At present, 44 species of Hippocampus have been found in 159 countries and regions worldwide (Figure 1 and Supplementary Table 1; CITES, 2021b; FishBase, 2021), with the highest diversity of species observed in Australia (Figure 2A). Oceania has the largest number of species on the continent, followed by Asia and Africa (Figure 2B). Hippocampus is primarily found in the Indo-Pacific Ocean and distributed in shallow waters with a depth of less than 30 m in temperate, subtropical, and tropical regions (Foster and Vincent, 2004). On the west coast of peninsular Malaysia, Hippocampus kelloggi Jordan et Snyder was collected by trawlers in deepwater below 65 m (Choo and Liew, 2003). The habitat of Hippocampus includes seagrass, coral reefs, mangroves, and seaweeds, with coral reefs representing the main habitat constituents of tropical Hippocampus (Foster and Vincent, 2004). Hippocampus coronatus Temminck and Schlegel and Hippocampus mohnikei Bleeker generally inhabit seagrass in the southern coastal waters of Korea. Temperate Hippocampus tends to inhabit seagrass beds, which provide food for a variety of flora and fauna (Choi et al., 2012). Among the five medicinal Hippocampus in China, only Hippocampus japonicus Kaup is mainly distributed in northern China. The other four species were mainly found in the tropical and subtropical regions of China, such as the East China Sea and South China Sea (Lin et al., 2008; Han, 2013).

The ease of identification of a Hippocampus specimen varies from species to species. Hippocampus specimens were generally identified using preparation tools (ruler, magnifying glass, calculator, etc.) by identifying characteristics, using measured and recorded data about an unidentified specimen, and using standards (Supplementary Tables 1–6 from a guide of Hippocampus identification) to determine possible species and identify specimens. The data mainly included the head length (HL) and snout length (SnL), length of the snout in relation to the length of the head (HL/SnL), number of tail rings (TaR), trunk rings (TrR), etc., (Lourie et al., 2004). The characteristics and morphological identification based on distinguishing characteristics are appropriate when performed by investigators who are experienced with Hippocampus identification. However, some Hippocampus specimens are particularly similar, and new species may be encountered; thus, deoxyribonucleic acid (DNA) barcoding technology may need to be applied for Hippocampus identification. The species phylogenetic tree is constructed based on the alignment results of the complete mitochondrial genome or mitochondrial cytochrome b gene from different species (Casey et al., 2004; Ge et al., 2018). The phylogenetic tree with the Cytochrome Oxidase I (COI) gene as a primer showed that different species of Hippocampus were in different branches with reasonable distances between them, and the accuracy was higher than that of other mitochondrial genes (Hou et al., 2016). Liu et al. (2018) used a multiplex polymerase chain reaction (PCR) method to simultaneously identify five medicinal Hippocampus species by judging the absence and size of DNA bands on the gel map in a single assay. DNA barcoding provides an accurate technique for the identification of Hippocampus specimens. A rapid detection kit should be developed in the future to reduce the complicated and time-consuming aspects of this technology. Fast and effective identification methods can be used to protect endangered Hippocampus species and monitor the Hippocampus trade (Luo et al., 2013). In TCM, metabolomics technology has been used to study differences in metabolites for various species and find metabolic markers, and such work has provided references for the identification of multisource medicinal materials (Fan, 2012). This may provide new ideas for the identification of Hippocampus.

Recent studies have revealed that the number of Hippocampus individuals has continuously declined over the past 20 years. The trade demand for Hippocampus has increased significantly because of its value for medicine, aquarium displays, and collectibles (Vincent et al., 2011). The export volume of medicinal Hippocampus is concentrated in Southeast Asian countries, with Thailand being the largest exporter (Figure 3A and Supplementary Table 2; CITES, 2021a). The export volume of Hippocampus kuda Bleeker, Hippocampus trimaculatus Leach, and Hippocampus kelloggi Jordan et Snyder accounted for 88% of the global export of dried Hippocampus (Loh et al., 2016). Regarding importing countries, China and its affiliated Hong Kong Special Administrative Region and Taiwan were the main importers of dried Hippocampus due to the influence of TCM (Figure 3B and Supplementary Table 3; CITES, 2021a). Exports of other Hippocampus species were mainly concentrated in Australia and some countries in the Americas (Convention on International Trade in Endangered Species of Wild Fauna and Flora, 2020). A large global trade volume is mainly associated with wild Hippocampus caught by trawl by catch. Huge fishing pressure has made Hippocampus populations unsustainable (Vincent et al., 2011). Moreover, the Hippocampus population is also threatened by habitat reduction and environmental pollution (Rosa et al., 2005). The International Union for Conservation of Nature (IUCN) listed Hippocampus on the red list, with five medicinal Hippocampus species listed as vulnerable species (IUCN, 2021). In addition, breeding techniques for Hippocampus similar to wild medicinal species are required. With the gradual solution to problems associated with rearing and bacterial infection in the breed, an increasing number of countries have tried to breed Hippocampus, such as Australia, Brazil, China, Sri Lanka, the United Kingdom, the United States, and Vietnam (Koldewey and Martin-Smith, 2010). The amount of wild Hippocampus cannot meet market needs, and artificial breeding is an important way to meet market demand and protect wild Hippocampus resources. In addition, we may seek alternative species. The phylogenetic tree of Hippocampus listed eight species of Hippocampus that were similar to the medicinal Hippocampus (Figure 4 and Supplementary Table 4; NCBI, 2020). As a next step, we may identify Hippocampus species with a large trade volume to study their ingredients and pharmacological activities and strive to find supplemental medical Hippocampus species.

Amino acids play vital roles in regulating metabolism, growth, and development. Among the 20 types of amino acids constituting human proteins, essential amino acids cannot be synthesized by humans (Mitsuhashi, 2014). Twenty-three types of amino acids have been found in Hippocampus (Supplementary Table 6). Lin et al. (2008) found that the total amount of amino acids was as high as 50–60% (based on a comparison of their retention times with those of amino acid standards) in six species of Hippocampus, and eight types of essential amino acids accounted for 17–20% of the total amino acid content, which indicated that the Hippocampus is a high-quality protein source. The content of glycine was the highest (83.98 mg/g) among the 15 types of amino acids in Hippocampus kuda Bleeker and Hippocampus trimaculatus Leach (Sun et al., 2020). In addition, Hippocampus is also rich in glutamic acid, aspartic acid, alanine, and arginine (Lin et al., 2009). Jia and Yao (1990) reported 22 types of amino acids in Hippocampus kuda Bleeker and Hippocampus japonicus Kaup, including taurine, a special amino acid with high medicinal value. The type and the content of amino acids in Hippocampus erectus Perry were similar to those in medicinal Hippocampus, and even the content of essential amino acids in Hippocampus erectus Perry was higher than that in medicinal Hippocampus (Yan Z. Z., 2019). Peptides are important marine natural products that widely exist in vast marine organisms. Most peptides extracted from Hippocampus have various pharmacological activities and can be classified as angiotensin-converting enzyme (ACE) inhibitory peptides, anti-inflammatory peptides, neuroprotective peptides, and antimicrobial peptides. Shi et al. (2020) isolated ACE inhibitory peptides with the amino acid sequence Pro-Ala-Gly-Pro-Arg-Gly-Pro-Ala from the enzymatic hydrolysate of Hippocampus trimaculatus Leach. Circular dichroism analysis showed that the secondary structure was mainly composed of a random coil. Three ACE inhibitory peptides were also isolated from cultured Hippocampus abdominalis Lesson. The amino acid sequence of Cys-ASN-Val-Pro-Leu-Ser-Pro was more stable in molecular docking with ACE, which may become the preferred candidate for antihypertensive peptides (Je et al., 2020). Ryu et al. (2010) isolated an anti-inflammatory peptide composed of 15 types of amino acids (including five aspartic acids) from Hippocampus. Recently, a novel neuroprotective peptide (HTP-1) rich in hydrophobic amino acids and a molecular weight of less than 1 kDa have been isolated from Hippocampus trimaculatus Leach. The N-terminal amino acid sequence of HTP-1 is similar to that of insulin-like growth factor-1 (IGF-1) and may have neuroprotective effects (Pangestuti et al., 2013). Sun et al. (2012) identified an antimicrobial peptide (HKPLP) from the brooding pouch of male Hippocampus Kuda Bleeker. Bioinformatics analyses of available genomic and transcriptomic data allowed us to find the distribution of antimicrobial peptides closely related to the male brooding pouch (Chen et al., 2019). In conclusion, the activity of low molecular weight peptides is affected by the composition and sequence of amino acids. In terms of favorable structure-activity relationships for high ACE-inhibitory activity, aromatic amino acids at the C-terminus end and hydrophobic amino acids at the N-terminus end were most potent (Xu, 2015). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging results indicated that low molecular weight (43–278 kDa) peptides from Hippocampus erectus Perry showed higher activity than peptides over 278 kDa (Chen et al., 2018). Elucidating the structure-activity relationships of these peptides may help develop peptides with enhanced bioactivity.

Steroids are among the major ingredients of Hippocampus, and 38 types of steroids have been isolated and identified to date (Supplementary Table 6). Gas chromatography–mass spectrometry (GC–MS) analysis identified 16 types of steroids from seven species of Hippocampus. Cholesterol and cholest-4-en-3-one were the major ingredients in the total steroids (Chen, 2015). Sun et al. (2020) reported that the average content of steroids in Hippocampus kuda Bleeker was 6.16 mg/g, and cholesterol accounted for 0.55 mg/g. The average ratios of cholest-4-en-3-one and cholest-3,6-dione in Hippocampus trimaculatus Leach, Hippocampus histrix Kaup, and Hippocampus japonicus Kaup were 0.0301 and 0.0096%, respectively (Zhang et al., 1998). In addition, Wu et al. (2017b) extracted 3β-hydroxycholest-5-en-7-one and cholest-5-en-3β,7β(7α)-diol from Hippocampus trimaculatus Leach. Cholesterol stearate and cholest-5-en-3β,7α-diol were also extracted from Hippocampus histrix Kaup (Wang et al., 1998). Yang et al. (2014) supplemented steroids of Hippocampus erinaceus Gunther, and six ingredients were identified: cholesterol, cholest-4-en-3-one, (22E,24R)-3β,5α,9α-trihydroxyergosta-7,22-dien-6-one, 24-methyl-cholesta-7,22-diene-3β,5α,6β-triol, 3β-hydroxy-7-methoxy-cholesta-5-en, and 3β-hydroxycholest-5-en-7-one. In addition, brassicasterol, which has antitumor activity, has been recently obtained from Hippocampus abdominalis Lesson (Xu et al., 2020). Although a large number of steroids have been isolated, marker ingredients have not been identified from Hippocampus quality evaluation standards. It is urgent to establish a stable and controllable method to scientifically evaluate and control the quality of Hippocampus.

Hippocampus contains 82 types of fatty acids, including 35 types of saturated fatty acids and 47 types of unsaturated fatty acids (Supplementary Table 6). Lin et al. (2008) found that there were 12 types of fatty acids distributed widely among six species of Hippocampus, and hexadecanoic acid, octadecanoic acid, oleic acid, 5,8,11,14,17-eicosapentaenoic acid (EPA), and 4,7,10,13,16,19-docosahexaenoic acid (DHA) were the main ingredients. The ratios of unsaturated fatty acids and EPA and DHA to total fatty acids were 48–59% and 14–35%, respectively. Studies have shown that EPA and DHA not only reduce the risk of cardiovascular diseases, such as atherosclerosis, arrhythmia, and hyperlipidemia (Watanabe and Tatsuno, 2017), but also improve sperm quality and sperm motility when used as food supplements (Hosseini et al., 2019). Chen (2015) identified 26 types of fatty acids from seven species of Hippocampus; among benzenepropanoic acids, 3,5-bis(1,1-dimethylethyl)-4-hydroxy was found for the first time. Tetradecanoic acid, hexadecanoic acid, octadecanoic acid, 9(Z)-octadecenoic acid, 5,8,11,14-eicosatetraenoic acid, and DHA were the main fatty acids in the five medicinal Hippocampus species. Zhang and Wang (1996) isolated 42 fatty acid peaks of Hippocampus japonicus Kaup by a rapid fat extraction method, among which EPA and DHA accounted for 14.1%. Amazingly, there were many other types of fatty acids in Hippocampus erectus Perry, such as heptadecanoic acid, tetracosenoic acid, 11-octadecenoic acid, 15-tetracosenic acid, and 7,10,13,16-docosatetraenoic acid (Nicolás et al., 2014).

Mineral elements play an important role in growth because they help to improve immunity and anti-fatigue (Sherman, 1992). Twenty-four types of mineral elements were found in five Hippocampus medicinal species. The contents of P, K, Na, and Mg were all above 100 mg/g, while the contents of mineral elements, including Al, Fe, Mn, Sr, Zn, etc., were at the level of a microgram. Lin et al. (2009) reported that the contents of Mn and Zn in Hippocampus trimaculatus Leach were 21.73 and 31.44 μg/g, respectively. The total contents of mineral elements in Hippocampus Kuda Bleeker were 84,873 μg/g, which also contained Cr, Co, Se, and Pb (Jia and Yao, 1990). The effect of Hippocampus on warming and tonifying kidney yang also benefited from mineral elements (Zhang et al., 1997). Clinical studies have shown that higher levels of mineral elements may reduce the likelihood of declines in cognitive function over 5 years (Gerardo et al., 2020). In the brain, various nervous activities require the participation of essential mineral elements, such as Fe and Zn. Some neurodegenerative diseases are related to an imbalance in the homeostasis of these cations (Bohic et al., 2011), which indicates that Hippocampus may have a therapeutic effect on neurodegenerative diseases.

Studies on Hippocampus mainly focus on amino acids, mineral elements, and fatty acids, while relatively few reports have focused on the analysis technologies and ingredient activities, such as that of phospholipids and nucleosides. Phospholipids are the main component of biomembranes. A study of the contents and types of phospholipids in five medicinal Hippocampus was carried out using molybdenum blue colorimetry and thin layer chromatography (TLC) scanning. The results showed that the total contents ranged from 3.28 to 7.82 mg/g, including LPC, SM, PC, and PE (Xu et al., 1994). Shen et al. (2016) identified 50 phospholipid molecules by hydrophilic interaction chromatography-quadrupole time of flight-mass spectrometry (HILIC-QTOF/MS). In addition to PC and PE, there were 12 PIs and 9 PSs. PC and PE accounted for most of the total phospholipids at 1.91–4.89 mg/g and 1.52–2.64 mg/g, respectively. Marine nucleosides were found to have antiviral, anticancer, and hypertensive activities (Huang et al., 2014). Zheng et al. (2012) reported that the contents of hypoxanthine extracted from Hippocampus Kuda Bleeker and Hippocampus japonicus Kaup were 0.73 and 0.52 mg/g, respectively. The average contents of nucleosides and nucleotides in the Hippocampus japonicus Kaup were 1.974 and 0.79 mg/g, respectively. However, uridine, cytosine nucleotides, and guanine nucleotides were not found in Hippocampus japonicus Kaup (Wei et al., 2015). Yan Z. Z., (2019) found six nucleosides in Hippocampus erectus Perry, more types and contents than those in Hippocampus japonicus Kaup.

Apart from the abovementioned ingredients, other ingredients have also been reported. Six phthalate derivatives have been isolated from ethanol extracts of Hippocampus Kuda Bleeker and Hippocampus trimaculatus Leach, including dibutyl phthalate, bis(2-ethylhexyl)phthalate (Wu et al., 2017b), bis(2-ethylheptyl)phthalate, bis(2-ethyldodecyl) phthalate, 2-ethyldecyl 2-ethylundecyl phthalate, and 2,12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecylphthalate (Li et al., 2008). As a part of ongoing research on extraction and isolation, paeonol was incidentally isolated from Hippocampus Kuda Bleeker (Himaya et al., 2012). The flavor profile was identified based on a set of volatile ingredients that rendered the characteristic taste and smell of Hippocampus. Based on headspace solid-phase microextraction combined with GC–MS, 84 potential volatile ingredients were identified, including acids, hydrocarbons, alcohols, aldehydes, ketones, esters, and amines. The main flavor ingredients with contributions over 70% were (E,E)-3,5-octadiene-2-one, trimethylamine, hexanoic acid, 2-nonone, etc., (Si et al., 2018). Glycoproteins of Hippocampus have rarely been reported because of their relatively complex composition. Box-Behnken and ultrasonic extraction technology were applied to separate glycoproteins HG-11 and HG-21 (Su and Xu, 2015a). Glycoproteins included in marine organisms are the main active ingredients and deserve additional attention in future research. In addition, the nuclear magnetic resonance (NMR) and GC–MS metabonomics methods (Zhao, 2018) and the Traditional Chinese Medicines Integrated Database (TCMID) (TCMID, 2020) have provided a large amount of information regarding the chemical ingredients of Hippocampus.

Lin et al. (2008) compared different ingredient constituents of six species of Hippocampus. The crude protein content was the most abundant in Hippocampus kelloggi Jordan et Snyder, while, in Hippocampus histrix Kaup, was the lowest. Notably, more than half of the fatty acids in Hippocampus comes Cantor were unsaturated fatty acids, resulting in approximately 1.25–1.8 times as much EPA and DHA in Hippocampus comes Cantor compared to those in medicinal Hippocampus species. Mg was the most abundant mineral element in Hippocampus trimaculatus Leach and Hippocampus histrix Kaup, and the contents were 1125.44 and 1076.57 μg/g, respectively. Chen (2015) compared steroids in seven species of Hippocampus. The most abundant steroids content was found in Hippocampus japonicus Kaup, and the least abundant steroid content was found in Hippocampus histrix Kaup and Hippocampus comes Canter. Xu et al. (1994) reported the total phospholipid contents of five medicinal Hippocampus species. The highest phospholipids content was observed in Hippocampus japonicus Kaup (7.82 mg/g), but PEs were hardly detected compared with that of any other Hippocampus species. ¹H-NMR metabonomics studies showed that the contents of glutamic acid, aspartic acid, glycerol, taurine, and acetic acid in Hippocampus trimaculatus Leach and Hippocampus kelloggi Jordan et Snyder were significantly higher than those in Hippocampus kuda Bleeker, while the contents of isoleucine and leucine were significantly low (Zhao, 2018). TCM theory about medicinal Hippocampus emphasizes that both male and female Hippocampus should be used together (Xu et al., 2002). Whether there was a difference in medicinal value between males and females was not clear. At the age of 6 months, female Hippocampus had a slightly higher proportion of steroids and unsaturated fatty acids than males (Sun et al., 2020). The fatty acid extraction rate of male Hippocampus was slightly higher than that of females (Su and Xu, 2015b). There was no significant difference in ingredient constituents between male and female Hippocampus erectus Perry and Hippocampus trimaculatus Leach, although the contents of cholesterol and hypoxanthine in female Hippocampus were significantly higher than that in males, and the content of uridine in female Hippocampus erectus Perry was significantly lower than that in males (Yan Z. Z., 2019). Such findings may explain why male and female Hippocampus would be used together. In conclusion, gender had a limited effect on the ingredient constitutions.

Eggs of Hippocampus guttulatus Cuvier are round and transparent, and the yolk sac provides nutrition for embryo development. Eggs and newborn Hippocampus represent different growth stages, and their ingredient constituents are different. Taking fatty acids as an example, the fatty acid contents in newborn Hippocampus were much lower than those in eggs. The content of unsaturated fatty acids in eggs was more than four times higher than that in newborn Hippocampus, and DHA and EPA were rich in eggs. Omega-3 unsaturated fatty acids were consumed during embryonic development, which explained the difference in fatty acid contents between newborn Hippocampus and eggs (Faleiro and Narciso, 2010). Sun et al. (2020) analyzed the ingredients of wild Hippocampus at the ages of 6 month, 1 year, and 2 years. It was obvious that crude protein content was higher in the ration of 1-year-old Hippocampus compared with the ration of 6- and 2-year-old. The Fe, Cu, and Zn contents increased with age, whereas unsaturated fatty acid contents decreased significantly. Contents of proteins and fatty acids were more abundant in 1-year-old Hippocampus; therefore, Hippocampus in this stage is proper for TCM applications.

The qualities of wild and cultured Hippocampus differed due to the different growth environments. The contents of essential amino acids and unsaturated fatty acids were much higher in cultured Hippocampus kuda Bleeker than in wild Hippocampus. In terms of mineral elements, Se, Fe, Cu, and Ca were much more abundant in wild Hippocampus (Sun et al., 2020). Lin et al. (2009) showed that the contents of DHA and EPA in cultured Hippocampus trimaculatus Leach were nearly two times as high as those in wild Hippocampus, and the proportions of polyunsaturated fatty acids in total fatty acids were 31.89 and 21.39%, respectively. The cultured Hippocampus was rich in polyunsaturated fatty acids, but there were some deficiencies in protein. In addition to Pb, the contents of Cd, Cr, Zn, and Mn in cultured Hippocampus were higher than those in wild Hippocampus. Additional nutritional supplements were added to the bait as required. Understanding the difference in nutritional characteristics between wild and cultured Hippocampus is helpful for optimizing the breeding technology and supplementing wild Hippocampus.

For cultured Hippocampus, different baits had a great influence on the ingredient constituents of Hippocampus. Hexadecanoic acid, octadecenoic acid, octadecatrienoic acid, eicosapentaenoic acid, etc., are rich in Artemia and rotifers. Several differences in fatty acid types have been observed for different baits, and obvious variations have been observed for EPA and DHA. Otero-Ferrer et al. (2010) fed Hippocampus hippocampus Linnaeus Artemia and rotifers, and, after 5 days of feeding, the rotifer group showed a higher level of omega-3 unsaturated fatty acids, and the fatty acid types were more abundant than those in the Artemia group. The contents of 9,12-octadecadienoic acid and 9,12,15-octadecatrienoic acid in the Artemia group were more prominent. After 34 days of feeding, fatty acids in the Artemia group were gradually enriched, and the contents of DHA and EPA in the Artemia group were higher than those in the rotifer group. In addition, the Artemia group also showed lower mortality and a higher growth rate. Segade et al. (2016) further compared the differences among Hippocampus-fed Artemia, frozen Artemia, and Mysis. The results showed that the crude protein content of the frozen Artemia group was the lowest among the three groups. The content of polyunsaturated fatty acids in the Artemia group was the highest, while that in the Mysis group was the lowest. Frozen bait may not fully meet the nutritional needs of Hippocampus. In addition, the Mysis group had the most abundant nucleosides, and thymine was not detected in the frozen Mysis group or the Artemia group (Yan Z. Z., 2019). In the selection of baits, mixing of different baits may meet different nutritional needs.

The hydrophilic ingredients of Hippocampus are mainly amino acids, peptides, glycoproteins, nucleosides, etc., Chen et al. (1997) compared amino acids in water and ethanol extracts of Hippocampus. The results showed that the Hippocampus water extract contained 17 types of amino acids, while the ethanol extract contained only 10 types of amino acids. The essential amino acid content of the Hippocampus water extract was also higher than that of the ethanol extract. Hippocampus peptides were obtained by enzyme hydrolysis and chromatography technologies. For bioactive peptides from marine organisms, the first step was the trituration of the tissues, followed by initial treatment (organic extraction, concentration, and/or partitioning). Under the guidance of activity assays, chromatographic technology was used for prepurification, and purified peptides were finally obtained by high-performance liquid chromatography using reversed-phase (RP-HPLC). Alternatively, after tissue separation, enzymatic hydrolysis could be employed to identify bioactive peptides (Macedo et al., 2021). Enzymatic hydrolysis is a commonly used method to extract Hippocampus peptides. Commonly selected enzymes include pronase E, alcalase, protamex, trypsin, papain, and pepsin (Table 1). Enzymes and substrates at a ratio of 1:100 (w/w) were mixed under certain conditions, and then the mixture was inactivated at 100°C for 10 min and stored at −80°C (Pangestuti et al., 2013). In addition, Hippocampus was defatted with ethyl acetate or CO₂ supercritical fluid extraction technology and then mixed with enzyme (Jiang et al., 2014). Jiang et al. (2014) studied the condition of compound enzyme hydrolysis and found that the hydrolysis rate of alcalase and trypsin was 2–3% higher than that of single enzyme hydrolysis. We summarized the enzyme reaction under different conditions to obtain optimized reaction conditions (Table 1). Enzymes were selected according to the different needs of research. For example, Hippocampus trimaculatus Leach protein was hydrolyzed by four different proteases (trypsin, pepsin, papain, and alcalase) to prepare peptides, and, compared with other hydrolysates, alcalase hydrolysates exhibited the highest ACE inhibitory activity (Xu, 2015). Nucleosides of Hippocampus are generally extracted with 50% ethanol or water, followed by HPLC analysis (Wei et al., 2015; Yan Z. Z., 2019). Chen et al. (2010) developed an online rapid method for the identification of hypoxanthine, and the antioxidant hypoxanthine was identified from a water extract of Hippocampus japonicus Kaup by HPLC-ESI-TOF/MS combined with an ABTS free radical scavenging online detection system. Zheng et al. (2012) used different antioxidant evaluation experiments for DPPH free radical scavenging, and an online detection system also identified hypoxanthine from water extracts of Hippocampus kuda Bleeker and Hippocampus japonicus Kaup. The online detection system combines biological activity and chemical separation, and the target ingredient may be quickly separated.

More reports have focused on Hippocampus hydrophobic ingredients than other types, and these hydrophobic ingredients include fatty acids, steroids, and phospholipids. The extraction of fatty acids in Hippocampus is performed by the traditional solvent extraction method. Nicolás et al. (2014) directly soaked Hippocampus powder in chloroform:methanol (2:1) for 24 h to extract total lipids. The extraction solvent of chloroform:methanol (2:1) was also used for ultrasonic extraction (Xu et al., 1994). The results showed that there were more types of fatty acids in the ultrasonic extraction. Extraction methods using ether as the extraction solvent include Soxhlet extraction (Zhang et al., 1997), rapid fat extraction (Zhang and Wang, 1996), and ultrasonic extraction (Chen, 2015). Among them, the rapid fat extraction method greatly shortened the extraction period and effectively reduced the oxidation of Hippocampus unsaturated fatty acids (Zhang and Wang, 1996). In summary, ultrasonic extraction and rapid fat extraction are generally used in the extraction of fatty acids. Compared with traditional solvent extraction methods with the problems of solvent residue and heating damage to fatty acids, CO₂ supercritical fluid extraction can extract total lipids at low temperatures and without organic solvents (Jiang et al., 2013). Huang and Xu (2016) compared the effects of chloroform-methanol extraction, absolute ethanol extraction, petroleum ether Soxhlet extraction, and CO₂ supercritical fluid extraction on the lipid and fatty acid constituents of Hippocampus. The results showed that the types of fatty acids by the four methods were almost the same while the contents of unsaturated fatty acids in chloroform-methanol extraction and absolute ethanol extraction were the highest, with saturated fatty acids in CO₂ supercritical fluid extraction the highest. The extraction of Hippocampus steroids has generally been performed by ethanol extraction followed by organic solvent (n-butanol, ethyl acetate, and petroleum ether) extraction. The fractions were then repeatedly chromatographed with silica gel column chromatography using a stepwise gradient elution of different solvents (Wu et al., 2017b). The total lipids of the Hippocampus were extracted by chloroform-methanol (2:1), 75% ethanol, and water, and the content of total phospholipids was identified by molybdenum blue colorimetry. Studies have shown that the chloroform-methanol (2:1) extraction method has the highest content of total phospholipids (Xu et al., 1994; Sheng et al., 2011).

The pharmacological activities of Hippocampus peptides include antihypertensive, antioxidation, and anti-inflammation activities (Figure 6). The ACE inhibitory peptide obtained from Hippocampus abdominalis Lesson induced an increase in NO content in blood vessels through the expression of phosphor-endothelial nitric oxide synthase (p-eNOS) to promote vasodilation and lower blood pressure (Je et al., 2020). ACE is part of the renin-angiotensin system and plays a vital role in regulating blood pressure, and ACE inhibitors are effective in the treatment of hypertension (Li et al., 2014). In addition, free radicals are in a special state of balance in the human body, and when this balance is disrupted, they may cause oxidative stress, which can lead to the damage of blood vessels and cardiovascular diseases. Interestingly, Gu and Xu (2016) found that Hippocampus ACE inhibitory peptide showed DPPH radical scavenging activity. One of the medicinal candidates for the treatment of hypertension may be bifunctional peptides with both ACE inhibitory and antioxidant activity. The therapeutic effects of peptide SHP-1 isolated from Hippocampus kuda Bleeker on arthritis were observed with human chondrocyte-like SW-1353 and human osteoblast-like MG-63 cells. The inhibition rates of SHP-1 on collagen release in SW-1353 and MG-63 cells were 40.35–59.96% and 60.82–85.82%, respectively. SHP-1 inhibited the expression of collagenase 1,13 and NO induced by the proinflammatory cytokine 12-O-tetradecanoylphorbol-13-acetate (TPA) and blocked NF-KB/p38 kinase (Ryu et al., 2010). Ryu (2010) further isolated a Hippocampus peptide with alkaline phosphatase activity and showed that it not only promoted the differentiation of MG-63 osteoblasts and SW-1353 chondrocytes but also inhibited the expression of the inflammatory factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). A peptide showed remarkable anti-inflammatory activity by regulating the NF-κB and MAPK signaling pathways. Patients with arthritis have persistent joint swelling and restricted movement due to pain (Mandl, 2019). The anti-inflammatory effect of peptides may be associated with the traditional efficacy of Hippocampus in reducing swelling.

Hippocampus also presents an anti-fatigue effect (Hu et al., 2000). Kang et al. (2017) found that enzymatic hydrolysates of Hippocampus abdominalis Lesson promoted the proliferation of C2C12 myoblasts and increased the contents of physical activity markers, such as ATP and glycogen. Hippocampus enzymatic hydrolysates not only promoted the elimination of lactic acid and urea nitrogen during exercise but also strengthened the antifatigue activity with increasing exercise intensity. Compared with the antifatigue effect of the one-time fatigue model, the incremental exercise fatigue model was more effective. For normal mice, Hippocampus enzymatic hydrolysates improved the survival time under hypoxia and cold conditions (Peng and Chen, 2005). Accordingly, supplementation with Hippocampus enzymatic hydrolysates may be a natural health product that has an improved effect on the human body. For nervous diseases, Hippocampus enzymatic hydrolysates promoted the recovery of neurological function and reduced the volume of cerebral infarction and brain edema in animal models of ischemia-reperfusion brain damage (Feng et al., 2005). The peptide HTP-1 had a protective effect on Aβ₄₂-induced PC12 cells and may be an active ingredient of Hippocampus for the treatment of neurodegenerative disease (Pangestuti et al., 2013). However, the neuroprotective mechanisms of Hippocampus require further investigation. In a coculture system with PC12 cells and BV-2 cells induced by Aβ₄₂, HTP-1-protected PC12 cells were not subjected to the neurotoxicity of BV-2 cells. HTP-1 attenuated the neurotoxic mediators released by BV-2 cells and activated PI3K/Akt by inducing transforming growth factor-β (TGF-β), which is the main signaling pathway of neuronal survival (Pangestuti and Kim, 2015).

The Hippocampus activity of warming and tonifying kidney yang was closely related to the alcohol extract of Hippocampus. Chen et al. (2009) studied the effect of the n-butanol extract from Hippocampus on a mouse model of kidney yang deficiency. After 15 days of continuous administration, the results showed that the n-butanol group improved the rectal temperature, grip strength, testis index, and seminal vesicle index of the model mouse. The ethanol extract of Hippocampus was orally administered to normal and cyclophosphamide model mice, and it was found to increase the overall sperm number and viability. From the perspective of modern science, Hippocampus improves the symptoms of kidney deficiency, such as weakness at the waist and knees, frigidity, chilliness, and cold limbs (Zhang et al., 1995). The total sterol content in Hippocampus kuda Bleeker not only increased the content of testosterone in vivo but also changed the structure of the seminiferous duct and promoted the formation of sperm. However, long-term high-dose intake of total sterols might change the shape of the kidney and testis (Sun, 2015). Over thousands of years, traditional Chinese clinical practices have supported the effect of Hippocampus on improving sexual function, although the material basis and concrete mechanism of efficacy were not clear. The enzymatic hydrolysate of Hippocampus had a certain therapeutic effect on arthritis, while the alcohol extract was more effective in the treatment of neuroinflammation. Himaya et al. (2011, 2012) isolated 1-(5-bromo-2-hydroxy-4-methoxyphenyl)ethanone (SE1) and paeonol from Hippocampus kuda Bleeker and showed that it inhibited the expression of proinflammatory cytokines in BV-2 microglial cells induced by lipopolysaccharide (LPS). This compound played an anti-inflammatory role by inhibiting the phosphorylation of JNK and p38 in the MAPK pathway and nuclear heterotrophs in the NF-kB pathway (Figure 7). Yan et al. (2019) further found that SE1 and paeonol improved the morphology of activated microglia with enlarged cell bodies and shorter synapses. Recent studies have shown that neurodegenerative diseases are closely related to neuroinflammation caused by activated microglia (Sarlus and Heneka, 2017). SE1 and paeonol extracted from Hippocampus-modulated neuroinflammation and may be potential candidates for treating neurodegenerative diseases, such as Alzheimer’s disease (AD). To verify the effects and mechanisms of SE1 and paeonol on AD, in vitro or in vivo experiments with pharmacological approaches are needed in the future. Recently, patients with schizophrenia have been shown to have an increased inflammatory response, and autophagy dysfunction caused by inflammation might continue to increase the inflammatory response. Studies have shown that the alcohol extract of Hippocampus kelloggi Jordan et Snyder exerts anti-inflammatory effects by improving autophagy, thus revealing a new possible target for Hippocampus-based therapeutics to combat schizophrenia (Yan L., 2019). In addition, the methanol extract of Hippocampus increased the concentration of neurotransmitters and the expression of brain-derived neurotransmitters by inhibiting the secretion of proinflammatory factor interleukin-1β (1L-1β), which improved depressive behavior in mice and restored normal neurotransmitter function (Li et al., 2020). The Hippocampus anti-inflammatory effect is associated with the regulation of the MAPK and NF-κB pathways, which are primarily associated with inflammation (Wu et al., 2017a). Wu et al. (2020) identified miR-98-5p from the cellular RNA library, and 3β-hydroxycholest-5-en-7-one isolated from Hippocampus regulated the expression of the miR-98-5p target gene TNFAIP3 to inhibit the excessive secretion of proinflammatory factors and produce an anti-inflammatory effect. The screening of antitumor active ingredients from TCM has become a hot field. Studies have shown that the alcohol extract of Hippocampus has an inhibitory effect on tumor cells. In a study of dihydrotestosterone-induced LNCaP cells of prostate cancer, dual-targeting AKT and AR signaling showed that brassicasterol from Hippocampus abdominalis Lesson exerted an anticancer effect (Xu et al., 2020). Sphingoid bases were purified from Hippocampus using lipid extraction, saponification, and acid hydrolysis (Yu, 2018). The IC₅₀ of human chronic myeloid leukemia cells treated with sphingoid bases for 24 h was 37.17 μg/ml. Further investigation of the mechanisms by Western blot and RT–PCR analysis revealed that sphingoid bases exerted their antitumor activity by inducing apoptosis via both the mitochondrial apoptosis pathway and the death receptor pathway. In addition, sphingoid bases also inhibited the formation of neovascularization and promoted cell apoptosis by inhibiting the expression of COX-2. The antitumor activity of Hippocampus is mainly caused by low- and medium-polarity ingredients and has great research prospects in cancer therapy.

The antioxidant activity of Hippocampus was also explored. Three phthalic acid derivatives isolated by Qian et al. (2012) from the methanol extract of Hippocampus kuda Bleeker had antioxidant activity, among which 2,12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecylphthalate had the strongest antioxidant activity and exhibited dose-dependent effects; moreover, it is a safe and effective antioxidant that inhibited reactive oxygen in mouse macrophages (RAW264.7) and had no toxic effect on cells. The ethanol extract of Hippocampus had a certain immunomodulatory effect. On the one hand, it increased the spleen weight of mice and promoted lymphocyte proliferation and phagocyte function (Chen et al., 1995). On the other hand, it inhibited the delayed-type hypersensitivity induced by dinitrochlorobenzene in mice (Zhu, 2005). Brain monoamine oxidase B (MAO-B) increases during aging, which is considered a sign of aging. The alcohol extract of Hippocampus Kuda Bleeker had a significant inhibitory effect on MAO-B activity in the mouse brain, suggesting that the Hippocampus delayed aging (She et al., 1995). The methanol extract of Hippocampus had an obvious inhibitory effect on the experimental common carotid artery and cerebral thrombosis in mice. The inhibition rate of 200 mg/kg extract on cerebral thrombosis was 46.8%. The main active ingredients were unsaturated fatty acids, and the content of 9,12-octadecadienoic acid accounted for 18.5% (Xu and Xu, 1997). In addition, the ethanol extracts from five species of Hippocampus promoted the differentiation of osteoblasts and the formation of bone nodules and had a certain therapeutic effect on osteoporosis (Wang, 2015).

Antioxidant activity was found in both the water extracts of Hippocampus japonicus Kaup and Hippocampus Kuda Bleeker (Chen et al., 2010; Zheng et al., 2012), suggesting that the water extract of Hippocampus had antioxidant activities. The most important key factor affecting the degree of pharmacological activity was the extraction time, followed by the extraction temperature (Yi et al., 2006). Qian et al. (2008) compared the antioxidant activities of the methanol extract, ethanol extract, and water extract. The antioxidant activity degree of the methanol extract was higher than that of the other two extracts, and its activity degree was between that of α-tocopherol and vitamin C. In general, moderately and less hydrophilic ingredients showed strong antioxidant activity. The warming and tonifying kidney yang effect has been associated with the alcohol extract of Hippocampus. However, the water extract of Hippocampus also improved the ability of the testicles in the mice model with kidney yang deficiency, and the testis index was higher than that observed for the petroleum ether extract at the same dose (Chen et al., 2009).

Sun et al. (2012) synthesized the antimicrobial peptide HKPLP from the brooding pouch cDNA library of Hippocampus kuda Bleeker. The results indicated that the most sensitive bacterium of HKPLP was Staphylococcus aureus. In addition, HKPLP appeared to have a minor inhibitory effect on fungi. Ko et al. (2016) identified a goose-type lysozyme (ShLysG) from Hippocampus abdominalis Lesson that had a certain inhibitory effect against gram-positive/negative bacteria and a potential immune-protecting effect against pathogens. Bacterial pathogen factors are one of the important factors affecting the survival of marine organisms. Isolation of marine microorganisms with antimicrobial activity from the intestinal contents and skin mucus of Hippocampus guttulatus Cuvier promoted the development of breeding and rearing of Hippocampus (Balcazar et al., 2010). In addition, the ethyl acetate extract from Hippocampus significantly improved benign prostatic hyperplasia in castration and testosterone mouse models (Xu et al., 2014).