGTCAGTGGA
R:AGAGTCCCAGGCCTTTACTACTTTC		 136 [21]
β-肌动蛋白
β-actin		 NM_205518 F:GCCAACAGAGAGAAGATGACAC
R:GTAACACCATCACCAGAGTCCA		 140 [19]

1.4 数据分析

数据经Excel 2010整理后,使用SPSS 26.0软件进行单因素方差分析(one-way ANOVA),并用Duncan氏法进行多重比较,以P<0.05为显著性判断水平,结果以平均值±标准差表示。

2 结果

2.1 桑叶提取物体外抗氧化活性

还原能力通常用吸光度值来表示,吸光度值越大,还原能力越强。由图1可见,桑叶提取物浓度在0.2~1.5 mg/mL时对羟自由基、ABTS自由基和DPPH自由基均有清除作用,呈现剂量依赖性,且桑叶提取物的还原能力、羟自由基清除率和ABTS自由基清除率随着桑叶提取物浓度的增加显著升高(P<0.05)。桑叶提取物浓度在1.5 mg/mL时,对羟自由基、ABTS自由基和DPPH自由基的清除率分别达到39.10%、95.21%和27.65%。桑叶提取物清除羟自由基、ABTS自由基和DPPH自由基的半抑制浓度(IC50)分别为5.076、0.669和15.872 mg/mL。由此可见,桑叶提取物清除自由基的能力的大小为:ABTS自由基>羟自由基>DPPH自由基。
图1 桑叶提取物体外抗氧化活性

数据标相同小写字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。

Fig.1 In vitro antioxidant activity of mulberry leaf extract

Value with the same small letter mean no significant difference (P>0.05), while with different small letters mean significant difference (P<0.05).

2.2 桑叶提取物对黄羽肉鸡抗氧化能力的影响

2.2.1 血清抗氧化指标

由表3可见,与CON组相比,0.05%、0.10%和0.20%组血清MDA含量和SOD活性无显著差异(P>0.05),血清ROS含量显著降低(P<0.05);其中,0.10%组血清ROS含量比CON组显著降低了57.45%(P<0.05)。0.20%组血清CAT活性显著高于CON组和0.05%、0.10%组(P<0.05),分别显著升高了96.50%、91.34%和67.52%。与CON组相比,0.05%、0.10%和0.20%组血清GPx活性分别显著升高了142.48%、99.39%和46.95%(P<0.05),血清T-AOC分别显著升高了32.51%、25.93%和21.06%(P<0.05)。
表3 桑叶提取物对黄羽肉鸡血清抗氧化指标的影响

Table 3 Effects of mulberry leaf extract on serum antioxidant indexes of yellow feather broilers

项目
Items		 组别Groups P值
P-value
CON 0.05% 0.10% 0.20%
过氧化氢酶CAT/(U/mL) 6.30±2.03b 6.47±2.29b 7.39±0.48b 12.38±1.89a 0.002
超氧化物歧化酶SOD/(U/mL) 289.65±17.60 289.29±22.10 296.81±89.00 268.53±44.60 0.772
谷胱甘肽过氧化物酶GPx/(U/mL) 369.02±74.91d 894.81±83.83a 735.80±77.71b 542.29±62.30c <0.001
丙二醛MDA/(nmol/mL) 1.55±0.40 1.89±0.60 1.72±0.46 1.76±0.44 0.642
活性氧ROS/(ng/mL) 148.10±14.54a 104.83±9.45b 63.02±12.62d 91.69±11.52c <0.001
总抗氧化能力T-AOC/(μmol/L) 730.63±25.33b 968.18±24.64a 920.10±55.92a 884.47±131.54a 0.021

同行数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。下表同。

In the same row, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as below.

2.2.2 肝脏抗氧化指标

由表4可见,与CON组相比,0.05%、0.10%和0.20%组肝脏SOD活性无显著差异(P>0.05),肝脏MDA和ROS含量显著降低(P<0.05)。与CON组相比,0.10%和0.20%组肝脏CAT活性显著升高了25.12%和51.57%(P<0.05),0.05%、0.10%和0.20%组肝脏GPx活性分别显著升高了207.58%、121.21%和81.43%(P<0.05)。0.20%组肝脏T-AOC显著高于CON组和0.05%、0.10%组(P<0.05)。
表4 桑叶提取物对黄羽肉鸡肝脏抗氧化指标的影响

Table 4 Effects of mulberry leaf extract on liver antioxidant indexes of yellow feather broilers

项目
Items		 组别Groups P值
P-value
CON 0.05% 0.10% 0.20%
过氧化氢酶CAT/(U/mg) 226.03±21.17c 246.35±17.98bc 282.80±36.48b 341.66±16.21a 0.002
超氧化物歧化酶SOD/(U/mg) 698.01±60.53 694.44±60.19 690.83±14.53 715.28±27.49 0.913
谷胱甘肽过氧化物酶GPx/(U/mg) 2.64±0.77d 8.12±0.72a 5.84±0.63b 4.79±0.78c <0.001
丙二醛MDA/(nmol/mg) 28.23±1.27a 20.93±0.84b 19.42±2.37b 22.35±1.05b <0.001
活性氧ROS/(ng/mg) 1.07±0.09a 0.76±0.09b 0.42±0.07d 0.69±0.08c <0.001
总抗氧化能力T-AOC/(μmol/g) 6.71±0.78b 8.05±0.92b 8.29±1.53b 10.48±1.10a 0.021

2.2.3 肝脏抗氧化基因mRNA相对表达量

由图2可见,与CON组相比,0.10%组肝脏核因子E2相关因子2(Nrf2)mRNA相对表达量显著升高了70%(P<0.05),0.20%组肝脏SOD1 mRNA相对表达量显著升高了35.64%(P<0.05),0.10%和0.20%组肝脏CAT mRNA相对表达量分别显著升高了52.52%和121.21%(P<0.05);0.05%、0.10%和0.20%组肝脏GPx mRNA相对表达量分别显著升高了414.85%、153.46%和102.91%(P<0.05),且0.05%组肝脏GPx mRNA相对表达量显著高于0.10%和0.20%组。
图2 桑叶提取物对黄羽肉鸡肝脏抗氧化基因mRNA相对表达量的影响

数据柱标相同小写字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。

Fig.2 Effects of mulberry leaf extract on mRNA relative expression levels of antioxidant genes of yellow feather broilers

Value columns with the same small letter mean no significant difference (P>0.05), while with different small letters mean significant difference (P<0.05).

3 讨论

3.1 桑叶提取物体外抗氧化活性

3.1.1 桑叶提取物的还原能力

自由基通过捕获或抢夺电子保持自身的稳定,还原能力可以反映抗氧化剂的给电子能力,给电子能力越强,其抗氧化活性也就越强。桑叶提取物中总酚和黄酮含量与抗氧化活性之间存在显著的线性关系[22]。本试验发现,桑叶提取物浓度在0.2~1.5 mg/mL时,还原能力随桑叶提取物浓度的增加而显著上升,且增加幅度较大,说明桑叶提取物的抗氧化活性也随桑叶提取物浓度增加而升高,与Cui等[23]研究结果一致。

3.1.2 桑叶提取物对自由基的清除活性

羟自由基是ROS中最具活性的自由基,细胞内源性过氧化氢(H2O2)产生的羟自由基启动脂质过氧化链反应,导致ROS和脂质过氧化物增加[24]。ABTS是用来测定总抗氧化能力的一种有效的方法。DPPH自由基是一种稳定的以氮为中心的自由基,其清除能力的测定已被广泛用于评估体外抗氧化活性。研究发现,桑叶乙醇提取物清除羟基自由基的IC50为0.524 2 mg/mL[11]。1.0 mg/mL的桑叶多糖对羟基自由基的清除率达到83.46%[17]。提纯后的桑叶黄酮浓度为2.4 mg/mL时,对羟自由基的清除能力达97.12%[25]。桑叶提取物浓度在0~1 mg/mL时,对ABTS自由基的清除率呈现缓慢的上升趋势,但是在浓度为1 mg/mL时对ABTS自由基的清除率高达99.94%[25]。不同桑叶提取物单体清除ABTS自由基活性也不相同,桑叶提取物中芦丁清除ABTS自由基活性相比于异槲皮苷更强,芦丁的IC50为0.017 2 mg/mL[26]。桑叶提取物以浓度依赖的方式清除DPPH自由基[27]。有研究发现,桑叶提取物浓度为1 mg/mL时,对DPPH自由基清除率达到68.16%[28]。桑叶多糖浓度为4.0 mg/mL时,对DPPH自由基的清除率达到66.34%[17]。桑叶水提取物对DPPH自由基的清除率在34.60%~61.70%[22]。有研究发现,DPPH自由基清除活性与桑叶提取物的总酚含量密切相关[29]。
综上可知,桑叶提取物中的多糖、黄酮和多酚都具有一定的还原能力和自由基清除活性。本试验结果表明,桑叶提取物浓度在1.5 mg/mL时对羟自由基的清除率在39.10%,可在一定程度反映桑叶提取物对机体内产生的羟自由基也具有清除能力,从而间接减少脂质过氧化物和ROS的产生。本试验中,1.0和1.5 mg/mL桑叶提取物对ABTS自由基清除率分别达到57.76%和95.21%,IC50为0.669 mg/mL。桑叶提取物浓度为1.5 mg/mL时对DPPH自由基的清除率可达27.65%,IC50为15.872 mg/mL。本研究所用桑叶提取物对羟自由基、ABTS自由基和DPPH自由基均有清除活性,尤其对ABTS自由基的清除活性最高。与他人研究相比,本试验桑叶提取物的自由基清除活性较低,这可能由于提取方法和提取溶剂等的不同,各种活性成分此消彼长,最终会影响到桑叶提取物的抗氧化活性[8],这也就是不同研究结果体外抗氧化活性出现差异的原因所在。总的来说,从本试验对桑叶提取物体外抗氧化活性的测定结果来看,证实了桑叶提取物作为外源性抗氧化剂具有现实可行性。

3.2 桑叶提取物对黄羽肉鸡抗氧化能力的影响

当氧化和抗氧化系统之间的平衡被打破,机体内自由基的累积和这些化合物导致的细胞损伤引起氧化应激,威胁到机体健康。持续的氧化应激会导致机体的慢性炎症[30]。MDA是脂质过氧化的碳基终产物,MDA含量的增加反映了脂质过氧化和组织损伤的增强[31]。需氧生物在正常代谢过程中会产生ROS,是一系列基本生化过程产生的副产品,低水平的ROS在氧化还原信号中发挥重要作用,但其过量累积对细胞和组织具有高度毒性和有害性[32]。为了保护机体免受氧化应激伤害,生物体已经进化出几种防御机制,例如抗氧化酶或直接清除自由基和相关化合物的小分子质量分子[33]。其中,Nrf2是一种转录因子,可以调节如抗氧化酶、炎症相关蛋白、药物转运蛋白和代谢酶的表达来应对环境应激,维持机体内环境的稳态[34]。Nrf2对保护细胞免受氧化应激至关重要,可激活GPx[32]、CAT和SOD[35]以防止过度氧化损伤。Nrf2的缺乏导致抗氧化酶的表达下降,ROS含量因此上升[36]。可见Nrf2对机体维持正常抗氧化能力至关重要。抗氧化酶如SOD、GPx和CAT可将一些有毒的自由基代谢为无害的副产物,T-AOC反映机体总抗氧化水平。
桑叶提取物可以提高机体抗氧化能力。一方面,桑叶提取物对于外界因素刺激引起的氧化应激具有治疗作用。例如,桑叶提取物可显著改善LPS和D-氨基半乳糖(D-GalN)诱导的大鼠肝脏组织的病理状态,Nrf2蛋白质表达水平增加,提高SOD和CAT活性,降低MDA含量[37]。乙酰氨基苯酚(PTL)诱导的大鼠氧化应激[38]、高淀粉诱导的鱼氧化应激[39]、乙醇饮食诱导的小鼠肝脏氧化应激[40]在添加桑叶提取后也发现了相同的结果。另一方面,桑叶提取物通过增加机体抗氧化酶的活性和抗氧化基因mRNA相对表达量,降低自由基和相关化合物的含量,提高机体抗氧化能力。例如,桑叶黄酮可显著增加黄鳝肝脏GPx1、GPx8、CAT、SOD和Nrf2 mRNA相对表达量,同时发现CAT和GPx活性显著升高,ROS含量显著降低[41]。Wen等[42]从桑叶中提取出新的异戊二烯黄酮类化合物morachalcone D可上调GPx4、CAT、SOD2和Nrf2 mRNA相对表达量,减少ROS的产生。蛋鸡饲粮中添加0.8%~1.2%桑叶提取物可显著提高血清GPx和SOD活性[6]。本试验也发现,饲粮中添加桑叶提取物后,黄羽肉鸡肝脏Nrf2、CAT、SOD1和GPx mRNA相对表达量升高,与血清和肝脏CAT、SOD和GPx活性升高的结果一致,同时降低了血清和肝脏ROS和MDA含量。

4 结论

① 桑叶提取物具有良好的体外抗氧化活性,对ABTS自由基的清除率最高。
② 桑叶提取物可提高黄羽肉鸡肝脏抗氧化基因的mRNA相对表达量,提高血清和肝脏CAT、SOD和GPx活性,并降低MDA和ROS含量。
③ 综合考虑,本试验条件下桑叶提取物在黄羽肉鸡饲粮中的添加量以0.10%~0.20%为宜。

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