中药在疾病防治、养生保健等方面发挥独特且重要的作用，其中以黄曲霉毒素真菌毒素的污染引起的中药品质与安全问题显得尤为突出。黄曲霉毒素（AFs） 是 主 要 由 黄 曲 霉 （Aspergillus flavus） 和 寄 生 曲 霉 （Aspergillus parasiticus）产生的次生代谢产物。天然存在的 AFs 主要有四种类型，其中AFB₁ 的毒性最大。黄曲霉毒素污染可导致严重的经济与健康危害。虽然真菌毒素检测方法和限量标准可以保证消费者的使用安全性，但从源头上控制污染更为重要。目前的常规控制方法（物理、化学和生物方法）仍旧存在一些局限性，如残留毒性强、成本高、菌株不相容等。因此，开发天然、环保、安全的“绿色化学品”——植物精油，具有广阔的应用前景。精油（EO）是真菌生长和霉菌毒素产生的有效抑制剂，在病原微生物中产生耐药性的风险较低。课题组在前期的研究中发现柠檬（Citrus limon L.）精油可以显著抑制黄曲霉的生长，但作用机制不清楚。

槟榔为高油脂的种子类中药材，在收获、储存和运输的各个阶段极易受到病原真菌的污染，严重影响了槟榔的药用安全性和有效性。然而，目前关于柠檬精油在抑制黄曲霉及黄曲霉毒素机理上的研究较少，其在中药防霉中的应用也未见报道，槟榔的防霉措施更是鲜有研究。因此，本研究拟通过多指标表征和多组学联用，阐明柠檬叶精油（LLEO）抑制黄曲霉的机制，并探索通过添加外源精油控制槟榔发霉的可行性。主要研究思路和结论如下：

一、体外抑菌实验和气相色谱-质谱（GC-MS）分析表明，柠檬叶精油（LLEO）的抑菌效果优于柠檬果精油（LPEO），主要活性成分为香茅醛

通过 GC-MS 分析，LPEO 主要成分为(+)-柠檬烯（74.465%），LLEO 主要成分为香茅醛（52.679%）和(+)-柠檬烯（20.238%）。LLEO 对于黄曲霉生长具有更强的抑制作用，香茅醛的抑菌活性显著强于(+)-柠檬烯，且香茅醛与 LLEO 的最低抑菌浓度（MIC）相近。因此认为香茅醛是 LLEO 中的主要抑菌活性成分。

二、通过电镜观察、麦角甾醇测定、细胞内容物外漏分析、酶活检测及多组学分析等方法，阐明 LLEO 抑制黄曲霉的机理

LLEO 可有效地抑制黄曲霉的生长和黄曲霉毒素的产生，机制可能与质膜的破坏，抗氧化防御系统的调节和能量代谢的改变有关。LLEO 在较低浓度下表现出抗黄曲霉毒素的特性，表明 LLEO 抑制真菌生长和 AFs 产生的机制不同，但具体差异尚未清晰。通过扫描电镜（SEM）和电子显微镜（TEM）观察，发现 LLEO 处理组的分生孢子发生了明显萎缩和凹陷，菌丝体细胞中的细胞膜被破坏，细胞器出现萎缩或解离，细胞质液泡化等。进一步分析表明，LLEO 可降低细胞膜重要组成成分麦角甾醇含量，随着细胞膜通透性和完整性的破坏，
细胞内容物如核酸和蛋白质的泄露增强。此外，LLEO 可诱导 A. flavus 孢子产生大量 ROS，扰乱真菌的抗氧化防御系统，且导致膜脂过氧化产生丙二醛（MDA）。在轻度氧化应激的背景下，LLEO 处理组中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx）活性增加。

通过对 LLEO 处理前后的黄曲霉菌丝体进行转录组、蛋白质组和代谢组分析，发现差异基因、差异蛋白和差异代谢物多呈现下调趋势。综合多组学分析结果，LLEO 可通过碳水化合物代谢、能量代谢、脂质代谢、氨基酸代谢、过氧化物酶体代谢以及黄曲霉毒素合成等生物途径抑制黄曲霉的菌丝生长和黄曲
霉毒素合成。多组学分析结果与前期观察到的生化指标具有一致性，初步揭示了 LLEO 抑制黄曲霉的分子机制，精确的分子靶标仍需通过分子对接、基因敲除/过表达、酶动力学等实验方法进一步解析和验证。

三、建立了离心辅助-固相萃取结合超高效液相色谱-串联质谱（UHPLC-MS/MS）检测槟榔多毒素的检测方法，AFs 是污染槟榔的主要真菌毒素

本研究开发了一种离心辅助-固相萃取结合 UHPLC-MS/MS 的槟榔及其饮片中多毒素的高通量检测方法，目标毒素 0.1 ~ 1000 μg/kg 范围内线性良好（r²>0.99），精密度（RSD<8%）和回收率（70 ~ 120%）良好，基质效应可控。大腹皮样品中未发现真菌毒素污染情况，而在槟榔（13.3%）、槟榔饮片（43.3%）和焦槟榔（40.0%）样品中均检测到不同程度的毒素污染，其中 AFs为主要污染物（最高值：槟榔 5.43 μg/kg，槟榔饮片 7.55 μg/kg，焦槟榔 4.02μg/kg），16.7%槟榔饮片样品检出 ST（最高 2.17 μg/kg），两个样品（WBL-4 和BLS-2）毒素超标。

四、通过槟榔的质量评价，探索 LLEO 在槟榔防霉贮藏中应用的可行性

通过反式培养实验结果表明，LLEO 可以显著抑制槟榔上黄曲霉的生长和AFs 的累积。槟榔霉变后，多糖（降低 38.46%）和酚类物质（儿茶素降低52.18%）含量显著减少，生物碱类成分未发生明显变化。LLEO 处理对槟榔中主要有效成分的含量无显著影响。通过 GC-MS 以香茅醛为特征标志物进行分析，结果表明 LLEO 处理后可在槟榔表面检测到精油残留，其残留量随时间递减，18 天后完全挥发。

本研究初步揭示了 LLEO 抑制黄曲霉的分子机制，并验证了其在槟榔防霉应用中的有效性，为中药的防霉措施提供了新见解，并将指导精油的合理应用。

 

Traditional Chinese Medicine (TCM) plays a unique and important role in disease prevention, treatment, and health maintenance. However, mycotoxin contamination such as aflatoxins (AFs), poses significant challenges to the quality and safety of TCM. Aflatoxins are secondary metabolites predominantly produced by Aspergillus flavus and Aspergillus parasiticus. Among naturally occurring AFs, AFB₁ is the most toxic. Aflatoxin contamination not only causes significant economic losses in crops but also poses serious health risks to consumers. Although mycotoxin detection methods and regulatory limits can safeguard consumer safety, it is more imperative to mitigate contamination and waste risks at the source. Conventional control methods (physical, chemical, and biological) are often constrained by limitations, such as residual toxicity, high costs, and strain incompatibility. In this context, essential oils (EOs), as natural, eco-friendly, and safe "green chemicals," show great potential for controlling AFs. EOs are effective inhibitors of fungal growth and mycotoxin production, with a low risk of inducing microbial resistance. Previous studies by our team have demonstrated that Citrus limon L. EO exhibits significant inhibitory effects on the growth of Aspergillus flavus, although the underlying mechanism remains unclear.

Arecae semen, a high-fat seed-based TCM, is particularly susceptible to fungal contamination and mycotoxin production during harvesting, storage, and transportation, severely compromising its medicinal safety and efficacy. Despite this, there is limited research on the mechanism of lemon EO in inhibiting A. flavus and AFs, and its application in TCM mold prevention remains unexplored. Moreover, studies on mold
prevention in A. semen are scarce. Therefore, this study aims to elucidate the mechanism by which lemon leaf EO (LLEO) inhibits A. flavus through multi-omics analysis and explore its feasibility in controlling fungal contamination and AFs accumulation in A. semen. Key Findings are as follow:

1. In vitro antimicrobial assays and GC-MS analysis reveal superior efficacy of lemon leaf EO (LLEO) over lemon fruit EO (LPEO) with citronellal as the primary active constituent.

Gas chromatography-mass spectrometry (GC-MS) analysis revealed that lemon peel EO (LPEO) and LLEO differ in composition. LPEO primarily contains (+)-limonene (74.465%), while LLEO is rich in citronellal (52.679%) and (+)-limonene
(20.238%). LLEO demonstrated superior antifungal activity against A. flavu,  with citronellal being the primary active component.

2. Mechanistic elucidation of LLEO-mediated A. flavus inhibition: A multi-omics approach integrating electron microscopy, ergosterol quantification, membrane integrity assays, and enzymatic activity profiling.

LLEO effectively inhibited A. flavus growth and aflatoxin production， with mechanism potentially involving disruption of the plasma membrane, alteration of antioxidant defense systems, and interference with energy metabolism. LLEO shows
antiaflatoxin properties at relatively low concentrations. This suggests that the mechanisms of its inhibition of fungal growth and AFs production are distinct, though the specific differences remain to be clearly elucidated. Scanning electron microscopy
(SEM) and transmission electron microscopy (TEM) revealed significant morphological damage, including spore shrinkage, membrane disruption, organelle disintegration, and cytoplasmic vacuolization. LLEO reduced ergosterol content, increased membrane permeability, and enhanced leakage of cellular contents such as nucleic acids and proteins. Additionally, LLEO induced reactive oxygen species (ROS) production, leading to lipid peroxidation and malondialdehyde (MDA) formation. Under mild oxidative stress, the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) increased.

Multi-omics analysis (transcriptomics, proteomics, and metabolomics) revealed that LLEO downregulated genes, proteins, and metabolites associated with carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid
metabolism, peroxisome metabolism, and aflatoxin biosynthesis. These findings align with biochemical observations, further validating inhibitory mechanisms of LLEO. However, the precise molecular targets still require further elucidation and validation
through molecular docking, gene knockout/overexpression, and enzyme kinetics assays.

3. Development of a centrifugal-assisted solid-phase extraction combined with UHPLC-MS/MS method for multi-mycotoxin detection in A. semen products: AFs identified as primary contaminants.

A centrifugal-assisted solid-phase extraction combined with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for rapid and sensitive detection of multiple mycotoxins in areca nuts and their processed products. The method demonstrated excellent linearity (0.1-1000 μg/kg, r²>0.99), precision (RSD<8%), satisfactory recovery rates (70-120%), and controllable matrix effects. Results showed no contamination in A. pericarpium (Dafupi) samples, while contamination was detected in whole A. Semen (WBL, 13.3%), sliced A. semen (BLS, 43.3%), and A. semen Tostum (JBL, 40.0%). AFs emerged as predominant contaminants, with maximum concentrations of 5.43 μg/kg (WBL), 7.55 μg/kg (BLS), and 4.02 μg/kg (JBL). Notably, sterigmatocystin (ST) was identified in 16.7% BLS (maximum: 2.17 μg/kg), with two samples (WBL-4 and BLS-2) surpassing regulatory limits.

4.Application of LLEO in A. semen preservation.

LLEO demonstrated significant efficacy in preventing A. flavus contamination in A. semen, while preserving quality - preventing polysaccharide (38.46% decrease in controls) and phenolic (52.18% catechin reduction) degradation without affecting
alkaloids. GC-MS tracking confirmed citronellal-marked LLEO residues dissipated completely within 18 days, demonstrating its dual antifungal/quality-preserving potential for areca nut storage.

In conclusion, this study comprehensively elucidates the molecular mechanisms by which LLEO inhibits A. flavus growth and aflatoxin production. It also validates the effectiveness of LLEO in A. semen preservation, offering new insights into mycotoxin control in TCM and guiding the rational application of EOs.