**1. Introduction**

Recently, studies on the discovery and treatment of new diseases have been accelerated by the dramatic development of medical technologies. As our understanding of the human brain has widened, studies on the causes and treatments of diseases related to brain diseases have been intensively conducted [1–3]. Studies on the treatment and prevention of dementia, which is among the most threatening diseases to humans both socially and medically and seriously deteriorates patients' quality of life, have been intensively conducted, substantially advancing our mechanistic understanding of dementia and the development of diverse methods and drugs to treat dementia [3–5]. Specifically, many studies on cognitive function deterioration and preventive drugs have widely accepted the theory that a major cause of cognitive dysfunction is brain nerve cell death, due to persistent exogenous stress or accumulation of oxidants, which lead to cognitive function deterioration and consequent brain diseases, such as dementia [6–8]. Therefore, many studies on cognitive and memory impairments, such as dementia and Parkinson's disease, have concentrated on mechanisms that protect brain nerve cells by preventing the accumulation of metabolic oxidants through antioxidant activity or rapid decomposition of gene oxidants and the development of drugs for such mechanisms [9–11]. Therefore, drugs, such as donepezil, rivastigmine, and galantamine have been developed to control and/or enhance the neuronal signaling pathway linked to p-ERK/p-CREB/BDNF. However, after extended use, these synthetic drugs show adverse side effects, such as vomiting, hand tremors, and heart disease [12–14]. Accordingly, remarkable findings have been reported from studies on natural products that protect cranial nerves by exerting antioxidant activity, and these products have relatively fewer side effects [15–17].

Most of these studies have fundamentally focused on suppressing cognitive function and memory loss through antioxidant-mediated cranial nerve protection. However, rather than exhibiting intense antioxidative effects in the brain nerve cells and their activation sites, natural products suppress the deterioration of cognitive function resulting from overall antioxidative activity throughout the body, so the actual effects have been remarkably lower than expected in most cases [18,19]. Among the recent studies on suppressing cognitive function and memory loss, those featuring natural products with functions beyond simple antioxidant activity to directly suppress memory deterioration by inhibiting inflammation in brain nerve cells have attracted substantial attention [20]. In particular, the brain damage-induced activation of pro-inflammatory cytokines, such as TNF-α and IL-6, can evoke brain inflammation, which results in various neurodegenerative diseases like ischemia, trauma, infection, Alzheimer's disease and Parkinson's disease [20–22]. There is recent strong evidence that continuous stress and diverse inflammation in the brain could be a major and direct cause of dementia and memory impairment, due to the continuous accumulation of inflammatory cytokines in the brain. While many efforts have been made to reduce the production of these inflammatory cytokines to indirectly attenuate degenerative brain diseases, more detailed studies on the mechanism underlying delayed neurodegeneration should be carried out based on our understanding of the blood-brain barrier [23,24]. Therefore, the development of functional natural materials that more efficiently prevent and treat dementia is possible if the materials can selectively inhibit brain nerve cell inflammation and exert antioxidative activity [25,26].

To this end, a variety of natural resources have been developed recently, including *Spirulina* which has attracted much attention. *Spirulina* has been consumed by humans for thousands of years without side effects and has been selected as space food by the US National Aeronautics and Space Administration (NASA). Since *Spirulina* has diverse active substances, such as C-phycocyanin, beta-carotene, chlorophyll, and functional fatty acids, it has been reported to have excellent anti-cancer, immunity enhancing, skin improving, and anti-inflammatory effects and to be useful in treating hypertension, diabetes, and diseases related to metabolism, such as liver dysfunctions [27–29]. In particular, *Spirulina* contains at least 50–100% more beta-carotene and chlorophyll, which are highly antioxidative, than other natural products [27,30]. Therefore, many study findings have been reported promising outcomes of *Spirulina* on brain nerve cell protection from these strong antioxidants, and many positive effects of *Spirulina* in cranial nerve protection are associated with the strong antioxidant activities of chlorophylls [31,32]. Interestingly, *Spirulina* is also known to contain at least 70–90% pure chlorophyll a, which has relatively higher activity than the other subtypes chlorophyll b and c which normally exist in a mixture in most plants. Therefore, studies using the antioxidant activity of *Spirulina* are expected to have great potential [33,34]. However, most of these studies have focused on the antioxidant activity and regulation of antioxidative signaling pathways in the body [32,35,36], while studies on enhancing memory by suppressing brain nerve cell inflammation via chlorophyll derived from *Spirulina* are extremely rare.

Although studies on brain cell anti-inflammation using chlorophylls from *Spirulina* are promising, obtaining a high concentration of chlorophylls from *Spirulina* using existing conventional extraction processes is difficult because chlorophylls are extremely vulnerable to heat and fat solubility [37,38]. Low-temperature extraction is essential to obtain high-concentration chlorophyll that maintains its activity. However, it is difficult to obtain chlorophyll at a sufficient concentration by ordinary low-temperature extraction processes, due to very low extraction yields and longer process time [37,38]. To overcome this difficulty, an ultrasonic low-temperature extraction process will be an excellent alternative to treat heat-sensitive *Spirulina* extract. The ultrasonic process (UE) is a typical low-temperature extraction process that does not apply heat and can efficiently extract heat-sensitive active components through efficient destruction of the cell walls using air cavities generated by ultrasonic vibration even at room temperature or temperatures below 40 ◦C [39,40]. Therefore, this study investigated the anti-inflammatory mechanism of brain nerve cells associated with the antioxidant activity of chlorophylls in the *Spirulina* extract obtained by low-temperature ultrasonic treatment.
