*2.1. Ech A Mitigates Cerebral Ischemic Injury*

To confirm the mitigative effect of Ech A on cerebral ischemic disease, rat MCAo models were exposed to 10 μM Ech A and subsequently assessed for brain infarct volume and water content. We used 2,3,5-triphenyltetrazolium chloride (TTC) staining, one of the most conventional methods, to visualize the infarct region of the ischemic cerebrum. Ech A substantially shows the visual recovery reducing the brain infarct volumes at 7 days after reperfusion (Figure 1A). Alleviation of the injured area after exposure to Ech A was confirmed by comparing the percentage of white region in the control brain with the Ech A treatment group through TTC staining (Sham: 0.0%, control group: 52.7%, 10 μM Ech A: 21.9%) (Figure 1B). Next, we investigated the water content of rat MCAo model brain tissue, which is a marker of severe ischemic injury [13]. The water content is used to signify the ischemic brain edema in the infarct hemisphere [14], and to examine the effect of Ech A treatment on the blood–brain barrier (BBB) leakage in this study. Cerebral edema plays a key role in fatal outcomes associated with numerous neurological conditions, including ischemic stroke [15]. The percentage of water content in the control group was increased, as compared to the sham group. The Ech A-treated group revealed decreased water content as compared to the control group (sham: 79.4%, control: 81.7%, 10 μM Ech A: 80.3%) (Figure 1C). Taken together, we construe that Ech A presents the visual restoration in the infarct cerebrum of rat MCAo models. However, the functional recovery and the reparative mechanism by Ech A treatment on brain ischemic disease remain to be identified.
