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Title: 冰片的抗菌与抑菌应用研究

天然来源龙脑的抗菌活性及定量数据

不同植物来源龙脑的抗菌活性及定量数据

天然来源的龙脑作为多种植物精油的主要成分之一,具有明确的抗菌活性,其抑菌作用在不同植物来源的精油中存在差异[1][2]。单一龙脑成分的抗菌活性亦得到验证,但研究结论存在一定分歧;同时,龙脑的抗菌活性在复合体系中也有所体现,例如C. ornifolia精油的抗菌活性部分归因于龙脑等含氧单萜的存在[3],且在精油成分抗菌谱排序中,龙脑的活性位于胸腺醇、香芹酚等成分之后[4]

不同来源龙脑及含龙脑精油的抗菌活性对比

来源类型 具体来源/成分 龙脑含量 抗菌对象 抗菌活性指标(MIC/MBC/抑菌圈) 文献来源
植物精油 A. argyi精油 - 未明确具体微生物 与樟脑等单萜类化合物共同表现出有效抗菌效果 [2]
植物精油 P. barbatus精油 20.7% Brevibacillus laterosporus、Cryptococcus neoformans MIC值0.137–0.55 mg/mL [5]
植物精油 C. ornifolia精油 2.9% 革兰氏阳性菌 MIC值0.40–3.25 mg/mL [3]
植物精油 C. parvifoila精油 - 革兰氏阳性菌 MIC值2.18–8.75 mg/mL [3]
植物精油 S. satureioides精油 12.4% E. coli ATCC 8729、P. aeruginosa ATCC 9027等 MIC值低于0.25%,对部分菌株有杀菌作用 [6]
植物精油 某植物精油 10.8% M. smegmatis、C. perfringens、Ent. aerogenes等10种微生物 MIC值0.56 mg/mL(前两者)、72.00 mg/mL(后者) [7]
植物精油 Satureja montana L.精油 含龙脑成分 E. coli、S. aureus MIC值6.25 μL/ml [8]
植物精油 另一植物精油 2.34% S. aureus(ATCC 29213、ATCC 95923)、M. luteus(NRLL B-4375)等7种细菌 中等抑制活性 [9]
植物精油 D. graveolens精油 18.7% 13种模式细菌 MIC值569.4–18220.8 μg/mL,革兰氏阳性菌更敏感 [10]
植物精油 T. argyrophyllum var. argyrophyllum茎油 15.0% Bacillus cereus MIC值125 μg/mL,与氯霉素活性相当 [11]
植物精油 T. argyrophyllum var. argyrophyllum花油 12.0% B. subtilis、耐甲氧西林S. aureus MIC值125 μg/mL,活性弱于氯霉素 [11]
植物精油 迷迭香精油 7.7% Xanthomonas axonopodis pv. phaseoli(XapG2、Xap3) 对XapG2抑制效果优于Xap3,是抗Xap活性的主要贡献成分 [12]
植物精油 A. canescens精油 6.9% 真菌Candida albicans 抑菌圈直径7.16 ± 0.20 mm,弱抗菌活性 [13]
植物精油 R. beesianus精油 15.0% B. subtilis、Proteus vulgaris、S. aureus等 MIC值3.13 mg/mL(前两者)、6.25 mg/mL(后几种);MBC值6.25 mg/mL(前两者)、6.25–12.50 mg/mL(后几种) [14]
植物精油 O. vulgare L.精油 6.52% 所有测试细菌菌株 抑菌圈直径36.60–40.88 mm;MIC值0.01–0.05 mg/mL;MBC值0.1–0.2 mg/mL [15]
单一龙脑成分 单一龙脑 100% Listeria monocytogenes 4b 抑制浓度和杀菌浓度与1,8-桉叶素相同 [16]
单一龙脑成分 单一龙脑 100% 李斯特菌 MIC值5 mg/mL [17]
单一龙脑成分 单一龙脑 100% 多种测试细菌 MIC值0.05~3.2 mg/mL;MBC值1~3.2 mg/mL [18]
单一龙脑成分 单一龙脑 100% Escherichia coli、Acinetobacter baumanii、Enterobacter cloacae等 抑制活性优于庆大霉素,最低MIC值0.47 mg/L [19]
单一龙脑成分 单一龙脑 100% S. aureus、B型Streptococcus等5种细胞 MIC值1.0–2.0%;最低杀菌浓度(MFC)1.5–2.0% [20]
单一龙脑成分 单一龙脑 100% 测试细菌 抗菌活性均高于128 µg/mL(0.557–0.842 mM),无有效抑制 [21]
单一龙脑成分 D. benenica提取物分离的龙脑 100% S. aureus、Bacillus subtilis MIC值6.49 mM,活性弱于反式邻香豆酸和反式肉桂酸 [22]

龙脑在植物精油抗菌活性中的贡献及成分关联分析

天然来源龙脑作为含氧单萜类化合物,广泛存在于多种植物精油中并具有明确的抗菌活性[23][24][3][25]。其在不同植物精油中的含量差异显著,且含量与精油的抗菌活性存在一定关联——通常龙脑含量较高的精油(如T. vulgaris、T. satureioides)表现出更强的抑菌效果[5][26][27][28][29][30]。此外,龙脑常与其他萜类化合物(如樟脑、1,8-桉叶素、α-蒎烯等)通过协同作用增强精油的抗菌活性[3][31][32],部分研究还指出其与芳樟醇在OEOs中存在强相关性(R = 0.98)[33]

不同植物精油中龙脑的含量及对应抗菌活性

植物精油 龙脑含量 抗菌活性相关数据 文献来源
D. viscosa 第二丰富化合物 - [24]
T. vulgaris挥发油 76.42% - [34]
P. barbatus 20.7% 对Brevibacillus laterosporus等的MIC值0.137-0.55 mg/mL [5]
C. ornifolia 2.9% 对革兰氏阳性菌的MIC值0.40-3.25 mg/mL,活性部分归因于龙脑 [3]
M. cristata subsp. phrygia 主要成分 对革兰氏阴性和阳性致病菌的抑制作用由龙脑主导 [35]
T. satureioides 27% 对微生物的MIC值1.1-11.2 μg/mL,活性与龙脑相关 [27]
T. satureioides 21.2% MIC值0.15 mg/mL,抑菌圈30.3 ± 0.02 mm,活性与龙脑相关 [29]
T. satureioides 34.26% 对分枝杆菌的MIC值0.015-0.062% v/v,活性与龙脑相关 [28]
T. satureioides 21.56%-25.04% - [30]
A. millefolium花/叶 主要成分之一 花精油对P. glaucum的MIC值0.45 mg/mL,活性与龙脑等成分相关 [36][37]
C. urucurana茎皮 较高 对S. epidermidis和E. coli的MIC值1.25 mg/mL,活性与龙脑含量相关 [38]
R. officinalis 26.48 wt.%(首要化合物) - [39]
R. officinalis 10.39% 对白色念珠菌的强活性可能与龙脑相关 [40]
薰衣草杂交品种('Abrialis'等) 较高 对L. monocytogenes的MIC值低至0.3 μL/mL [26]
T. pectinatus 2.7% 分馏组分IV(龙脑62.3%)参与抗菌活性;对Cl. perfringens的MIC值0.30 mg/mL [41]
S. satureioides 12.4% 对E. coli等的MIC值<0.25%,活性与龙脑相关 [6]
S. eremophila 21.83%(主要成分之一) 对敏感微生物的MIC值7.8至>500 μg/mL,活性与龙脑相关 [42]
T. arduini 5.4% 对细菌的MIC值6.25-37.50 mg/mL,对真菌的MIC值7.81-25.00 mg/mL,活性与龙脑相关 [43]
X. hypolampra茎皮 7.8% 对S. aureus等的MIC值>500 μg/mL [44]
Artemisia属部分植物 2.3-8.1% - [45]
S. officinalis 5-4.5% 对微生物的完全抑制浓度20 μl/ml,活性与龙脑相关 [46]
S. officinalis 7.6% 对C. acutatum的MIC值1800 ppm,对B. cinerea的MIC值10000 ppm,活性与龙脑相关 [47]
Kythrean Sage 6.2% 抗菌活性可能与龙脑相关 [48]
部分未明确植物 0.7-4.7% - [49]
部分未明确植物 2.6% - [50]
T. argyrophyllum var. argyrophyllum花 12.0% - [11]
T. argyrophyllum var. argyrophyllum茎 15.0% 对B. cereus的MIC值125 μg/mL(与氯霉素相当),活性与龙脑相关 [11]
D. graveolens 18.7% 对革兰氏阳性菌的MIC值569.4-18220.8 μg/mL,活性与龙脑相关 [10]
A. canescens 6.9% 对革兰氏阳性菌的抑制活性可能与龙脑相关 [13]
R. beesianus 15.0% 对S. aureus等的MIC值3.13-6.25 mg/mL,广谱抗菌活性与龙脑相关 [14]
O. vulgare L. 6.52% 对细菌的MIC值0.01-0.05 mg/mL,强抑制活性与龙脑相关 [15]
H. chrysotricha 32.7% 对革兰氏阳性菌的强抑制活性可能与龙脑相关 [51]
部分未明确植物 1.4-27.7% 含量变化显著影响抗菌活性 [52]

龙脑的抗菌活性具有一定选择性,多数研究指出其对革兰氏阳性菌的抑制作用更显著[53],例如对金黄色葡萄球菌的活性高于大肠杆菌,且对白色念珠菌具有抑制作用[54]。偏最小二乘(PLS)模型分析证实,龙脑是与植物精油抗菌活性相关的重要化合物(VIP>1)[31],其含量变化会显著影响精油的抗菌效果[52]

具体机制方面,龙脑可通过形成氢键发挥对革兰氏阳性菌的活性[55],既可单独抑制致病菌生长,也可与其他成分协同作用[56]。部分研究显示,龙脑含量较低时可能导致精油抗菌活性减弱(如CbEO对细菌的低效性[57]),而富含龙脑的组分(如T. pectinatus精油分馏组分IV,龙脑含量62.3%[41])或精油(如YSF[58])则表现出更强的抗菌效果。此外,不同来源的龙脑(如(+)-龙脑)也可能影响抑菌活性,例如EO 3对枯草芽孢杆菌等的中等抑制作用与其中的(+)-龙脑相关[59]

在定量数据上,含龙脑的植物精油抑菌效果差异较大:薰衣草杂交品种精油对L. monocytogenes的MIC值低至0.3 μL/mL[26],而X. hypolampra茎皮精油对S. aureus的MIC值则>500 μg/mL[44]。部分单一龙脑或含龙脑精油的抗菌数据显示,其对特定菌株具有较强活性,如Marzeh bakhtiari精油(含13.4%龙脑)的MIC范围为6.25至25 µg/ml[60],X. strumarium精油对金黄色葡萄球菌的MIC值为0.5 ± 0.1 µg/mL[61]等,进一步验证了龙脑作为关键抗菌成分的作用[61][62][59]

龙脑对特定致病菌的抑菌杀菌作用及机制初探

龙脑作为单萜类化合物,其抗菌活性已在多项研究中得到证实[1][63],可抑制革兰氏阳性菌、革兰氏阴性菌及真菌,不同研究中其对特定菌株的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)或最低真菌浓度(MFC)存在差异。此外,含有较高水平龙脑的蓍草(A. millefolium L.)花部精油,对革兰氏阳性菌(如蜡样芽孢杆菌、金黄色葡萄球菌、表皮葡萄球菌)和革兰氏阴性菌(如鼠伤寒沙门氏菌、阿贡纳沙门氏菌、大肠杆菌)均表现出抗菌作用,且其抗菌活性强于叶部精油[37]

龙脑对不同微生物的抗菌活性及浓度参数

微生物类别 菌株(或比较场景) MIC值 MBC/MFC值 参考 citations
革兰氏阳性菌 Listeria monocytogenes 4b血清型 与MBC相同 与MIC相同 [16]
革兰氏阳性菌 L. monocytogenes(迷迭香成分比较) 5 mg/ml - [17]
革兰氏阳性菌 金黄色葡萄球菌(Staphylococcus aureus 7.00 mg/ml - [64]
革兰氏阳性菌 金黄色葡萄球菌(ATCC 29213、ATCC 95923) 6.25 mg/mL 6.25 mg/mL [65][14]
革兰氏阳性菌 粪肠球菌(Enterococcus faecalis 6.25 mg/mL 6.25 mg/mL [65][14]
革兰氏阳性菌 枯草芽孢杆菌(Bacillus subtilis 3.13 mg/mL 6.25 mg/mL [65][14]
革兰氏阳性菌 B型链球菌等5种常见革兰氏阳性菌 1.0–2.0% 1.5–2.0%(MFC) [20]
革兰氏阴性菌 大肠杆菌(Escherichia coli 2.5 µg/mL 2.5 µg/mL [66]
革兰氏阴性菌 大肠杆菌(E. coli 6.25 mg/mL 12.50 mg/mL [65][14]
革兰氏阴性菌 铜绿假单胞菌(Pseudomonas aeruginosa 25.0±0.11 µg/mL - [66]
革兰氏阴性菌 铜绿假单胞菌(P. aeruginosa 6.25 mg/mL 12.50 mg/mL [65][14]
革兰氏阴性菌 克雷伯菌(Klebsiella 1250 µg/mL - [67]
革兰氏阴性菌 普通变形杆菌(Proteus vulgaris 3.13 mg/mL 6.25 mg/mL [65][14]
真菌 白色念珠菌(Candida albicans 3.0 µg/mL 3.0 µg/mL(MFC) [66]
真菌 烟曲霉(Aspergillus fumigatus 4.5 µg/mL 4.5 µg/mL(MFC) [66]

在抗菌活性比较中,龙脑对李斯特菌的抑菌效果弱于马鞭草酮、樟脑和乙酸龙脑酯,但强于石竹烯[17];对革兰氏阳性菌的活性广谱性排名位于百里酚、香芹酚、α-松油醇等单萜类成分之后[4];对鲍曼不动杆菌(Acinetobacter baumanii)、阴沟肠杆菌(Enterobacter cloacae)的活性甚至优于庆大霉素[19],同时可抑制白色念珠菌生长[54][13]

关于龙脑的抗菌机制,相关研究初步表明其可能通过破坏细菌细胞壁完整性发挥作用:经龙脑处理的金黄色葡萄球菌和枯草芽孢杆菌(B. subtilis)细胞出现细胞壁破裂、细胞溶解等现象,同时伴随电导率升高、核酸和水溶性蛋白质泄漏增加[64],提示龙脑可通过损伤细胞膜结构,导致细胞内容物外泄,最终抑制或杀灭细菌。进一步研究发现,龙脑作为单萜类化合物,可通过破坏细菌细胞膜完整性发挥抗菌作用[68][65],具体机制包括限制疏水性化合物通过脂多糖层的扩散速率、耗散离子梯度,从而损害细菌细胞内的必要过程并最终导致细胞死亡[68]。此外,龙脑的亲脂性使其能够穿透细胞膜并导致细胞裂解[54],这一机制可能是其对革兰氏阳性菌敏感性高于革兰氏阴性菌的原因之一[54][10]

龙脑抗菌活性的影响因素及检测方法应用

天然来源龙脑作为多种精油的主要成分,对革兰氏阳性菌、革兰氏阴性菌及真菌均表现出抗菌活性。不同来源的含龙脑精油对各类微生物的抑制效果存在差异,其最低抑菌浓度(MIC)因精油种类、龙脑含量及目标菌株而异。此外,龙脑可抑制单核细胞增生李斯特菌(L. monocytogenes),且对不同血清型菌株的抑制作用差异较小[16]

含龙脑精油的抗菌活性及对应最低抑菌浓度(MIC)

精油来源 龙脑含量 目标菌株 MIC值 参考文献
迷迭香精油 26.48 wt.% 金黄色葡萄球菌、大肠杆菌等 低于7.50 mg/mL [39]
Achillea millefolium 花精油 主要成分之一 金黄色葡萄球菌(S. aureus) 0.45 mg/ml [36]
Achillea millefolium 叶精油 主要成分之一 金黄色葡萄球菌(S. aureus) 1.67 mg/ml [36]
Marzeh bakhtiari 精油 13.4% 志贺氏菌(S. flexneri)、大肠杆菌等 6.25–25 µg/ml [60]
Lavandula cv 'Sumiens' 精油 高龙脑含量 李斯特菌 0.6 μL/mL [26]
Satureja cuneifolia 精油 - 白色念珠菌(C. albicans)、光滑念珠菌(C. glabrata) 与龙脑含量呈正相关 [69]

龙脑的抗菌活性受多种因素影响,包括其在精油中的含量、提取方法及检测方法的选择。在提取方法方面,经改良水蒸馏法(MAHD)提取的精油中龙脑等含氧单萜含量更高,其对无害李斯特菌(L. innocua)的抑菌圈直径可达30.00 ± 3.46 mm,显著高于传统水蒸馏法(HD)提取的精油[70];铜处理植物的乙酸乙酯提取物中龙脑含量增加,对蜡样芽孢杆菌(B. cereus)的抑菌圈直径显著增大[71]

龙脑抗菌活性的常用检测方法及操作要点

检测方法 溶剂/操作要点 结果判断依据 应用示例 参考文献
肉汤稀释法 乙醇制备10%溶液,系列稀释 2,3,5-三苯基四氮唑氯化物(TTC)显色 龙脑对单核细胞增生李斯特菌的MIC测定 [16]
微量稀释法 二甲基亚砜(DMSO)为溶剂,制备系列稀释液,96孔板培养 浊度(turbidity)变化 MIC值确定 [72]
纸片扩散法 纸片负载样品,置于培养基上培养 抑菌圈直径 铜处理植物提取物对蜡样芽孢杆菌的活性评估 [71]
琼脂稀释法 精油与含吐温80的培养基混合,涂布菌株培养 菌落生长情况 MIC值确定 [73]

这些检测方法的应用为龙脑抗菌活性的定量分析提供了可靠依据,进一步支撑了其作为天然抗菌成分的应用潜力。

龙脑与其他活性成分的协同抗菌效应

龙脑作为一种双环单萜醇,单独抗菌活性较弱[74][75][76][77],对金黄色葡萄球菌、大肠杆菌等菌株抑制作用有限,其抗菌活性与水芹烯、樟脑、橙花叔醇相当,弱于α-蒎烯[75]。尽管如此,龙脑与其他抗菌成分协同作用时展现出显著增效效应,机制可能与穿透细菌细胞膜、抑制外排泵活性有关,可增强萜品烯-4-醇、柠檬烯等低浓度强效抗菌成分的效果[74];其协同效应还与其抗黏附作用相关,通过减少细菌附着和生物膜形成,为其他抗菌成分创造作用条件[74][78]

龙脑单独及协同抗菌活性的关键数据

抗菌类型 研究对象 关键指标及数值 参考文献
单独抗菌 大肠杆菌O157:H7 MIC = 0.5 μl/ml [77]
单独抗菌 单核细胞增生李斯特菌 MBC = 1 μl/ml [77]
协同抗菌(单萜组合) 白色念珠菌、热带假丝酵母(龙脑+γ-萜品烯/α-萜品烯) 部分比例FICI低至0.133 [76]
协同抗菌(精油成分) 金黄色葡萄球菌(含龙脑13.0–19.7%的薰衣草叶茎精油) FICI低至0.076 [79]
协同抗菌(精油+抗生素) 龙脑所在精油+美罗培南 精油MIC从10%降至0.63%(v/v),美罗培南MIC从32 μg/mL降至8 μg/mL,FICI=0.3125 [80][81]
协同抗菌(精油+抗生素) 含龙脑18.7%的D. graveolens精油+抗生素 42.2%组合协同,与氯霉素组合协同达60.0%,对奇异变形杆菌ATCC 12453效果显著 [10]
协同抗菌(涂层) 龙脑基聚合物涂层 释放龙脑抑制细菌附着,与涂层成分协同增强效果 [78]

具体协同组合研究显示,龙脑与对伞花烃、γ-萜品烯、α-萜品烯等成分的组合具有明确协同作用,对伞花烃也常作为龙脑协同作用的共同组分[76]。例如龙脑基聚合物涂层可通过释放龙脑抑制细菌附着,同时与涂层中其他成分协同增强抗菌效果[78]。这些研究表明,龙脑虽单独抗菌活性有限,但通过与其他活性成分的协同作用,可显著提升整体抗菌效能,为其在抗菌领域的应用提供了重要依据。

龙脑的抗菌机制及生物膜抑制作用

龙脑作为含氧单萜类化合物具有一定抗菌活性,但其单独作用时效果相对较弱[74][75]。研究显示,龙脑对金黄色葡萄球菌(S. aureus)等革兰氏阳性菌的敏感性高于革兰氏阴性菌,与水芹烯、樟脑、橙花叔醇的抗菌效果相当[75]。其抗菌机制主要包括两方面:一是破坏细菌细胞膜完整性,具体表现为增加细胞膜孔隙率[82],导致细胞内电解质(如电导率升高)、核酸(A260值上升)及蛋白质等重要成分泄漏,最终造成细胞壁破裂和细胞裂解[64];二是通过与特定细菌的酪氨酸-tRNA合成酶(TyrRS)形成氢键(如结合GLY47、ALA51残基),抑制氨基酰化过程以干扰细菌生命活动[83]

在生物膜抑制与协同抗菌方面,龙脑表现出显著应用潜力。低浓度龙脑即可有效抑制白色念珠菌(C. albicans)生物膜形成,且能增强对部分细菌的抗生物膜活性;尽管单独抗菌效果有限,它可通过穿透细胞膜、抑制外排泵活性的方式,与其他抗菌化合物产生协同作用,显著提升整体抗菌活性。具体数据如下:

龙脑的生物膜抑制及协同抗菌活性

作用类型 作用对象 关键效果描述 相关文献
生物膜抑制 白色念珠菌(C. albicans) 0.005%浓度下抑制率超过80% [84]
生物膜抑制 单核细胞增生李斯特菌、铜绿假单胞菌(P. aeruginosa) 增强抗生物膜活性 [82]
协同抗菌 α-蒎烯、柠檬烯、松油烯-4-醇等 穿透细胞膜并抑制外排泵活性,增强整体抗菌活性 [74]

这种多靶点作用机制与协同效应,使得龙脑在复方抗菌体系中具有重要应用价值。

龙脑基复合材料的抗菌应用特性

龙脑基复合材料的抗菌应用特性研究表明,其抗菌效能可通过结构修饰或复合策略显著提升。针对AAVO、AAVO-Ne和AAVO-NeCs三种材料的抗菌性能测试显示,三者对金黄色葡萄球菌、大肠杆菌、鼠伤寒沙门氏菌及白色念珠菌均具有抑制作用,且抑制效果因菌种和材料类型而异;其中AAVO-NeCs通过复合策略实现了抗菌活性的显著增强,在食品保鲜和货架期延长领域具有应用潜力[85]

AAVO、AAVO-Ne与AAVO-NeCs的抗菌性能对比(大肠杆菌及鼠伤寒沙门氏菌相关数据)

测试指标 材料 数值/结果 提升倍数/对比情况 文献
鼠伤寒沙门氏菌抑菌圈直径 AAVO 10.7 mm - [85]
鼠伤寒沙门氏菌抑菌圈直径 AAVO-NeCs 20 mm 较AAVO提高1.87倍 [85]
大肠杆菌MIC值 AAVO 20.995 μg/mL - [85]
大肠杆菌MIC值 AAVO-Ne 17.765 μg/mL - [85]
大肠杆菌MIC值 AAVO-NeCs 4.307 μg/mL 较AAVO降低4.8倍 [85]
大肠杆菌抗菌率(5 min) AAVO-NeCs 36.49% 优于AAVO和AAVO-Ne [85]
大肠杆菌抗菌率(10 min) AAVO-NeCs 51.11% 优于AAVO和AAVO-Ne [85]
大肠杆菌抗菌率(20 min) AAVO-NeCs 91.06% 优于AAVO和AAVO-Ne [85]
大肠杆菌抗菌率(40 min) AAVO-NeCs 97.33% 优于AAVO和AAVO-Ne [85]
大肠杆菌抗菌率(60 min) AAVO-NeCs 98.94% 优于AAVO和AAVO-Ne [85]

除复合纳米材料外,龙脑基聚合物也可通过特定机制实现抗菌功能。例如,IBOMA基聚合物可通过酯键水解释放龙脑,抑制细菌附着与生长,进而用于构建生物相容性抗菌涂层界面;其防污性能依赖涂层自更新,以及水解释放的龙脑与天然防污剂樟脑的协同作用[78]。此外,含有10%龙脑的复方萜烯制剂Rowatinex也表现出抗菌作用,进一步证实龙脑是其抗菌活性的重要组分之一[86]

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