Ginsenosides as dietary supplements with immunomodulatory effects: a review

Immune disorders have become one of the public health problems and imposes a serious economic and social burden worldwide. Ginsenosides, the main active constituents of ginseng, are regarded as a novel supplementary strategy for preventing and improving immune disorders and related diseases. This review summarized the recent research progress of ginsenosides in immunomodulation and proposed future directions to promote the development and application of ginsenosides. After critically reviewing the immunomodulatory potential of ginsenosides both in vitro and in vivo and even in clinical data of humans, we provided a perspective that ginsenosides regulated the immune system through activation of immune cells, cytokines, and signaling pathways such as MAPK, PI3K/ Akt, STAT, and AMPK, as well as positively affected immune organs, gut flora structure, and systemic inflammatory responses. However, the evidence for the safety and efficacy of ginsenosides is insufficient, and the immune pathways of ginsenosides remain incompletely characterized. We believe that this review will provide a valuable reference for further research on ginsenosides as dietary supplements with immunomodulatory effects.


Introduction
Genetic factors, environmental pollution, mental pressure, and unhealthy lifestyles are responsible for immune dysfunction.In addition, with the population aging and the outbreak of various epidemics, diseases caused by immune dysfunction such as allergies, rheumatoid arthritis (RA), rheumatic heart disease and systemic lupus erythematosus (SLE) are emerging [1].
As a research study published by The Lancet showed an increasing trend in the incidence and prevalence of immune diseases, and the world is facing severe economic and social pressures [2].Immunity is a selfdefense function of the human body, and the immune system is a significant system of immune response and immune function.The balance and coordination within the immune system are critical to maintain body health.The response of the immune system can be categorized into two categories: immunoenhancement and immunosuppression.Immunoenhancement refers to an enhanced immune response to pathogenic microbial infections, immunodeficiencies and other diseases.However, the hyperactivity of its function will cause damage to organs or tissues.Many autoimmune diseases, such as RA, SLE and ulcerative colitis (UC), are caused by over-immunization, which requires drug intervention to suppress the body's immune response.Immunosuppression means that the immune response is downregulated, which can lead to increased susceptibility to infection, even cancers and tumors.Immunomodulation, a treatment strategy that uses natural or synthetic drugs and microorganisms to activate or inhibit the immune system to fight disease, has become a research hotspot and major challenge in the contemporary healthcare area.Immunomodulators are classified as natural and synthetic drugs.The application of immunomodulators has a positive effect on various types of infections, allergies or cancers.These are natural and synthetic agents used to adapt to the immune system response.Clinically, levamisole, cyclophosphamide (CTX), dexamethasone (DEX), methotrexate (MTX), hydrocortisone, and other chemical synthetic drugs are widely used in immunomodulation, the use of such immunomodulators has been controversial due to their typical side effects such as alopecia, diarrhea, hematological diseases, bdelygmia, immune organ failure [3].Natural agents consisting of botanicals and dietary supplements are known to be comparatively safe but less studied.This review is intended to raise the interest of researchers in the use of ginsenosides as an alternative remedy to traditional therapy, especially in cases where the immune system is partially impaired or specific immunotherapy is needed to minimize adverse events.
Ginseng (Panax ginseng C.A. Meyer) is a widely known medicinal and food plant, that contains more than 100 functional compounds with physiological functions against cancer, viruses, inflammation, aging, diabetes, dementia and fatigue [4].Ginsenosides are the main active components extracted from the fruits, flowers, leaves, stems and roots of ginseng.QYResearch showed that the global ginseng industry market size stayed above and below $7.9 billion, with China and South Korea being the major ginseng markets, followed by Japan and the EU region.Currently, the global ginsenoside head manufacturers mainly include Folotto, BTGin, Dr. Ginseng, Onstin, and Mkule.On the basis of different product types, it is categorized into capsules, oral liquids, drops, tablets and powders.The price increases with the purity of ginsenosides.Total ginsenoside power costs roughly $166.8/kg,rare ginsenoside Rg3 power costs $69,514.1/kg,and ginsenoside Rh2 power costs $136,247.6/kg.Generally, the more homogeneous the ingredient, the clearer the efficacy.Products in the market have different specifications and advertise different efficacy.Some products confused the concept of total ginsenosides and ginseng powder, and are very vague about the labeling of purity.Emphasis on the development of standardized extracts and quality control can augment consumer reliance.Exploring and elucidating the regulatory mechanisms is a critical step in the development of ginsenosides with immunomodulatory effects as nutritional supplements.
Ginsenosides belong to triterpenoids, which are composed of steroid skeleton (17 carbon) containing sugar.To date, more than 300 natural and transformed ginsenosides have been identified and studied [5].Depending on the difference between aglycones, ginsenosides can be classified into protopanaxadiol (PPD), protopanaxatriol (PPT), ocotillol and oleanolic acid types (Fig. 1A).Ginsenosides Rb1, Rc, Rb2, Re, Rb3, Rg1 and Rf are considered to be the major ginsenosides as they account for more often than 90% of all ginsenosides.The main ginsenosides could be converted into rare ginsenosides by some factors in the processing process [6][7][8][9] (Fig. 1B and C), and the factors include biological factors such as enzymes and microorganisms, chemical factors such as acid and base, and physical factors such as high temperature and high pressure.The bioactivity and bioavailability of ginsenosides vary with the diversity of their chemical structures [10].The rare ginsenosides are readily absorbed by the human body and have high bioactivity as the sugar groups are partially removed.Therefore, rare ginsenosides are the main active compounds in the research of immunomodulation.
Extensive in vitro and in vivo studies on ginsenosides have been reported with rewarding results.The details of the immunomodulatory effects of ginsenosides as shown in Fig. 2.Among these studies, researchers used a variety of cell lines, including RAW264.7,PBMC, HepG2, B16F10 and others, and reported both direct cytotoxic effects and indirect cytotoxic effects.A variety of in vivo cancer models including rats, ferrets, BALB/c mice, thymus mice, C57BL/6 mice and several others were stimulated by lipopolysaccharide (LPS), cyclophosphamide (CTX), gemcitabine (GEM) and other drugs were used to establish the animal models of immune enhancement and immunosuppression in vivo study.Therefore, the mechanisms of ginsenosides regulating immune dysfunction can be studied from cellular and organism levels.PPT-type and PPD-type ginsenosides are currently the most studied.Ginsenosides as the main source of nutritional and functional foods with the characteristics of nature, efficiency and security, have developed into an inventive strategy to preclude or improve immune dysfunction under the particular background of chronic diseases and infectious diseases.As a result, it is necessary to elucidate the mechanisms of ginsenoside modulating the immune system.
This review focuses on the underlying the current state of knowledge on the potential mechanisms of ginsenosides on immune disorders in the last decade.Literature search was conducted in peer-reviewed and clinical databases, which include PubMed (https:// www.ncbi.nlm.nih.gov/ pubmed), Scopus (https:// www.scopus.com), Web of Science (http:// www.webof knowl edge.com), Medline (https:// www.medli ne.com), and Clinical Trials (https:// clini caltr ials.gov) using the following keywords: Ginsenoside, Ginseng, Immune, Cancer, Tumor, Stress, Liver, Spleen, Intestine, Bone marrow.To provide new insights into the critical path ahead, the immunomodulatory effects, mechanism

The effects of ginsenosides on innate and adaptive immunity
The effects of ginsenosides on innate and adaptive immunity are summarized and shown in Table 1.During the process of inflammation, injury, infection, and aging, immune cells regulate the availability of various cytokines.Therefore, immune cells were viewed as highly dynamic partners of the immune system.Ginsenoside Rg1 regulated cytokine levels in RAW264.7 cells and enhanced the innate immune response of mouse peritoneal macrophages through differential regulation of phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor kappa-B (NF-κB) signaling pathways [11].Compound K (CK) and 20S-dihydroprotopanaxadiol (2H-PPD), promoted the innate immune response and motivated macrophages and monocytes by raising the level of phagocyte uptake, cell-to-cell adhesion and the apparent level of CD80 and CD86, costimulatory molecules [12,13].Ginsenoside Rg3 intervention inhibited M1-type macrophage marker gene expression and induced M2-type macrophage polarization in LPS-induced mice, suggesting that Rg3 promoted the elimination of inflammation in mouse peritonitis model [14].Dendritic cells (DCs) are the most potent specialized immunogenpresenting cells in the body, which are essential for initiating adaptive immune responses.Rg1 was effective in promoting phenotypic maturation and eliciting adaptive immune responses in human iDCs [15].Another study reported that Rg1 raised the number of DCs, B and T cells in DEX-treated mice [16].
Ginsenosides also play a regulatory role in adaptive immunity.A study reported that 20(S)-Rg3 and 20(R)-Rg3 significantly enhanced the secretion of interleukin (IL)-2 and interferon (IFN)-γ by activating the proliferation of lymphocytes and natural killing (NK) cells, which greatly contribute to antitumor effects [17].Ginsenoside Re activated T and B lymphocytes, and increased the concentration of antibodies in mice [18].Similarly, ginseng stem-leaf saponins (GSLS) promoted the activation of T and B lymphocytes and the production of Fig. 2 The details of the immunomodulatory effects of ginsenosides.Various factors can lead to human immune system disorders.Currently, chemical drugs commonly used in clinical practice to improve immune imbalance include levamisole, cyclophosphamide, dexamethasone, etc.However, the natural active ingredient ginsenoside is more advantageous than chemical drugs.Extensive in vivo and in vitro experiments were used to investigate the mechanisms of ginsenosides regulating immunity.A variety of cell lines and animal models were used.Ginsenoside, as a potential immune regulator, has a protective function on human organs and can effectively prevent and alleviate the occurrence and development of a variety of immune diseases Agents to relieve CIA [23] "↑" indicates upregulation and "↓" indicates downregulation IgG and IgG titers in serum, indicating that both cell and humoral immunity responses were enhanced [19].Oral administration of ginsenoside Rg1 significantly enhanced the expressions of specific antibodies and promoted the proliferation of lymphocytes [20].Ginsenoside Rd upregulated Foxp3 expression to drive regulatory T cell (Treg) differentiation and induced the generation of IL-10, TGF-β and IL-35 [21].Rg1 increased the count of neutrophils in the peritoneal cavity and reversed the overexpression of cytokines and suppressed the apoptosis of lymphocytes in septic mice spleen and thymus [22].CK restored the swollen joint count, polyarthritis index, spleen index and serum antibody (IgG1, IgG2a) and other levels to normal in collagen-induced arthritis (CIA) mice.CK treatment also restored the proliferation of B cells and subsets [23].

Ginsenosides and cancer immunity
As shown in Fig. 3, ginsenosides were reported to elucidate the immunizing effect against cancer.
Ginsenosides exert anticancer effects in hepatocellular carcinoma, colorectal carcinoma, breast carcinoma, and lung carcinoma by inhibiting cell proliferation and migration, angiogenesis, and reversing drug resistance [24][25][26].Ginsenoside Rh2 inhibited breast cancer cell Moreover, 20(S)-Rh2 also weakened the infusion of leukemia cells into the spleen by blocking the PI3K/ AKT/mammalian target of rapamycin (mTOR) signaling pathway [29].Ginsenoside Rh2 significantly improved the survival time in melanoma mice, enhanced the infiltration of CD4 + T and CD8 + T cells in tumors and triggered cytotoxicity in spleen lymphocytes [30].Notably, ginsenoside Rk3 blocked the cell cycle in hepatocellular carcinoma (HCC) at the G1 phase to induce autophagy and apoptosis.Meanwhile, Rk3 regulated the expression of PI3K and AKT proteins to inhibit the growth of HCC [31].20(S)-Rh2 lowered cancer cell survival and inhibited the phosphorylation of transcription activator 3 (STAT3) and the expression of matrix metalloproteinase Fig. 3 Ginsenosides modulate cancer immunity.Ginsenosides induced cell-mediated immune response-related pathways, increased uptake of cancer cells by DC cells, increased the cytotoxicity of NK cells, regulated macrophages, T cells and Th1/Th2 balance, so as to reduce organ damage and improve the immune capacity of cancer chemotherapy patients (MMP), resulting in suppressing tumor invasion in human colorectal cancer (CRC) cells [32].Similarly, it was reported that ginsenoside Rg3 could repress the growth and stemness of CRC cells both in vitro and in vivo.Rg3 impaired the migration of CRC cells in vitro.
Rg3 repressed the vascularization of CRC xenografts by downregulating the expressions of angiogenesis-related genes.In addition, Rg3 strengthened the cytotoxicity of oxaliplatin and 5-Fluorouracil against orthotopic xenografts in vivo [33].High expression of Fibroblast growth factors FGF8 is commonly linked to tumor generation and vasculogenesis.Treatment of ovarian cancer mice with Rg5 for one month resulted in a significant reduction of tumor size and restraint of tumor metastasis.Ginsenoside Rg5 significantly reduced FGF8b expression in ovarian cancer cell line OCI-P9a cells, suggesting that anticancer and antimetastatic effects of ginsenoside Rg5 were possibly related to the FGF8brelated pathway [34].CK inhibited tumor growth by inducing apoptosis and tumor cell differentiation through multiple signaling pathways such as 5' AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK) and NF-κB.Moreover, CK regulated the tumor microenvironment by inhibiting tumor angiogenesisrelated proteins [35].

Ginsenosides as immune modulator
Sorafenib (SFN) and GEM remain the first-line treatment choice for cancer therapy.Ginsenoside Rg3 and combined SFN treatment relieved the hepatocellular carcinoma progression via the mediation of HK2mediated gluconeogenesis and PI3K/Akt pathway [36].GEM combined with Rh2 boosted the invasion of DCs to tumors and reduced the expression level of immunosuppressive factors, such as VEGF, IL-6, and TGF-β against trypanic cancer via the NF-κB pathway [37].Ginsenoside Rg3 increased NK cells' cytotoxicity via the mitogen activated protein kinase (MAPK) pathway and also raised the expression of NK-activated reactors [38].

Reduction of chemo or radiotherapy-induced side effects
CTX has been widely used as a traditional alkylating chemotherapeutic agent for the treatment of tumors in clinical practice for over 50 years.The use of high doses of CTX during chemotherapy often results in severe allocytopenia, which can lead to life-threatening conditions.CK is a bioactive derivative of ginsenoside Rb1.Ginsenoside CK improved the decline of spleen and thymus indices caused by CTX.CK controlled the apoptosis of BM nucleated cells (BMNCs) by the mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) and Bcl-2 assaciated X protein (Bax)/Bcl-2 pathways and promoted the BMNCs to enter the cell cycle, proliferate and differentiate normally [39].Panaxadiol saponins component (PDS-C) promoted the proliferation and differentiation of hematopoietic progenitor cells in CTX-induced myelosuppressed mice, which mediated by p-MEK and p-ERK, as well as the C-kit and GATA-1 transcription factors [40].
Rg3 intervention significantly increased the body weight, the organ indices of immune organs, thymus and spleen in mice, and alleviated the pathological damage of organs by regulating macrophages and Th1/Th2 balance, suggesting that Rg3 could boost immunity in chemotherapy recipients [41].

Immunomodulators against viruses, bacteria and other parasites
The effects of ginsenosides as immunomodulators against viruses, bacteria and other parasites are shown in Table 2.
When humans or animals are infected with a virus, various types of cytokines are known to be activated to prevent the virus from replicating.Ginsenosides Rb1 and Rg3 regulated the expression of immune factors and inflammatory factors, enhancing the phagocytosis of bacteria by phagocytes [42,43].Ginsenoside Rg3 inhibited lytic replication and the proliferation of γ herps viral via p38/JNK -related pathways [44].Ginsenoside Rb1 remarkably reduced the expression of enterovirus 71 (EV71)-induced viral protein-1 (VP-1) [45].Rg3 attenuated the replication of hepatitis B virus DNA by stimulating TRAF6/TAK1 degradation and repressing JNK/AP-1 signaling.Among them, TRAF6 and TAK1 are adaptor molecules that signal through a toll-like receptor (TLR)-myeloiddifferentiationfactor88 (MyD88)dependent pathway.Ginsenoside Rg3 dose-dependent inhibited IL-8 and TNF-α levels in HepG2.2.15 cells [46].Toxoplasma gondii (T.gondii), a neurotropic specialized cytosolic parasite, is responsible for central nervous system disorders.GRh2 inhibited T. gondii infection-induced microglial activation and neuronal damage through the downregulation of the TLR4/NF-κB signaling pathway [47].

Ginsenosides in autoimmune disorders and allergies
Autoimmune disorders and allergic reactions are manifestations of excessive immunity, and ginsenosides gained attention as a hopeful preventive and therapeutic adjuvant in combating excessive response of the immune system.Rh2 significantly attenuated the symptoms of weight reduction, intestinal damage and shortened colon length in dextran sodium sulfate (DSS)-induced ulcerative colitis mice.Rh2 could lower the secretion of IL-6, TNF-α, IL-1β and inhibit STAT3/miR-214 activation induced by IL-6 [48].Ginsenosides Rb1, Rh1, Rg1 and Rg3 inhibited the proliferation of B cells and the secretion of antibodies (IgG, IgM) by promoting caspase 3 and Fas/FasL expression in SLE mice [49].The process of allergic immune responses regulated by ginsenosides is closely related to its effects on humoral and cellular immune aspects.Rh2 inhibited the levels of TNF-α, IL-4, IL-1β and IL-8 in ovalbumin (OVA)-induced asthmatic mice, as well as the activation of BCR signaling molecules in lung tissue.Rh2 suppressed the release and degranulation of histamine from IgE-sensitized MCs, which might be related to NF-κB/AKT/Nrf2 and NF-κB/p38 MAPK/Nrf2 pathways [50].Ginsenoside Rg3 could ameliorate allergic airway oxidative stress and inflammation sensitized with OVA by reducing the levels of ROS, Th2 cytokine and chemokine [51].

Ginsenoside as radioprotectant via immunomodulation
Prolonged exposure to ultraviolet (UV) radiation, especially UVB, can cause damaging effects within cells, involving DNA damage, inflammatory responses and oxidative stress, resulting in skin aging.Ginsenoside Rk1 significantly inhibited the excessive production of ROS and enhanced antioxidant enzyme activity to attenuate oxidative damage.In addition, Masson staining and histological confirmed that Rk1 remarkably improved UVB-induced epidermal thickening, skin roughness, disorganized collagen fiber arrangement and wrinkles in BALB/c nude mice.The above results indicated that ginsenoside Rk1 could be used to develop natural dietary supplements for skin health [52].Rh2 inhibited mitochondrial mitophagy in UV-exposed normal human dermal fibroblast (NHDF) cells damaged by reinstating membrane electrical potential and mitochondrial ATP production, causing restoration of cell proliferation, extracellular matrix (ECM) and antioxidant capacity [53].Similarly, ginsenoside Rk1 could reduce intestinal epithelial cells apoptosis in RIII rats by inhibiting PI3K/AKT/ mTOR pathway [54].

Immunity modulation in stress
Ginsenoside Rk1 protected human melanocytes from H 2 O 2 -induced oxidative stress by modulating the PI3K/ Nrf2 pathway [55].Rg3 regulated the oxidative phosphorylation pathway and augmented CD4 + CD25 + Foxp3 Treg cells function in RA mice [56].Rg3 improved the immune response of LPS-stressed broilers, inhibiting pro-inflammatory cytokines and inflammatory mediators production [57].Ginsenoside Rg6 promoted the expression of IL-10 and miR-146a expression in septic mice.The miR-146a is an anti-inflammation operational miRNA and is in charge of suppressing LPS-induced production of pro-inflammatory cytokines [58].Ginsenoside Rg1 prevented complement-mediated damage to podocytes by repressing MAPK activation and reducing ROS production in membranous nephropathy (MN) podocyte injury mice [59].Ginsenoside Rb1 reversed the excessive splenic apoptosis induced by Deoxynivalenol (DON) via regulating the mitochondria-mediated apoptosis pathway, down-regulated caspase-3, caspase-9 and Bax in mice.In addition, Rb1 promoted the accumulation of IgA, IgG and IgM [60].F1-enhanced mixture improved mitotic and endoplasmic reticulum stress-related autophagic fluxes and inhibited apoptosis thus protecting against HFD-induced oxidative damage and cell aging in brain.F1-enhanced mixture declined the secretion of 24 factors in the hippocampus of mouse brain through NF-κB signaling pathway, such as several chemokines, growth factors and pro-inflammatory cytokines.The results provided explicit evidence for its potential as a functional food [61].The anti-aging gene SIRT1 has a regulatory role in mitochondrial biogenesis, energy homeostasis, and prevention of oxidative stress [62,63].Ginsenoside Rh2 ameliorated mitochondrial dysfunction induced by H 2 O 2 by promoting the expression of SIRT1 and PGC-1α genes [64].Rg1 protected NSCs from OGDinduced oxidative stress by modulating Bax/Caspase3 and p38/JNK phosphorylation [65] (Table 3).

Role in hepatic disorders
Numerous studies showed that systemic inflammation was related to liver immune regulation (Fig. 4).
Ginsenosides can inhibit inflammatory molecules and protect liver through ROS, MAPKs, NF-κB/AP-1, Keap1/ Nrf2 and HO-1/ARE signaling pathways.Researchers suggested that the pathology of hepatic diseases is closely related to TLR-4 and the immune cascade [66], and a variety of liver injury models have been used to evaluate the immunomodulatory role of ginsenosides in liver diseases.For instance, ginsenoside Re mitigated alcohol hepatic injury by down-regulating TLRs [67].Ginsenoside Rc relieved the damage of hepatocytes and oxidative stress in alcoholic liver disease (ALD) by up-regulating the SIRT6/NRF2 pathway [68].Ginsenoside Rg1 could significantly reduce the liver weight and improve the structure of liver lobule in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-induced liver injury mice by inhibiting CYP1A1 through the aryl hydrocarbon receptor [69].20(R)-Rg3 effectively reduced acetaminophen (APAP)-induced necrosis, apoptosis, and inflammatory infiltration of liver tissue cells by activating the PI3K/AKT signaling pathway [70].Rg1 significantly reduced APAP-induced oxidative stress and hepatotoxicity by suppressing Keap-1 expression and up-regulating Nrf2 target genes.In addition, Rg1 could inhibit the activity of enzymes in the forming of the APAP toxic metabolite [71].Similarly, ginsenoside Rg1 promoted the survival rates of hepatocytes and liver repair in carbon tetrachloride-induced acute liver injury mice.Rg1 reduced the levels of AST, ALT, and ALP in serum and increased GSH, SOD and CAT in liver, which were related to the up-regulation of the Nrf2 pathway [72].Ginsenoside 20(S)-Rh1 maintained the normal levels of FBG and insulin, reducing the apoptosis of liver tissue by inhibiting the activation of the Akt/ FoxO1 pathway, effectively improving the liver injury caused by type 2 diabetes [73].Ginsenoside Rg2 inhibited the expression of liver autophagy-related proteins by triggering the Akt/mTOR signaling pathway, improving liver fibrosis induced by a high-fat diet (CDAHFD).The same results were obtained in vitro experiments [74].

Role in hematopoietic disorders
PDS-C could improve the myelosuppression state of mice and promote the production of BM hematopoietic cells.It found that after the intervention of PDS-C, the counts of white blood cells, platelets, and neutrophils in mice were remarkably increased in a concentrationdependent manner, the myelosuppression state of AA was significantly reduced, and the number of hematopoietic cells in BM was enriched.In addition, PDS-C treatment increased CD4 + CD25 + FoxP3 + cells.
These results indicated that PDS-C has dual activities of boosting hematopoiesis and modulating immunity [75].Ginsenoside Rg1 could also improve hematopoietic stem cells by inhibiting mitochondrial apoptosis caused by Bax translocation, thereby restoring hematopoietic function [76].Ginsenoside Rb1 inhibited NLK expression and promoted erythropoiesis in models of diamond-Beckfan anemia (DBA).Among them, NLK is a fragment of the MAPK family, whose activation contributes to the pathogenesis of DBA.Therefore, NLK is considered a potential therapeutic target [77].These data indicated that ginsenosides can regulate immunity positively to restore hematopoietic function (Fig. 5A).

Role in intestinal disorders
The intestine holds the vast majority of immune cells in the human body.Intestinal ecological disorders can destroy the intestinal epithelial barrier, cause host immune dysfunction, and lead to cancer.Ginsenosides can regulate intestinal immunity through various pathways (Fig. 5B).Ginsenoside Rb1 inhibited inflammatory response and oxidative stress by regulating the PI3K/Akt/Nrf2 pathway in ischemia/reperfusion (IR) damage mice [78].Rk1 alleviated the apoptosis of intestinal epithelial cells induced by oxidative stress in RIII rats.The pathway of the anti-apoptotic effect may concern the inhibition of PI3K/AKT/mTOR [54].With the emergence of intestinal flora research, there is no doubt that it provides a new thought to study the immune mechanisms of ginsenosides.Intestinal microbiota cooperate with the immune system to maintain the balance and homeostasis of the internal environment, and normal microbiota can stimulate the host to produce immunity or elimination function.Ginsenoside Rg1 improved effectively the body weight, colon length, change rate of colon mass index and colon mucosal injury in UC mice.Rg1 also increased the variety of colon flora and the proportion of beneficial bacteria [79].Ginsenoside Rb1 reduced the proportion of Bacteroides and promoted the proportion of phylum Verrucomicrobia and the genus Akkermansia [80].Rg1 regulated the structure of gut microbiota and increased the relative abundance of gut microbiota in mice, such as Roseburia, Lachnospiraceae, Ruminococcaceae and Alistipes, while the relative abundance of potential pathogens such as Alloprevotella, Helicobacteraceae, Allobaculum, Dubosiella and Mycoplasma was decreased.Meanwhile, Rg1 improved the inflammation of colon tissue and repaired the damaged mucosal barrier.This indicates that Rg1 can enhance immunity by regulating the homeostasis of intestinal microflora in mice [16].Ginsenoside Rg1 significantly increased the abundance of Bacteroidetes and Firmicutes and reduced the relative abundance of Proteobacteria and Cyanobacteria in morphine-induced intestinal flora dysbiosis mice [81].

Clinical trials about ginsenoside immune modulation
The ameliorative effects of ginsenosides on immunerelated diseases have been shown not only in cellular and animal experiments but also in available human data.Clinically, 414 patients suffering from NSCLC improved their symptoms, reduced chemotherapy-induced myelosuppression and prolonged their survival after receiving a combination of Rg3 and chemotherapy [82].Additionally, Shengmai injection containing ginsenoside Re, Rg1, and Rb1 is used as adjunctive therapy for chronic obstructive pulmonary disease (COPD) in combination with western medications to improve overall clinical effectiveness and lung function according to the results of 23 rationalized controlled trials involving 1804 participants [83].
A study evaluated the effectiveness of ginseng extracts GS-3K8 and GINST in the prophylaxis of acute respiratory illness (ARI), where GS-3K8 was a ginseng extract enriched with PPD-type ginsenosides, and GINST was a pectinase-processed ginseng extract.A total of 45 participants in the study were directed to take two caplets after each meal for 12 weeks, with the incidence of ARI and duration of symptoms as the primary clinical indicators.The results indicated that GS-3K8 and GINST had a preventive effect on ARI [84].It was reported that ginsenosides alleviated the adverse reactions caused by chemotherapy, and has been prescribed as an adjuvant to TACE for hepatocellular carcinoma (HCC) [85].A clinical study included 1308 patients and explored the efficacy of TACE combined with ginsenosides Rg3, Rh2, CK and total ginsenoside GS in the treatment of HCC, with the quality of life, tumor response, liver function, survival rates and adverse reactions as the main indicators.The results showed that both short-term and longterm treatment of ginsenosides improved liver function and reduced adverse reactions during TACE treatment.It also showed that ginsenoside was an effective and reliable adjuvant in tumor therapy [86].PI3K/AKT pathway was thought to play an influential role in inhibiting HCC growth by Rk3.It was reported that Rk3 prolonged the survival of HCC patients by inhibiting PI3K/AKT pathway signaling in clinical samples [31].The research found a remarkable drop in central and peripheral arterial blood pressure in healthy people after ingesting Rg3 isolated from red ginseng, suggesting that ginsenosides have a preventive effect against cardiovascular disease [87].Ginsenoside Rg3 restored the morphology and function of normal human dermal fibroblasts under UV irradiation and promoted protein expression related to antioxidant activity and cell proliferation [88].
Although a considerable amount of preclinical information has been available to support the concept that ginsenosides may modulate human immunity through multiple pathways, unfortunately, the efficacy and safety of the clinical application of ginsenosides are still controversial and even contradictory.With disappointment, the translation process from accomplished preclinical results to operative clinical settings, and useful clinical evidence is often missing.Therefore, much work has to be done in this regard to adequately define the potential of ginsenosides as dietary supplements.

Bioavailability of ginsenosides
Absorption, distribution, metabolism and excretion of ginsenosides are crucial for interpreting their immunomodulatory effects.It was reported that ginsenosides could be rapidly absorbed in the gastrointestinal tract [89] and metabolized in liver microsomes [90].In addition, less than 2% of orally administered ginsenosides were recovered in human urine, suggesting that most ginsenosides are not absorbed in their original form [91]. Ginsenosides come into contact with gastrointestinal fluids containing gastric acid and gastric enzymes, enteric enzymes, and colonic bacteria after oral administration, and most of the intact ginsenosides are metabolized and converted in the gastrointestinal tract to ginsenosides with more biological effects [92].Taking PPT-type ginsenoside Re as an example, Re was converted to secondary metabolites Rg2, Rh1, F1 and PPT by intestinal flora, and oxidation and deglycosylation were the main metabolic processes of Re.These secondary metabolites could be more readily absorbed into the bloodstream and enter the body circulation.Moreover, Re was hardly detected in the feces of rats, while its metabolites could be detected [93].Another study reported that PPT-type ginsenoside Rg2 was converted to four metabolites, M1-M4, in rat liver microsomes, and these metabolites had enhanced biological activity [90].Kang et al. reported that the biotransformation of PPD-type ginsenoside Rb1 was mainly realized by various enzymes secreted by microorganisms in the intestine.Ginsenoside Rb1 was rapidly hydrolyzed by intestinal flora to Rd, which is then mostly deglycosylated to F2. Rd and F2 were further hydrolyzed to CK, a major deglycosylated metabolite readily absorbed into the systemic circulation [94].Hong et al. proposed a feasible metabolic pathway for PPD-type ginsenoside Rg5.A total of 17 metabolites were detected in biological samples, including rat liver microsomes (13), rat urine (5), feces (5) and plasma (5).Oxidation, deglycosylation, deoxygenation, glucuronidation, demethylation, and dehydration were the major metabolic reactions of Rg5, and fecal clearance was the major excretion route of Rg5 and its metabolites [95].
The dammarane backbone and glycosyl structure of ginsenosides leads to poor cell permeability and low bioavailability in the human body, resulting in limiting the utilization of ginsenosides [96].Current methods on improvements of the poor oral bioavailability of ginsenosides include pretreatment, structural modification, drug combination, and micro-and nano-delivery.Specifically, the bioavailability of ginsenosides can be effectively improved by using genetic engineering techniques to obtain strains with high deglycosylation activity or heat treatment to pretreat ginsenosides [97,98].Cell membranes are mainly composed of lipids, so the structural modification mainly improves the membrane permeability of ginsenosides by increasing the lipophilicity of ginsenosides [99,100].Numerous studies have revealed that the combination of ginsenosides with certain drugs, such as prebiotics, borneol and verapamil, could effectively improve their oral bioavailability [101,102].Tiny particles are easily absorbed by the body.The tiny droplet size of a micro (or nano)-system have large interfacial surface areas that controls the release and absorption of ginsenoside.In addition, micro-or nano-delivery system also modify the lipophilicity or hydrophilcity of ginsenoside, thereby, enhance the penetrability of ginsenoside [103,104].In the context of dietary support and functional foods, these technologies may tremendously improve the effectiveness, safety and bioavailability of ginsenosides, but their anticipated amplification of immune potential, increased utilization and reduced toxicity still need to be optimized and discovered.

Further considerations and conclusive remarks
Recently, many studies have preliminarily elucidated the immunomodulatory activities of ginsenosides, but there are still some problems to be addressed.First of all, the modernization of plant active components is an arduous task.Ginsenosides may vary significantly in nature, active ingredient content, and efficacy due to the use of different extraction, isolation and purification methods.Therefore, professionals must strive to bring about improvements in production and detection systems, and more clinical studies are needed to confirm the efficacy of ginsenosides in modulating the body's immune system and to explore precise immunomodulatory mechanisms.Secondly, the current research on the immunocompetence of ginsenosides mainly focuses on the detection of immune organs or peripheral blood indicators but does not combine the actual absorption and utilization of different ginsenosides in the human body, which is not conducive to the overall description of the immune pathway of ginsenosides, and it is difficult to conclude a reasonable recommended dosage through such studies.Furthermore, most of the experiments were conducted in young mice over a short period of time and mostly under pathological conditions.However, they are not adequate to elucidate the complex diversity of pathological changes during the evolution of human immune disorders.Large animal models (guinea pigs, rabbits, dogs, and monkeys) due to their significant genetic, physiological, biochemical and metabolic similarities to humans, can close the gap between fundamental research and cautious clinical applications and also facilitate the establishment of an appropriate ginsenoside delivery system, which would alleviate bioavailability barriers.Currently, some investigators provide shallow results, whereas others refuse to offer suitable information related to clinical outcomes or quantify clinical significance.Therefore, more research in this area is urgently needed to eliminate inconsistencies and to contribute worthwhile information for the prospective development of ginsenoside functional foods.Although few studies reported negative effects of ginsenosides, it is crucial to assess their safety.A dose-dependent increase in the toxicity of ginsenosides in animals has been reported [105].And ginsenosides caused different degrees of toxicity in different experimental animals [106,107].Therefore, professionals need to carefully investigate the underlying toxigenicity of ginsenosides, which will help in framing safety and efficacy studies in the future.
The research on the immunocompetence of ginsenosides is still a vast uncultivated land.It is necessary to further explore the specific molecular targets and pathways of ginsenosides from multiple perspectives and in an all-round way, which is of great value for formulating novel dietary strategies to prevent and improve immune disorders.

Fig. 1
Fig. 1 Types of ginsenosides and the conversion pathway between some ginsenosides.The ginsenosides treated by physical, chemical or biological methods can be transformed into new ginsenosides by deacylation, deglycosylation and dehydration proliferation and enhanced immunogenicity by downregulating the expression of hypermethylated genes such as INSL5, CASP1 and OR52A1 and up-regulating hypomethylated genes such as ST3GAL4, C1orf198 and CLINT1[27].Rh2 and its octyl derivative Rh2-O increased the levels of TNF-α, IL-2, T lymphocyte and NK cell, and facilitated tumor cell apoptosis by controlling the phosphorylation of caspase and B-cell lymphoma-2 (Bcl-2)[28].Ginsenoside 20(S)-Rh2 significantly decreased the spleen index in acute lymphoblastic leukemia (T-ALL) mice, and the spleen immunity was enhanced by regulating immune factors.

Fig. 5
Fig. 5 Ginsenosides promoted the proliferation and differentiation of hematopoietic stem cells in bone marrow, which differentiated into neutrophils and lymphocytes, enhancing the recovery of hematopoietic and immune functions.Ginsenosides participated in the intestinal immune process by regulating the functions of IgG + plasma cells, macrophages, DC cells and T cells, which effectively improved microbial dysbiosis, dysfunction of tight junctions and impaired mucosal barrier

Table 1
Effect of ginsenosides on innate and