Optimization of extraction and purification process of total flavonoids from safflower leaves and study on their anti acute liver injury activity
The dried flower of the Asteraceae plant Carthamus tinctorius L. is a precious traditional Chinese medicinal herb and one of the most widely used medicinal herbs in Mongolian medicine clinical practice. This medicine is cool in nature, slightly bitter in taste, and has a nourishing effect on the heart and liver meridians. It is a good medicine for promoting blood circulation, clearing blood stasis, and relieving pain. Traditionally, the use and research on plant safflower have only focused on its medicinal flowers. However, the flower yield of plant safflower is very low, which leads to a large amount of leaves produced after harvesting its medicinal parts – “flowers” – being discarded, resulting in resource waste. Scholars have studied the chemical composition of the aboveground parts of safflower, but most of them focus on alkaloids and terpenes. However, there is relatively little research on its flavonoids, which have pharmacological activities such as antioxidant, antiviral, hepatoprotective, anticancer, and anti-inflammatory. Plant derived flavonoids also have natural and low toxicity characteristics, and have broad prospects in the development of efficient and safe natural medicines, skincare products, and health products. At present, there is no research on the extraction and purification process and biological activity of flavonoids from safflower leaves. Related studies have shown that the alcohol extract of safflower stems has a significant anti CCI4 induced acute liver injury effect. Therefore, it is worth exploring whether safflower leaf extract also has liver protective effects. Therefore, from the perspective of fully utilizing the non medicinal resources of safflower, this experiment optimized the extraction and purification process to obtain total flavonoids from Carthamus leaves tinctorius L. leaf,TFCTLL)， And further explore its hepatoprotective effect, providing data support for the full utilization of non medicinal parts resources of safflower and its pharmacological activity research.

 

This study used safflower waste safflower leaves as experimental materials for the first time, and extracted total flavonoids from safflower leaves using heating reflux method. Based on single factor experiments, a Box Behnken response surface test was designed to optimize the optimal extraction process for total flavonoids from safflower leaves. The solid-liquid ratio was determined to be 1:27, ethanol concentration was 80%, and extraction time was 65 minutes. The actual extraction rate was 4.921%, which was 0.014% lower than the theoretical value. Statistical analysis was conducted on single factor and response surface results, and it was found that there was no significant difference between the two (P>0.05). This model predicts well and stably, and can be used for extracting total flavonoids from safflower leaves. Due to the low extraction rate of the crude extract containing total flavonoids from safflower leaves obtained through the optimal extraction process, further enrichment and purification of the crude extract are required for subsequent activity studies.

Macroporous resin has been widely used in the separation and purification of natural products in recent years, especially for the enrichment of flavonoids. Due to its simple operation, no pollution, low energy consumption, and good separation effect, it has been widely used in the purification research of flavonoids. Most flavonoids are weakly polar, so weakly polar and non-polar resins have better purification effects on flavonoids. Hou et al. studied the enrichment and purification of total flavonoids in Artemisia annua using macroporous adsorption resin. Static adsorption experiments were conducted to investigate the adsorption and desorption abilities of NKA-9, GPD100, HPD400, HPD600, AB-8, D101, X-5, and GM301 macroporous adsorption resins for total flavonoids. The results showed that AB-8 macroporous adsorption resin had the best adsorption and desorption ability. Related literature has also reported four types of macroporous adsorption resins, HPD-100, HPD-600, D101, and HP-20, which have significant advantages in adsorption and resolution of flavonoids compared to other types of resins. Therefore, in this experiment, five types of macroporous adsorption resins suitable for enriching total flavonoids, AB-8, HPD-100, HPD-600, D101, and HP-20, were selected for the purification of TFCTLL. After obtaining the optimal purification process, the extract of safflower leaves extracted under the optimal process was adsorbed onto a pre treated macroporous resin chromatography column AB-8 and eluted with 95% ethanol. The total flavonoid purity reached 61.42%, and the eluent was collected and concentrated into a dry extract. Provide a foundation for subsequent activity research.

Related studies have shown that alcohol extracts from safflower stems have significant anti CCI4 induced acute liver injury effects. Does safflower leaf extract also have liver protective effects? This study identified four flavonoids in TFCTLL, including verbascoside, quercetin-7-O – β – D-glucoside, verbascoside, and vitexin. The relevant research results have shown that it has specific anti-inflammatory, antioxidant, and anti fibrotic effects, which can reduce serum ALT and AST activity, inhibit the levels of inflammatory factors such as TNF – α and IL-1 β, inhibit the activation of antioxidant enzymes and NF – κ B pathway, and alleviate liver damage by regulating lipid metabolism, antioxidant, anti-inflammatory, reducing iron deposition, regulating tumor cell proliferation, anti apoptosis, promoting autophagy, and other pathways. It is often used to treat acute liver injury and related diseases.

Acute liver injury is a sudden abnormal liver function disease caused by various reasons in the short term. Its clinical prognosis is poor, and if not actively intervened and treated, it may further develop into cirrhosis and liver cancer, threatening human life and health. There are various factors that can cause liver damage, including chemical liver damage, bacterial and viral induced biological liver damage. CCI4 is the most commonly used hepatotoxin in experimental animal research to induce chemical liver injury, and a single dose of CCI4 can prepare liver cell injury type ALI. It can cause damage to the structure and function of liver cells, leading to the overflow of intracellular ALT and AST into the bloodstream, resulting in an increase in ALT and AST activity in the blood, which is positively correlated with the degree of liver injury. Therefore, ALT and AST activity are often used as indicators to evaluate the degree of liver injury. The animal experiment results of this study showed that after intraperitoneal injection of CCI4, the activities of ALT, AST, and TBA in serum significantly increased, and liver tissue lesions occurred, indicating the successful establishment of a CCI4 induced acute liver injury mouse model. After intervention with TFCTLL, the activities of ALT, AST, and TBA in mouse serum were significantly reduced, and the degree of liver tissue lesions was alleviated, indicating that TFCTLL has a protective effect on CCI4 induced acute liver injury in mice.

This study starts from the perspective of safflower medicinal resources, converts traditional non medicinal waste and by-products into resource-based drugs, fully utilizes safflower medicinal materials, and explores the hepatoprotective effects of safflower leaves. The results indicate that the TFCTLL obtained after extraction and purification has an effect on acute liver injury mice. This study can provide reference for the subsequent development and utilization of TFCTLL, and is also of great significance for the high-value utilization of safflower resources.