Study on Echinacea and Immune System

Echinacea purpurea, also known as purple coneflower, is a perennial herb belonging to the family Asteraceae, which is in the division Magnoliophyta, class Dicotyledoneae, order Asterales, and family Asteraceae [1]. There are nine species in the genus, of which Echinacea purpurea, 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 for 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 the body's immunity [2], resisting infection and reducing inflammatory damage [3-5], and is often used to treat influenza and promote wound healing [6-7]. A large number of in vitro and in vivo experiments have shown that Echinacea can enhance the body's immunity, and its immunostimulatory and anti-inflammatory effects have a good preventive 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 immunomodulatory effects in aquatic animals such as fish and shrimp. In June 2012, Echinacea and its preparations were approved as a national Class I new veterinary drug by the Ministry of Agriculture and Rural Affairs of China, which has led to extensive research and application prospects for Echinacea in aquaculture. In this study, the main active ingredients, immunomodulatory effects and mechanisms of Echinacea purpurea, as well as research on its application in aquaculture, are reviewed. The prospects and development trends of its application in aquaculture are also discussed, with the aim of providing a reference for its promotion and application in aquaculture.

1 Active ingredients

immunomodulatory effects and mechanisms of Echinacea purpurea extracts

The chemical composition of Echinacea purpurea is very complex. Its main active ingredients include polar components (caffeic acid and its derivatives), non-polar components (alkyl amides), and high-molecular-weight components such as polysaccharides and glycoproteins [8]. The content and function of Echinacea purpurea's active ingredients vary greatly depending on the place of origin, growth period, part, and processing method. In 2004, Gertsch et al. [9] first explored the possible molecular mechanism of the anti-inflammatory effect of Echinacea, and concluded that the active ingredient alkylamide in Echinacea can bind to cannabinoid type 2 receptors (CB2) and regulate a series of signal pathways, thereby regulating the expression of tumor necrosis factor-alpha (TNF-α) and improving the body's anti-inflammatory ability. Subsequently, many teams studied the mechanism of the immune-modulating effect of Echinacea, but there are still many unclear areas [10]. It is currently believed that the immune-modulating 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 in Echinacea

1. 1. 1 Caffeic acid and its derivatives

Caffeic acid is one of the common phenolic acids in Echinacea purpurea [11]. Caffeic acid derivatives (CADs) are a class of metabolites containing the basic structural unit of caffeic acid, hydroxycinnamate, and are the main polar active ingredients in Echinacea purpurea. CADs are widely distributed in plants. In addition to Echinacea purpurea, they are also found in high concentrations in Salvia miltiorrhiza, Salvia japonica, Rosmarinus officinalis, blueberries Vaccinium spp., apples Malus domestica, coffee Coffea Linn, etc. all contain relatively high levels of CADs [11-13]. At present, the main methods of obtaining CADs include extraction and isolation from natural products and chemical synthesis. So far, 15 CADs have been isolated and identified from Echinacea purpurea, including caffeic acid, chicoric acid, chlorogenic acid, echinacoside, and articholic acid [14]. CADs have a variety of biological activities, including the ability to inhibit the key enzyme hyaluronidase involved in bacterial infection, significant immunostimulatory effects and anti-inflammatory effects, as well as strong antioxidant effects [8, 15-16].

1. 1. 2 Alkylamides

Alkylamides are a large class of non-polar active ingredients in Echinacea, mainly found in the roots. More than 20 Echinacea-derived alkylamides have been identified. These substances are mainly isobutyronamides of straight-chain fatty acids with an alkene and/or acetylene bond [17-18], with alkyl groups of 4 or 5 carbons. Alkylamide substances are also found in many plants such as Gerbera jamesonii and Zanthoxylum bungeanum Maxim. [19], and are closely related to the anti-inflammatory activity of plants [20]. Their content can be used as an important quality control indicator for Echinacea preparations [21].

The alkyl amides in echinacea are difficult to isolate and are not easily obtained. At present, the sample recovery rate can be improved by using high-speed countercurrent chromatography combined with high-performance liquid chromatography, which can effectively control the quality of echinacea products [22]. During the extraction process, the part where the alkyl amides are located, the freshness of the drying, and the ratio of the extraction solvent used, etc., will all affect the content and biological activity of the alkyl amides. Alkyl amides themselves have anti-inflammatory and immune-modulating effects, and are therefore one of the key factors affecting the pharmacological activity of Echinacea.

1. 1. 3 Polysaccharides and glycoproteins

Echinacea contains a variety of macromolecules such as polysaccharides and glycoproteins. Two immunopolysaccharides, 4-methoxy-glucurono-arabino-xylan polysaccharide with a relative molecular mass of 35,000 and arabino-rhamno-galactan polysaccharide with a relative molecular mass of 50,000, were found in the aqueous extract of the aerial parts of Echinacea [23–24]. Three glycoproteins with relative molecular masses of 17,000, 21,000 and 30,000 can be isolated from the roots of Echinacea purpurea. The main sugar component of the above-mentioned glycoproteins is arabinose, which can account for more than 80%, followed by galactose and glucosamine. The protein component is mainly aspartic acid, glycine, glutamic acid and alanine [23, 25]. The content and activity of echinacea polysaccharides are related to a variety of factors. Some studies have shown that the polysaccharide content of tetraploid Echinacea is higher than that of diploid Echinacea [26]. Echinacea polysaccharides and glycoproteins have strong immunological activity, anti-inflammatory, antioxidant and immune-enhancing functions.

1. 2 Immune regulation

1. 2. 1 Reduce the body's oxidation level

Free radicals are reactive oxygen species, including hydroxyl radicals, superoxide radicals and lipid radicals. They have extremely strong oxidizing power and can interact with biological macromolecules such as nucleic acids and sugars in the body, destroying cell structure and function and damaging health. The active ingredients in Echinacea purpurea and its extracts can improve the body's antioxidant capacity. A DPPH radical scavenging test based on TLC-DPPH thin-layer chromatography confirmed that carp feed containing Echinacea polyphenols has strong free radical scavenging capacity, and that the scavenging capacity is positively correlated with the amount of Echinacea polyphenols in the feed [27]. Further studies have confirmed that Echinacea exerts its antioxidant function mainly through the following two pathways:

(1) Echinacea directly scavenges free radicals in the body. The CADs contained in Echinacea are polyphenolic compounds that can supply hydrogen to lipid radical compounds and transform themselves into stable phenolic oxygen radicals, directly absorbing free radicals to achieve antioxidant effects and slowing down the oxidation process [14].

(2) Echinacea prevents further oxidation in the body by regulating the activity of antioxidant enzymes. Echinacea can regulate the activity of antioxidant enzymes in animals such as superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), etc., to remove organic peroxides in the body, reduce the content of malondialdehyde (MDA), and reduce or prevent lipid peroxidation [28]. In addition, the polysaccharide components of Echinacea purpurea not only exert an antioxidant effect through the above-mentioned pathways, but also can complex with metal ions such as Fe2+ and Cu2+ to inhibit the production of free radicals. Moreover, the antioxidant effect of polysaccharides is enhanced when they act synergistically with alkylamides and CADs [29].

1. 2. 2 Anti-inflammatory effect

Echinacea and its extracts can improve the body's anti-inflammatory ability. Oskoii et al. [30] found that the addition of echinacea extract to the feed can increase the total number of leukocytes, the number of lymphocytes, and the hematocrit value in the blood of rainbow trout Oncorhynchus mykiss, and enhance the anti-inflammatory ability of rainbow trout. The anti-inflammatory mechanism of echinacea and its extracts may include the following pathways:

(1) The main active ingredient in Echinacea, alkylamides, can bind to CB2 and activate a series of related signal transduction pathways to improve the animal's anti-inflammatory function [31]. Manayi et al. [10] showed that after alkylamides bind to the G protein-coupled receptor CB2 as a ligand, regulates cyclic adenosine monophosphate (cAMP), activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways, and downstream activation of the nuclear factor-κB (NF-κB) and the activity of the transcription activator-2/cAMP response element binding protein regulates the expression of TNF-α and thus regulates the body's immune response. In addition, echinacoside can stimulate the expression of interleukin-10 and inhibit the production of cyclooxygenase and 5-lipoxygenase, thereby inducing a series of immune response reactions and activating anti-inflammatory activity [32].

(2) Echinacea CADs can enhance arginase activity, inhibit the synthesis of inflammatory mediators nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and the secretion of pro-inflammatory cytokines, thereby exerting an anti-inflammatory effect [33-34].

(2) The anti-inflammatory effect is achieved through its active ingredient, polysaccharides. Fast et al. [35] found that the aqueous extract of Echinacea purpurea root inhibits the production of TNF-α by triacylglycerol peptides stimulated monocyte-macrophage cells (THP-1) by activating the phosphoinositide 3-kinase/protein kinase B signaling pathway, thereby phosphorylating protein kinase B and ribosomal S6 protein, promoting anti-inflammatory effects. Since polyphenols and alkylamides are not present in the water extract, its anti-inflammatory effect is likely to be achieved through its active ingredient, polysaccharides. This may be a new mechanism by which Echinacea and its extracts exert their immunomodulatory effects.

1. 2. 3 Enhancing immune function

The enhancement of the body's immune function by Echinacea and its extracts involves the following pathways:

(1) Enhancement of the body's humoral immune function by increasing the level of humoral antibodies and the conversion rate of B lymphocytes in immune organs, thereby promoting the growth and development of immune organs. Gavage experiments have shown that Echinacea and its extracts can enhance the antibody secretion function of mice, up-regulate the expression of non-specific immune genes, increase the spleen index of normal mice, thereby promoting the growth and development of the spleen and enhancing the immune function of the body [36]. It also has a relieving effect on the immune function of mice in an inhibited state after cyclophosphamide induction, and can also increase the thymus index and spleen index of mice [37]. There have been no in-depth research reports on the effects of Echinacea and its extracts on the development of immune organs in aquatic animals.

(2) It directly acts on immune cells to enhance cellular immune function. Echinacea and its extracts increase the number and activity of lymphocytes in the body, induce an increase in the content of interleukin-2 and γ-interferon, and significantly improve cellular immunity [38].

(3) Echinacea and its extracts can regulate Toll-like receptor (TLR) signal transduction, activates the downstream key signaling molecule MAPK pathway, causing phosphorylation of extracellular regulated protein kinases (ERK) and JNK, which then activates NF-κB, upregulates the expression of costimulatory molecules CD80 and CD86 and major histocompatibility complex class II molecules (Major histocompatibility complex class II,  MHC Ⅱ )  expression, thereby upregulating macrophage function [39]. In 2017, Fu et al. [40] found that Echinacea purpurea extract polarizes mouse bone marrow-derived macrophages to the classic activated macrophage phenotype by activating the JNK signaling pathway, inducing macrophage activation and antigen presentation, and exerting its anti-infection function.

(4) The active ingredients of Echinacea, polysaccharides and glycoproteins, can promote the production and secretion of macrophage γ-interferon, β-interferon, interleukin-2 and interleukin-6, enhance respiratory burst activity, thereby further enhancing phagocytic ability and improving immune function [41-42].

2 Application of Echinacea in aquaculture

In 2008, Ren Yonglin [43] first used Echinacea purpurea in carp, Cyprinus carpio L., and found that Echinacea purpurea extract could promote the growth of carp and improve their immune ability. Since then, the application of Echinacea purpurea and its extracts in aquaculture animals has been increasing, and it has been shown to have a good effect on antioxidant, improving immune ability, anti-infection and promoting growth in a variety of fish and shrimp.

2. 1 Enhancing the antioxidant function of aquatic animals

The level of oxidation in the body is an important factor affecting the health of animals [28], and the active ingredients in Echinacea have a strong antioxidant effect, which can reduce the level of oxidation in the body. Echinacea and its extracts have been shown to improve the oxidation level and health status of organisms by regulating the level of antioxidant enzymes and directly degrading free radicals. This has been demonstrated in fish such as Carassius auratus and Scophthalmus maximus, and the antioxidant capacity of echinacea extract is optimal when the addition amount is in the range of 2–4 g/kg [44–45]. Adding 4 g/kg Echinacea purpurea extract to the feed can significantly increase the activity of SOD, CAT and GR in the serum and tissue of crucian carp (P<0.05), and significantly reduce the content of MDA and hydroxyl radicals (P<0.05) [44]. At the same time, it can also significantly up-regulate the expression of 8 miRNAs in the liver of crucian carp, enhancing the antioxidant function of the crucian carp and thus improving its health [46].

Qin Zhihua et al. [45] also obtained similar results in turbot. A 3.2 g/kg (fish body mass) injection of Echinacea purpurea extract can significantly increase the serum SOD activity of turbot (P<0.01). In addition, the addition of Echinacea extract to carp feed 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 [28].

2. 2 Improving the immune capacity of aquatic animals

Echinacea and its extracts can be used as an “immune trigger” for aquatic animals [47], which can improve the immune capacity of aquatic animals by improving biochemical and hematological indicators and increasing the level of non-specific immunity.

Aly et al. [48] found that Echinacea extracts can improve the neutrophil adhesion rate and red blood cell hematocrit values of tilapia Orechromis niloticus and gray mullet Mugil cephalus, significantly increase the number of white blood cells, especially lymphocytes, thereby improving fish survival. In terms of blood biochemical indicators, feeding Echinacea purpurea extract can significantly increase the serum albumin, globulin and total protein content of gray mullet (P < 0.05) [47].

In addition, feeding or injecting Echinacea purpurea extract can also increase the lysozyme activity, phagocytic activity and respiratory burst activity of some fish, such as silver carp Barbus grypus [49], gray mullet [47] and turbot [45], and enhance nonspecific immunity. Feeding Echinacea purpurea extract can increase the immune levels of turbot and gray mullet, and the effect of the increase is positively correlated with the dose. Further studies have found that injecting Echinacea purpurea extract can significantly enhance the lysozyme activity and respiratory burst activity in turbot serum (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 gene expression, thereby improving the immune function of turbot [50].

2. 3 Improving the resistance of aquatic animals to infection

Infection with pathogenic microorganisms is one of the factors limiting the sustainable development of aquatic animal health. Echinacea extract improves the immune response and survival rate of aquatic animals by enhancing the ability of the non-specific immune system to inhibit viruses and bacteria [51-52].

Echinacea extract can prevent or reduce the damage caused by certain pathogenic microorganisms to rainbow trout. The protective effect is related to the amount added: adding 1.5 g/kg of echinacea extract to the feed can enhance the disease resistance of rainbow trout to Streptococcus iniae [53], while the best protection rate for rainbow trout infected with Vibrio anguillarum was achieved with an addition of 0.1 g/kg Echinacea extract in the feed, which reduced mortality by 32% compared to the control group [42].

In mullets, Photobacterium disease caused by Photobacterium damselae subsp. damselae is one of the main diseases in marine aquaculture, with extremely high mortality rates. Adding 0.2 g/kg of Echinacea extract to the feed can enhance the mullet's resistance to Photobacterium damselae subsp. damselae, reducing the mortality rate by 46% [47]. Similar results were also found in turbot, where the injection of 3.2 g/kg (body weight) Echinacea extract could enhance the turbot's resistance to Edwardsiella tarda and reduce mortality [45]. In terms of antiviral infection, Medina-Beltrán et al. [54] found that when 0–4 g/kg of Echinacea purpurea extract was added to the feed of Litopenaeus vannamei, the infection rate of white spot syndrome virus (WSSV) in Litopenaeus vannamei significantly decreased (P<0.05) with the increase of the addition amount. At an addition of 4 g/kg, the infection rate decreased from 100% in the control group to 8.3%. This indicates that Echinacea extract can effectively reduce the chance of Vannamei shrimp being infected with WSSV.

2. 4 Promoting the growth of aquatic animals

Echinacea and its extracts have been shown to have a significant growth-promoting effect in various aquatic animals. When Ren Yonglin [43] first applied echinacea to aquatic animals, he found that echinacea extract promoted the growth of carp, as evidenced by an increase in the weight gain rate and specific growth rate of carp and a decrease in the feed conversion ratio. Since then, more studies have confirmed that echinacea extract has a good growth-promoting effect on different fish. The optimal feed addition amount of Echinacea purpurea extract for feeding rainbow trout [30] and European smelt Husohuso [55] is 0.5 g/kg, and the growth-promoting effect is optimal at this dose. Kasiri et al. [56] used the angelfish Pterophyllum scalare as the research object and compared the growth-promoting effect of Echinacea purpurea extract with that of the commonly used growth promoter levamisole. It was found that the final body mass, weight gain rate and specific growth rate of the guppies fed feed with added Echinacea purpurea extract were all better than those of the levamisole group.

Oniszczuk [27] first used extrusion cooking to prepare fish feed containing Echinacea, which is a balanced diet 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 by fish. Akbary et al. [57] fed mullet with Echinacea extract added to the feed, which significantly increased the weight gain rate and protein efficiency ratio (P<0.05), indicating that Echinacea can improve the utilization rate of protein in the feed by mullet, thereby improving the growth performance of mullet.

In addition, Echinacea and its extracts can reduce the feed coefficient and feed conversion rate, promote the increase in body mass of fish, possibly through affecting the intestinal flora, inducing the secretion of digestive enzymes [58], regulating amino acid metabolism, etc. Some studies have shown that Echinacea polysaccharides have potential application value in regulating intestinal microecology [59], which may be one of the reasons why Echinacea and its extracts can improve the function of the digestive system, increase energy utilization, and promote growth [60]. Guz et al. [61] found that adding Echinacea purpurea extract to the diet of guppies (Poecilia reticulata) increased the levels of glycine, cysteine, histidine and serine in the fish. This suggests that Echinacea purpurea can promote fish growth and maintain health by regulating amino acid metabolism.

3 Problems and prospects

Echinacea is rich in a variety of bioactive substances and has significant immunomodulatory and growth-promoting effects. It has been widely used in human medicine and animal husbandry. In human medicine, there have been in-depth studies on the active ingredients, pharmacological effects and mechanisms of action of Echinacea. In contrast, research on Echinacea in animal husbandry, especially aquaculture, is still in its infancy, and there are still many problems that need to be solved urgently.

(1) The diversity of species in which Echinacea is used should be further explored. The types of aquatic animals that have been used with Echinacea are still relatively few, and research on invertebrates in particular is almost non-existent. Given the large number of types of aquatic animals that are farmed, it is necessary to further strengthen research on the application of Echinacea in different aquatic animals.

(2) The mechanism of Echinacea purpurea promoting growth and regulating immunity in aquatic animals needs to be further explored. The detailed mechanism of Echinacea purpurea on different aquatic animals is not yet fully understood. For example, there have been almost no reports on the research of Echinacea purpurea on the immunity of aquatic animals, especially mucosal immunity. The effect of Echinacea purpurea on regulating the intestinal flora of aquatic animals and its detailed mechanism in the process of promoting growth need to be further elucidated. After the application of Echinacea, there are significant differences in the biological activity of Echinacea on different species, and the specific mechanism behind this also needs to be further explored.

(3) The use strategy of Echinacea also needs to be further improved. The optimal timing, dosage and method of addition need to be explored for different cultured objects, growth stages, living environments and different pathogen types.

(4) The effect of the combined use of Echinacea needs to be further studied. Whether Echinacea can be used in combination with other aquatic inputs such as Chinese herbal medicines, oligosaccharides or polysaccharide immune enhancers and probiotics, as well as the medicinal effect, safety and mechanism of action after combined use, still needs to be further verified and discussed.

(5) The influence of the source and extraction process of Echinacea purpurea on its active ingredients deserves further exploration. The introduction, planting and extraction process of Echinacea purpurea will have a significant impact on the content and effect of its active ingredients. Related research still needs to be further promoted. It is believed that with the continuous deepening of related research, Echinacea purpurea will have broad application prospects in aquaculture.

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