Ginsenoside Compound K: Insights into Recent Studies on Pharmacokinetics and Health-Promoting Activities

Ginseng (Panax ginseng) is an herb popular for its medicinal and health properties. Compound K (CK) is a secondary ginsenoside biotransformed from major ginsenosides. Compound K is more bioavailable and soluble than its parent ginsenosides and hence of immense importance. The review summarizes health-promoting in vitro and in vivo studies of CK between 2015 and 2020, including hepatoprotective, anti-inflammatory, anti-atherosclerosis, anti-diabetic, anti-cancer, neuroprotective, anti-aging/skin protective, and others. Clinical trial data are minimal and are primarily based on CK-rich fermented ginseng. Besides, numerous preclinical and clinical studies indicating the pharmacokinetic behavior of CK, its parent compound (Rb1), and processed ginseng extracts are also summarized. With the limited evidence available from animal and clinical studies, it can be stated that CK is safe and well-tolerated. However, lower water solubility, membrane permeability, and efflux significantly diminish the efficacy of CK and restrict its clinical application. We found that the use of nanocarriers and cyclodextrin for CK delivery could overcome these limitations as well as improve the health benefits associated with them. However, these derivatives have not been clinically evaluated, thus requiring a safety assessment for human therapy application. Future studies should be aimed at investigating clinical evidence of CK.


Introduction
Ginseng (Panax ginseng) of the Araliaceae family is a perennial plant which has been conventionally used as a functional food. It is commonly consumed as a health supplement in Korea, Japan, China, the United Kingdom, Canada, Germany, France, and Austria [1]. Ginseng's bioactive constituents, including ginsenosides, phenolic compounds, and polysaccharides, have several medical uses [2]. Ginsenosides (or panaxosides) are the key pharmacologically significant bioactive constituents of ginseng. Nearly 150 ginsenosides isolated from roots, fruits, leaves, flower buds, processed items of ginseng, and other species have been identified [3]. A category of triterpene and saponin ginsenosides are divided into two forms: tetracyclic triterpenoids (four-ring dammarane type) and pentacyclic triterpenoids (five-ring oleanolic type) [4]. The dammarane type saponins are further categorized into protopanaxadiol (PPD) and protopanaxatriol (PPT) saponins [5]. The ginsenosides of PPD group constitute Compound K (CK), Rg3, Ra1, Ra3, Ra2, Rh2, Rb1, Rb3, Rb2, F2, Rc, and Rd, while PPT group constitutes of F1, Rg1, Rg2, Rf, Re, and Rh1 [3,6]. The oleanolic type saponins, such as Ro, are very low in concentration and thus rarely detected [4,6]. Examples of rare ginsenosides are CK, Rg3, F2, and Rh2, which are either absent in unprocessed ginseng or available at low concentrations [7]. It is

Literature Search
A precise literature search was carried out with Google Scholar, PubMed, and the Science Direct repositories for related findings between January 2015 and May 2020. The following keywords: "Ginsenoside compound K" or "Compound K" or "20-O-D-glucopyranosyl-20(S)-protopanaxadiol" or "IH901" or "M1" or "health-promoting activities of compound K," or "Ginsenoside Compound K and  [30] *** The study was conducted on both rats and humans. # analysis was compared with other ginsenosides, including Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, Rf, F1 and F2. ##  Furthermore, a study on healthy Chinese participants has established the relationship between ABCB1 gene polymorphisms and CK pharmacokinetics. The results indicated that the gene NR1I2 (rs1464602 and rs2472682) allied primarily to the pharmacokinetics of CK. While ABCC4 (rs1751034 and rs1189437) influenced the pharmacokinetic behavior of both CK and its metabolite 20(S)-PPD. Such hereditary variations could thus partially describe the inter-individual variances in the pharmacokinetic behavior of CK [31].

Solubility, Permeability, and Efflux
Many health-promoting activities of CK have been reported. However, low water solubility, low membrane permeability, and efflux phenomenon critically weaken its efficacy and restrict its clinical application. The use of cyclodextrin (CyD) and nanocarriers have been implemented to improve the bioavailability of CK. Table 2 summarizes various modifications of CK with their outcomes.
The use of CyD has been duly recognized to improve the pharmacological behavior of drugs. In this line, an inclusion complex, K/γ-CyD, with improved oral bioavailability and solubility [21], compared to an earlier finding (in β-CyD only solubility was improved) has been described [32]. In another study, the use of ginsenoside CK with TPGS (d-alpha-tocopheryl polyethylene glycol (PEG) 1000 succinate) (GCKT)-liposomes has been described to improve solubility, targeting tumor cells, and minimizing efflux. The d-alpha-tocopheryl polyethylene glycol (PEG) 1000 succinate and phospholipid could increase the solubility of CK in the form of GCKT-liposome, leading to significant repression of tumor growth [33]. Phospholipid use improves biocompatibility, which could restore permeability and increase the process of ADME [34]. The d-alpha-tocopheryl polyethylene glycol (PEG) 1000 succinate has widely been documented as an inhibitor of P-glycoprotein (P-gp)-mediated efflux in drug delivery systems [35], and P-gp-mediated efflux was reported to be a significant limiting factor for the efficacy of CK [36].
In another study, CK-micelles (CK-M) from TPGS, PEG, and PCL (polycaprolactone) showed enhanced solubility and improved bioactivities. After 48 h, the CK was released slowly from CK-M with a drug release percentage of more than 42.1 ± 3.2% and without bursting. In the first eight hours, the rate of in vitro drug release for free CK with bursting was 84.4 ± 4.2%. Additionally, the P-gp-mediated efflux in the CK-M group was substantially inhibited compared to free CK, suggesting drug uptake by the target cells [36]. Likewise, CK ascorbyl palmitate (AP)/TPGS micelles enhanced solubility of CK and significantly inhibited P-gp-mediated efflux [37]. Similarly, the micellar system based on phosphatidylcholine (PC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine polyethylene glycol 2000 (DP) showed improved solubility and continued release of CK [38]. The water solubility of the CK nanoparticles (NPs)/bovine serum albumin (BSA) and CK, was compared, and BSA was found to augment the water solubility of CK. The high biocompatibility, dispersive nature, and conjugative ability to several target molecules make BSA a useful carrier molecule [39].
In another study, CK was loaded onto gold(G)NPs synthesized using probiotic bacteria (Lactobacillus kimchicus DCY51 T ) and evaluated for the effectiveness of drug loading [40]. Furthermore, the use of deoxycholic acid (DA)-O-carboxymethyl chitosan (OCMC) has been advocated to increase solubility and ability for CK. For example, CK-NPs conjugated with DA-OCMC showed increased solubility and improved drug entrapping and drug loading efficiencies. The release pattern of CK was pH-dependent and faster at lower pH. The collective release of CK at pH 7.4 and 5.8 was 10.7 ± 0.71%, and 16.3 ± 1.4%, respectively, after the first 48 h, without bursting. Notably, over 120 h of the study, a significant increase in the release of CK was observed. These findings indicate that CK was released slowly (the pH of blood), thus the system could be used for target delivery of CK [41]. Table 2. Solubility, permeability, and efflux of CK and its derivatives.

Modified CK
Model Major Findings Ref.

Safety
As per the new clinical guidelines, drug safety tests should be screened with two animal types, inclusive of non-rodents (usually dogs) and rodents (mice or rats) [44]. For preclinical safety evaluation, rats and mice were assessed for acute and 26-week recurrent-dose toxicity of CK.
Single oral supplementation of CK for rats (8 g/kg) and mice (10 g/kg) did not induce toxicity or mortality in the same. On the other hand, for 26-week toxicity (e.g., clinical symptoms, biochemical and hematological parameters, urine analysis, the body weights, food consumption, and histopathology of rats) were evaluated at 13, 40, or 120 mg/kg doses of CK. The NOAEL (no observed adverse effect levels) doses were 40 mg/kg and 120 mg/kg for females and males, respectively. However, a decrease in body weight, fur-loss, reduced activity, and lack of energy were transiently observed in the 120 mg/kg male test group [45]. Oral preclinical safety of CK was evaluated on Beagle dogs (4, 12, or 36 mg/kg) for 26-weeks. The NOAEL dose for dogs was 12 mg/kg [46].
Considering clinical perspective, in a randomized, double-blind trial on healthy Chinese subjects, compared to placebo, the treatment group were orally administered CK at 100, 200, or 400 mg doses for up to nine times a week. The results of this documented study showed the safety of CK during the intervention period [47]. However, it has been suggested that further evaluations are necessary to affirm the safety of CK administration in humans.

Health-Promoting Activities
Compound K, in terms of its bioactivity, has gained much interest as a rarely known ginsenoside [48]. Investigation on the CK metabolism is beneficial to gain better insights into the pharmacological activities of CK. Concerning this, a recent study used an ultra-performance LC quadrupole time-of-flight tandem MS to characterize CK (oral dose 50 mg/kg) in feces and urine of SD rats, resulting in the detection of tentative twelve (M1-M12) metabolites. The authors suggested sequential oxidation, deglycosylation and conjugation as the key metabolic pathways for CK metabolic profile characterization [49]. This section summarizes recent studies on various health-promoting activities of CK (Tables 3 and 4 and Figure 2).
Similarly, Choi et al. showed the ameliorating effects of GBCK25 (fermented ginseng, rich in CK) on nonalcoholic steatohepatitis (NASH). They found that GBCK25 was capable of downregulating cytochrome P450 2E1 (CYP2E1) levels alongside reduced activation of cellular c-Jun N-terminal kinase (JNK) [55]. These findings indicate that CK can be used for liver disease prevention and/or treatment.

Anti-Inflammatory
From previous studies, the anti-inflammatory activity of CK was ascribed to decreased synthesis of pro-inflammatory cytokines ((interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α)), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) [11]. However, recent studies have strengthened our understanding of the mechanistic implications at molecular and cellular levels ( Table 3). In one study, CK attenuated NF-κB by modulating the Akt1-mediated inflammatory gene expression in LPS-induced macrophages [2]. Compound K-rich fraction (BIOGF1K), consisted of 3.2 g of CK and 1.5 g of saponin F1, and was examined for its anti-inflammatory activity. Compound K-rich fraction has down-regulated LPS-stimulated nitric oxide (NO) production in RAW264.7 cells. Furthermore, expressions of iNOS and IFN-β were reduced by suppressing stimulation of NF-κB and interferon regulatory factor 3, respectively. The inhibitory mechanism of BIOGF1K was due to the blockage of an inhibitor of kB kinase (IKK) and TANK-binding kinase 1 (TBK1), leading to reduced production of NO and IFN-β [56]. Likewise, in another study BIOGF1K inhibited COX-2 and iNOS mRNA expressions in LPS-induced RAW264.7 cells. Mechanistically, BIOGF1 K blocked activation of activator protein-1 (AP-1) pathway by targeting mitogen-activated protein kinases (MAPKs) such as ERK (extracellular signal-regulated kinase) and p38, and MAPK kinases (MAPKKs) such as MAPK/ERK kinase 1/2 and MAPK kinase 3/6 [57]. Together, these findings indicate that BIOGF1 K plays a protective role in macrophage-mediated inflammatory responses. In addition, the use of CK as BSA-CK NPs [39] and CK-conjugated superparamagnetic iron oxide nanoparticles [58], has been shown to have anti-inflammatory activity against RAW 264.7 cells induced by LPS. In another study, CK-mediated modulation of sodium/glucose cotransporter one via the epidermal growth factor receptor (EGFR) pathway was found to reduce intestinal inflammation [59].
Inflammation commonly follows pain. In this line, the effect of CK on inflammation and pain was represented using in vivo models of xylene-induced ear swelling, and paw oedema stimulated with carrageen. The anti-inflammatory and pain-reducing effects of CK were due to the decreased production of prostaglandin E2 by downregulating COX-2 expression (Table 3) [60]. Referring to arthritis, the attenuating role of CK has been shown by inhibiting the production of inflammatory cytokines, suppressing T-cell activation, inhibiting the multiplication of B-cells, macrophages regulation, and reducing the level of autoantibodies [11]. Among T cells, the potential mechanism of CK treatment involves suppression of dendritic cells (DCs) priming of T-cell activation, suppression of chemokine CCL21 (with receptor CCR7) associated with DC movement and signaling between T cells and DCs in collagen-induced arthritis animal model. Notably, a positive correlation (R 2 = 0.9830, p = 0.0009) was found in percentages of activated T-cells and DCs, while a negative correlation (R 2 = 0.8348, p = 0.03) in percentages of naïve T cells and DCs [61]. In another study, CK suppressed humoral immune response of T helper type 1 (Th1) cells and significantly suppressed expressions of matrix metalloproteinases (MMP)-3 and-13 and receptor activator of NF-κB ligand (RANKL) [62]. Concerning effects on B cells, CK was described as reducing the percentage of memory B cells. Authors suggested that the reduction in memory B cells may be dependent upon T-cells [63]. Previously, CK displayed anti-arithic effects on multifunctional macrophages by reducing the development of pro-inflammatory cytokines. In a recent study, however, the function of CK was shown to inhibit β-arrestin2, thus hindering the transition of macrophages from type M1 to type M2 [64]. The protective role of CK was also reported against osteoarthritis using in vitro and silico studies. Compound K displayed high binding affinity to a cytokine-activated kinase (IKK) compared to other ginsenosides as revealed in the molecular docking analysis. Thus, the anti-osteoarthritic effect of CK was due to inhibition of IKK activity in vitro [65]. Interestingly, in a recent study, GNPs were made intracellularly using Gluconacetobacter liquefaciens kh-1 (a probiotic strain) and used for synthesizing peptide (CopA3) conjugated nanoparticle (GNP-CK-CopA3) hybrids. Compound K, as peptide-nanoparticle hybrids showed anti-inflammatory effects by inhibiting the activation of NF-kB and MAPK signaling pathways [66].

Anti-Atherosclerosis
Atherosclerosis is well known to be an inflammatory disease; the anti-inflammatory effects of CK are more or less directly linked to its anti-atherogenic effects. In terms of anti-atherosclerosis, an important feature of CK was found to be associated with liver X receptor alpha (LXRα) ( Table 3). Targeting LXRα, a study showed that CK treatment resulted in a dose-dependent reduction of atherosclerotic plaques by activating the reverse cholesterol transport (RCT) pathway, reducing inflammatory cytokines, and inhibition of inflammasome activity with LXR activation in apoE-/-C57BL/6 mice. Compound K triggered the RCT pathway by upregulating ATP-binding cassette transporter (ABC) A1, ABCG1, LXRα, ABCG5, and ABCG8. In addition, CK supplementation increased the cholesterol efflux and reduced the inflammasome activity in peritoneal macrophages of mice [67]. Another research demonstrated the use of CK and its derivatives in the activation of LXRα. The study documented the synthesis of six CK derivatives by adding short-chain fatty acids into the carbohydrate chain of CK at different locations. Effects on the foam cell model were evaluated, and the biological activities of all derivatives were found to be at par or better than their parent CK. All derivatives were capable of activating LXRα. Compound K derivative 1 displayed the best potency amongst all [68].
Similarly, it was found that the CK prevents inflammation and apoptosis in human umbilical vein endothelial cells, induced through oxidized low-density lipoprotein (ox-LDL). In endothelial cells, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) uptakes ox-LDL leading to pro-inflammatory effects. Compound K decreased LOX-1 expression and inhibited the nuclear translocation of NF-kB, and phosphorylation of JNK and p38 [69]. The results indicate that CK [67,69] and its derivatives [68] have the anti-atherosclerosis effect.

Anti-Diabetic
Ginsenosides play an important anti-diabetic role by modulating insulin resistance, regulating lipid and glucose metabolism, protecting from the inflammatory response, and oxidative stress. In this line, a study showed that CK administration suppressed liver gluconeogenesis by inhibiting glucose-6-phosphatase and phosphoenolpyruvate carboxykinase expressions in HFD-fed ICR mouse model and HepG2 cell line. Meanwhile, the expressions of hepatocyte nuclear factor 4 alpha, peroxisome proliferator-activated receptor 1-alpha, and forkhead transcription factor O1 were decreased while AMPK phosphorylation was increased significantly [70]. Furthermore, the management of insulin resistance is essential for controlling diabetes. In this line, CK was found to be able to inhibit inflammation and modulate insulin resistance in adipose tissue by repressing the activation of NOD-like receptor family, pyrin-containing protein 3 (NLRP3) associated with endoplasmic reticulum stress (ERS) ( Table 3) [71]. In another study, beta-cyclodextrin-conjugated CK (β-CD) was used to modulate diabetes against an alloxan-induced zebrafish model. The recovery of affected pancreatic islets in CD-CK conjugate was significantly higher (EC 50 = 2.16 µM) than in CK (EC 50 = 7.22 µM) [72]. Furthermore, the protective effect of CK on diabetic nephropathy in HFD/ streptozotocin-induced mice has been demonstrated through significant reduction of oxidative stress and down-regulating expressions of NADPH oxidase (Nox)-1 and-4 proteins. Additionally, the reactive oxygen species (ROS)-mediated activation of the inflammasome assembly was reduced, and renal p38 MAPK phosphorylation was inhibited (Table 3) [73].

Anti-Cancer
The promising anti-cancer activity CK has been previously identified in various types of cell lines, including lung carcinoma, leukemia, breast cancer, colorectal cancer, prostate cancer, gastric carcinoma, nasopharyngeal carcinoma and pulmonary adenocarcinoma [11,74]. Among recent findings (Table 4), a study documented the suppressing effect of CK on COX-2 and Arg-1 genes linked to immunosuppression, apoptosis, and pro-inflammatory cytokines production by myeloid-derived suppressor cells (MDSCs) from the xenografted colorectal (CT26) cancer mice. Compound K could act as a promising therapeutic molecule by targeting MDSCs [75]. Another study elucidated the inhibitory action of CK on the development and metastasis of glioblastoma cell lines (U87MG and U373MG). The effects were due to cell cycle arrest, decreased cyclins (D1 and D3) levels, apoptosis through nuclear condensation, activation of apoptotic enzymes, increased production of ROS, and the depolarized potential of the mitochondrial membrane. The anti-proliferative effect was due to the blockage of the phosphatidylinositol three kinase (PI3K)/Akt signaling pathway in glioblastoma [76]. Likewise, CK was found to block glycogen synthase kinase 3β signaling [77] and the PI3K/Akt signaling pathway [78] in breast cancer cells (MCF-7). Additionally, the combined CK and cisplatin had better effects than either molecule alone [78]. A later study gave in vivo evidence of CK's protecting effects against hormone-independent breast cancer by degrading cyclin D1 protein [79] (Table 4). Recently, Li et al. synthesized ester derivatives (1c, 2c, 3c) of M1, and found that among all, compounds 2c, 3c had an effective growth inhibitory effect on MCF-7 cells [80]. Another study shed light on the biological mechanism of CK against breast cancer using SKBR3 cells. Compound K displayed anti-cancer effects in SKBR3 cells by enhancing apoptosis through downregulation of Akt-1. In addition, CK was found to reduce invasion and metastasis [81].
Compound K inhibited proliferation, augmented autophagy, and apoptosis of non-small cell lung cancer (NSCLC) (A549 and H1975) cells through the mammalian target of rapamycin (mTOR)/AMPK and JNK signaling pathways (Table 4) [82]. Furthermore, the suppression of the growth of NSCLC cells was studied by targeting the metabolism of glucose. Compound K suppressed the levels of hypoxia-inducible factor 1-alpha and its downstream glucose transporter1 gene [83]. Another study showed the anti-cancer effect of CK against HepG2 cells and xenografted (HepG2) BALB/c nude mice. Compound K resulted in cell cycle arrest, blocked cell cycle progression, and apoptosis induction by modulating B-cell lymphoma 2 (Bcl2) to Bcl2 associated X (an apoptosis regulator) ratio in HepG2 cells. Furthermore, a substantial reduction in tumor proliferation was observed in the CK-supplemented mice group [84]. Also, CK induced apoptosis and ERS in liver cancer cells and xenografted mice by modulating signal transducers and activators of transcription-3 (STAT3) activation [85]. Another study showed for the first time that CK targeted annexin A2, which leads to inhibition of NF-κB [86]. Compound K enhanced ERS and calcium release by ryanodine receptors leading to apoptosis in lung cancer cells of humans. In particular, the use of an ER stress inhibitor (4-phenylbutyrate) enhanced CK-mediated apoptosis [87]. Another research provided the first proof that the CK usage results in the TNF-related apoptosis-inducing ligand (TRAIL) sensitization in TRAIL-resistant HT-29 cells and potentiated TRAIL-stimulated apoptosis in HCT116 by autophagy-linked death receptor (DR) 5 stimulation. The upregulated expression of DR5 was dependent upon ROS mediated JNK-autophagy-activation and CCAAT/enhancer-binding protein (C/EBP) homologous protein/p53 pathway (autophagy-independent) [88]. In neuroblastoma cells, CK enhanced ROS-linked apoptosis and impaired the autophagic flux. In addition, CK with chloroquine (combination approach) stimulated apoptosis in cell line and mouse models and may, therefore, be a potential approach for treating neuroblastoma [16]. The protective role of CK was also investigated against glioma (inveterate brain tumor). Compound K was observed to inhibit the stromal cell-derived growth factor 1 migration of C6 glioma cells by controlling protein kinase C alpha, ERK1/2, and MMP signaling molecules (Table 4) [89]. Recent studies showed the bioactivity of CK against human osteosarcoma cell (MG63 and U2-OS) lines. ↑ anti-proliferative and apoptotic activities ↑ levels of cleaved caspase-7, -8, and caspase-9 ↓ Bcl2 levels and AKT-1 levels, no effect on AKT-2 levels [81]   GCKT-liposomes group, ↓mean tumor size from 219.0 ± 17.0 mm 3 to 45.8 ± 3.2 mm 3 slow ↑ in body weight in the initial days later no change [33] In vitro A549 GCK + GCKT-liposomes different concentrations, 24 h IC 50 , GCKT-liposomes (16.3 ± 0.8 µg/ml) and CK (24.9 ± 1.0 µg/ml) No cytotoxicity to A549 with T-liposomes alone    Significant ↓ in neurobehavioral scores ↓water content in brain tissue at 60 mg/kg/day ↓ brain infarct volume ratio ↑SOD and GSH-Px activities and ↓MDA levels ↓expressions of inflammatory molecules [101] CK In vivo Kunming mice SD rats CK (30, 10, and 3 mg/kg), daily once, 4 weeks CK (30, 10, and 3 mg/kg), daily once, 2 weeks Improved depressive-like activities in mice In rats, ↑sucrose preference and body weight Improved food consumption and crossings in CUMS rats ↑dopamine and 5-HT (serotonin) levels and no effect on norepinephrine ↓ expression of neurotransmitter degrading enzymes ↑BDNF, NGF levels and SOD, GPx, and GSH activities [102] CK In vivo Kunming mice CK (30, 10 and 3 mg/kg), 4 weeks Prevented depressive-and anxiety-like behaviors ↓ MDA level and ↑ SOD expression ↓ IL-1β and IL-18 Inhibited expressions of NLRP3 and cleaved caspase-1 [103]   Anti-aging/skin protection
Similarly, CK-M described earlier in this review, were used for in vitro (A549 and PC-9) and in vivo models. The apoptosis percentage of CK-M was higher than free CK. Meanwhile, CK-M also displayed an improved tumor inhibitory effect in vivo [36]. The anti-lung cancer effects (in vitro and in vivo) of CK AP/TPGS mixed micelles [37] and CK PC/DP micellar system [38] have also been documented. Moreover, using in vitro models, BSA-CK NPs showed more significant cytotoxic effects in the liver carcinoma (HepG2), skin cell line (HaCaT), A549 cells, and colon cancer cell line (HT29) compared to monomer CK. However, for in vivo application, authors advocated the use of human serum albumin as an alternative to BSA to evade plausible immunologic concerns in humans [39]. In addition, CK-loaded GNPs have been identified as effectual photothermal and chemotherapeutic agents [40]. On a similar line, a higher dose-dependent inhibitory effect of chitosan nanoparticles loaded with CK (CK-NPs) was observed compared to CK. Authors described a lower (IC 50 = 16.58 µg/mL) value for CK-NPs compared to CK (IC 50 = 23.33 µg/mL) in HepG2 cells [41]. Likewise, CK loaded O-OCMC nanoparticles showed inhibitory effects against prostate cancer (PC3) cells through enhanced cytotoxicity and uptake of CK [42]. Chitosan polymer micelles decorated using A54 peptide, known as APD-CK, were utilized against Huh-7 and HepG2 cells. APD-CK showed higher cytotoxicity compared to CK and promoted apoptosis of HepG2 cells (Table 4) [43]. In another study, tLyp-1 (tumor-homing peptide) decorated liposomes loaded with parthenolide (active anti-tumor agent isolated from Tanacetum parthenium) and CK have been evaluated as lung cancer-targeting system. Enhanced anti-tumor activity was observed against both the cell line and animal model with limited adverse effects. In A549 cells, CK/parthenolide tLyp-1 liposomes decreased mitochondrial membrane potential and allowed greater Ca 2+ efflux as well as significant inhibition of cell migration. From in vivo study, it was found that the complex has greater anti-tumor activity than the combination of these substances [91].

Neuroprotection
Several studies have documented the therapeutic effects of ginseng and ginsenosides in many central nervous system (CNS) ailments, for instance, Alzheimer's disease, Parkinson's disease, depression, and other ailments [6]. The protection could be ascribed to reducing neuroinflammation, neuroprotection, and regulating neurotransmitter release. In previous articles, the cognitive and neuroprotective role of CK has been described [3,11]. The current review summarizes the recent progress in neuroprotection effects of CK (Table 4).
In a study, CK (derived from red ginseng) was able to exert neuroprotective effects in memoryimpaired mouse (scopolamine-induced) model by inducing nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-facilitated antioxidants. No effect was observed on the acetylcholine esterase (AChE) activity. In addition to that, CK defense against glutamate-induced cytotoxicity was also observed in HT-22 cells [93]. Recently, CK was found to be protective against memory and cognitive impairment in db/db mice by suppressing inflammation and oxidative stress, ameliorating dyslipidemia, insulin sensitivity, glucose tolerance as well as modulating ER stress and NLRP3 inflammasome pathway (Table 4) [94]. Moreover, in another study, CK minimized the morphine dependency by decreasing N-methyl-D-aspartate acid receptor subunit 1 activation in cultured cortical neurons from mice and frontal cortex of rat brains [95]. Another recent research documented the protective effects of CK against epilepsy in status epilepticus (SE) rat model. Compound K stimulated the release of gamma amino-butyric acid (GABA) and enhanced the GABA type A receptor subunit alpha1-facilitated inhibitory synaptic transmission [96]. In addition to the anti-atherosclerosis effects of CK and its derivatives as described above [68], the role of LXRα in immunomodulation has also been described in continuous research. Researchers demonstrated that CK was able to mitigate post-operative cognitive dysfunction (POCD). They found that CK inhibited hippocampal inflammation by activating LXRα. CK attenuated memory dysfunction by modulating Morris water-maze (MWM) test scores in an aged mouse model (Table 4) [97].
Regulation of aggregated amyloid-β (Aβ) is an important part of Alzheimer's disease (AD) treatment. Concerning this, CK was found to enhance memory function, reduce Aβ expression and aggregation, and neuronal apoptosis by activating the Nrf2/Kelch-like ECH-associated protein-1 signaling pathway in ICR mice with diminished memory. Furthermore, the defensive effects were also due to the activation of the antioxidant system [98]. Likewise, the influence of the CK was elucidated using Aβ peptide-induced HT22 cells by improving viability, growth, and apoptosis of HT22 cells, as well as localization and expression of Aβ peptide in cells. Besides, ATP levels of cells were enhanced by increasing the activity of glucose transporters. Compound K restored abnormal energy metabolism (Aβ induced) by modulating the expressions of several enzymes (Table 4) [99]. Zong et al. first found that the CK mitigates cerebral discrepancies and Aβ 1-42 accumulation in the hippocampus of chronic cerebral hypoperfusion-induced vascular dementia SD rats by enhancing the pSer9Glycogen synthase kinase-3b expression. To add further, CK upregulated PI3K/Akt pathway, resulting in increased insulin-degrading enzyme activity, a central enzyme accountable for degrading Aβ 1-42 in the brain (Table 4) [100].
In an in vivo research, CK pretreatment resulted in the reduction of neurobehavioral score, water in brain tissue, and the cerebral infarct volume ratio against cerebral ischemia/reperfusion (I/R) injury in Wistar rats. Compound K enhanced activities of antioxidant enzymes, decreased levels of IL-1β, TNF-α, and declined level of high mobility group box 1 protein. Generally, the protective effects of CK against cerebral I/R damage may be due to anti-inflammatory and antioxidant bioactivities [101]. The earlier reviews have described the neuroprotective role of CK [3]. However, its antidepressant effect has been described recently. Depression is a global societal health concern. Concerning the effect of CK on depression, a study used two models: behavioral despair model (mice) and the chronic unpredictable mild stress (CUMS, rats) model. The antidepressant role may be due to regulation of the concentration of monoamine neurotransmitters, enhanced antioxidant capacity, and increased expressions of neuronal growth factor and brain-derived neurotrophic factor in the CNS [102]. In a follow-up study, the defensive effect of CK on CUMS depression was evaluated by inhibiting oxidative stress, inflammatory cytokines and NLRP3 expression using a mouse model [103].
Amongst others, recent work on explaining the beneficial effects of CK on hippocampal neurogenesis has been published. In one study, CK was found to induce neurogenesis and decrease apoptosis in thrombin-induced endogenous neural stem cells (EnNSCs) and improve animal prognosis by stimulating LXRα activation [104]. In another study, CK treatment triggered the proliferation of fresh cells and substantially increased their differentiation in the hippocampus (dentate gyrus) by activating brain derived neurotrophic factor (BDNF) signaling. The higher dose of CK was found to be more effective in aged mice compared to young ones (Table 4) [105].

Anti-Aging/Skin Protection
It is well-known that the production and synthesis of hyaluronic acid (HA) decreases with age. A study by Lim et al. identified that CK led to increased production of HA by activating Src (tyrosine kinase)-dependent Akt and ERK [106]. Ultraviolet type B (UVB) radiation induces photo-aging due to collagen degradation (type I and III) and increased production of iNOS, COX-2, and MMPs. Compound K supplementation diminished production of COX-2 and MMP-1 in NIH3T3 cells treated with UVB, and expression of type I collagen was modulated. Compound K also displayed the ability to improve the skin's moisture level. The study also found skin hydration effects of CK in HaCaT cells (Table 4) [107]. Compound K-rich fraction applied to UVB-treated cells was equally successful in the prevention of UVB-mediated aging [108]. Interestingly, the effectiveness against reducing mRNA expression of MMP-1 was more in a synergistic approach by CK and maclurin (a natural compound) compared to individual compounds [109]. Another study assessed the role of CK in improving skin barrier function by upregulating the expression of serine protease inhibitor Kazal type-5 in atopic dermatitis-like mice and UVB-irradiated mouse model [110]. In recent research, CK improved imiquimod-induced psoriasis by inhibiting regenerating islet-derived protein 3 (RegIII) gamma expression in the mouse model and IL-36γ-induced Reg3A expression in human keratinocytes (Table 4) [111]. These findings advocate that CK plays a key role in the defense and anti-aging effects.

Others
Recently, a patented study investigating the beneficial effects of CK on renal tubulointerstitial lesions in C57BL/6 mice with unilateral ureteral obstruction identified the kidney-protective effects of CK. The results showed reduced production of pro-inflammatory cytokines and the prevention of leukocytes infiltration and fibrosis in the kidney. The positive outcomes were showcased by inhibiting NF-κB-associated priming, and by modulating STAT3 signaling and NLRP3 inflammasome activation (Table 4) [112]. Likewise, another study showed the protecting effects of CK on accelerated and severe lupus nephritis by hindering activation of NLRP3 inflammasome [113]. Ren and coworkers evaluated the protecting effects of CK and its analogues against asthma. Compound K and its analogues displayed a significant impact by reducing IgE and airway resistance [114]. Compound K pretreatment was reported to protect against cardiac I/R by activating the PI3K-Akt signaling pathway, which is crucial for autophagy-triggered apoptosis (Table 4) [115].

Anti-Diabetic
Clinical studies evaluated the bioactivities of hydrolyzed ginseng extract. Recently, a double-blind, randomized controlled trial assessed the anti-glycaemic effects of hydrolyzed ginseng extract (GINST15, rich in CK) on prediabetic participants for 12 months. GINST15 resulted in improved fasting and 1-hour postprandial glucose levels. No effect was observed on 2-hour postprandial glucose levels [116]. An earlier clinical study also confirmed these results in which fermented red ginseng extract supplementation decreased fasting glucose, postprandial glucose levels and improved insulin levels in type 2 diabetic subjects compared to placebo [117].

Neuroprotection
Flanagan et al. have documented the effects of GINST15 on hypo-pituitary-adrenal (HPA) and antioxidant activity clinically. In this double-blind, placebo-controlled, counterbalanced within-group study design different doses (high dose-960 mg and low dose-160 mg) of CK were given for 2 weeks. They found that, in response to severe exercise, CK supplementation resulted in dose-dependent declines in circulating cortisol and augmented antioxidant activity. This study first provided insights into the impact of ginseng treatment on the reactions to the stress associated with work [118]. A recent continued study by the same research group demonstrated direct evidence on task-related brain activity by evaluating CK's treatment on behavioral performance and electroencephalography measures of cortical activity. After exercise, the upper and lower body response times were improved. Compound K augmented activity in cortical regions accountable for sustained attention, whereas exercise-triggered increases in arousal were diminished. In short, CK has been found to have inducible effects on the activity of the brain [119].

Liver Protection
As mentioned above, in an in vivo study, GBCK25 has been found to have defensive effects against NASH [55]. On a similar line, recent research evaluated the protective effects of GBCK25 on liver function in a 12-week, randomized, double-blind clinical trial. The supplementation includes GBCK25 tablets (high (500) and low (125) mg/day) and placebo. Treatment at a low dose significantly reduced gamma-glutamyl transferase and high-sensitivity C-reactive protein levels in male participants, while high dose abridged fatigue score significantly. No side effects were observed for the supplementation. The study indicated that GBCK25 is safe and can improve liver function [120].

Concluding Remarks
The review aimed at providing current information concerning the pharmacokinetics, safety, and health-promoting activities of CK and its derivatives for preventing and managing diseases. It is well known that the CK is more bioavailable than its parent saponins and has several health benefits. Although more bioavailable than major ginsenosides, CK has certain drawbacks that restrict its clinical use. The use of CK derivatives as nanocarriers were shown to have better permeability, solubility, and efflux, including enhanced health-promoting activities. The review provides new insight into CK derivatives to increase the metabolic proficiency of CK. Most health-promoting activities were in vitro and in vivo, including hepatoprotective, anti-inflammatory, anti-atherosclerosis, anti-cancer, neuroprotection, skin protection, and anti-aging. Besides, the limited number of clinical studies have also been documented. The diverse bioactivities of CK were based on modulating complicated signaling pathways and targeting various molecules. Compound K has been found to attenuate the activities of AMPK, MAPK, NF-kB, PI3K/Akt, mTOR/AMPK, JNK PI3K/mTOR/p70S6K1 signaling pathways. Overall, pharmacokinetic studies on monomer CK and its preclinical and clinical safety information data are limited. Further investigations are warranted to appraise the efficacy and safety of CK and its derivatives, especially in clinical studies.