柴胡桂枝汤挥发油提取工艺优化及体内抗流感病毒活性研究

Optimization of Volatile Oil Extraction from Bupleuri and Ramuli Cinnamomi Decoction and Investigation of Its In Vivo Anti-Influenza Virus Activity

关键词:  柴胡桂枝汤  挥发油  流感病毒  气相色谱-质谱  最佳提取工艺

Key words:  bupleuri and ramuli cinnamomi decoction  volatile oil  influenza virus  gas chromatography-mass spectrometry  optimum extraction processo

摘要:

目的 优化柴胡桂枝汤挥发油提取工艺，分析成分组成及评估挥发油在柴胡桂枝汤抗病毒活性中的作用。方法 通过单因素试验、响应面分析法优化提取工艺，并进行验证，利用GC-MS对所提挥发油进行定性分析。其次，将36只Balb/c小鼠随机分为6组：空白对照组、模型组、达菲组（19.5 mg·kg-1·d-1）、水提液组、挥发油组、水提液+挥发油组（各中药干预组给予相同剂量：24.7 g·kg-1·d-1），除对照组外，其余各组小鼠滴鼻感染H1N1流感病毒以复制流感小鼠模型，待体重下降后开始给药。观察小鼠的一般体征指标，实验结束留取胸腺、脾、肺称重，计算脏器指数；利用RT-qPCR测定肺组织病毒载量和细胞因子。结果 最佳提取工艺为收集8倍药材量的馏出液体积，加水量为药材量15倍，浸泡时间4 h，通过GC-MS分析，表明柴胡桂枝汤挥发油主要包括桂皮醛、棕榈酸、2-甲氧基肉桂醛等41种成分。药效学结果表明柴胡桂枝汤水提液和挥发油均能改善流感病毒感染后小鼠体重降低程度，降低小鼠肺内病毒载量，其中，与水提液组相比，挥发油+水提液组在降低流感小鼠的肺内病毒载量，抑制细胞因子IFN-γ、TNF-α、IL-6 mRNA的表达方面效果更优。结论 挥发油在柴胡桂枝汤干预流感病毒感染小鼠中可能发挥着重要作用，优化后的挥发油提取工艺稳定可行，有助于柴胡桂枝汤挥发油的进一步开发利用。

Abstract:

OBJECTIVE This study aims to optimize the extraction process of volatile oil from Bupleuri and Ramuli Cinnamomi Decoction (BCD), analyze its composition, and evaluate the role of volatile oil in the antiviral activity of the decoction. METHODS The extraction process was optimized through single-factor experiments and response surface methodology. The volatile oil was then qualitatively analyzed using gas chromatography-mass spectrometry (GC-MS). Subsequently, 36 Balb/c mice were randomly divided into six groups: a blank control group, a model group, an oseltamivir group (19.5 mg·kg-1·d-1), a water extract group, a volatile oil group, and a water extract + volatile oil group (Each traditional Chinese medicine intervention group received the same dosage of 24.7 g·kg-1·d-1). With the exception of the control group, the other groups of mice were infected with H1N1 influenza virus via nasal drip to replicate the influenza mouse model. Medication was administered after weight loss. The general physiological indicators of mice were observed, and organs such as the thymus, spleen, and lungs were collected, weighed at the conclusion of the experiment, and the organ index was calculated. viral load and cytokines in lung tissue were determined by RT-qPCR. RESULTS The optimal extraction process involves collecting a distillate volume that is eight times the number of medicinal materials, adding fifteen times the number of medicinal materials, and soaking for four hours. GC-MS analysis revealed that the volatile oil of BCD primarily consists of 41 components, including cinnamaldehyde, palmitic acid, 2-methoxycinnamaldehyde. Pharmacodynamic results demonstrated that both the water extract and volatile oil of BCD were effective in improving weight loss in mice infected with influenza virus, reducing the viral load in the lungs of mice. Compared to the water extract group, the volatile oil + water extract group exhibited superior effects in reducing the viral load in the lungs of influenza-infected mice and inhibiting the expression of cytokines IFN-γ, TNF-α, and IL-6 mRNA. CONCLUSION The volatile oil may have a synergistic effect in the intervention of BCD on influenza virus infection in mice. The optimized process for extracting volatile oils is stable and feasible, which contributes to the advancement and utilization of volatile oils from BCD.