Optimization of the preparation process of saffron acid from gardenia yellow pigment based on orthogonal experimental method
Saffron acid (structural formula shown in Figure 1) is one of the main active ingredients in the Iris plant Crocus sativus L., which has pharmacological effects such as treating neurological diseases, anti cardiovascular diseases, antioxidation, and anti-tumor effects. A large number of studies have shown that the traditional Chinese medicine Gardenia jasminoides Elli is rich in saffron glycosides with saffron acid as the aglycone, and the plant resources are abundant. The project team established a preparation process for enriching and extracting gardenia yellow pigment from gardenia using polyamide column chromatography in the early stage, and for the first time proved that gardenia yellow pigment has good antioxidant, anti hypoxia, and anti fatigue biological activities, and has good development value. However, pharmacokinetic studies have shown that saffron glycosides cannot be absorbed into the bloodstream in their original form, and their bioavailability is low after oral administration, ultimately leading to hydrolysis into saffron acid. In order to further conduct relevant research, it is necessary to prepare high-purity saffron acid. Zhang separated and enriched saffron total saponins through macroporous adsorption resin, and hydrolyzed them with 10% KOH to obtain crude saffron acid. Fang et al. extracted gardenia yellow pigment from gardenia and hydrolyzed it with 10% KOH solution to prepare crude saffron acid extract. The preparation of high-purity saffron acid usually requires further complex purification processes such as column chromatography or recrystallization. However, due to the difficulty in dissolving saffron acid in water and its poor solubility in general organic reagents such as ethanol and methanol, the actual purification process often requires the use of organic reagents such as pyridine and DMF, making it difficult to avoid organic residues; At the same time, the use of a large amount of organic reagents can also cause serious environmental pollution. The existing preparation process has complicated operational steps and long preparation cycles, making it difficult to achieve industrial production of high-purity saffron acid.

This study designed an orthogonal experiment to systematically investigate the process conditions of alkaline hydrolysis. The content and yield of crocetin acid were used as the evaluation indicators to optimize the alkaline hydrolysis process of gardenia yellow pigment through orthogonal design. At the same time, the treatment method of gardenia yellow pigment after alkaline hydrolysis was improved to obtain a high yield, high purity, and preparation method with fewer steps and no residual organic solvents. This provides a reference for the extraction and industrial production of crocetin acid in gardenia, and is conducive to the wide development and application of crocetin acid in the food and medicine fields.

The research group quickly extracted and enriched high-purity gardenia yellow pigment from gardenia using polyamide column chromatography in the early stage. The absorption peak at 440nm was detected by HPLC, and it was found that its main components were crocin-1 and crocin-2. Based on this, gardenia yellow pigment was used as the raw material for preparing crocetin acid.

At present, the methods for preparing saffron acid mostly use alkaline hydrolysis to hydrolyze saffron glycosides in gardenia into saffron acid, and subsequent purification of saffron acid commonly uses column chromatography or recrystallization methods. However, due to the insoluble physical properties of crocetin, the preparation process using column chromatography or recrystallization is not only cumbersome, time-consuming, consumes a large amount of reagents, and seriously pollutes the environment, but also difficult to avoid organic residues when using organic reagents such as DMF.

This study investigated and optimized the preparation process of crocetin acid using different treatment methods for the hydrolyzed reaction solution. After optimization, the process steps were simple, and high-purity crocetin acid was prepared in one step from gardenia yellow pigment, eliminating the tedious purification processes such as column separation or recrystallization, avoiding the consumption of organic reagents and the problem of residual reagents in the process. The preparation process was green and pollution-free, and the obtained crocetin acid content could reach over 95%. At the same time, in order to improve the yield of saffron acid, the alkaline hydrolysis process of gardenia yellow pigment was investigated on the basis of optimizing the process. Single factor investigation was conducted by selecting the material to liquid ratio, NaOH concentration, hydrolysis temperature, and hydrolysis time. Orthogonal experiments were designed based on the investigation results, and the optimal conditions for alkaline hydrolysis were determined using content and yield as evaluation indicators.

This experiment optimized the route for preparing saffron acid from gardenia yellow pigment, and designed single factor and orthogonal experiments to optimize the optimal conditions for alkaline hydrolysis. A rapid, high-yield, and high-purity method for preparing saffron acid was obtained. Compared with commonly used methods, it does not require subsequent purification treatment, does not involve organic solvents, saves production time and costs, and has good reference significance for extracting and preparing saffron acid from gardenia.