北桑寄生产黄酮内生菌的分离鉴定及其抗氧化和抑菌活性评价

Isolation and identification of flavonoid-producing endophytes with antioxidant and antibacterial activities from Loranthus tanakae

摘要:

【背景】北桑寄生(Loranthus tanakae)植物中黄酮类化合物成分含量较高，生物活性较好。由于植物资源短缺，导致其生物活性成分的研究受限。【目的】从北桑寄生中分离、筛选和鉴定产黄酮类化合物的内生菌，并评估其体外抗氧化和抑菌活性。【方法】以北桑寄生枝条为研究对象分离内生菌，通过显色反应和薄层色谱法(thin-layer chromatography，TLC)筛选产黄酮类化合物的内生菌，并采用NaNO₂-Al (NO₃)₃比色法测定其总黄酮含量。利用清除自由基和抑菌的体外实验来初步评估产黄酮内生菌的生物活性。【结果】共筛选出4株产黄酮内生菌，其中3株为内生真菌，1株为内生细菌，通过形态学和分子生物学方法将其分别鉴定为Botryosphaeria sp.(ZC020)、Phoma sp.(ZZ105)、Nemania sp.(ZS042)和Pseudomonas sp.(ZC026)。4株菌中ZC020和ZS042的总黄酮含量较高，分别为(44.58±0.72) mg/L和(31.98±0.18) mg/L (P<0.05，n=3)，并且ZS042可产生与北桑寄生植物相同类型的黄酮类化合物。ZC026和ZS042表现出优异的抗氧化活性，其1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl，DPPH)和2,2'-联氮-二(3-乙基苯并噻唑啉-6-磺酸)二铵盐[2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt，ABTS]自由基清除率分别为72.85%±0.66%(ZC026)、57.01%±0.89%(ZS042)和85.36%±0.75%(ZC026)、88.17%±0.15%(ZS042)(P<0.05，n=3)。ZZ105对革兰氏阳性菌和革兰氏阴性菌均有抑制作用。【结论】从北桑寄生枝条中分离出4株产黄酮类化合物的内生菌，其中有一株为内生细菌。这项工作为北桑寄生黄酮类化合物的生产提供了新的资源，也为其他珍稀药用植物和材料的开发和保护提供了参考。

Abstract:

[Background] Loranthus tanakae has rich flavonoids with good pharmacological activities, whereas the studies of the flavonoids are limited because of the shortage of plant resources. [Objective] To isolate and identify the flavonoid-producing endophytes from L. tanakae and study their antioxidant and antibacterial activities in vitro. [Methods] The flavonoid-producing endophytes were isolated from the twigs of L. tanakae and screened by color reaction and thin-layer chromatography (TLC). The total flavonoid content was determined via NaNO₂-Al(NO₃)₃ colorimetry. The biological activities of flavonoid-producing endophytes were preliminarily evaluated by experiments of scavenging free radicals and inhibiting bacteria in vitro. [Results] A total of four flavonoid-producing endophytes, including three fungal strains and one bacterial strain, were screened out and identified as Botryosphaeria sp. (ZC020), Phoma sp. (ZZ105), Nemania sp. (ZS042), and Pseudomonas sp. (ZC026) based on morphological and molecular characteristics. ZC020 and ZS042 had higher total flavonoid content, which was (44.58±0.72) mg/L and (31.98±0.18) mg/L (P<0.05, n=3), respectively. Notably, ZS042 produced the same flavonoids as L. tanakae. ZC026 and ZS042 exhibited excellent antioxidant activity, which showed the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rates of 72.85%±0.66% and 57.01%±0.89% and the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging rates of 85.36%±0.75% and 88.17%±0.15%, respectively (P<0.05, n=3). ZZ105 had inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria. [Conclusion] Four flavonoid-producing endophytes were isolated from L. tanakae and one of them was a bacterial strain. This work provides a new resource for the production of flavonoids by L. tanakae and a reference for developing and conserving other rare and precious medicinal plants and materials.