Abstract: Echinacea Extract is a perennial Compositae that is rich in caffeic acid derivatives, alkylamides, polysaccharides and glycoproteins and other active substances. Echinacea and its extracts have significant immunomodulatory and growth-promoting effects , widely used in human medicine, and increasingly used in animal husbandry and aquaculture. In this study, the main active components, immunomodulatory effects and mechanisms of Echinacea purpurea, as well as the application status of Echinacea purpurea in aquaculture, were systematically reviewed. The diversity of aquatic animals, the specific mechanism of promoting growth and regulating immunity, as well as the use strategy, compound effect, extraction process, etc. should be strengthened. The results of this study can provide reference for the promotion and application of Echinacea in aquaculture.
Key words : echinacea; growth promotion; immune regulation; aquaculture
Echinacea Extract , also known as Echinacea , is a perennial herb belonging to the phylum Angiosperms, Dicotyledon, Platycodon, Compositae and Echinacea . There are 9 species in this genus, among which E. Extract , E. angustifolia and E. pallida have been developed into medicines and are widely used in medicine . Native to North America, Echinacea is one of the most widely used dietary supplements in Europe and the United States. It is often used in the prevention or early treatment of colds and is a world-famous “immune” herb. Modern pharmacological studies have shown that Echinacea has the functions of improving immunity , resisting infection and reducing inflammatory damage , and is often used to treat influenza and promote wound healing . A large number of in vitro and in vivo experiments have shown that Echinacea can enhance the body’s immunity, and its immune stimulation and anti-inflammatory effects have a good prevention and control effect on bacterial and viral diseases.
In recent years, Echinacea and its extracts have been widely used as a feed additive in livestock and poultry production, and have also shown good growth-promoting and immune-regulating effects in aquatic animals such as fish and shrimp. In June 2012, Echinacea and its preparations were approved by the Ministry of Agriculture and Rural Affairs of China as a national first-class new veterinary drug, which made Echinacea have a wide range of research and application prospects in aquaculture. In this study, the main active components, immunomodulatory effects and mechanisms of Echinacea, as well as its application in aquaculture, were reviewed, and its application prospects and development trends in aquaculture were prospected. Provide reference for the promotion and application of aquaculture.
1 Active components, immunomodulatory effects and mechanisms of Echinacea Extract
The chemical composition of Echinacea is very complex, and its main active components include polar components (caffeic acid and its derivatives), non-polar components (alkylamides), and high molecular weight components such as polysaccharides and glycoproteins . The contents and effects of active ingredients of Echinacea vary greatly with the different origins, growth periods, parts and processing methods. In 2004, Gertsch et al. first explored the possible molecular mechanism of the anti-inflammatory effect of Echinacea, and believed that the active components of Echinacea, alkylamides, could bind to cannabinoid type 2 (CB2) receptors and regulate a series of signaling pathways. , thereby regulating the expression of tumor necrosis factor-alpha (TNF-α) and improving the body’s anti-inflammatory ability. Subsequently, many groups have studied the immunomodulatory mechanism of Echinacea, but there are still many unclear places . At present, it is believed that the immunomodulatory effect of echinacea is mainly achieved by improving the body’s antioxidant capacity, reducing inflammatory damage and enhancing the body’s immune function.
1.1 Active ingredients of Echinacea
1.1.1 Caffeic acid and its derivatives Caffeic acid is one of the common phenolic acids of Echinacea  . Caffeic acid derivatives (CADs) are a class of hydroxycinnamate containing basic structural units of caffeic acid. Metabolites are the main polar active components of Echinacea purpurea. CADs are widely distributed in plants. In addition to Echinacea, there are more contents in Salviae miltiorrhiza , Salvia japonica , Rosmarinus officinalis , blueberry Vaccinium spp., apple Malus domestica , coffee Coffea Linn, etc. . At present, the main ways to obtain CADs include extraction and isolation from natural products and enrichment by chemical synthesis. So far, 15 kinds of CADs have been isolated and identified from Echinacea, such as caffeic acid, cichoric acid, chlorogenic acid, echinacea, and artichoke acid. CADs have a variety of biological activities, can inhibit the key enzyme hyaluronidase involved in bacterial infection, have obvious immune stimulation and anti-inflammatory effects, and also have strong antioxidant effects .
1.1.2 Alkyl amide substances Alkyl amide substances are a large class of non-polar active ingredients of Echinacea purpurea, which mainly exist in the roots. More than 20 echinacea-derived alkyl amides have been identified so far, which are mainly methacrylamides with olefinic and/or acetylenic straight-chain fatty acids , alkyl moieties with 4 or 5 carbons alkanes base. Alkylamides also exist in various plants such as Gerbera jamesonii and Zanthoxylum bungeanum Maxim , which are closely related to the anti-inflammatory activity of plants , and their content can be used as an important quality control index for Echinacea preparations. . Alkylamides in Echinacea are difficult to separate and difficult to obtain. At present, high-speed countercurrent chromatography combined with high-performance liquid chromatography can improve the sample recovery rate, which can effectively control the quality of Echinacea products. During the extraction process, the location of the alkyl amide substances, the degree of drying freshness and the ratio of the extraction solvent used will affect the content and biological activity of the alkyl amide substances. Alkylamides themselves have anti-inflammatory and immune-regulating effects, therefore, they are one of the key factors affecting the pharmacological activity of Echinacea purpurea.
1.1.3 Polysaccharides and glycoproteins Echinacea contains a variety of macromolecular substances such as polysaccharides and glycoproteins. 4-Methoxy-glucaldehyde-arabinose-xylan with a relative molecular mass of 35 000 and arabinose-rhamnogalactose with a relative molecular mass of 50 000 were found in the water extract of the aerial parts of Echinacea purpurea These two immune polysaccharides. Three glycoproteins with relative molecular weights of 17 000, 21 000 and 30 000 were isolated from Echinacea purpurea root. The sugar components in the above glycoproteins are mainly arabinose, up to more than 80%, followed by galactose and glucosamine, and the protein parts are mainly aspartic acid, glycine, glutamic acid and alanine. . The content and activity of Echinacea polysaccharide are related to many factors. Studies have shown that the polysaccharide content of tetraploid Echinacea is higher than that of diploid Echinacea . Echinacea polysaccharides and glycoproteins have strong immune activity, and have anti-inflammatory, antioxidant and immune-enhancing functions.
1.2 Immunomodulatory effects
1.2.1 Reduce the level of oxidation in the body Free radicals are active oxygen free radicals, including hydroxyl free radicals, superoxide free radicals and lipid free radicals, which have extremely strong oxidative power and can interact with biological macromolecules such as nucleic acids and carbohydrates in the body to destroy cells. structure and function, impairing the health of the body. The active ingredients in Echinacea and its extracts can improve the body’s antioxidant capacity. The DPPH free radical scavenging test based on TLC-DPPH thin layer chromatography confirmed that the carp feed containing Echinacea polyphenols has a strong free radical scavenging ability, and its scavenging ability is positively correlated with the content of Echinacea polyphenols in the feed . More studies have confirmed that Echinacea purpurea’s antioxidant function is mainly achieved through the following two ways:
(1) Echinacea directly scavenges free radicals in the body. The CADs contained in Echinacea are polyphenolic compounds, which can supply hydrogen to lipid compound free radicals, and at the same time transform themselves into stable phenolic oxygen free radicals, which directly absorb free radicals to achieve anti-oxidation and delay the oxidation process .
(2) Echinacea prevents further oxidation of the body by regulating the activity of antioxidant enzymes. Echinacea can remove organic peroxides in the body by regulating the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) in animals, and reduce the production of Dialdehyde (MDA) content, reduce or prevent the occurrence of lipid peroxidation . In addition, the polysaccharides of Echinacea can not only exert antioxidant effects through the above-mentioned ways, but also can complex reactions with metal ions such as Fe 2+ and Cu 2+ to inhibit the generation of free radicals, and the polysaccharides cooperate with alkylamides and CADs. The antioxidant effect is stronger when it acts.
1.2.2 Anti-inflammatory effect Echinacea and its extracts can improve the body’s anti-inflammatory ability. Oskoii et al. found that adding Echinacea extract to the feed could increase the blood leukocyte count, lymphocyte count and hematocrit value of rainbow trout Oncorhynchus mykiss and other parameters, and enhance the anti-inflammatory ability of rainbow trout. The anti-inflammatory mechanism of action of echinacea and its extracts may include the following pathways:
(1) Alkylamides, the main active components of Echinacea, can combine with CB2 and activate a series of related signal transduction pathways to improve the anti-inflammatory function of animals . Manayi et al showed that alkylamides act as ligands to bind G protein-coupled receptor CB2, regulate cyclic adenosine monophosphate (cAMP), and activate p38 mitogen-activated protein kinase (mitogen- activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways, while downstream activation of nuclear factor-κB (NF-kappaB, NF-κB) and transcriptional activator-2/cAMP The activity of the response element-binding protein regulates the expression of TNF -α , thereby regulating the immune response of the body. In addition, echinacea can stimulate the expression of interleukin-10, inhibit the production of cyclooxygenase and 5-lipoxygenase, so that the body produces a series of immune responses and activates anti-inflammatory activity .
(2) Echinacea CADs can enhance arginase activity, inhibit the synthesis of inflammatory mediators nitric oxide (NO), inducible nitric oxide synthase (iNOS), and the secretion of pro-inflammatory cytokines. Thus play an anti-inflammatory effect.
(3) Anti-inflammatory effect is achieved through its active ingredient polysaccharide. Fast et al.found that Echinacea root water extract inhibited triacyl lipopeptide-stimulated mononuclear macrophages (THP-1) to produce TNF- α by activating the phosphatidylinositol-3-kinase/protein kinase B signaling pathway , Thereby phosphorylation of protein kinase B and ribosomal S6 protein promotes anti-inflammatory effect. Since there are no substances such as polyphenols and alkylamides in the water extract, its anti-inflammatory effect should be achieved through its active ingredient polysaccharide, which may be a new immunomodulatory effect of Echinacea and its extracts. mechanism.
1.2.3 Enhance immune function Echinacea and its extracts enhance immune function in the following ways:
(1) Improve the humoral immune function of animals by increasing the level of humoral antibodies and the transformation rate of B lymphocytes in immune organs, thereby promoting the growth and development of immune organs. The gavage test shows that Echinacea and its extracts can enhance the antibody secretion function of the mouse body, up-regulate the expression of non-specific immune genes, and increase the spleen index of normal mice, thereby promoting the growth and development of the mouse spleen and enhancing the immune function of the body . At the same time, it can relieve the immune function of mice under the inhibition state induced by cyclophosphamide, and can also improve the thymus index and spleen index of mice . There is no in-depth research report on the effects of echinacea and its extracts on the development of immune organs in aquatic animals.
(2) Directly act on immune cells to enhance cellular immune function. Echinacea and its extracts increase the number and activity of lymphocytes in the body, induce the content of interleukin-2 and γ -interferon to increase, and significantly improve the level of cellular immunity .
(3) Echinacea and its extracts can regulate Toll-like receptors (TLR) signal transduction, activate the downstream key signaling molecule MAPK pathway, make extracellular regulated protein kinases (ERK), JNK Phosphorylation, then activation of NF-κB, up-regulates the expression of costimulatory molecules CD80, CD86 and major histocompatibility complex class II (Major histocompatibility complex class II, MHC II), thereby up-regulating macrophage function . In 2017, Fu et al. found that Echinacea extract polarized mouse bone marrow-derived macrophages to a classically activated macrophage phenotype by activating the JNK signaling pathway, inducing macrophage activation and antigen presentation, and exerting its anti-inflammatory properties. Infection function.
(4) The polysaccharides and glycoproteins of Echinacea purpurea can promote the production and secretion of γ -interferon, β -interferon, interleukin-2 and interleukin-6 in macrophages, and enhance respiratory burst activity, thereby further enhancing phagocytosis ability to improve immune function .
2 Application of Echinacea in aquaculture
In 2008, Ren Yonglin used Echinacea for the first time in the carp Cyprinuscarpio L. and found that Echinacea extract could promote the growth of carp and improve the immunity of carp. Since then, the application of echinacea and its extracts in aquaculture animals has been increasing, and it has shown good anti-oxidative, immune-enhancing, anti-infection and growth-promoting effects in a variety of fish and shrimp.
2.1 Enhance the antioxidant function of aquatic animals
The oxidation level of the body is an important factor affecting the health status of animals , and the active components of Echinacea have a good antioxidant effect, which can reduce the oxidation level of the body. Echinacea and its extracts can improve the oxidation level and health of the body by regulating the level of antioxidant enzymes and directly degrading free radicals. It has been confirmed in fish such as Carassius auratus and Turbot Scophthalmus maximus , and Echinacea extract added Antioxidative capacity reaches the best in the range of 2-4 g/kg . Adding 4 g/kg Echinacea extract to the feed can significantly increase the activities of SOD, CAT and GR in the serum and organ tissues of crucian carp ( P < 0.05), and significantly reduce the content of MDA and hydroxyl radicals ( P < 0.05) . , can also significantly up-regulate the expression of 8 miRNAs in the liver of crucian carp, enhance the antioxidant function of crucian carp, thereby improving the health status . Qin Zhihua et al. also obtained similar results on turbot, and the injection of 3.2 g/kg (fish body weight) of Echinacea extract could significantly improve the serum SOD activity of turbot ( P < 0.01). In addition, carp feed supplemented with echinacea extract can directly scavenge free radicals. In production, echinacea extract can be used as a natural free radical scavenger to prevent fish diseases caused by oxidative stress .
2.2 Improve the immunity of aquatic animals
Echinacea and its extracts can be used as “immune triggers” for aquatic animals , which can improve the immune capacity of aquatic animals by improving fish biochemical indicators, hematological indicators, and increasing the level of non-specific immunity.
Aly et al. found that Echinacea extract could improve the neutrophil adhesion rate and hematocrit value of tilapia Orechromis niloticus and gray mullet Mugil cephalus , significantly increase the number of white blood cells, especially lymphocytes, thereby improving fish survival rate. In terms of blood biochemical indicators, feeding Echinacea extract can significantly increase the serum albumin, globulin and total protein content of gray mullet ( P <0.05) . In addition, feeding or injecting Echinacea extract can also increase the lysozyme activity, phagocytic activity and respiratory burst activity of certain fish such as Barbus grypus , gray mullet and turbot, enhance non-specific Heterosexual immunity. Feeding echinacea extract can improve the immune level of mullet and gray mullet, and the improvement effect is positively correlated with the dose; further research found that injection of echinacea extract can significantly enhance the lysozyme activity and respiratory burst activity in the serum of turbot ( P < 0.01), while up-regulating the expression of immune genes such as lysozyme, C3 complement, transforming growth factor β1 (TGF-β1), interleukin-1h (IL-1h) and transferrin genes in the head kidney of turbot, Improve the immune function of turbot .
2.3 Improve the anti-infection ability of aquatic animals
Infection by pathogenic microorganisms is one of the factors restricting the sustainable development of aquatic animals. Echinacea extract improves the anti-infection ability and survival rate of aquatic animals by enhancing the non-specific immune system’s ability to inhibit viruses and bacteria .
Echinacea extract can prevent or reduce the damage of some pathogenic microorganisms to rainbow trout, and its protective effect is related to the addition amount: adding 1.5 g/kg of echinacea extract to the feed can enhance the resistance of rainbow trout to Streptococcus iniae However, adding 0.1 g/kg of Echinacea extract to the feed had the best protection rate for rainbow trout infected with Vibrio anguillarum , and its mortality was 32% lower than that of the control group . For mullet, photobacterial disease caused by Photobacterium damselae , is one of the main diseases of marine aquaculture, and the mortality rate is extremely high. Adding 0.2 g/kg of Echinacea extract to the feed can enhance the disease resistance of mullet to L. mermaidii subsp. ichthyicidal, and reduce the mortality of mullet by 46% . Similar results were also found in turbot, where injection of 3.2 g/kg (fish body weight) of Echinacea extract could enhance the resistance of turbot to Edwardsiella lentus and reduce mortality. In terms of anti-viral infection, Medina-Beltrán et al. found that when 0-4 g/kg of Echinacea extract was added to the feed of Litopenaeus vannamei , with the increase of the addition amount, the effect of Echinacea vannamei on Vitiligo was reduced. The infection rate of syndrome virus (WSSV) was significantly reduced ( P < 0.05), and the infection rate decreased from 100% in the control group to 8.3% when the addition amount was 4 g/kg. This indicates that Echinacea extract can effectively reduce the probability of WSSV infection in L. vannamei.
2.4 Promote the growth of aquatic animals
Echinacea and its extracts have shown significant growth-promoting effects in different aquatic animals. When Ren Yonglin first applied Echinacea purpurea to aquatic animals, he found that Echinacea purpurea extract can promote the growth of carp, which is reflected in the increase of the weight gain rate and specific growth rate of carp, and the decrease of the bait coefficient. Since then, more studies have confirmed that echinacea extract has a better growth-promoting effect on different fish. Echinacea extract fed rainbow trout and European carp Husohuso at an optimum feed dosage of 0.5 g/kg, and the growth-promoting effect was the best at this dose. Kasiri et al. took the angelfish Pterophyllum scalare as the research object, and compared the growth-promoting effects of echinacea extract and the commonly used growth promoter levamisole, and found that the angelfish fed with the echinacea extract supplemented the diet, increased the final body mass, increased The weight rate and specific growth rate were better than those of the levamisole group. Oniszczuk used extrusion cooking to prepare fish feed containing Echinacea for the first time. This fish feed has a balanced composition and is rich in nutrients such as protein, fat, calcium, nitrogen and phosphorus, and can be used as a nutritional supplement to promote the growth of aquatic animals. In addition, Echinacea itself has the ability to provide nutrients, and can improve the utilization rate of feed nutrients for fish. Akbary et al. added Echinacea extract in the feed to feed mullets, which significantly improved the fish weight gain rate and protein efficiency ratio ( P < 0.05), indicating that Echinacea can improve the utilization rate of protein in the feed by mullets, thereby Improve mullet growth performance.
In addition, Echinacea and its extracts can reduce the feed coefficient and feed conversion rate, and promote the increase of fish body weight, which may be achieved by affecting the intestinal flora, inducing the secretion of digestive enzymes , and regulating amino acid metabolism. . Studies have shown that echinacea polysaccharides have potential application value in regulating intestinal microecology , which may be one of the reasons why echinacea and its extracts can improve the function of the digestive system, improve energy utilization, and promote growth found that adding Echinacea extract to the feed of guppy Poecilia reticulata could increase the content of glycine, cysteine, histidine and serine in the fish. This suggests that Echinacea can promote fish growth and maintain health by regulating amino acid metabolism.
3 Problems and prospects
Echinacea is rich in a variety of biologically active substances, has significant effects on regulating immunity and promoting growth, and has been widely used in human medicine and animal breeding. In human medicine, there have been in-depth studies on the active ingredients, pharmacological efficacy and mechanism of action of Echinacea purpurea. In comparison, the research on echinacea in animal breeding, especially in aquaculture, is still in its infancy, and there are still many problems to be solved.
(1) The diversity of applied species of Echinacea should be further explored. There are few aquatic animal species that have been applied to Echinacea, especially the research on invertebrates is almost blank. In view of the large number of aquaculture species, it is necessary to further strengthen the related application research of Echinacea in different aquaculture animals.
(2) The mechanism of echinacea’s growth-promoting and immune-regulating effects on aquatic animals needs to be further explored. The detailed mechanism of action of Echinacea on different aquatic animals is not very clear. For example, the research on Echinacea’s immunity, especially mucosal immunity, has hardly been reported. The detailed mechanism of action needs to be further elucidated. After the application of Echinacea, its biological activities on different species are quite different, and the specific mechanism behind it also needs to be further explored.
(3) The use strategy of Echinacea also needs to be further improved. According to different breeding objects, growth stages, living environments and different pathogen species, the optimal timing, dosage and adding methods are discussed.
(4) It is necessary to strengthen the research on the compound use effect of Echinacea purpurea. Whether echinacea can be used in combination with other aquatic inputs such as Chinese herbal medicine, oligosaccharide or polysaccharide immune enhancers and probiotics, and the medicinal effects, safety and mechanism of action after compound use still need to be further verified and discussed. .
(5) The source of Echinacea and the influence of extraction process on its active components deserve further discussion. The introduction, cultivation, and extraction process of Echinacea have a significant impact on the content and effect of its active ingredients, and related research still needs to be further advanced. It is believed that with the continuous deepening of related research, echinacea will have broad application prospects in aquaculture.