Extraction of Total Flavonoids from Shaanxi Huangjing by Enzyme Assisted Ultrasound and Study on Its Antioxidant and Anti A549 Cell Proliferation Activities
Polygonatum sibiricum (PS) is a famous medicinal and edible herb in China, mainly produced in Hebei, Shaanxi, Anhui, Yunnan, Zhejiang, Heilongjiang and other regions. Its dried roots and stems are used for nourishing yin and qi, tonifying kidneys and moistening lungs. Research has shown that Polygonatum sibiricum exerts pharmacological effects such as anti-tumor, anti-aging, and immune regulation through polysaccharides, flavonoids, saponins, lignans, amino acids, and volatile oils. It is widely used in clinical practice to treat malignant tumors such as esophageal cancer, gastric cancer, rectal cancer, and lung cancer.
Our previous research based on network pharmacology found that flavonoids in Polygonatum sibiricum can exert anti non-small cell lung cancer (NSCLC) effects through signaling pathways such as apoptosis, hypoxia inducible factor-1, and tumor necrosis factor. The specific material basis and molecular mechanism need to be studied by extracting total flavonoids from Polygonatum sibiricum. Research has shown that Shaanxi produced Polygonatum sibiricum has the highest total flavonoid content, while Yunnan produced Polygonatum sibiricum has the lowest total flavonoid content. The extraction methods of total flavonoids from Polygonatum sibiricum include extraction method, ultrasound method, and enzyme assisted ultrasound method, but there has been no optimization of the extraction process and analysis of flavonoid components. There is even less research on the content of each component and its antioxidant and anti-tumor effects.
In recent years, ultrasound combined with enzymatic method has gradually been applied to the extraction of active ingredients from traditional Chinese medicine. Ultrasonic waves can propagate in liquid media to produce a special “cavitation effect”, which accelerates the leaching of active ingredients in traditional Chinese medicine through powerful shock waves. Cellulose enzymes mainly degrade plant cell walls, increase cell wall permeability, promote rapid dissolution and diffusion of active ingredients in Chinese herbal medicine, and thus improve the yield of active compounds. Therefore, enzyme assisted ultrasound extraction of active ingredients in traditional Chinese medicine can reduce solvent usage, shorten extraction time, and improve the yield of medicinal ingredients. This study aims to use enzyme assisted ultrasound to extract total flavonoids from Shaanxi produced Polygonatum sibiricum, and optimize the optimal extraction process of flavonoids by combining key factors such as solid-liquid ratio, ethanol concentration, enzyme dosage, and ultrasound time. The response surface methodology will be used to detect the flavonoid components of Shaanxi produced Polygonatum sibiricum by high performance liquid chromatography (HPLC), and the in vitro antioxidant and anti A549 cell proliferation effects of the extract will be studied, providing data support for the development of more diverse pharmacological parts of Shaanxi produced Polygonatum sibiricum.

This article uses ultrasound assisted cellulase method to extract total flavonoids from Polygonatum sibiricum produced in Shaanxi. The extraction process of total flavonoids from Polygonatum sibiricum was optimized using response surface methodology as follows: cellulase dosage of 2%, ethanol concentration of 90%, ultrasound time of 20 minutes, and solid-liquid ratio of 1:16 (g/mL). Under these conditions, the yield of total flavonoids from Polygonatum sibiricum produced in Shaanxi was (103.10 ± 0.02) μ g/g. According to literature, the yield of total flavonoids in Polygonatum sibiricum produced in Acheng, Heilongjiang is lower than that of Polygonatum sibiricum produced in Jiuhua Mountain, Anhui, and higher than that of Polygonatum sibiricum produced in Baise, Guangxi and Lincang, Yunnan. The most influential factor on yield in multi factor analysis is the amount of cellulase used, indicating that the wall breaking rate of Polygonatum sibiricum is directly proportional to the dissolution of its flavonoids. This result is consistent with literature reports. The study on the interaction between various factors showed that the interaction between ultrasound time and solid-liquid ratio, as well as the interaction between ethanol concentration and ultrasound time, had the most significant effect on the total flavonoid yield of Polygonatum sibiricum. This study applied HPLC method for the first time to analyze and determine the flavonoid components and content in Shaanxi Huangjing. Unlike the Huangjing decoction pieces from Tongji Tang of Guizhou National Pharmaceutical Group and Tongrentang of Beijing in Bozhou, the Huangjing flavonoids produced in Shaanxi are mainly composed of quercetin dihydrate and baicalein, both of which have good anti-tumor and antioxidant effects. It was first discovered that Huangjing flavonoids produced in Shaanxi are rich in baicalein.

The results of antioxidant experiments showed that the total flavonoids of Polygonatum sibiricum produced in Shaanxi have strong antioxidant capacity, and their scavenging rate of DPPH free radicals is comparable to that of vitamin C. When used in small doses, their antioxidant capacity is not as good as that of vitamin C and BHT, but as the dose increases, their ability to scavenge free radicals gradually increases, basically equivalent to that of vitamin C and BHT. This article also confirms for the first time that flavonoids extracted from Polygonatum sibiricum produced in Shaanxi can exhibit dose-dependent and time-dependent anti A549 cell proliferation effects in vitro. This is consistent with our previous network pharmacology and evidence-based medicine research results, which can provide a material basis for further studying the flavonoid components of Shaanxi Huangjing and its anti NSCLC effects.