Antimicrobial activity of <i>Carya illinoensis</i> leaves

YE Shenyi; RUAN Xiayun; FAN Ying; ZHANG Huanshuai; JING Changyi; MAO Shengfeng; ZHANG Ailian

doi:10.11833/j.issn.2095-0756.2014.04.025

Volume 31 Issue 4

Jul. 2014

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YE Shenyi, RUAN Xiayun, FAN Ying, ZHANG Huanshuai, JING Changyi, MAO Shengfeng, ZHANG Ailian. Antimicrobial activity of Carya illinoensis leaves[J]. Journal of Zhejiang A&F University, 2014, 31(4): 658-662. doi: 10.11833/j.issn.2095-0756.2014.04.025

Citation:

YE Shenyi, RUAN Xiayun, FAN Ying, ZHANG Huanshuai, JING Changyi, MAO Shengfeng, ZHANG Ailian. Antimicrobial activity of Carya illinoensis leaves[J]. Journal of Zhejiang A&F University, 2014, 31(4): 658-662. doi: 10.11833/j.issn.2095-0756.2014.04.025

Antimicrobial activity of Carya illinoensis leaves

doi: 10.11833/j.issn.2095-0756.2014.04.025

School of Forestry and Biotechnology, Zhejiang A & F University, Lin'an 311300, Zhejiang, China

Received Date: 2013-07-29
Rev Recd Date: 2013-12-02
Publish Date: 2014-08-20

Abstract

The purpose is to provide theoretical basis for developing a new type of natural antiseptic products. The antimicrobial activity of Carya illinoensis leaves was determined through extraction, in vitro, with 75% ethanol and then fractionated with petroleum ether, chloroform, ethyl acetate, n-butanol, and water in turn. The cup plate method was used to determine antimicrobial activity of the extract and extractants with cultures of Aspergillus flavus, Rhizopus oryzae, Penicillium sp., Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. Then, the half inhibitory concentration (C_E50) were calculated. Results showed that the mean inhibition zone diameter for the ethyl acetate extract of Penicillium sp. was 9.18 mm, of R. oryzae was 10.86 mm, of A. flavus was 8.26 mm, and of B. subtilis was 10.18 mm exhibiting a strong inhibitory effect(P < 0.05, P < 0.01). However, for A. flavus, the inhibitory effect of the petroleum ether extract (C_E50 0.91 g·L-1) was better than the ethyl acetate extract (C_E50>200 g·L-1). The mean inhibition zone diameter for the n-butanol extract of S. cerevisiae (11.13 mm) and E. coli (8.83 mm) also showed a strong inhibitory effect(P < 0.05, P < 0.01); nevertheless, the inhibitory effect of the aqueous extract(9.13 mm) for E. coli was better than the n-butanol extract(8.83 mm). Thus, the n-butanol extract and the ethyl acetate extract of Carya illinoensis leaves have the best inhibitory effect. For the experimental results, according to different control object, we can use different extraction methods and technology to research and develop the exclusive antiseptic, in order to improve the effect of antiseptic.
- forest protection,
- Carya illinoensis,
- extract,
- antimicrobial activity,
- half inhibitory concentration

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美国山核桃Carya illinoensis，又名薄壳山核桃或长山核桃，原产于美国和墨西哥北部，世界多个国家都有引种栽培。中国江苏省于20世纪引入栽培于淮安、南京、泰州、江阴等地。近年来，浙江农林大学开展了薄壳山核桃引种、育种与推广栽培。胡桃科Juglandacecae植物含有黄酮类、萜类、萘醌苷类、多酚类和有机酸类等成分，具有抑制醛糖还原酶，抑制细菌DNA起始复合物的形成，抗肿瘤，镇痛消炎等作用^[1-3]。胡旭姣等^[5]的研究表明:山核桃叶片提取物具有抑制胃癌SGC-7901细胞增殖和诱导该肿瘤细胞凋亡的作用。美国山核桃与山核桃Carya cathayensis同属，两者亲缘关系相近。一般认为，植物之间亲缘关系相近的个体常具有类似的化学成分和相似临床疗效的规律。殷舒等[５]对山核桃叶片提取物的抑菌作用研究表明:山核桃叶片提取物对金黄色葡萄球菌Staphylococcus aureus, 大肠埃希菌Escherichia coli，酵母菌Saccharomyces cerevisiae，黄曲霉菌Aspergillus flavus，青霉菌Penicillium sp.和黑曲霉菌Aspergillus niger均有抑制作用。本实验采用滤纸片法，首次对美国山核桃叶的总提取物，以及石油醚、氯仿、乙酸乙酯、正丁醇、水5个不同极性的提取物进行抗菌作用研究，以期为今后农业上的新型植物农药开发和医学上的临床应用提供科学依据。

1. 材料与仪器

1.1. 材料和供试菌种

美国山核桃叶，2012年10月采于浙江农林大学东湖校区。菌种根霉菌Rhzopus oryzae，酵母菌Saccharomyces cerevisiae，黄曲霉菌Aspergillus flavus，青霉菌Penicillium sp.，大肠埃希菌Escherichia coli和枯草杆菌Bacillus subtilis均由浙江农林大学森林保护实验室提供。

1.2. 试剂与仪器

供试有机溶剂及所用生化试剂均为国产分析纯。使用下列仪器:SHB-III循环水式多用真空泵（河南省予华仪器有限公司）；旋转蒸发仪（河南省予华仪器有限公司）；AB104-N分析天平；MLS-3750全自动蒸汽灭菌锅（SANYO）；PRX-350C智能人工气候箱（宁波海曙赛福实验仪器厂）；MCV-B131FCT超净工作台（SANYO）。

2. 方法

2.1. 药液的制备^[5]

新鲜美国山核桃叶（5.00 kg）于50 ℃烘箱中烘48 h至干，粉碎，过40目筛，取粉500 g，用体积分数为75%乙醇回流提取3次，2 h·次^-1，滤液合并，减压浓缩至无醇味，烘干，得总浸膏72.6 g。取30.0 g总浸膏用水分散后分别用石油醚、氯仿、乙酸乙酯、正丁醇萃取，各萃取液及水相减压浓缩回收溶剂，烘干，得到不同极性提取物。随后用二甲基亚砜将总浸膏和不同提取物浸膏分别配制成质量浓度为10.000，5.000，2.500，1.250和0.625 g·L^-1的药液，备用。

2.2. 菌种活化

将各供试细菌分别移接入牛肉膏斜面培养基上，真菌接入马铃薯葡萄糖琼脂（PDA）斜面培养基上，细菌置于37 ℃的培养箱中培养48 h，真菌置于25 ℃的培养箱中培养96 h。

2.3. 菌悬液的制备^[6]

将活化好的各菌种制成比例为1.0×10⁸～1.0×10⁹菌落形成单位（CFU）·L^-1的菌悬液，备用。

2.4. 抑菌试验-纸片法^[7]

无菌条件下，取菌悬液0.1 mL，用无菌涂布棒均匀涂布在平板培养基上，将灭菌后直径为6 mm的滤纸小圆片在上述各抽提液中浸泡20 min后，取出晾干，等距离贴于平板上，4片·皿^-1，3个重复·菌种^-1，分别以各自无浸膏的萃取液为空白对照。细菌置于31 ℃培养箱中培养24 h，真菌置于25 ℃培养箱中培养72 h后，用十字交叉法，测定抑菌圈直径，并按以下公式计算抑菌率:抑菌率=（对照菌落数-处理菌落数）/对照菌落数×100%。

2.5. 数据处理分析

2.5.1. 抑菌直径均值方差分析

抑菌圈直径原始数据用DPS数据处理系统软件进行多因素方差分析，并对平均数用最小显著性差异法（LSD）进行多重比较。

2.5.2. 真菌半数抑菌质量浓度的计算^[8]

根据提取物质量浓度与抑制率获得回归方程，计算各真菌抑制率在50%时的各提取物质量浓度，即为各提取物对各真菌的半数抑菌质量浓度（C_E50）。

3. 结果与分析

3.1. 美国山核桃叶不同提取物的抑菌活性

美国山核桃叶各极性提取物对6株供试菌均表现出一定的抑菌活性，其中正丁醇提取物与乙酸乙酯提取物对除大肠埃希菌外的5株供试菌均表现出较强的抑菌活性，故可认为美国山核桃叶正丁醇提取物与乙酸乙酯提取物的抑菌效果最佳。而对于黄曲霉菌，石油醚提取物表现出较强的抑菌活性（表 1）。

酵母菌		青霉菌		黄曲霉菌		根霉囷		枯草杆菌		大肠埃希菌
提取液	抑菌圈直径/mm	提取液	抑菌圈直径/mm	提取液	抑菌圈直径/mm	提取液	抑菌圈直径/mm	提取液	抑菌圈直径/mm	提取液	抑菌圈直径/mm
正丁醇	11.13 aA	乙酸乙酯	9.18aA	石油醚	8.35 aA	乙酸乙酯	10.86 aA	乙酸乙酯	10.18 aA	水	9.13 aA
水	10.48 aAB	水	8.71 aAB	乙酸乙酯	8.26 abAB	氯仿	9.36 bAB	氯仿	9.88 aA	正丁醇	8.83 abAB
氯仿	10.19abAB	氯仿	8.59abAB	总浸膏	8.22 abAB	正丁醇	9.32 bBC	正丁醇	9.30 aA	石油醚	8.65 bcAB
总浸膏	10.07abAB	石油醚	8.40abAB	正丁醇	7.47 bcBC	石油醚	8.25 cCD	石油醚	8.53 bB	氯仿	8.23 bcB
石油醚	10.04 bBC	总浸膏	8.26abAB	水	7.35 bcBC	水	8.64 cD	水	8.09 bcB	乙酸乙酯	7.88 bcB
乙酸乙酯	8.79 cC	正丁醇	7.92 bB	氯仿	7.18 cC	总浸膏	7.44 dD	总浸膏	7.77 cC	总浸膏	7.77 cB
说明：数据后不同小写字母表示差异达0.05显著水平，不同大写字母表示差异达0.01显著水平。

Table 1. Variance analysis of inhibition zone of different extracts from Carya illinoensis against 6 species of strains

由表 1可知:美国山核桃叶不同极性提取物及总浸膏对细菌和真菌皆有抑菌作用，但各提取物的抑菌效果存在差异。对真菌的抑菌作用:石油醚提取物对黄曲霉菌的抑菌作用显著大于其他4种提取物及总浸膏，在0.01及0.05水平上均存在显著差异，故对于黄曲霉菌，石油醚提取物的抑菌效果最佳，抑菌圈直径均值为8.35 mm；对于青霉菌，乙酸乙酯提取物的抑菌效果最佳，抑菌圈直径均值为9.19 mm；对于酵母菌，正丁醇提取物的抑菌效果最佳，抑菌圈直径均值为11.13 mm；对于根霉菌，乙酸乙酯提取物的抑菌效果最佳，抑菌圈直径均值为10.86 mm。在对细菌的抑菌试验中:正丁醇提取物对枯草杆菌的抑菌效果最佳，抑菌圈直径均值为10.19 mm；而对于大肠埃希菌，水提取物的抑菌效果最佳，抑菌圈直径均值为9.13 mm。由此可知:美国山核桃叶的乙酸乙酯提取物和正丁醇提取物均表现出较为广谱的抑菌作用，对真菌与细菌都有不同程度的抑菌作用，且差异显著；乙酸乙酯提取物对于根霉菌的抑菌作用显著大于其他供试菌种，正丁醇提取物对于酵母菌和枯草杆菌的抑菌作用显著大于其他供试菌种。故美国山核桃叶的正丁醇提取物和乙酸乙酯提取物的抑菌效果最佳。

3.2. 美国山核桃叶不同浓度各极性提取物对各菌种的半数抑菌质量浓度

由表 2可知:美国山核桃叶的正丁醇提取物和乙酸乙酯提取物对各真菌均表现出较强的抑菌作用。对于酵母菌，正丁醇提取物表现出较其他提取物最强的抑菌活性，其半数抑制质量浓度（C_E50）值为3.68 g·L^-1。对于根霉菌，氯仿提取物表现出较乙酸乙酯提取物更强的抑菌活性，其C_E50值为9.44 g·L^-1。对于黄曲霉菌，石油醚提取物表现出较正丁醇提取物和乙酸乙酯提取物更强的抑菌作用，C_E50值为0.91 g·L^-1。对于青霉菌，水提取物表现出较正丁醇提取物和乙酸乙酯提取物更强的抑菌作用，C_E50值为0.01 g·L^-1。

菌种	提取物	不同药液质量浓度死亡率/%					回归方程	相关系数	C_E50/(g.L¹)
菌种	提取物	10.00	5.00	2.50	1.25	0.65	回归方程	相关系数	C_E50/(g.L¹)
黄曲霉菌	总浸膏	55.00	69.44	64.44	65.37	70.19	y=5.46-0.237 2x	-0.682 1	1.94
	水	64.52	66.81	70.48	68.19	67.07	y=5.46-0.059 7 x	-0.463 9	> 200.00
	正丁醇	63.15	68.89	70.19	67.41	68.15	y=5.47-0.077 7x	-0.497 5	> 200.00
	石油醚	57.78	66.85	65.19	65.74	62.22	y=5.35+9.000 0x	-0.328 1	0.91
	氯仿	68.37	69.48	71.74	69.63	68.85	y=5.50-0.010 6x	-0.133 3	> 200.00
	乙酸乙酯	61.30	61.30	68.15	67.04	65.74	y=5.41-0.131 7x	-0.712 6	> 200.00
酵母菌	总浸膏	52.?6	59.26	60.56	57.96	52.59	y=5.14+0.017 6 x	0.088 0	0.00
	水	55.00	58.15	59.63	52.96	50.74	y=5.06+0.117 1 x	0.591 8	0.30
	正丁醇	35.56	56.30	56.30	63.33	60.19	y=5.28-0.489 9 x	-0.816 0	3.68
	石油醚	5?.07	50.74	59.44	58.70	65.19	y=5.27-0.176 8 x	-0.624 6	31.79
	氯仿	58.33	54.07	57.04	53.70	59.44	y=5.15-0.016 1 x	-0.116 4	> 200.00
	乙酸乙酯	43.70	69.07	69.26	65.93	61.30	y=5.39-0.273 0 x	-0.463 3	27.63
青霉菌	总浸膏	57.41	64.44	61.11	69.63	68.70	y=5.45-0.251 2 x	-0.855 9	59.82
	水	37.78	34.44	34.63	38.52	44.26	y=4.71-0.151 8x	-0.672 5	0.01
	正丁醇	42.59	33.52	34.07	32.22	30.56	y=4.47+0.234 3x	0.865 1	188.21
	石油醚	42.04	35.00	35.44	31.04	35.74	y=4.54+0.150 9x	0.658 5	> 200.00
	氯仿	63.15	66.11	67.59	56.48	63.33	y=5.29+0.081 9x	0.341 4	0.00
	乙酸乙酯	57.59	58.52	62.22	62.41	66.48	y=5.35-0.192 2x	-0.964 9	62.82
根霉菌	总浸膏	62.41	62.52	57.96	59.26	59.04	y=5.20+0.087 9x	0.757 7	0.01
	水	66.37	70.63	66.41	68.70	65.78	y=5.42+0.029 5x	0.241 6	0.00
	正丁醇	56.96	62.48	66.93	65.33	68.30	y=5.43-0.228 5x	-0.900 9	75.73
	石油醚	65.19	66.11	64.00	65.56	60.74	y=5.31+0.845 0x	0.695 3	0.00
	氯仿	45.22	59.63	64.07	61.11	65.19	y=5.35-0.358 8x	-0.816 5	9.44
	乙酸乙酯	49.44	57.41	52.04	54.63	62.78	y=5.18-0.133 9x	-0.746 5	22.21

Table 2. C_E50 of different extracts of Carya illinoensis against 6 species of strains

4. 结论与讨论

美国山核桃叶的各提取物中，正丁醇提取物和乙酸乙酯提取物具有最佳抑菌效果。两者均对革兰氏阳性菌（枯草杆菌）具有明显抑制作用，其中正丁醇提取物抑菌效果优于乙酸乙酯提取物；正丁醇提取物对革兰氏阴性菌（大肠埃希菌）抑制作用不明显。各提取物对酵母型真菌和霉菌型真菌也有明显抑菌作用，其中正丁醇提取物对酵母菌型真菌的抑菌作用明显强于乙酸乙酯提取物，而乙酸乙酯提取物对霉菌型真菌的抑菌作用明显强于正丁醇提取物。此外，石油醚提取物对黄曲霉菌具有最佳抑菌作用。由此推断，美国山核桃叶所含抑菌成分复杂，可分布于不同有机相，且对不同的供试微生物有不同的抑菌效力。

张野平等^[9]对胡桃醌对肿瘤细胞的增殖抑制作用和抗菌作用研究表明，胡桃醌对许多革兰氏阳性菌和阴性菌均有抑制作用。潘亚琴等^[10]对山核桃叶与美国山核桃叶中化学成分的初步分析研究表明，美国山核桃叶与山核桃叶中所含化学成分类型基本相同。由此可见，美国山核桃叶中也可能含有抑菌的有效成分。本实验结果亦表明美国山核桃叶提取物对革兰氏阳性菌和革兰氏阴性菌具有明显抑制作用。美国山核桃、山核桃和核桃楸（含有胡桃醌药效成分）^[11]为同属植物, 而胡桃醌对许多革兰氏阳性菌和阴性菌均有抑制作用^{[9, 12]}。利用植物亲缘关系来寻找新的药效成分的思路来分析，同属的美国山核桃叶极可能含有类似的药效成分。此外，美国山核桃叶还对新型隐球菌（酵母型真菌）和霉菌型真菌具明显抗菌作用，对此种抗菌作用的有效成分有待于进一步研究。

生物农药是天然存在的或者经过基因修饰的药剂，与常规农药的区别在于其独特的作用方式、低使用剂量和靶标种类的专一性。随着科学技术的迅速发展，生物农药的范畴不断扩大，涉及到动物、植物、微生物中的许多种类及多种与生物有关的具有农药功能的物质。日益成长的有机农业使得生物农药的需求逐渐上扬，而美国山核桃在长江流域以南各省林储丰富，从资源利用的角度可考虑在保护基础上进行合理开发。美国山核桃是落叶乔木，秋末冬初，叶会全部凋落，次年重新长出新叶，叶资源可不断再生，因此，开发利用美国山核桃叶资源切实可行。

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Antimicrobial activity of Carya illinoensis leaves

doi: 10.11833/j.issn.2095-0756.2014.04.025