*3.1. Total Chlorophyll Concentrations in the Extracts from Two Di*ff*erent Extraction Processes*

Table 1 shows the comparison of the total chlorophyll in the extracts and the difference in extraction yields between the conventional 70% ethanol at 80 ◦C for 12 h extraction and the UE extraction. The amount of chlorophyll in the UE extract was 17.56 mg/g, which is at least three-fold higher than the concentration obtained through extraction with 70% ethanol at 80 ◦C which was 6.24 mg/g. This result clearly proved that most of the chlorophyll in the extract was destroyed at temperatures exceeding 60 ◦C resulting in extremely low chlorophyll content. Therefore, this result reconfirms that heat-sensitive substances, 'such as chlorophyll can be extracted' in their intact forms with the least loss of their activities only when they are extracted at low temperatures associated with UE. Additionally, low-temperature extraction does not affect the activity of the target substance, and the extraction yield is reduced to below half of that of the UE. This confirms that simple low-temperature extractions cannot extract substances with the targeted activity; however, the UE extraction produces higher concentrations than the concentrations ranging from 8 to 15 mg/g reported in other works using chlorophyll extractions with other solvents, process temperatures, or complex processes in *Spirulina* [33,38]. Therefore, we could conclude that *Spirulina* extract from UE results in relatively high extract yields, and the activity of the targeted bioactive substances is maintained. These results were similar to other studies that mentioned the excellence of UE in low-temperature extraction processes [39,48].



\* EE, 70% ethanol extraction at 80 ◦C for 12 h. \*\* UE, ultrasonic pretreatment with 70% ethanol at 40 kHz and room temperature for 8 h, and further extraction at 65 ◦C for 4 h.

As shown in Table 1, the extracts from the UE are expected to contain intact forms of chlorophylls at high concentrations and have many beneficial biological activities. In particular, 70–80% of chlorophyll in *Spirulina* is known to be in a form of chlorophyll a, which reportedly has stronger antioxidant activity than other chlorophylls [33,34]. Therefore, the extract obtained through this process is expected to have strong antioxidative activity. The extract has also been reported in various to protect cranial nerves. However, most of these studies showed that the antioxidant activity amplifies the p-ERK/p-CREB/BDNF signaling pathway and enhances cognitive functions through inhibition of the acetylcholinesterase (AChE) enzyme [10,32,35]. Furthermore, studies on the anti-inflammatory action of the *Spirulina*

extracts, or their chlorophylls on the resultant protection of nerve cells, are very rare. Therefore, the effects of the *Spirulina* extracts, obtained from the ultrasonic process on suppressing the inflammation of mouse nerve cells through antioxidation, are shown as follows.
