The time for saponins to reach T_max in rat plasma was less than 2 h, indicating that saponins are rapidly absorbed and readily distributed in tissues (Li et al., 2006; Gui et al., 2007). In humans, Liu et al. (2011) reported that the T_max of Re was 1.19 ± 0.44 h after oral ingestion. Another study showed that the T_max of Re was 0.75 h after oral administration of total P. notoginsenoside powder in rats, suggesting rapid absorption of Re in the gastrointestinal tract. The absolute bioavailability of Re was 7.06% (Li et al., 2006). Joo et al. (2010) revealed that the T_max of Re was 0.4 ± 0.2 h in ICR mice. The same study also showed that the oral bioavailability was 0.19–0.28%, suggesting that the absorption rate of Re was lower after oral administration. Shi et al. (2013) demonstrated that Re (12.5, 25 and 50 mg/kg, s.c. injection) was rapidly distributed to the cerebrospinal fluid and exhibited linear pharmacokinetics in rats, and that the T_max of Re was 1 h for all doses. However, for the lowest dose of 12.5 mg/kg, Re was not detectable in dialysates after 4 h. Extensive gastrointestinal metabolism, poor membrane permeability, and low solubility of deglycosylated products may limit the absorption of ginsenosides in the intestines. Therefore, the dose of test compounds must be high to detect ginsenoside content in plasma (Qi et al., 2011).

According to preclinical trials, several types of saponins, including ginsenosides Rg2, Rh1, F1, Rg1, and protopanaxatriol, may be metabolites of Re in human plasma and urine samples (Liu et al., 2011). After administration of Re (200 mg/kg, p.o. for 24 h), the major excreted ginsenoside metabolites in rat urine included Rg1 and Re. In feces, the main metabolite was Rg1, but other deglycosylated metabolites, including F1 and protopanaxatriol, were also detected (Kim et al., 2013). Yang et al. (2009) identified 11 and nine metabolites together with Re in rat urine collected after intravenous (50 mg/kg, i.v.) and oral (100 mg/kg, p.o.) administration of Re, respectively. The metabolites included Rg1, Rg2, Rh1, and F1. Oral and intravenous doses of Re showed distinct metabolism patterns in the rat, but there were also certain characteristics in common. Deglycosylation was found to be the major metabolic pathway of Re in rats, indicating that a large part of Re was metabolized and transformed in the gastrointestinal tract to ginsenosides with more biological effects (Christensen, 2009). The Re may be metabolized into ginsenosides Rh1 and F1 by human intestinal microflora, and subsequently absorbed into the blood (Bae et al., 2005). After oral administration of 100 mg/kg Re to rats, Chen et al. (2009) detected six metabolites of Re in feces, including ginsenosides Rg₂, Rh₁, Rh1, F1, Rh1, and PPT. In general, Re may be hydrolyzed by gastric fluids to ginsenoside Rg2 that is then converted in the intestine into ginsenoside Rh1 by the elimination of rhamnose through intestinal bacteria. Intact Re also reaches the large intestine where it can be metabolized by bacteria into ginsenoside F1 and 20(S)-PPT via ginsenoside Rg1. Like intestinal bacteria, several food microorganisms produce specific forms of ginsenosides. (Chi and Ji, 2005) tested the biotransformation of Re by cell extracts from various food-grade edible microorganisms, and found Re was transformed into Rh₁ via Rg₂ by Bifidobacterium sp. Int57 and SJ32, Re was transformed into Rh₁ via Rg₁ by Aspergillus niger KCTC 6906, and Re was transformed into Rg₂ by A. usamii var. shirousamii KCTC 6956.

Joo et al. (2010) found that Re was rapidly cleared from the bodies of male or female mice within 0.2 ± 0.03 and 0.5 ± 0.08 h, respectively, after intravenous administration. Chen et al. (1980) estimated that the half-life of Ren in rabbits, after intravenous administration, was about 0.83 h, and the elimination half-life of Re after i.p. injection could be measured from urine (1.165 h) but not plasma samples. In healthy volunteers, the half-life of Re after oral ingestion of Re tablets (200 mg/tablets, p.o.) was reported to be 1.82 ± 0.75 h (Liu et al., 2011). A randomized, double-blind, placebo-controlled trial reported that researchers were unable to detect Re in plasma of obese adults, even though the subjects were prescribed large daily oral doses of P. ginseng and Re for 30 days and ingested the last dose 30 min before collection of blood samples to assess Re concentrations. The absence of Re may be explained by the quick elimination of ginsenoside (Reeds et al., 2011). Pharmacokinetic studies of Re in rats and human volunteers were consistent with this statement. After intragastric (i.g.) administration of Banxia Xiexin Decoction in rats, plasma concentrations of Re at most time points were lower than the lower limit of quantification (Wang et al., 2008a). Pharmacokinetic studies of Re in rats and volunteers following i.v. administration of Shen Mai indicated that Re was quickly eliminated in the body, and that pharmacokinetic characteristics fitted the two-compartment model (Liu et al., 2005; Xia et al., 2008). Altogether, evidence from pharmacokinetic and metabolic studies of Re demonstrated that 1) the absorption of Re was fast in the gastrointestinal tract; 2) Re may be metabolized mainly into Rh1 and F1 by intestinal microflora before absorption into blood; and 3) Re was quickly cleared from the body (Peng et al., 2012).

Attele et al. (2002) found that Re (20 mg/kg, i.p. for 12 days) had marked anti-hyperglycemic activities, with no effect on the body weight of C57BL/6J ob/ob mice. This finding suggests that Re has potential as an anti-diabetic agent. Re (10 mg/kg, i.p. for 12 days) significantly reduced fasting blood glucose levels and promoted glucose tolerance (GT) and systemic insulin sensitivity (IS) in ob/ob mice without affecting body weight (Xie et al., 2005a). These findings suggest Re may provide a therapeutic role in ameliorating GT and insulin resistance (IR) in patients with type 2 diabetes mellitus (T2DM). Administration of Re (0.2 mg/ml for 90 min) rapidly normalized IR and muscle glucose transport induced by high-fat diet (HFD) in the epitrochlearis and soleus muscles of rats (Han et al., 2012). Re may have specifically acted to ameliorate IR in muscles of rats because it failed to modify HFD-induced muscle glucose transport resistance following stimulation by contraction or hypoxia. Muscle contraction and hypoxia exert an insulin-like-stimulating effect on glucose transport. However, Re did not affect basal or insulin-stimulated muscle glucose transport in chow-fed rats. According to these animal studies, P. ginseng or ginsenoside appeared to improve oral GT and accelerate insulin-stimulated glucose disposal (Xie et al., 2004). The Re-induced improvement in IS may or may not be associated with weight loss. Therefore, it remains unclear whether the amelioration was due to weight loss or insulin-sensitizing traits. These studies demonstrated the association between the anti-hyperglycemic activity of Re and improved IS, whereas body weight was unaffected. The improvement may be attributed to the insulin-sensitizing properties of Re. Quan et al. (2012) studied the potential anti-glycemic role of Re in HFD-induced diabetes in mice. Administration of Re (20 mg/kg, i.g. for 3 weeks) markedly lowered BG and triglyceride levels and prevented hepatic steatosis in C57BL/6J mice on a HFD. The hypoglycemic effect was associated with suppression of hepatic gluconeogenesis, possibly associated with AMP-activated protein kinase (AMPK) activation. In rats on a HFD, Re (40 mg/kg, i.p. for 2 weeks, twice a day) improved IR by inhibiting c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-kB activation (Zhang et al., 2008). Several studies have concluded that the anti-hyperglycemic effect of Re was primarily responsible for improved microvasculopathy or reduced cognitive impairment in HFD-induced diabetic mouse models. In such models, Re (20 mg/kg, i.g. for 8 weeks) exerted a protective and anti-angiopathy effect in DM, such as the initial stages of high-sucrose-HFD (HSHF)-induced diabetes, HSHF+alloxan monohydrate-induced Type 1 diabetes mellitus (T1DM), and HSHF+streptozotocin (STZ)-induced T2DM. Administration of Re reduced BG levels, regulated increasing insulin levels, improved lipid metabolism, and reduced endothelial cell dysfunction. The underlying mechanism was possibly associated with p38 mitogen-activated protein kinase (MAPK) activation, and extracellular signal-regulated kinase (ERK) 1/2 and JNK signaling (Shi et al., 2016). In addition, Re (20 mg/kg, i.g. for 2 weeks) had an anti-diabetic microvasculopathy effect, including protective actions against oxidative stress in the kidneys and eyes, and increased BG and lipid levels in rats with STZ-induced diabetes (Cho et al., 2006). In rats with STZ-induced T1DM, Re (40 mg/kg, i.g. for 8 weeks) improved diabetes-related cognitive decline while decreasing fasting BG levels, although it did not affect BG, which was associated with oxidative stress and inflammation (Liu et al., 2012). In mice, Re improved HFD-induced IR through amelioration of hyperglycemia by protecting the brain cholinergic and antioxidant systems (Kim et al., 2017). Specifically, Re (5, 10 and 20 mg/kg/d, i.g. for 4 weeks) improved diabetes-associated cognitive impairment, and was possibly associated with improvement of the anti-oxidant and cholinergic systems in brain tissue. In HFD-induced hyperglycemic C57BL/6 mice, Re played a positive role through amelioration of insulin tolerance and BG levels. Re possibly improved learning and memory disorders related to HFD-induced diabetes. As the major ginsenoside in the P. ginseng berry ethyl acetate fraction (blended with drinking water 20 and 50 mg/kg, p.o. for 4 weeks), Re ameliorated cognitive decline in a dose-dependent manner because of its cholinergic activity, and it decreased oxidative stress in mice with HFD-induced T2DM and behavioral deficiency (Park et al., 2015).

In 3T3-L1 adipocytes, Re (10 μM for 24 h) improved IR by inhibiting the inflammatory signaling cascade and activating the insulin signaling pathway (Zhang et al., 2008). Further results demonstrated that Re (1–10 μΜ for 0.5 h) increased glucose uptake in mature 3T3-L1 cells by significantly enhancing glucose transporter 4 (GLUT4) mRNA expression through the phosphoinositide 3-kinase (PI3K)-dependent pathway involving insulin receptor substrate-1 (IRS-1) in the glucose transport system cascade (Lee et al., 2011). Gao et al. (2013) demonstrated that Re (30, 60 μM for 5 days) reduced IR in adipocytes by directly enhancing the expression of peroxisome proliferator-activated receptor-γ (PPARγ)-2 and the corresponding AP2 genes, increasing adiponectin and IRS-1 expression, inhibiting inflammatory cytokine tumor nuclear factor-α (TNF-α) expression and production, and promoting GLUT4 translocation. The regulation of these factors facilitated adipocyte glucose uptake and disposal, although it failed to enhance GLUT4 expression. Another study found that Re (20 μM for 3 h) suppressed glucose generation in HepG2 cells, possibly by triggering the expression of the orphan nuclear receptor small heterodimer partner gene via AMPK activation (Quan et al., 2012). These results indicate that Re improved IR through reduction of lipotoxicity in the muscles and liver by enhancing adipocyte lipid storage capacity and promoting GLUT4 translocation to plasma membranes. Thus, Re compound regulation of insulin-stimulated glucose ingestion led to improved IR. Furthermore, Re (3 μM for 24 h) was proposed to exert anti-angiogenetic effects in diabetic retinopathy through the PI3K/Akt-mediated hypoxia-inducible factor-1-alpha (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway in high-glucose-induced retinal endothelial RF/6A cells (Xie et al., 2020).

Overall, in vivo and in vitro data suggest four possible mechanisms underlying Re-induced improvement of diabetes and diabetes-related complications: 1) regulation of insulin resistance and insulin secretion, 2) modulation of glucose or lipid metabolism, 3) modulation of inflammatory cytokines, and 4) activation of oxidative stress.

In rats with sciatic nerve crush injury, Re (2.0 mg/kg, i.p. for 4 weeks) promoted functional recovery, nerve regeneration, and proliferation of injured sciatic nerves. The Re compound promoted Schwann cell proliferation, differentiation, and migration during the course of peripheral neural repair after crush injury. This effect was possibly mediated by the regulation of ERK1/2 and JNK1/2 signaling pathways (Wang et al., 2015).

One study reported the anti-oxidant effects of Re (5, 10 and 20 mg/kg, i.g. for 1 week) in rats with cerebral ischemia-reperfusion (I/R) injury. The Re compound considerably increased membrane fluidity of brain mitochondria, activated anti-oxidative enzymes, and decreased lipid peroxidation products, including malondialdehyde (Zhou et al., 2006). Neuroprotective effects of Re (5, 10 and 20 mg/kg, i.g. for 1 week) against cerebral I/R injury in rats were associated with a reduction in malondialdehyde levels and mitochondrial swelling, leading to an increase in H⁺-ATPase activity (Chen et al., 2008).

Tu et al. (2017) reported that Re (20 mg/kg, i.p. for 3 days) attenuated convulsive behaviors, oxidative damage, pro-apoptotic potential and neuronal degeneration through the interleukin-6 (IL-6)-dependent PI3K/Akt signaling pathway in mice with trimethyltin-induced neurotoxicity. Treatment with Re (20 mg/kg, i.p. for 1 day) markedly decreased phencyclidine-induced neurotoxic alterations, including behavioral changes and mitochondrial dysfunction. These Re-mediated alterations were due to interactive modulation between glutathione peroxidase-1 (GPx-1) and NADPH oxidase in mice (Tran et al., 2017).

Administration of Re (50 mg/kg, i.p. for 10 days) before immobilization stress markedly improved body weight, serum corticosterone levels, behavioral alterations, and cognitive deficits in rats. These effects were mediated through modulation of the central noradrenergic system and hypothalamic corticotrophin-releasing factor in the brain (Lee B et al., 2012). Another study showed Re (20, 40 mg/kg, i.p. for 3 weeks) inhibited memory deficits induced by chronic restraint stress (Wang et al., 2021). The protective effects were related to anti-inflammatory and anti-oxidant activities of the Re compound, as well as positive regulation of brain-derived neurotrophic factor and plasticity-associated proteins in the hippocampus.

Administration of Re can effectively prevent onset of Alzheimer’s disease (AD) by improving the activity of dopamine (DA) neurons. One study found that Re (6.5, 13 and 26 mg/kg, i.g. for 13 days) prevented apoptosis of substantia nigra dopaminergic neurons induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in C57BL mice (Xu et al., 2005). The effect was mediated by reversing the abnormal expression of apoptosis regulatory proteins and inhibiting caspase-3 activation. Administration of Re (10, 20 mg/kg, i.p. for 2 weeks, twice a day) rescued methamphetamine-induced dopaminergic neurotoxicity. The effect was associated with potentiating oxidative burdens, compensative induction of GPx activity, mitochondrial dysfunction, pro-inflammatory changes, apoptotic cellular degeneration, and dopaminergic degeneration through inactivation of the protein kinase Cẟ (PKCδ) gene (Shin et al., 2014). Another study reported that Re (20 mg/kg, i.p. for 5 days, twice a day) protected methamphetamine-treated prodynorphin knockout mice against dopaminergic neurotoxicity through anti-oxidant, anti-inflammatory, and anti-apoptotic actions. The effects were facilitated by dynorphin-induced upregulation of the κ-opioid receptor, followed by substance P-mediated downregulation of the NK1 receptor (Dang et al., 2018).

Administration of Re (10 µM) and ginsenoside Rd (5 µM for 48 h) provided considerable neuroprotective effects on primary dopaminergic midbrain neurons treated with CCl₄. The neuroprotective effects were in part due to the lowering of oxidative stress and alleviation of inflammatory responses (Zhang et al., 2016). In addition, Re treatment (50, 100 μM for 24 h) of SH-SY5Y cells rescued methamphetamine-induced mitochondrial burden (compensative induction of cytosolic and mitochondrial GPx activity, mitochondrial oxidative stress, mitochondrial dysfunction, and mitochondrial translocation of cleaved PKCδ, and pro-apoptosis through genetic inhibition of PKCδ) (Nam et al., 2015). Kim et al. (2012) investigated the actions of Re on mitochondrial dysfunction in a PD model. They found that Re (3 µM) targeted mitochondrial dysfunction and rescued the defective PINK1-Hsp90/LRPPRC-Hsp60-complex IV signaling axis of PINK1-null neurons by restoring nitric oxide (NO) levels. Co-treatment using Rd and Re (0.5, 1 μM for 24 h) protected SH-SY5Y cells against rotenone-induced toxicity by regulating molecular mechanisms that enhanced cell viability, including prevention of morphological changes, lowered oxidative stress, improved mitochondrial integrity and function, and inhibited apoptosis owing to oxidative stress (Gonzalez-Burgos et al., 2017). The anti-oxidant mechanism of Re in PD remains unclear. In SH-SY5Y cells treated with 6-hydroxydopamine to induce oxidative stress, the Re compound (25 µM for 9 h) mediated its anti-oxidant effect by upregulating a key antioxidant gene GPX4 via PI3K/Akt and ERK cascades (Lee et al., 2020).

Kai-Xin-San, a Chinese herbal formula, has been clinically administered at 3 g/kg (i.g. for 4 weeks) to treat animals with AD and neurosis. P. ginseng, a component of Kai-Xin-San, is known to enhance learning ability and memory. In addition, positive effects of Re and Rb1, the most abundant saponins, on learning ability and memory were reported (Wang et al., 2010). Amyloid β (Aβ) peptide plays an important role in AD. Zhou et al. reported that Re may interfere with AD progression by affecting the Aβ peptide (Zhou et al., 2020). Oral administration of Re (25 mg/kg, i.g. for 18 h) considerably reduced Aβ1-40 and Aβ1-42 levels in brains of Tg2576 mice (Chen et al., 2006). Furthermore, Li et al. (2018) demonstrated that Re (4 mg/kg, i.g. for 40 days) improved cognitive impairment, reduced Aβ accumulation, and restored biomarker levels, including amino acids, lecithin, and sphingolipids in the plasma of AD mice. Because of its effect on Aβ peptides, Re is increasingly considered a potential alternative drug for AD treatment. In addition, Re exhibits anti-dementia activity. The Re compound improved extracellular levels of DA and acetylcholine (Ach), particularly in the hippocampus. Also, Re (12.5, 25 and 50 mg/kg, s.c.) increased extracellular levels of DA and Ach in the medial prefrontal cortex (Shi et al., 2013).

Treatment with Re has been reported to improve AD by affecting Aβ peptide levels in several cell models. Liang et al. reported that Re markedly reduced the generation of Aβ proteins in N2a/APP695 cells. The effect of Re (50–100 μM for 24 h) on Aβ generation was mediated by PPARγ activation in combination with Aβ-site precursor protein-cleaving enzyme 1 inhibition (Cao et al., 2016). Treatment with Re (0.1–100 μM for 2 h) considerably reduced cell toxicity and increased the release of lactate dehydrogenase, thereby attenuating PC12 cell damage induced by Aβ peptides (Ji et al., 2006). In addition, Re (25 µM for 48 h) exhibited neuroprotective activity against neurotoxicity arising from Aβ25-35 in SH-SY5Y cells by reducing oxidative damage and neuronal cell apoptosis. The neuroprotective activity was associated with the activation of nuclear factor erythroid-2 associated factor 2/heme oxygenase-1 anti-oxidant response pathways and inhibition of reactive oxygen species (ROS)-dependent apoptosis signal-regulated kinase 1/JNK/Bax apoptosis pathways (Liu et al., 2019). Furthermore, Kim et al. demonstrated that Re (5 μg/ml for 48 h) effectively upregulated the expression of choline acetyltransferase and vesicular acetylcholine transporter, and Ach production in Neuro-2a cells, thus countering symptoms during AD progression (Kim J et al., 2014).

In vivo and in vitro data suggest six possible mechanisms of Re-mediated improvement of complications associated with nervous system diseases: 1) regulation of central cholinergic pathways, 2) modulation of the apoptotic signaling pathway, 3) modulation of inflammatory responses, 4) modulation of mitochondrial burden, 5) regulation of anti-oxidant signaling pathways, and 6) reduction of Aβ peptide accumulation and loss of midbrain DA neurons.

Treatment with Re considerably inhibited neutrophil infiltration in a model of skin inflammation arising from 12-O-tetradecanoylphorbol-13-acetate. It also improved paw and ear oedema, increased malondialdehyde levels in paw fluid during c-carrageenan-induced edema, and suppressed interleukin-1β (IL-1β) and TNF-α expression in lipopolysaccharide (LPS)-stimulated murine Raw 264.7 macrophages (Paul et al., 2012). Moreover, Re (1 mg/kg, i.v. for 15 min) suppressed the LPS-induced increase in body temperature, white blood cell count, and pro-inflammatory mediators (Su et al., 2015). In LPS-induced systemic inflammation, Re (10, 20 mg/kg, i.g. for 4 h) suppressed serum levels of IL-1β and TNF-α in mice. Similarly, in 2,4,6-trinitrobenzene sulfonic acid-induced colitic mice, Re (10, 20 mg/kg, i.g. for 3 days) suppressed the expression of IL-1β, TNF-α, cyclooxygenase-2, and inducible nitric oxide synthase, and the activation of transcription factor NF-κB. However, it enhanced the expression of anti-inflammatory cytokine IL-10, indicating that Re can suppress Th1 rather than Th2 cell activation (Lee I et al., 2012). Administration of Re (15 mg/kg, i.g. for 1 week) also prevented NF-κB activation and LPS-induced myocardial inflammation in mice. The action of Re in cardiac dysfunction involves both MAPK inhibition and preserved activation of estrogen receptors and the PI3K/Akt signaling pathway (Chen et al., 2016). Treatment with Re (6–50 mg/kg, p.o. for 2 h) produced strong and significant inhibitory actions against LPS-induced lung inflammation in mice, and decreased inflammatory cell infiltration into lung tissue. The effect was mediated by inhibiting the activation of MAPK and transcription factors NF-κB and c-Fos (Lee et al., 2018).

An in vitro investigation of the anti-inflammatory effects of Re (5, 10 μΜ for 30 min) in macrophages showed that it suppressed the expression of pro-inflammatory cytokines (TNF-α and IL-1β) and activation of transcription factor NF-κB by preventing the binding between LPS and toll-like receptor 4 (TLR4). However, Re did not suppress pro-inflammatory cytokines in peptidoglycan- or TNF-α-stimulated peritoneal macrophages (Lee J et al., 2012), highlighting its action in reducing inflammation by suppressing the LPS and TLR4 interaction in macrophages. Su et al. (2015) demonstrated that Re (50 μg/ml for 1 h) competed with LPS binding to the TLR4, and blocked the LPS-triggered signaling pathway in LPS-stimulated RAW264.7 cells. Extracellular Re was shown to compete with LPS binding to the TLR4, consistent with its role in the activation of extracellular TLR4 (Su et al., 2012). In addition, Wu et al. reported an anti-inflammatory role of Re (10–100 μΜ for 48 h) in LPS-induced activated N9 microglial cells. Re mediated its effects by inhibiting the generation of NO and TNF-α through downregulation of NF-κB activation (Wu et al., 2007). Treatment with Re (2 μg/ml for 24 h) reduced neuroinflammation by reducing the levels of inducible nitric oxide synthase and cyclooxygenase-2, and activating p38 MAPK in LPS-treated BV2 microglial cells (Lee K et al., 2012). Moreover, Quan H et al. (2019) reported that Re (10–40 μΜ for 24 h) inhibited LPS-induced TNF-α and IL-6 production in RAW264.7 cells, and reduced IL-6, NO, prostaglandin E2, and TNF-α secretion in primary rat hepatocytes via MAPK and NF-κB signaling pathways. Re is an effective component of Shen Fu, and was reported to exert anti-inflammatory effects by suppressing the NF-κB signaling pathway in TNF-α-stimulated EAhy926 cells (Li P et al., 2016). Incubation with Re (1.7 μg/ml for 24 h) decreased histamine secretion in human mast cells, and reduced IL-1α, IL-8, and IL-10 levels, and regulated T-cell-expressed and secreted protein secretion in A549 cells (Bae et al., 2012).

Altogether, in vivo and in vitro study data indicate that the possible mechanism of anti-inflammatory activities of Re involves NF-κB inactivation and reduced inflammatory cytokine release.

Kim et al. (2011) showed that Re improved ischemia/reperfusion (I/R) dysfunction by reversing the hemodynamic change (aortic flow, coronary flow, perfusion pressure, and cardiac output) and inhibiting the level of intracellular Ca²⁺ ([Ca²⁺]_i). This study indicated that the anti-ischemic effect of Re was mediated by inhibiting an increase of [Ca²⁺]_i. Additionally, Re prevented heart mitochondrial Ca²⁺ accumulation in I/R injury. In isolated single cardiomyocytes, Re suppressed the L-type Ca²⁺ current and strengthened the slowly activating delayed rectifier K⁺ current (I_Ks). This may be the underlying mechanism that prevented mitochondrial Ca²⁺ overload (Bai et al., 2003; Bai et al., 2004).

A rat model showed that Re (20 mg/kg, i.g. for 15 days) provided an effective treatment for myocardial infraction arising from left anterior descending coronary artery ligation. Treatment with Re improved the parameters of myocardial injury by downregulating the expression of intercellular adhesion molecule-1 and inhibiting polymorphonuclear leukocyte infiltration (Jing et al., 2010; Li et al., 2013). In this research, Re was reported to exhibit a protective role in ischemia-induced myocardial injury by regulating calcium transport, preserving mitochondrial structure and function, enhancing anti-oxidant capacity, and recovering myocardial blood flow.

In addition, Re lowered myocardial injury and suppressed cardiac hypertrophy in experimental models with cardiac dysfunction. Lim et al. (2013) proposed that Re (100 μM, injected into the aortic line for 3 min) exerted beneficial effects on cardiac function in rats with I/R injury, considerably improved hemodynamic functions and left ventricular developed pressure, ameliorated electrocardiographic abnormalities, and decreased the production of TNF-α. Treatment with Re (5, 20 mg/kg, i.g. for 4 weeks) also reduced isoproterenol-induced myocardial fibrosis, increased heart weight and hydroxyproline content, and reduced heart failure. The molecular mechanisms underlying the protective role of Re were possibly related to regulation of the transforming growth factor-beta 1 (TGF-β1)/Smad3 pathway (Wang et al., 2019). In a rat model of myocardial injury, Re (135 mg/kg, i.g. for 4 weeks) preserved cardiac function and structure, reduced myocardial injury and stress, and decreased left ventricular fibrosis by regulating the AMPK/TGF-β1/Smad2/3 and FAK/PI3K/Akt signaling pathways (Yu et al., 2020). These findings suggest a possible therapeutic role for Re in suppressing ventricular remodeling and promoting postinfarction healing. Overall, Re restored blood supply quickly and also delayed detrimental ventricular remodeling during chronic myocardial infraction rehabilitation.

Wang et al. found that Re (200 μg/ml for 24 h) increased H9c2 cell viability after tertbutyl hydroperoxide treatment and reduced lactate dehydrogenase release and cell apoptosis (Wang et al., 2020). Treatment with Re (100 μM for 3 h) inhibited glucose deprivation-induced autophagy of H9c2 cardiac muscle cells, an effect which may be associated with the inhibition of autophagy, increase in cellular ATP content and viability, and alleviation of oxidative stress (Zhang et al., 2020). In addition, in the hypoxia/reoxygenation injury model, Re (100 µM for 21 h) increased HL-1 cell viability and ATP levels. The possible mechanism was that Re acted on the binding interface between HIF-1α and von Hippel-Lindau protein to prevent the binding of these proteins, thereby suppressing HIF-1α ubiquitination (Sun et al., 2020).

Administration of Re (≥10 nM) effectively suppressed the electromechanical alternans of cardiomyocytes in cats and humans by increasing sarcoplasmic reticulum Ca²⁺-release channels, and thereby improving arrhythmia (Wang et al., 2008b). Furukawa et al. (2006) showed that Re (3 μM) increased I_Ks, [Ca²⁺]_i, activation of eNOS, and NO production through a c-Src/PI3K/Akt-dependent mechanism related to the non-genomic pathway of sex steroid receptors. Similarly, in vascular smooth muscle cells (VSMCs), Re non-genomically and dose dependently activated K_Ca currents and eNOS (EC₅₀ = 4.1 ± 0.3 μM) through the c-Src/PI3-kinase/Akt pathway of the estrogen receptor (Nakaya et al., 2007). A study on human umbilical vein endothelial cells (HUVECs) revealed that Re augmented [Ca²⁺]_i and NO production in a dose-dependent manner (EC₅₀ of 316 and 615 nM, respectively) (Leung et al., 2007). In human coronary artery endothelial cells, Re (1 μM) induced vasorelaxation by increasing small-conductance Ca²⁺-activated K⁺ (SK_Ca) channel activity, stimulating NO production, and promoting vasodilation (Sukrittanon et al., 2014).

Abnormal structure and function of VSMCs may result in the development and progression of arteriosclerosis. Enhanced proliferation and migration of VSMCs represent critical events during the course of atherosclerotic lesion development (Bennett et al., 2016). Gao et al. (2018) demonstrated that Re (25 or 50 mg/kg, i.g. for 2 weeks) inhibited VSMC proliferation by suppressing phenotypic modulation and inhibiting vascular neointimal hyperplasia in balloon-injured rats through the eNOS/NO/cyclic guanosine monophosphate (cGMP) pathway. Re improved platelet-derived growth factor-BB-induced VSMC proliferation through G₀/G₁ cell cycle arrest, which was associated with eNOS/NO/cGMP pathway activation (Gao et al., 2019).

In contrast, endothelial cells provide an interface between circulating blood in the lumen and other vessel walls. Endothelial cells exhibit great sensitivity and vulnerability to toxic substances circulating in blood vessels. Endothelial dysfunction is an important contributor to the pathobiology of atherosclerosis (Gimbrone and García-Cardeña, 2016). Huang et al. (2016) found that Re (4, 16, and 64 μmol/L for 24 h) attenuated oxidative damage in H₂O₂-induced HUVECs and increased the production of NO and eNOS, superoxide dismutase (SOD), and GPx activities. The protective effects were associated with an oxidative stress response, protein synthesis and mitochondrial function. In addition, Yang et al. demonstrated that Re (120 μg/ml for 12 h) improved oxidized low-density lipoprotein-induced endothelial cell apoptosis. The effect was possibly elicited through regulation of oxidative stress, inhibition of inflammatory mediators, and recovery of balanced pro- and anti-apoptotic protein expression via p38/MAPK/NF-κB and PI3K/Akt/NF-κB pathways. These pathways may be regulated by the lectin-like oxidized low-density lipoprotein receptor-1, NADPH oxidase, and estrogen receptor α (Yang et al., 2018). Therefore, Re is a potential anti-oxidant that may be used to protect HUVECs from damage by oxidative stress through the anti-oxidant defense system. The Re compound also inhibited VSMC proliferation, attenuated endothelial dysfunction, and possibly promoted NO production, thereby reducing atherosclerosis.

Re is a pro-angiogenic compound with high stability that upregulates in vitro proliferation, migration, chemo-invasion, and tube formation of HUVECs. It also affects ex vivo aortic sprouting and in vivo neovascularization. In vitro results revealed that Re (10–30 μg/ml for 48 h) dose dependently enhanced the proliferation, migration, and tube formation of HUVECs (Huang et al., 2005). Additionally, extracellular matrix incorporating Re (70 μg for 1 week and 1 month) induced angiogenesis and enhanced tissue regeneration by increasing neocapillary density and tissue hemoglobin in a rat model (Yu et al., 2007). These findings indicate that Re can serve as an angiogenic agent to accelerate tissue regeneration.

In summary, in vivo and in vitro reports suggest five possible mechanisms by which Re may improve the cardiovascular system: 1) attenuation of myocardial ischemia, 2) inhibition of [Ca²⁺]_i and activation of I_Ks, 3) increased NO production, 4) reduced cardiomyocyte apoptosis autophagy, and 5) the regulation of oxidative stress.

A combination of Re and cisplatin increased the survival rate of LLC-PK1 cells by 21.4%. However, the renoprotective effects of Re were weaker than that of Maillard reaction products in Re-leucine/serine and glucose-leucine mixtures. Moreover, Maillard reaction products reduced cisplatin-induced oxidative kidney damage by increasing 1,1-diphenyl-2 picrylhydrazyl radical-scavenging activity and decreasing the expression of cleaved caspase-3 protein in rats (Lee W et al., 2012; Kim M et al., 2014). (Wang et al., 2008c) found that Re (25 mg/kg, i.g. for 10 days) considerably suppressed acute kidney injury induced by cisplatin in mice, by inhibiting the oxidative stress damage, inflammatory response, and apoptosis. Re (5, 10 mg/kg, i.p. for 1 week) also improved cyclophosphamide-induced myelosuppression, alleviated clinical symptoms of myelosuppression, and promoted recovery of bone marrow hematopoietic functions. The possible mechanisms involved the regulation of hematopoiesis-related cytokine levels, promotion of cellular entry to the normal cell cycle, and improvement of bone marrow nucleated cell apoptosis-related protein expression (Han et al., 2019).

One mg/mL of American P. ginseng berry extract (containing 15.1 mg/g of Re for 72 h) exhibited strong anti-proliferative effects and triggered morphological alterations in SW480 human colorectal cancer cells (Xie et al., 2011). Re-carbon dots (0.5 mg/ml for 4 h) inhibited cancer cell proliferation (A375, HepG2, and MCF-7 cells) through the ROS-mediated pathway. However, the inhibitory effect on A375 cells was higher than that on other cells. Re induced apoptosis via the ROS- and caspase-mediated pathways (Yao et al., 2018). These findings demonstrate that Re can be used as a potential anti-cancer adjuvant for preventing and treating various cancers.

Altogether, in vivo and in vitro data show three possible mechanisms underlying the anti-cancer activities of Re: 1) inhibition of cell proliferation, 2) induction of cell apoptosis and 3) modulation of oxidative damage.

Song et al. (2014) demonstrated that Re (100 μg/ml for 48 h) had potential therapeutic efficacy in CVB3 and HRV3 infections in HeLa and Vero cells, respectively. Su et al. (2014) showed that co-administration of Re (5.0 mg/kg. s.c. for 3 weeks) with the rabies virus vaccine remarkably increased the serum antibody response in mice. Other studies have shown that co-administration of Re (50 μg, s.c. for 3 weeks) with inactivated influenza virus A/Fujian/411/2002 (H3N2) markedly amplified serum-specific antibody responses (IgG, IgG1, IgG2a, and IgG2b), hemagglutination inhibition titers, lymphocyte proliferation responses, and IL-5 and IFN-γ production (Song et al., 2010). Chan et al. (2011) reported that Re (50 μΜ for 16 h) protected HUVECs from H9N2/G1 influenza virus-induced apoptosis. CD4⁺ T cells are important immune cells in the human immune system. Son et al. found that Re (10, 20 and 40 μg/ml for 24 h) enhanced the viability of activated CD4⁺ T cells by downregulating IFN-γ production, which interfered with autophagy by reducing immunity-associated GTPase family proteins (Son et al., 2010). Re also enhanced the expression of Th1-type-related and Th2-type-related cytokines (Su et al., 2014). Administration of Re (10, 25 and 50 μg, s.c. for 2 weeks) had considerable adjuvant effects on specific antibody and cellular responses in ovalbumin-immunized mice, affecting the immune system favoring Th1- or Th2-type responses, as shown by enhanced titers of IgG1 and IgG2b isotypes (Sun et al., 2006). These results indicated Re-mediated activation of Th1 and Th2 immune responses in mouse models. Therefore, these studies indicate that Re can enhance the host immune system as a vaccine adjuvant.

An optimal balance of osteoblasts and osteoclasts is crucial for bone remodeling. Impaired bone homeostasis potentially causes bone disease, such as bone fracture and osteoporosis (Feng and McDonald, 2011). It was demonstrated that Re had dual effects promoting osteoblast differentiation and inhibiting osteoclast differentiation. This research showed that Re (2.5, 5 and 10 μM for 48 h) dose dependently inhibited osteoclast differentiation and decreased nuclear factor of activated T cell cytoplasmic 1 and tartrate-resistant acid phosphatase mRNA levels, which are osteoclast differentiation markers. These effects were elicited by blocking the ERK signaling pathway in bone marrow-derived macrophages stimulated with the receptor activator of NF-κB ligand. Osteoclast generation in zebrafish scales was inhibited by Re (10 μM for 5 weeks), shown by reduced expression of osteoclast marker genes tartrate-resistant acid phosphatase and cathepsin K (Park et al., 2016). Kim et al. (2016) found that Re affected the differentiation and mineralization of osteoblasts both in vitro and in vivo models. Treatment with Re (50 µM for 5 weeks) promoted the expression of osteoblastic markers, including alkaline phosphatase activity, and mRNA levels of alkaline phosphatase, type 1 collagen, and osteocalcin in mouse osteoblast precursor MC3T3-E1 cells. Moreover, Re amplified the mineralization of osteoblasts in mouse MC3T3-E1 cells and zebrafish scales.

Treatment with Re (5, 12, and 30 μM for 0.5 h) provided potential anti-photo-ageing activity in HaCaT keratinocytes under UVB radiation. This activity was possibly elicited through downregulation of UVB-induced intracellular ROS formation, production and secretion of pro-matrix metalloproteinase-2 and -9, and upregulation of total GPx levels and SOD activity (Shin et al., 2018). In addition, Oh et al. (2016) found that Re (5, 12 and 30 μM for 1 h) improved skin barrier functions, shown by enhanced cornified cell envelope formation, filaggrin levels and caspase-14 activity in HaCaT keratinocytes. Furthermore, Re (5, 12 and 30 μM for 24 h) demonstrated anti-oxidative activity through the upregulation of anti-oxidant components including total GPx and SOD under normal conditions. Re also prevented oxidative stress in HaCaT keratinocytes (Lim et al., 2016).

Re (0.05, 0.1 and 0.5 mg/ml for 2 h) protected chick cardiomyocytes from exogenous H₂O₂- and endogenous antimycin A-induced oxidative stress. The underlying mechanism for this protective effect involved scavenging of H₂O₂ and hydroxyl radicals. However, in an electron spin resonance spectroscopy study, Re did not reduce the 1,1-diphenyl-2 picrylhydrazyl-induced electron spin resonance signals for xanthine oxidase or H₂O₂ (Xie et al., 2006). Therefore, direct scavenging of free radicals was impossible through a single anti-oxidation pathway in vivo. The anti-oxidative effects of Re were achieved through activation or enhancement of the intracellular anti-oxidant system.

Kawase et al. (2014) reported that Re (0.1–1 μM for 24 h) exerted a positive effect on cholesterol metabolism, increasing the expression level of sterol 12a-hydroxylase mRNA in rat primary hepatocytes, thereby facilitating cholic acid generation within bile acids.

Jang et al. (2012) reported that Re (25 mg/kg, p.o. for 6 h) potently alleviated scratching behavior in mice with histamine-induced itch, by inhibiting the activation of transcription factors (NF-κB and c-jun), as well as the expression of IL-4 and TNF-α.

Zhang et al. (2006) demonstrated that Re (100 μM for 2 h) improved sperm motility from fertile and asthenozoospermic infertile human subjects by enhancing NOS activity to promote endogenous NO generation.

The Re-enriched fraction (containing 109.0 mg/g of Re, 54.5 mg/kg, i.g. for 5 weeks) of P. ginseng berries effectively restored ethanol-induced erectile dysfunction in male rats through the NO-cGMP pathway (Pyo et al., 2016).

(Li Z et al., 2016) reported that topical treatment (5 mg/day, topical application on the back for 9 weeks) with Re markedly triggered hair shaft growth through selective suppression of hair growth phase transition-associated signaling pathways and TGF-β signaling cascades in nude mice.

Re-mediated bidirectional regulation is dependent on the jejunal contractile status and requires the co-existence of the enteric nervous system, Ca²⁺, and Cajal interstitial cells. The stimulatory role of Re (10 μM) on jejunal contractility of rat isolated jejunal segments was associated with cholinergic stimulation, whereas its inhibitory role was associated with adrenergic activation and the NO-relaxing mechanism (Xiong et al., 2014). In addition, Re (40 μΜ) inhibited pacemaker potentials through ATP-sensitive K⁺ channels and the cGMP/NO-dependent pathway in cultured Cajal interstitial cells obtained from the small intestine of mice (Hong et al., 2015). Re (20, 100 mg/kg, i.g. for 30 min) ameliorated acute gastric mucosal lesions induced by compound 48/80, possibly by triggering mucus secretion and decreasing neutrophil infiltration, inflammation, and oxidative stress in gastric mucosa (Lee et al., 2014).

In vitro rat embryo cultures found that 50 μg/ml Re induced severe developmental delay and significantly reduced the morphological scores of all organ systems, but was not teratogenic to specific organ systems (Chan et al., 2004). However, in vitro embryotoxicity may not reflect the human situation, and limited information about the blood concentration of Re in humans was available from the medical literature. Further investigations are necessary to evaluate the pharmacokinetics and placental transfer of ginsenosides in humans.

No chronic carcinogenicity studies of Re in experimental animals have been found in the literature.

Several studies reported that some patients had vaginal bleeding and breast pain owing to the estrogen-like effects of P. ginseng (Palmer et al., 1978; Greenspan, 1983; Kabalak et al., 2004). The Re compound has an estrogen-like effect (Bae et al., 2005), and may have similar side effects, but these have not been reported in the literature.