3.1.1 Influence of temperature on the components of leaves
As a perennial herb, S. cusia has broad application prospects in antibacterial medication, especially in antiviral and tumor treatments (Xu et al., 2021). Indole alkaloids are mainly synthesized by the indole pathway in S. cusia (Yu et al., 2019). Indigo and indirubin are the main active substances in the leaves of S. cusia (Yang et al., 2020). According to the traditional extraction methods, the temperature has a great impact on the extraction efficiency of bioactive substances from different biomass. In this study, the fresh leaves ofS. cusia were treated with hot water at different temperatures, and the effect of temperature on the components of the leaves was investigated by determining indigo, indirubin and their precursors.
As shown in Fig. 1a, even though the increase in temperature could improve the extraction rate of indigo below 80 ℃, the indigo content extracted at higher temperatures was significantly less than that at lower temperatures along with time. At 100 ℃, no indigo was detected in the solution. The extraction results of indirubin were significantly different from those of indigo (Fig. 1b). At 80 ℃, indirubin was detected after 15 min; meanwhile, the content of indirubin increased with the increase in temperature. However, indirubin was not detected at 100 ℃. This result indicates that the extraction temperature had a great influence on what was extracted. In general, an increase in temperature aids in the extraction of substances. This tendency of indigo indicates that temperature not only affected the extraction efficiency of active substances but also affected the formation or decomposition of indigo. Because indigo and indirubin still maintained good thermal stability after short-time heat treatment (Sun et al., 2021), it could be considered that the change in temperature would affect the formation of indigo and indirubin.
It was found that the content of indigo in the solution was the highest at about 1.91 mg/g when S. cusia was treated at 60 ℃ for 30 min (Fig. 1a). The contents of indirubin in the solution were the highest at about 0.12 mg/g after it was treated at 80 ℃ for 30 min (Fig. 1b). Indigo and indirubin were not detected in the 100 ℃ treatment group. Indigo and indirubin do not exist in the leaves of S. cusia , so these two bioactive substances must be produced in the extraction process.
Indican is a precursor of indigo and indirubin. At 80 and 100 ℃, indican was detected, whereas there was no indican present in the solution at 60 and 40 ℃ (Fig. 1c). On the other hand, the intermediate in the conversion of indican to indigo and indirubin, indoxyl, was found under such conditions (Fig. 1d). The contents of indican and indoxyl in the solution were inversely proportional (Fig. 1c, d). In the general understanding, the extraction conditions affect the yield of the extracts with little effect on their components. In this study, the extraction conditions could not only change the yield of bioactives, but also the components and proportions of the bioactives, indicating that some reactions that affected the components of the biologically active substances took place during the extraction process. We assumed that revealing the reaction mechanism would help us to provide a theoretical and methodological basis for the evaluation and application of the bioactives in the future.