3.1.3 Influence of temperature on indican content in leaves
To further study the effect of β-glucosidase on indican, the leaves treated at different temperatures for 10 min were covered with boiled water and the released indican from the leaves was determined using HPLC. As shown in Fig. 1e, when the leaves were treated at lower temperatures (40 ℃ and 50 ℃), larger amounts of indican were detected even though the enzyme activity was higher. This could be because β-glucosidase cannot efficiently react with indican in vacuoles to produce indoxyl under such conditions so that the residual indican was high, reaching 1201.02 mg/L and 1336.31 mg/L, respectively (Fig. 1e). With an increase in temperature, the leaves were seriously damaged, and the indican released from vacuoles would be hydrolyzed by β-glucosidase even with low activity. When the leaves were treated at 100 ℃ for 10 min, the enzyme activity was too low to hydrolyze indican released from the vacuoles. Therefore, the largest amount of indican (376.16 g/L) was obtained from leaves.
According to the experimental results and previous studies, it could be considered that little indigo and indirubin were present in leaves before extraction. When suffering from the stresses from the outside, the plant starts an emergency mechanism to avoid the stress of the environment, i.e., indican is released from plant vacuoles and hydrolyzed by enzymes to form indoxyl and glucose. Previous studies have shown that indican is stored in plant vacuoles and released by chloroplasts. During natural fermentation, β-glucosidase hydrolyzes indican to form indole groups and glucose (Inoue et al., 2018). Hence, the morphology of some vacuoles would be destroyed under the temperature stress, resulting in the release of indican, to form indoxyl under the action of β-glucosidase.