Ginsenoside Rk1 bioactivity: a systematic review

Anti-cancer activity

Cell viability was measured by different assays through the studies including four studies used Cell Counting Kit-8 (CCK8) assay (Kim et al., 2012; Kim et al., 2008; Kim et al., 2013b; Ko et al., 2009), five studies used 3-(4,5-dimethyl-thiazol-2yl) -2,5-diphenyl tetrazolium bromide (MTT) assay (Kwak & Pyo, 2016; Lee et al., 2010; Park et al., 2002; Quan et al., 2015; Siddiqi et al., 2014), and one study that used WST-1 assay (Toh et al., 2011) while the final one used EZ-CytoTox cell assay kit (Park et al., 2015) (Table 2).

Antiplatelet aggregation activity

Two studies evaluated the anti-aggregation effects of G-Rk1 both in vivo and in vitro (Ju et al., 2012; Lee et al., 2009) respectively. Ju et al. (2012) compared the antiplatelet aggregation activity of G-Rk1 and acetylsalicylic acid (ASA). The results indicated that G-Rk1 exhibits a stronger antiplatelet aggregation activity than ASA in which the action of G-Rk1 in platelets might be related to arachidonic acid (AA) metabolism. In addition, the alteration of (S) hydroxyl eicosatetraenoic acids and thromboxane B2 levels were determined using an immunoassay kit and UPLC/Q-TOF MS system, respectively. The 12-hydroxyleicosatetraenoic acid level was remarkably decreased in the G-Rk1 group but increased in the ASA-treated group. The thromboxane B2 level in the washed platelets decreased significantly by 66% when treated with 100 µM ASA and 77% when treated with 10 µM G-Rk1 (Ju et al., 2012). They used the colorimetric COX inhibitor screening assay to measure the inhibitory effects of G-Rk1 on COX-1 and COX-2. It was found that G-Rk1 inhibits both COX-1 and COX-2 activities. However, at a concentration of 20 µM, G-Rk1-derived inhibition was higher on COX-2 than on COX-1 (Ju et al., 2012).

Lee et al. (2009) explained in his study that the effect of G-Rk1 on adenosine diphosphate (3–4 µM) induced platelet aggregation was monitored turbidimetrically by using ASA as a positive control. Both ASA and G-Rk1 showed the dose-dependent inhibitory effect on collagen, AA, and U46619 (9,11-dideoxy-11a,9a-epoxymethanoprostaglandin F2a) (thromboxane A2 mimetic drug)-induced platelet aggregation. However, they showed a negligible effect on adenosine diphosphate-induced aggregation. G-Rk1 exhibited the strongest inhibitory effect on collagen, AA, and U46619-induced platelet aggregation. In particular, it presented a 22-fold activity of ASA on AA-induced aggregation (Lee et al., 2009). G-Rk1 was found to be a potent inhibitor of AA and U46619 -induced platelet aggregation (Table 3).

Anti-inflammatory activity

G-Rk1 was found to have an anti-inflammatory effect by inhibiting NF-κB levels in the in vitro models (Lee, 2014). These results were assessed using luciferase assay. HepG2 cells were seeded at 1 × 10⁵ cells/well in a 12-well plate and grown for 24 h. While G-Rk1 was pretreated with dimethyl sulphoxide for 1 h and then it was treated with tumor necrosis factor-α (10 ng/mL), the sulfasalazine was used as positive control. Their data demonstrated the strong inhibitory activity of G-Rk1 on NF-κB expression with 50% (IC50) value from 0.75 µM. However, the results revealed that G-Rk1 had cytotoxic effects, which occur in concentrations higher than 10 µM. Another evaluation of G-Rk1 anti-inflammatory activity (Kim et al., 2010) was its suppressing effect on 12-O-tetradecanoyl- phorbol-13-acetate (TPA) induced mouse ear edema. The right ear of ICR mouse was treated with red ginseng saponin extract, G-Rg3, G-Rg5, and G-Rk1 of 10, and 50 mg/kg and after 30 min, ear edema in both ears was induced by topical application of TPA, which is a potent inflammatory agent. They measured the extent of edema and noticed that the pretreatment with red ginseng saponin extract or G-Rk1 suppresses TPA-induced mouse ear edema, and when administering G-Rk1 orally, the formation of edema was blocked. Hu et al. (2017) showed that in acetaminophen (APAP) induced liver injury in mice, G-Rk1 can be used as a protective agent, as it significantly reduced the levels of tumor necrosis factor (TNF-α) to 87 ng/L and when treated with 10 mg/kg G-Rk1 compared to 156 ng/L when treated with 250 mg/kg APAP. A significant reduction of interleukin-1β (IL-1β) was observed with G-Rk1 (Hu et al., 2017). Atopic dermatitis in which keratinocytes and macrophages produce excess chemokines and cytokines, especially thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), as well as nitric oxide (NO), Ahn’s results using G-Rg5/G-Rk1 on TNF-α/ IFN-γ stimulated human keratinocytes cell line (HaCaT cells) showed a significant reduction of TARC/CCL17 expression. Furthermore, using the same combination on the murine macrophage cell line RAW264.7, the secretion of lipopolysaccharide (LPS) mediated NO and reactive oxygen species were significantly reduced, suggesting G-Rg5/G-Rk1 as a promising natural therapy in the control of atopic dermatitis (Ahn et al., 2016) (Table 3).

Effect of G-Rk1 on vascular leakage

A study evaluated the G-Rk1 effect on VEGF (Maeng et al., 2013) by treating primary human retina microvascular endothelial cells with G-Rk1 at a concentration of (10 µg/ml) for 40 min then stimulating it with 20 µg/ml of VEGF to disrupt the cell membrane. Sucrose permeability assay was used to evaluate the endothelial permeability and the results showed that G-Rk1 inhibited VEGF-induced retinal endothelial permeability. They used reverse-transcription polymerase chain reaction (RT-PCR) and densitometric analysis was used to assess translocation of tight junctions (TJ) proteins, and immunostaining was used to evaluate disruption of TJ proteins after the cells were stained with anti-ZO-1, anti-ZO-2, and anti-occludin antibodies. The authors found that G-Rk1 inhibited VEGF effect on TJ protein localization but it did not affect the transcription of TJ proteins (Table 3).

Effect of G-Rk1 on lipid accumulation

Ginseng is known to have effects on obesity (Kim et al., 2009). In vitro treatment of mouse 3T3-L1 fibroblast cells with G-Rk1 resulted in reducing lipid accumulation, in which these cells differentiated into adipocytes after being treated with various G-Rk1 concentrations (10, 50, 100 µM) for 2 h at 490 nm optical absorbance (Kim et al., 2009) (Table 3).

Neuroprotective effect of G-Rk1

The combination of G-Rg5/G-Rk1 had a pronounced effect on the excitotoxic and oxidative stress-induced neuronal cell damage that was tested in primary cultured rat cortical cells (Bao et al., 2005). These cells were cultured in vitro for 12–20 days, then exposed to 100 µM glutamate or N-methyl-D-aspartate for 15 min in the absence or presence of G-Rg5/G-Rk1. The cell damage was assessed after 20–24 h by measuring LDH activity in the culture media. Data were calculated from cells exposed to the respective excitotoxic insults without ginsenosides. Data presented that approximately 70–80% of the cells were damaged by glutamate or N-methyl-D-aspartate compared to vehicle-treated control cells. The excitotoxic effect was significantly inhibited by G-Rg5/G-Rk1 in a concentration-dependent manner, in which 50% inhibition was achieved at 14.7 µg/mL of G-Rg5/G-Rk1.

In previous work, Bao et al. (2005) used a passive avoidance test to evaluate the effect of G-Rg5/G-Rk1. The latency in seconds was used to measure the cognitive performance of ethanol-induced amnesia in mice. The mice were orally treated with saline as vehicle and the ratio of G-Rg5/G-Rk1 equal 1:1 with a concentration of 10 mg/kg once a day for 4 days. The latency period of the mice administrated with ethanol was 24.9% less than the one of control mice (without ethanol-treatment), but it was significantly enhanced by the oral administration of G-Rg5/G-Rk1 with 1.2-fold increase than that of the control. The same steps were done, but this time after inducing amnesia with a single injection of scopolamine (3 mg/kg), also G-Rg5/G-Rk1 (10 mg/kg) provided the same enhancing significant result (p < 0.01). In another work, Jing et al. (2006) did the same tests of ethanol-induced amnesia in mice, which were given water as the control and ratio of G-Rg5/G-Rk1 equal 1:1 in the concentration of 10 mg/kg. They found that G-Rg5/G-Rk1 could significantly prolong the latency period by 2.97 folds more than that of the control. These two studies presented that G-Rg5/G-Rk1 would give beneficial results in the memory function of the normal, ethanol or scopolamine-induced amnesia in brains. G-Rk1 was reported to have a significant neurogenic activity in Epidermal growth factor-responsive neurosphere stem cells (erNSCs). However, this activity was less than G-Rg5 (Liu et al., 2007) (Table 3).

Nephroprotective effect of G-Rk1

Park et al. (2015) examined the effect of the G-Rg5/G-Rk1 combination on cisplatin-induced nephrotoxicity in mice at cisplatin concentration 25 µM and G-Rg5/G-Rk1 concentrations of (0, 50, 100, 250 µg/ml). Results with EZ-cytotoxic cell viability assay kit showed a significant reduction in cisplatin and induced a reduction in cell viability. This effect was higher than that of Epigallocatechin gallate at the same concentrations as G-Rk1 (Table 3).

Bone metabolism

Siddiqi et al. (2014) evaluated the osteogenic activity of G-Rg5/G-Rk1. MC3T3-E1 cells were treated with differentiation medium (either with or without G-Rg5/G-Rk1) for 12 days at different concentrations in which different substances were added to the culture medium in order to evaluate various effects of G-Rg5/G-Rk1 on differentiated fibroblast. The extent of calcium deposition, which is an indicator of osteoblasts mineralization, was measured by MTT assay. Data were expressed as a percentage of control, which showed that G-Rg5/G-Rk1 protected the extracellular matrix mineralization from antimycin A devastating effects. Besides, it turned out that alkaline phosphatase (ALP) activity evaluated by Smart BCA protein assay kit, increased by two folds after treatment with G-Rg5/G-Rk1 (30–50 µg/mL).

The effect of G-Rg5/G-Rk1 on cellular collagen was measured using Sirius Red-based colorimetric assay. Results were similar to that of ALP activity in which cellular collagen was markedly increased. When glutathione contents of the cells were measured by glutathione assay kit after exposure to various concentrations of G-Rg5/G-Rk1, data showed that G-Rg5/G-Rk1 increase the level of glutathione in a dose-dependent manner. In order to evaluate gene expression levels, a total RNA was isolated from the cells, which were treated with G-Rg5/G-Rk1 and was amplified by RT-PCR. The results indicated that the maturation and the differentiation of MC3T3-E1 cells were induced by G-Rg5/G-Rk1 mediated BMP-2/Runx2 and the level of expression of Runx2 increased by the action of G-Rg5/G-Rk1 (Table 3).

Anti-insulin resistance effect of G-Rk1

Ponnuraj et al. (2014) assessed the effect G-Rk1 on insulin resistance. 3T3-L1 cells were treated with G-Rg5/G-Rk1 complex at different concentrations where tunicamycin was used to induce stress on the endoplasmic reticulum (ER). As for cell viability, measured with MTT assay, results showed that cells treated with G-Rg5/G-Rk1 complex had overcome the stress which induced by tunicamycin. Cells were made insulin resistant by immersing them into a medium that contains insulin and by treating them with dexamethasone, then with the stress agent and G-Rg5/G-Rk1 complex and were analyzed by glucose oxidase reagent, while tunicamycin was used as a positive control. Results found that the amount of glucose left in the medium is high in the cells treated with tunicamycin and low in the cells treated with G-Rg5/G-Rk1 complex and this was achieved through C/EBP homologous protein-10 (CHOP)-mediated pathway and increase insulin-like growth factor receptor (IGF-2R) (Table 3).

Anti-oxidant effect of G-Rk1

Hydrothermal treatment of primary ginsenosides at 100 C transformed them into either deglycosylated and/or dehydrated ginsenoside. As the hypothermal reaction increase, it yields more 20 (S)-Rg3, Rk1, and Rg5. In addition, when they compared the antioxidant activity between the hydrothermally processed samples at 100 C and others processed by the steaming, they got results that showed that sun ginseng samples were higher in antioxidant activities. However, it results in fewer ginsenosides than these samples which reacted at 120 C (Ryu et al., 2016).

Antimicrobial effect of G-Rk1

Xue et al. (2017) assessed the antimicrobial effect of G-Rk1 measured by the minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC). Compared to Erythrocin (positive control), G-Rk1 exhibited higher MIC and MBC against different bacterial strains compared to Erythrocin (positive control) with (MIC: 31.3 vs 8 µg/ml; MBC: 125.0 vs 16.0 µg/ml) against Clostridium perfringens, (MIC: 16.0 vs 8 µg/ml; MBC: 125.0 vs 31.3 µg/ml) against Fusobacterium nucleatum, and (MIC: 62.5 vs 16.0 µg/ml; MBC: 125.0 vs 62.5 µg/ml) against Porphyromonas gingivalis. Therefore, G-Rk1 can be a promising cure for halitosis.