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.