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. 2023 Aug 19;16(3):344–357. doi: 10.1016/j.chmed.2023.04.001

Chemical constituents, pharmacological activities and quality evaluation methods of genus Hippocampus: A comprehensive review

Zhiyong Zhang a,b, Xiaoyang Zhang a,b, Xi Wang a,b, Xuting Guo a, Xinhao Yan a, Zheng Li a,b,c,⁎, Wenlong Li a,b,c,⁎
PMCID: PMC11283209  PMID: 39072207

Abstract

The genus Hippocampus is a multi-origin animal species with high medicinal and healthcare values. About 57 species of Hippocampus spread worldwide, of which about 14 species can be used as medicine, showing anti-oxidation, anti-inflammation, anti-depressant, anti-hypertension, anti-prostatic hyperplasia, antivirus, anti-apoptotic, antifatigue, and so on. And those pharmacological effects are mainly related to their active ingredients, including amino acids, abundant proteins (peptides and oligopeptides), fatty acids, nucleosides, steroids, and other small molecular compounds. The main means of authentication of Hippocampus species are morphological identification, microscopic identification, thin layer chromatography method, fingerprint method and genomics method. This review will provide useful insight for exploration, further study and precise medication of Hippocampus in the future.

Keywords: chemical constituents, Hippocampus, pharmacological activities, quality evaluation

1. Introduction

Marine biological resources, with their diversity, complexity and specificity, have become an important source of lead compounds for the development of new drugs (Gao et al., 2021, Kumaravel et al., 2012, Stuart et al., 2020). In addition to being a rich source for pharmaceutical drugs, marine natural products are increasingly recognized as a source in the discovery of functional foods and dietary supplements, and provide a useful exploration for breakthroughs in various scientific fields.

Hippocampus, commonly known as the seahorses, is a kind of precious marine medicine with high medicinal and healthcare values. In the traditional Chinese diet, it is often used to make medicated-cuisine and medicated-wines, such as Haima soup, Haima wine et al. (Chen, Shen, Chen, Gao, & Yang, 2015), which have the function of warming kidney to strengthen yang, dispersing nodules and detumescence, and relieving cough and asthma (Chinese Pharmacopoeia Commission, 2020). It is known as ‘South ginseng’ in traditional Chinese medicine (TCM) culture. Pharmacological studies have shown that Hippocampus have anti-oxidation (Chen et al., 2010, Kim et al., 2019, Zheng et al., 2012), anti-inflammation (Chen et al., 2015, Tharuka et al., 2019, Wu et al., 2020), anti-depressant (Li et al., 2020), anti-hypertension (Je et al., 2020), anti-prostatic hyperplasia (Xu et al., 2014), anti-virus (Sandamalika et al., 2021, Tharuka et al., 2019, Udayantha et al., 2021), anti-apoptotic (Kodagoda et al., 2022, Sellaththurai et al., 2020, Wijerathna et al., 2022), anti-fatigue (Guo et al., 2017, Zhang et al., 2019), and other functions (Pangestuti et al., 2013, Sellaththurai et al., 2020, Yuan et al., 2018). And those pharmacological action are mainly related to their active ingredients, including amino acids (Ge et al., 2019, Sari et al., 2017, Zhao, 2018), abundant proteins (peptides and oligopeptides) (Je et al., 2020, Pangestuti et al., 2013), fatty acids (Huang & Xu, 2016, Shen et al., 2016, Su & Xu, 2015), nucleosides (Wei et al., 2015, Zhao et al., 2011), steroids (Wu et al., 2017, Zhao, 2018), and other small molecular compounds (Si et al., 2018, Zhao, 2018).

The Hippocampus is a multi-origin species with the majority of its distribution in tropical, subtropical and temperate seas, of which 70% is found in the Indian, Pacific and Atlantic Ocean (Lourie, Pollom, & Foster, 2016). The Chinese Pharmacopoeia of 2020 edition includes five species of Hippocampus, namely H. kelloggi Jordan et Snyder, H. hitrix Kaup, H. kuda Bleeker, H. trimaculatus Leach, and H. japonicus Kaup (Chinese Pharmacopoeia Commission, 2020). The high economic value of Hippocampus has led to a large international trade (Jiang et al., 2018, Marín et al., 2021), which has led to the use of many similar species as commercial Hippocampus in TCM market. On the other hand, the identification of Hippocampus in the Chinese Pharmacopoeia (2020) only has morphological and microscopic methods, without content determination indicators, which poses a great challenge for the market supervision of Hippocampus herbs (Chinese Pharmacopoeia Commision, 2020). Therefore, it is crucial to develop an among-species identification method for distinguishing different species of Hippocampus.

The literature retrieved was conducted from a number of databases (e.g., PubMed, China National Knowledge Infrastructure (CNKI), Web of Science, Baidu Scholar, Elsevier, Scopus, Springer) to review the biological characterization, chemical constituents, pharmacology, and quality control methods of Hippocampus species, which is helpful to establish a more scientific and perfect quality control standard and provide references for the exploitation for new drug of this species. Even it is significant for finding reasonable substitutes species of medicinal Hippocampus based on this review.

2. Biological characterization

Genus Hippocampus, a kind of marine teleost fish, belongs to the Syngnatidae family, which also includes pipefish and seadragons (Vitturi & Catalano, 1988), and mainly distributed in the Indian, Atlantic and Pacific oceans (Perera, Dahanayaka, & Udagedara, 2017). Hippocampus generally inhabits shallow waters above 30 m in tropical and temperate regions, favoring seagrasses and macroalgal (Pereira, Silveira, & Abilhoa, 2018), which have a suite of unusual biological characteristics shared by these species including male pregnancy and monogamy (Holt, Fazeli, & Otero-Ferrer, 2021). In turn, these uncommon characteristics render them extremely vulnerable to environmental impacts, including climate change on the coral reef and bottom trawling causes to seabed habitats destruction (Scales, 2010, Wei et al., 2017).

The lifespans of Hippocampus species range from approximately one year of the species with small niche to approximately 3–5 years for the large niche. The Hippocampus species have similar shapes with unique body morphology includes a grasping, finless tail, the head positioned at a right angle to their trunk, a brood pouch sealed along the midline, and a raised dorsal fin base (Aylesworth, Loh, Rongrongmuang, & Vincent, 2017). More than 50 species of the genus Hippocampus distributed worldwide (Jiang et al., 2018), and Fig. 1 shows several common Hippocampus species in the TCM market.

Fig. 1.

Fig. 1

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Several common Hippocampus species (A, medicinal; B, adulterants) in TCM market.

3. Chemical constituents

With the upgrading and updating of analytical technology, and the continuous innovation of analytical instruments, the complex chemical components in TCM can be well separated and determined, which is of great help in elucidating their pharmacological mechanism of action (Shen, He, & Shi, 2021). Recent studies show that the main chemical constituents of Hippocampus include amino acids, polypeptides, steroids, phospholipids, fatty acids, nucleosides, and other compounds.

3.1. Amino acids and polypeptides

Amino acids are natural compounds that are involved in the regulation of various immune activities in the human body and can enhance the immunity of the organism (Bongioanni, Bueno, Mezzano, Longhi, & Garnero, 2021). Zhao et al. (2018) identified 33 compounds including 17 of amino acids and 16 other small molecules from three different Hippocampus (H. kelloggi, H. kuda and H. trimaculatus) using 1H NMR (nuclear magnetic resonance) technique. Lin et al. (2008) analyzed the chemical composition of six species of Hippocampus from the China coastal and found that the major amino acids (> 5% of the total) in Hippocampus were arginine, aspartic acid, glutamic acid, alanine, and glycine. Sari, Nurilmala, & Abdullah, 2017 identified 15 amino acids from the Hippocampus including nine essential and six non-essential amino acids. Je et al. (2020) isolated three angiotensin-converting enzyme (ACEs) inhibitory peptides from Hippocampus extract, and identified as Ala-Pro-Thr-Leu, Cys-Asn-Val-Pro-Leu-Ser-Pro-Pro-Leu, Pro-Trp-Thr-Pro-Leu with Q-TOF-MS. Kim, Kim, Fernando, & Sanjeewa, 2019 used quadrupole time-of-flight (Q-TOF) mass spectrometer combined with ESI source to study the chemical components with antioxidant activity in Hippocampus. The results showed that the tripeptide (Ala-Gly-Asp) had strong antioxidant activity. The chemical structures of amino acids and polypeptides in Hippocampus are shown in Table 1 and Fig. 2.

Table 1.

Amino acids and polypeptides isolated from Hippocampus.

No. Compounds Analytical techniques Hippocams species References
1 Isoleucine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
2 Leucine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
3 Valine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
4 Alanine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
5 Arginine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
6 Glutamic acid 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
7 Methionine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
8 Hydroxyproline 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
9 Proline 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
10 Aspartic acid 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
11 Ornithine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
12 Taurine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
13 Lysine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
14 Glycine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
15 Cysteine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
16 Tyrosine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
17 Phenylalanine 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
18 Threonine HPLC H. barbourin, H. comes Cantor, H. kuda Sari, Nurilmala, & Abdullah, 2017
19 Serine HPLC H. barbouri, H. comes, H. kuda Sari, Nurilmala, & Abdullah, 2017
20 Histidine HPLC H. barbouri, H. comes, H. kuda Sari, Nurilmala, & Abdullah, 2017
21 Ala-Pro-Thr-Leu Q-TOF-MS/MS H. abdominalis Je et al., 2020
22 Cys-Asn-Val-Pro-Leu-Ser-Pro-Pro-Leu Q-TOF-MS/MS H. abdominalis Je et al., 2020
23 Pro-Trp-Thr-Pro-Leu Q-TOF-MS/MS H. abdominalis Je et al., 2020
24 Ala-Gly-Asp Q-TOF-MS/MS H. abdominalis Kim, Kim, Fernando, & Sanjeewa, 2019
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Fig. 2.

Fig. 2

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Chemical structures of amino acids and polypeptides isolated from Hippocampus (compounds 21–23 are not listed in figure due to compound 24 sampling them).

3.2. Fatty acids

Fatty acid components include saturated fatty acids, unsaturated fatty acids, essential fatty acids, triglyceride oils, phospholipids and many other categories, which are the basic substances of life and have the functions of lowering serum cholesterol, improving blood circulation, and preventing cardiovascular diseases (Dyall et al., 2022, Ibarguren et al., 2014). Shen, Dai, Huang, & Cheng, 2016 established a new technology for extraction, visualization and quantitative analysis of phospholipids in Hippocampus. After being extracted and purified using solid phase extraction (SPE) technology, the samples were analyzed by hydrophilic interaction liquid chromatography (HILIC) coupled with Q-TOF-MS, and 50 kinds of phospholipid molecules were isolated and identified, including 15 kinds of PCs, 14 kinds of PEs, 12 kinds of PIs and nine kinds PSs. Su & Xu (2015) extracted the fatty acids from Hippocampus by CO2 supercritical fluid extraction technique, and the composition and distribution of fatty acids were determined by gas chromatography-mass spectrometry (GC–MS). Huang & Xu (2016) compared the extraction efficiency of fatty acids from Hippocampus under different extraction methods, and analyzed their fatty acid composition by GC–MS. Zhao (2018) used GC–MS analytical techniques combined with chemometric methods to compare the chemical composition of different species of Hippocampus. By comparing the standard substances, a total of 42 compounds were identified. It was found that fatty acids were the main factor affecting the difference in chemical compound between the H. histrix and the H. kuda. The chemical structures of fatty acids in Hippocampus are shown in Table 2 and Fig. 3.

Table 2.

Fatty acids isolated from Hippocampus.

No. Compounds Analytical techniques Hippocams species References
25 Sphingomyelin HILIC-Q-TOF-MS/MS H. histrix, H. trimaculatus, H. japonicus, H. kelloggi, H. spinosissimus Shen, Dai, Huang, & Cheng, 2016
26 Phosphatidyl cholines HILIC-Q-TOF-MS/MS H. histrix, H. trimaculatus, H. japonicus, H. kelloggi, H. spinosissimus Shen, Dai, Huang, & Cheng, 2016
27 Phosphatidyl ethanolamines HILIC-Q-TOF-MS/MS H. histrix, H. trimaculatus, H. japonicus, H. kelloggi, H. spinosissimus Shen, Dai, Huang, & Cheng, 2016
28 Phosphatidyl inositols HILIC-Q-TOF-MS/MS H. histrix, H. trimaculatus, H. japonicus, H. kelloggi, H. spinosissimus Shen, Dai, Huang, & Cheng, 2016
29 Phosphatidyl serines HILIC-Q-TOF-MS/MS H. histrix, H. trimaculatus, H. japonicus, H. kelloggi, H. spinosissimus Shen, Dai, Huang, & Cheng, 2016
30 Diphosphatidylglycerol HILIC-Q-TOF-MS/MS H. histrix, H. trimaculatus, H. japonicus, H. kelloggi, H. spinosissimus Shen, Dai, Huang, & Cheng, 2016
31 Phosphatidic acid HILIC-QTOF/MS H. histrix, H. trimaculatus, H. japonicus, H. kelloggi, H. spinosissimus Shen, Dai, Huang, & Cheng, 2016
32 Myristic acid GC–MS H. kelloggi Su & Xu, 2015
33 Palmitic acid 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
34 6-Hexadecenoic acid GC–MS H. kuda Su & Xu, 2015
35 9-Hexadecenoic acid GC–MS H. kuda Su & Xu, 2015
36 6,9-Hexadecadienoic acid 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
37 Heptadecanoic acid GC–MS H. kuda Huang et al., 2016
38 Stearic acid GC–MS H. kuda Huang et al., 2016
39 9-Octadecenoic acid 1H NMR H. trimaculatus Wu et al., 2017
40 12-Octadecenoic acid GC–MS H. kuda Su & Xu, 2015
41 8,11-Octadecadienoic acid GC–MS H. kuda Su & Xu, 2015
42 9,12-Octadecadienoic acid GC–MS H. kuda Su & Xu, 2015
43 6,9,12-Octadecatrienoic acid 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
44 Arachidic acid 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
45 11-Eicosenoic acid GC–MS H. kuda, Su & Xu, 2015
46 5,8-Eicosadienoic acid HPLC H. kuda, H. trimaculatus, H. kelloggi, H. spinosissimus, H. histrix, H. comes Lin et al., 2008
47 5,8,11,14-Eicosatetraenoic acid GC–MS H. kuda Su & Xu, 2015
48 Docosanoic acid GC–MS H. kuda Su & Xu, 2015
49 13-Docosenoic acid GC–MS H. kuda Su & Xu, 2015
50 Docosahexaenoic acid GC–MS H. kuda Su & Xu, 2015
51 Tetracosanoic acid 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
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Fig. 3.

Fig. 3

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Chemical structures of fatty acids isolated from Hippocampus.

3.3. Steroids

Steroids are a class of natural chemical components that are widely found in nature. Wu et al. (2017) isolated nine compounds from H. trimaculatus, including four steroids. Zhao (2018) found that the content of androst-4-ene-3,17-dione in H. histrix was significantly different from that of the other four species of Hippocampus in the Chinese Pharmacopoeia, and was more suitable for its aphrodisiac effect. The chemical structures of steroids in Hippocampus are shown in Table 3 and Fig. 4.

Table 3.

Steroids isolated from Hippocampus.

No. Compounds Analytical techniques Hippocams species References
52 Cholest-4-en-3-one 1H NMR H. trimaculatus Wu et al., 2017
53 3β-Hydroxycholest-5-en-7-one 1H NMR H. trimaculatus Wu et al., 2017
54 Cholest-5-ene-3β, 7β-diol 1H NMR H. trimaculatus Wu et al., 2017
55 Cholest-5-ene-3β, 7ɑ-diol 1H NMR H. trimaculatus Wu et al., 2017
56 Cholesterol 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
57 Cholestane-3,6-dione 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
58 Cholesteryl stearate 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
59 3β, 5α, 9α-Trihydroxy-ergosterol-7, 22-dien-6-one 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
60 24-Methyl-5α-cholest-7,22-dien-3β, 5, 6β-triol 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
61 3β-Hydroxy-7-methoxy-cholesta-5-en 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao, 2018
62 Androst-4-ene-3,17-dione 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
63 Cholest-4-ene-3, 6-dione 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
64 1, 2-Epoxycholestan-3-one 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
65 β-Sitosterol 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
66 Cholesta-3, 5-dien-7-one 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
67 Cholesta-4, 6-dien-3-one 1H NMR H. kelloggi, H. kuda, H. trimaculatus Zhao et al., 2018
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Fig. 4.

Fig. 4

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Chemical structures of steroids isolated from Hippocampus.

3.4. Nucleosides

Nucleosides are involved in mediating various physiological activities in the body and have a variety of biological activities. Zhao et al. (2011) developed a new method for the simultaneous determination of 16 nucleosides and nucleobases in various marine organism extracts based on ultrasound-assisted extraction (UAE), HILIC and ESI-TOF/MS. All 16 compounds were detected in Hippocampus extracts. Yan, Zhang, & Lin, 2019 investigated the nutritional and functional components of Hippocampus and identified a variety of components including amino acids, nucleosides, and fatty acids. Wei, Xu, Wei, Gao, & Wang, 2015 established a method for simultaneous determination of five nucleosides in Hippocampus, which provides assurance for their quality control. The chemical structures of nucleosides in Hippocampus are shown in Table 4 and Fig. 5.

Table 4.

Nucleosides isolated from Hippocampus.

No. Compounds Analytical techniques Hippocampus species References
68 Thymine HPLC-TOF/MS H. japonicus Zhao et al., 2011
69 Uracil HPLC-TOF/MS H. japonicus Zhao et al., 2011
70 Thymidine HPLC-TOF/MS H. japonicus Zhao et al., 2011
71 2′-Deoxyadenosine HPLC-TOF/MS H. japonicus Zhao et al., 2011
72 Adenine HPLC-TOF/MS H. japonicus Zhao et al., 2011
73 Uridine HPLC-TOF/MS H. japonicus Zhao et al., 2011
74 Adenosine HPLC-TOF/MS H. japonicus Zhao et al., 2011
75 Hypoxanthine HPLC-TOF/MS H. japonicus Zhao et al., 2011
76 Xanthine HPLC-TOF/MS H. japonicus Zhao et al., 2011
77 Cytosine HPLC-TOF/MS H. japonicus Zhao et al., 2011
78 Inosine HPLC-TOF/MS H. japonicus Zhao et al., 2011
79 2′-Deoxycytidine HPLC-TOF/MS H. japonicus Zhao et al., 2011
80 Guanine HPLC-TOF/MS H. japonicus Zhao et al., 2011
81 2′-Deoxyguanosine HPLC-TOF/MS H. japonicus Zhao et al., 2011
82 Cytidine HPLC-TOF/MS H. japonicus Zhao et al., 2011
83 Guanosine HPLC-TOF/MS H. japonicus Zhao et al., 2011
84 2′-Deoxyinosine HPLC-TOF/MS H. japonicus Zhao et al., 2011
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Fig. 5.

Fig. 5

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Chemical structures of nucleosides isolated from Hippocampus.

3.5. Others

In addition, compounds 85–102 (Table 5, Fig. 6) also were isolated from Hippocampus (Si et al., 2018, Wu et al., 2017, Zhao et al., 2018).

Table 5.

Others compounds isolated from Hippocampus.

No. Compounds Analytical techniques Hippocampus species References
85 Creatinine HS-SPME-GC–MS H. kuda Si, Ge, Xu, & Wang, 2018
86 Bis(2-ethylhexyl) phthalate 1H NMR H. trimaculatus Wu et al., 2017
87 Dibutylphthalate 1H NMR H. trimaculatus Wu et al., 2017
88 Glycerol 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao, 2018
89 Xylitol 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao, 2018
90 Inositol 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao, 2018
91 Glucitol 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao, 2018
92 Lactic acid 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
93 Amber acid 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
94 Citric acid 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
95 Creatine 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
96 Choline 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
97 Betaine 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
98 Taurine 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
99 Lactose 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
100 Fumaric acid 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
101 2-Hydroxy-4-methoxyacetophenone 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
102 2-Ethyl-11-methylhexadecyl phthalate 1H NMR H. kelloggi, H. kuda, H. trimaculats Zhao et al., 2018
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Fig. 6.

Fig. 6

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Chemical structures of other components isolated from Hippocampus.

4. Pharmacological activities

Hippocampus is a kind of tonic herb with the effects of warming kidney to strengthening yang, dispersing nodules and detumescence, relieving cough and asthma, relaxing muscles and activating collaterals, relieving pain and inflammation, calming nerves (Chen, Shen, Chen, Gao, & Yang, 2015). Modern pharmacological studies have shown that Hippocampus have anti-oxidation, anti-inflammation, anti-depressant, anti-hypertension, anti-prostatic hyperplasia, anti-virus, anti-apoptotic, anti-fatigue, and other functions (Fig. 7).

Fig. 7.

Fig. 7

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Health care function and active ingredients of Hippocampus.

4.1. Anti-oxidation

The active components in Hippocampus have scavenging free radicals scavenging and antioxidation effects. Ge, Gu, & Xu, 2019 investigated the antioxidant activity of different types of amino acids. It was found that polar and nonpolar amino acids synergistically increased the scavenging efficiency by increasing the effective concentration of free radical scavenging, and the antioxidant capacity per unit of polar amino acids was about 1.2 times higher than that of nonpolar amino acids. In addition, the antioxidant capacity per unit of aromatic amino acids was about two times higher than that of aliphatic amino acids. In assessing the antioxidant activity of Hippocampus, Kim, Kim, Fernando, & Sanjeewa, 2019 found that increasing the protein hydrolysate (HPH) of H. abdominalis was effective in reducing the level of reactive oxygen species (ROS) and cell death induced by 2,2‐azobis hydrochloride (AAPH) in zebrafish embryo cells. In the study on the change of antioxidant activity of polypeptide compounds in Hippocampus extracts during enzymatic hydrolysis, Guo et al. (2017) found that under the optimal conditions, the peptide induced by papain has high antioxidant activities. In the study of the protective and inhibitory effects of alkaline protease on low-density lipoprotein (LDL) oxidation in Hippocampus hydrolysate, Oh et al. (2018) found that H. abdominalia hydrolysates (SHAH) showed high antioxidant capacity. Nadarajapillai et al. (2021) analyzed the antioxidant properties of glutathione S-transferases alpha-4 from H. abdominalis (HaGSTA-4). Recombinant HaGSTA-4 was discovered to significantly protect cells from ROS inducers stress. In addition, overexpression of HaGSTA-4 protected cells from H2O2-induced oxidative stress. Thioredoxin domain-containing protein 17 (TXNDC17), which contains the structural domain of thioredoxin (Trx), can be involved in maintaining cellular redox homeostasis through thiol-disulfide reductase activity. Liyanage et al. (2019) identified TXNDC17 from H. abdominalis to determine free radical scavenging ability, antioxidant activity and cell viability. The study results indicate that HaTXNDC17 is involved in the immune mechanism of genus Hippocampus and has a role in maintaining cellular redox homeostasis. Trx plays a crucial role in the antioxidant defense system. Nadarajapillai, Sellaththurai, Liyanage, Yang, & Lee, 2020 characterized the mitochondrial Trx protein (HaTrx-2) from H. abdominalis, and the study results indicate that rHaTrx-2 had the ability to scavenge the free radicals. Peroxiredoxins (Prxs) are ubiquitously expressed antioxidant proteins that can protect aerobic organisms from oxidative stress. The studies have found that the recombinant H. abdominalis peroxiredoxins (HaPrx3, HaPrx4) protein exhibited insulin disulfide reduction activity, peroxidase activity and cell survival ability (Samaraweera et al., 2020, Samaraweera et al., 2021). Glutaredoxins (Grx) are redox enzymes conserved in viruses, eukaryotes, and prokaryotes. The studies have found that the recombinant H. abdominalis glutaredoxins (HaGrx1, HaGrx2) exhibited redox activity and cytoprotective activity (Omeka et al., 2019, Omeka et al., 2019).

4.2. Anti-inflammation

Inflammation is a defense response of the body to stimulation, characterized by redness, swelling, heat and pain. Wu et al. (2017) studied the anti-inflammation activity of 3β-hydroxycholesterin-5-en-7-one (HEO), which was isolated from H. trimaculatus. The results showed that HEO can significantly inhibit the expression of inflammatory factors of nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-1β (1L-1β), leading to achieve the purpose of anti-inflammation. On the other hand, Wu et al., (2020) showed that HEO exerted an anti-inflammatory effect via miR-98-5p. Cellular signaling analyses demonstrated that the HEO downregulated the nuclear factor κB (NF-κB) and extracellular signal-regulated kinase (ERK) of mitogen-activated protein kinase (MAPK) signaling pathways. These results lay a foundation for the development of new drug targets. IL-10 is a pleiotropic cytokine involved in the regulation of innate immunity and acquired immunity. In the study of Hippocampus IL-10, Tharuka, Priyathilaka, Yang, Pavithiran, & Lee, 2019 found that H. abdominalis IL-10 can significantly reduce the protein expression of inducible nitric oxide synthase and cyclooxygenase-2 in raw-264.7 cells induced by lipopolysaccharide (LPS). In the study of anti-inflammation active components in the extract of H. Trimaculatus Leach, Chen et al. (2015) found that lipid extracts of Hippocampus could inhibit the release of IL-6, IL-1β and TNF-α. Zhang et al. (2019) successfully isolated 2′-hydroxy-5′-methoxyacetophenone (2H5M) from Hippocampus extract, and observed the anti-inflammation effect of the compound in BV-2 cells and RAW264.7 cells stimulated by LPS. Molecular docking studies showed that 2H5M could form an active site with NF-κB, indicating that 2H5M has anti-inflammation effect.

4.3. Anti-depression

Hippocampus is an herb with sedative and sleep improving properties. In the study of the main mechanism of antidepressant effect on Hippocampus, Li et al. (2020) measured the concentrations of serum corticosterone, glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF), IL-1β, and monoamine neurotransmitters in CUMS-exposed mice after feeding hippocampus diet. It was found that feeding Hippocampus could increase the concentrations of neurotransmitters, BDNF, IL-1β and ROS significantly, while the concentrations of IL-10, antioxidant superoxide dismutase and glutathione peroxidase were reduced, and that dietary Hippocampus were effective in reversing anxiety and depression-like behaviors.

4.4. Anti-hypertension

Je et al. (2020) elucidated the vasodilation mechanism caused by the inhibition of ACE in Hippocampus. It was found that the peptides isolated from Hippocampus extracts could cause vasodilation through ACE inhibition, thus reducing the blood pressure of SHR (spontaneously hypertensive rat). Seahorse was hydrolyzed by Protamex (SHP) inhibits angiotensin-converting enzyme secretion and increases nitric oxide production, showing anti-hypertension effect.

4.5. Anti-prostatic hyperplasia

Hippocampus can enhance male function by dilating blood vessels. Xu et al. (2014) investigated the effects of Hippocampus spp. extracts in a rat model of benign prostatic hyperplasia (BPH) and oligospermia. It was found that Hippocampus extracts reduced the prostate index, increased penile NOS activity, decreased acid phosphatase (ACP) activity and prostatic proliferating cell nuclear antigen (PCNA) and basic fibroblast growth factor (bFGF) expression, and restored sperm viability and motility. Hippocampus extract may be a candidate marine drug for BPH.

4.6. Antivirus

Glutathione S-transferases (GSTs) are important enzymes involved in phase II detoxification. Udayantha et al. (2021) isolated an omega class GST from H. abdominalis (HaGSTO1) to study the defense ability against viral and bacterial infections. It was found that GSTO1 expression is significantly elevated after attack by bacteria and PAMPs, which indicated that HaGSTO1 is involved in the host defense mechanism in Hippocampus. Viperin is an antiviral protein. Tharuka, Priyathilaka, Yang, Pavithiran, & Lee, 2019 isolated a viperin homolog from H. abdominalis (HaVip) to determine its antiviral activity in vitro. It was found that HaVip could trigger antiviral and antibacterial responses, upon viral and bacterial pathogenic infections. Sandamalika, Samaraweera, Yang, & Lee, 2021 identified thioredoxin domain containing from Hippocampus abdominalis (ShTXNDC5), which was shown to have a potentially protective role against bacterial and viral invasions.

4.7. Antiapoptotic

Clusterin (CLU) is a glycoprotein that functions in different cell signaling pathways that are associated with various diseases. Wijerathna et al. (2022) investigate the bioactivity of CLU from H. abdominalis (HaCLU) on oxidative stress-induced cell death. The study found that HaCLU has anti-apoptotic function, inhibiting H2O2-induced oxidative stress and subsequent cell death. Cystatins play a crucial role in diverse pathophysiological conditions in animals. Kodagoda et al. (2022) showed that cystatin B from H. abdominalis (HaCSTB) increased the cell viability and reduced cell apoptosis upon VHSV infection. Sellaththurai et al. (2020) identified and characterized malectin from H. abdominalis (HaMLEC). It was found that over expression of HaMLEC can down regulate the viral transcription in vitro.

4.8. Antifatigue

In the study of anti-fatigue activity of Hippocampus, Guo et al. (2017) found that Hippocampal polypeptide could prolong the swimming time of mice by 33%−40%, stabilize blood glucose concentration, increase liver glycogen level and reduce blood lactate and blood urea nitrogen level. Zhang et al. (2019) purified and isolated a new peptide (SH200) from H. abdominalis. It was found that SH200 can prolong the time of cerebral ischemia–reperfusion in rats, which was a candidate natural preparation for alleviating body fatigue.

4.9. Other functions

Calreticulin (CRT) is a multi-functional, ubiquitous protein known for a variety of cellular functions. Sellaththurai et al. (2020) identified and characterized CRT from H. abdominalis (HaCRT) and analyzed the functional properties. The recombinant HaCRT demonstrated the detectable wound-healing ability. Pangestuti, Ryu, Himaya, & Kim, 2013 isolated and identified neuroprotective peptides from H. trimaculatus (HTP-1). HTP-1 has the ability to protect PC12 cells from Aβ42-induced neuronal death, which has the potential to be used in treatment of neurodegenerative diseases, particularly alzheimer disease. Oh et al. (2018) found that SHAH exhibited anti-atherogenic effects in oxidative stress-mediated human umbilical vein endothelial cell. Yuan et al. (2018) isolated active compound 1-(5-bromo-2-hydroxymethoxyphenyl)-ethenone (BHM) from Hippocampus and loaded it on TiO2/Sr doped hydroxyapatite (TiO2/Srha) composite scaffold to study the controlled release kinetics of BHM. The results showed that the TiO2/Srha/BHM composite exhibited good biocompatibility at a certain concentration of BHM (20 mol/L). The phenolic compound BHM mediated by the TiO2/Srha composite scaffold could be used for bone tissue repair. Ryu et al., 2010a, Ryu et al., 2010b found that a peptide (LEDPFDKDDWDNWK, 1821 Da) isolated from genus Hippocampus has the function of inducing the differentiation of osteoblast MG-63 and chondrocyte SW-1353 cells. In a study of collagen release in arthritis, Ryu et al., 2010a, Ryu et al., 2010b found that a novel peptide of Hippocampus hydrolysis product (SHP-1) has the function of inhibiting collagen and glycosaminoglycan (GAG) release.

5. Quality control methods

5.1. Market analysis of Hippocampus

As a multi-origin species, there are about 57 species of Hippocampus distributed worldwide, 14 of which are used as medicine. The high economic value of Hippocampus has led to a large international trade of it, and many similar morphological species are used as medicinal Hippocampus. For example, H. giraffe, which is difficult to distinguish by morphological identification, is often traded as a kind of adulterant of Hippocampus (Chen, 2015, Lai et al., 2019, Lai et al., 2019). Jiang et al. (2018) identified 23 different species from more than 1 000 Hippocampus samples collected from the TCM market, and the five species included in the pharmacopoeia accounted for 44.22% of the total number of Hippocampus, among which H. trimaculatus and H. japonicus accounted for the largest proportion, while the H. histrix was the rarest. Among the adulterants, H. spinosa and H. pacific are most commonly seen, accounting for 18.26% and 11.95% of the total, respectively.

5.2. Morphological identification

The unique body morphology of Hippocampus includes a grasping, finless tail, the head positioned at right angle to their trunk, a brood pouch sealed along the midline, and a raised dorsal fin base. He, Zhang, & Huang, 2021 identified different species of Hippocampus using the morphological identification method. This method was effective in distinguishing different species of Hippocampus circulating in the market, and was simple and fast. The morphological characteristics and geographical origins distribution of the five medicinal Hippocampus in Chinese Pharmacopoeia (2020) is shown in Table 6.

Table 6.

Morphological characteristics and geographical origins distribution of five medicinal Hippocampus in Chinese Pharmacopoeia.

Names Geographical origins Main morphological characteristics References
H. kelloggi Jordan et Snyder America, Australia, Caribbean, Cuddalore Coastal Water, Southeast Coastal of India, Coastal of China Body length 25–30 cm, yellowish white or dark brown Balasubramanian and Murugan, 2017, Chen, 2015, He et al., 2021; Harasti, 2017
H. histrix Kaup Japan, Singapore, Red Sea, Coastal of China, Hawaiian Islands, French Polynesia, Australia Body length 12–20 cm, Yellowish white or dark Chen, 2015, He et al., 2021, Stocks, Foster, & Bat, 2017, Wen, Li, Wan, Ren, & Guo, 2013
H. kuda Bleeker North Korea, Japan, Philippines, Northern Australia, Eastern Africa, Red Sea and Coastal of China Body length 12–25 cm, yellowish white or dark brown Chen, 2015, Harasti, 2015, He et al., 2021, Wang, 2012
H. trimaculatus Leach Coastal of China, East Africa and the Indian Body length 8–16 cm, yellowish white or dark brown Chen, 2015, Choo & Liew, 2006, He et al., 2021
H. japonicus Kaup Japan, North Korea, Western Pacific, Thailand and Cambodia, Coastal of China, Vietnam Body length 5–8 cm, dark brown Chen, 2015, He et al., 2021, Thangaraj, Lipton, & John, 2012
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Morphological identification can distinguish most species of Hippocampus, while a few species of Hippocampus are difficult to distinguish, for example, both H. histrix and H. spinosissimus have many spines on their bodies, which often cause confusion. Moreover, after processing, the appearance characteristics of the Hippocampus will change, it is difficult to identify the species by morphological identification alone (Jiang et al., 2017). Therefore, it is necessary to strengthen the basic research on Hippocampus, establish its quality control standards, and improve the confusing situation of Hippocampus sales.

5.3. Thin layer chromatography (TLC) method

TLC methods use the adsorbent's different adsorption capacity for each component of the sample to achieve the separation of each component (Kucherenko et al., 2019), and is always used for the quality control of TCM, which can reflect their internal quality directly. Wang (2015) used TLC method to study the differences of different Hippocampus samples. The results showed that individual variability existed between different Hippocampus samples, and for the problem of confusion in the sale of Hippocampus in the TCM market, the physical and chemical identification by TLC could achieve the purpose of differentiation, but to identify the variability of different species of Hippocampus needs to be studied in combination with other analytical methods.

5.4. Fingerprint method

Fingerprint of TCM is a comprehensive and quantifiable identification method, mainly including chromatographic fingerprints and spectral fingerprints (Wang et al., 2021). Among which, infrared spectroscopy is a qualitative and quantitative analysis method based on the absorption characteristics of infrared radiation (Simbizi, Gahungu, & Nguyen, 2020). Wang (2012) used Fourier transform infrared (FT-IR) combined with cluster analysis to perform spectral scanning of 33 batches of Hippocampus raw herbs and their extracts. The results of clustering analysis showed that the IR fingerprint spectra of the raw herbs showed unsatisfactory clustering results and the alcoholic extracts showed better results. High performance liquid chromatography (HPLC) method can separate the complex compound system and form a characteristic chromatogram (Staniak et al., 2020). Wang et al. (2009) established the HPLC fingerprints method for the analysis of H. japonicus, and used the similarity calculation software of Chinese medicine fingerprint profile to identify the authenticity and quality evaluation of H. japonicus. Li et al. (2009) established an HPLC fingerprints method to authenticity identification of H. trimaculatus.

5.5. Genomics method

Genomics technology refers to the combination of DNA sequences with a variety of molecular technical tools, thus elucidating the relationship between the structure and function of the whole genome and the interaction between genes (Gupta, 2008). Among them, DNA barcoding technology has great application potential in wildlife conservation (Galimberti et al., 2019). Sun, Fang, & Lai, 2019 systematically studied the Hippocampus by combining the morphological characteristics of Hippocampus with DNA barcodes. The results showed that CO-I and ATP6 barcodes could be used as indicators for studying the geographical ecology of vertebrates, which laid a foundation for rapid and accurate identification of medical Hippocampus species. Liu et al. (2018) developed a multiplex polymerase chain reaction (mPCR) method to identify the biological origin of Hippocampus. Based on the DNA sequence of mitochondria, specific primers for Hippocampus were designed. The multiplex PCR technology can be used for the simultaneous identification of complex multi-origin samples, which provides a new idea for quality control of Hippocampus. Hou, Wen, Peng, & Guo, 2018 identified nine different species of Hippocampus using the CO-I gene, and the results showed that the DNA barcode technology based on CO-I identify different Hippocampus species accurately. Lai et al., 2019, Lai et al., 2019 used morphological identification combined with DNA barcodes to conduct a biopharmacological study on H. japonicus, which provided support for the identification technology development and genetic diversity study of it. Chen et al. (2019) established a DNA barcoding database of CO-I, 16S rRNA and ATP6 sequences of H. trimaculatus and identify H. trimaculatus and other adulterants quickly and accurately. Lai et al., 2019, Lai et al., 2019 report the complete mitochondrial genome of H. camelopardalis Bianconi 1854, which provided essential and important molecular data for evolutionary analysis of genus Hippocampus.

Through reviewing the documents, it was found that the quality control methods of animal drugs are not complete, and the relevant literature only has the trait identification and microscopic identification items, lacking quantitative indicators, which may be related to the fact that the material basis of the pharmacological effect of animal drugs is still unclear, it will be an interesting research direction. The Chinese Pharmacopoeia of 2020 edition stipulates that adenosine can be used as a quantitative indicator of Cordyceps (Qian et al., 2021). Some scholars have similarly determined nucleosides in a variety of marine organisms, which points to a direction for quality control of animal drugs (Zhao et al., 2011).

6. Conclusion

Marine biological resources have become an important source of leading compounds in new drug research and development due to their diversity, complexity and specificity. As a precious marine animal medicine, Hippocampus have high medicinal and economic value. However, there is no comprehensive quality evaluation method for Hippocampus yet, which seriously affects the further processing of it. Therefore, it is necessary to strengthen the basic research of Hippocampus, establish its quality control standards, and improve the confusion status of Hippocampus sales. In the Chinese Pharmacopoeia of 2020 edition, there are only morphological and microscopic ways for the identification of Hippocampus, which requires high artificial experience and operation technology. This paper reviews the biological characterization, distribution, active ingredients, pharmacological actions, species identification and quality analysis methods of Hippocampus, which is helpful to establish a more scientific and perfect quality control standard and provide references for the exploitation for new drug of this species. And also, rapid analysis means of Hippocampus are needed, such as machine vision technology, spectroscopy and imaging technology, combined with artificial intelligence system, which can realize the rapid identification and automatic sorting of Hippocampus medicinal materials.

CRediT authorship contribution statement

Zhiyong Zhang: Conceptualization, Methodology, Software, Writing – original draft. Xiaoyang Zhang: Data curation, Software. Xin Gao: Data curation, Software. Xinhao Yan: Data curation. Xuting Guo: Data curation. Zheng Li: Writing – review & editing. Wenlong Li: Writing – review & editing. Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are extremely grateful for the financial support of the National key research and development program 2019YFC1711505, Hebei Industrial Innovation and Entrepreneurship Team (No. 215A2501D), Key Research and Development Projects in Hebei Province (No. 21372503D), Wuhan Science and Technology Project (No. 2020020602012116).

Contributor Information

Zheng Li, Email: lizheng@tjutcm.edu.cn.

Wenlong Li, Email: wshlwl@tjutcm.edu.cn.

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