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.