During the past decade, there was considerable interest in investigating black ginseng’s pharmacological actions using different biochemical, pharmacological and molecular biological techniques. The examined pharmacological activities included anticancer, hepatoprotective, antidiabetic, antioxidant and general tonic activities besides its effect on the central nervous and immune systems. Some of these reports were carried out to compare the biological activity of black ginseng with white and/or red ginseng. While most of these reports were designed to figure out the pharmacological potential of black ginseng in addition to its mechanism of action. Furthermore, there is extensive literature involving the biological activities of the major transformed ginsenosides, such as Rg3, Rk3, Rh2, Rk1 and Rg5, which are present in black ginseng in much higher concentrations than red ginseng and are totally absent in white ginseng. Additionally, the amounts of polyphenols, acidic polysaccharides and Maillard reaction products increase considerably by the steaming process to be in the highest concentrations in black ginseng. In the few next pages we will discuss some published biological studies reporting black ginseng total extract and pure fractions as well as the transformed ginsenosides and other highly concentrated metabolites.
4.1. Anticarcinogenic Effects
Black ginseng and its major ginsenosides exerted promising in vitro and in vivo anticarcinogenic effects against several cancer types through different mechanisms of action. Some of them showed strong clinical results and some others were used as anti-cancer products in the market.
Black ginseng’s crude saponin fraction exerted stronger in vitro cytotoxic activities than red ginseng against ACFIN, HCT-15 and PC-3 cell lines with IC
50 values ranging from 60.3–90.8 μg/mL [
22]. In another comparative study, black ginseng extract exhibited cytotoxic activity against colon 26-M3.1 carcinoma cell line with an IC
50 value of 800 μg/mL, while it was 2000 μg/mL for white ginseng [
41]. In addition, black ginseng extract could inhibit basic fibroblast growth factor (bFGF)-induced endothelial cell proliferation and migration dose-dependently through its ability to inhibit the angiogenesis process [
42]. Furthermore, breast cancer MCF-7 cell lines proliferation was inhibited by black ginseng due to cell cycle arrest induction in the G0/G1 phase in a dose-dependent manner [
43].
Black ginseng exhibited promising anticancer activities in different in vivo studies such as tumor inhibition in H
22 tumor-bearing mice dose-dependently via immune function improvement and tumor cell apoptosis induction [
24]. Moreover, it performed in vivo anticancer activities against hepatocellular carcinoma as it reduced the size and the volume of an HepG2 cell transplanted tumor in BALB/c nude mice [
44].
The anticancer activities of ginsenosides are inversely proportional to the number of sugar units in the molecule [
10]. For instance, ginsenosides with four or more sugar moieties, which present in white ginseng, with a lower ratio in red ginseng and are absent in black ginseng, such as Rb and Rc, did not exert significant anti-proliferative effects, while Rd (three sugar units) weakly inhibited cancer cells growth [
31]. On the other hand, the ginsenosides with one or two sugar residues (present in the highest ratio in black ginseng due to the steaming process), such as ginsenosides Rg
3 (two sugar units), Rh
2 (one sugar unit) and Rg
5 (two sugar units), exhibited promising anticancer activities against a wide array of cancer types in several in vitro, in vivo and clinical studies.
Rg
3 has been found to be a potent inhibitor of invasion of several tumor cell lines including gallbladder cancer (GBC-SD, Mz-ChA-1 and QBC939 cell lines) [
45], rat ascites hepatoma (MM1 cell line) [
46], melanoma (B16FE7, C8161 and A375 cell lines) [
46], human non-small lung carcinoma (H1650, H520 and H1963 cell lines) [
47] and human pancreatic adenocarcinoma (PSN-1 cell line) [
46], as well as human breast cancer (MDA-MB-231 and MCF-7 cell lines), human liver cancer (HepG2, Hep3B, Hep1-6 and SMMC-7721cell lines) and human colon cancer (HT-29, HCT116, SW480 and HCT116 cell lines) [
48].
The anticancer potential of Rg
3 has been associated with apoptosis induction which was evidenced in HepG2 cells through induction of calcium-dependent apoptosis [
49] and downregulation of the hypoxia-responsive transcription factor (HIF-1α) expression [
50]. In addition, apoptosis was confirmed in ovarian cancer cell lines via the reduction of (HIF-1α) expression [
51] and the downregulation of the Phosphatidylinositol-3 kinases (PI3K)/Akt pathway [
52]. Another important mechanism is the inhibition of tumor cells angiogenesis, which have been correlated mostly with the inhibition of the vascular endothelial growth factor (VEGF) expression in different human cancer cell lines such as esophageal carcinoma [
53] and Lewis lung carcinoma [
54].
The cytotoxic effects of Rg
3 were also exerted as a result of the induction of DNA double-strand breaks in human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS) [
55] and through the induction of cell detachment and modulation of MAP kinases in prostate cancer cell lines (LNCaP and PC3) with EC
50 values of 8.4 and 14.1 μM, respectively [
56]. In human glioma cells, Rg
3 induced senescence-like growth arrest by regulating Akt and p53/p21-dependent signaling pathways [
57] and altered cellular redox state in opposite directions by increasing the cellular GSH/GSSG ratio, enhancing the γ-GCS activity and suppressing ROS generation [
58], while in human colon cancer cell lines it inhibited micro-lymphatic metastasis [
59] and blocked the nuclear translocation by reason of downregulation of Wnt/β-catenin signaling [
60]. Human melanoma cell lines have been inhibited by Rg
3 through the down-regulation of histone deacetylase 3 (HDAC3) and the up-regulation of p53 acetylation [
61] in addition to the decrease of fucosyltransferase IV (FUT4) and its synthetic product Lewis Y (LeY) FUT4/LeY expression and the inhibition of EGFR/MAPK pathway activation [
62].
The in vivo anticancer activities of Rg
3 have been examined in several experiments; it could inhibit tumor growth and metastasis of human gastric cancer in SCID mice as it decreased intratumoral microvessel density [
63] and reduced lung tumor incidence in newborn mice injected with benzo(a)pyrene [
64]. Likewise, it reversed multidrug resistance (MDR) of lung adenocarcinoma in mice via the downregulation of MDR-mediated proteins, P-glycoprotein (P-gp), multidrug resistance-associated protein (MPR1) and lung resistance protein 1 (LPR1) [
65]. In hepatocellular carcinoma H22-bearing mice, both isomers of Rg
3, 20(S) and 20(R), improved cellular immunity, stimulated conA-induced lymphocyte proliferation and augmented Th1-type cytokines IL-2 and IFN-γ levels, with a notice that 20(R)-Rg
3 exhibited stronger activities [
66].
The synergetic effect of Rg
3 with different anticancer agents has been verified in different studies with several drugs, such as paclitaxel [
67], docetaxel [
68], 5-fluorouracil (5-FU) [
69], doxorubicin [
70], arsenic trioxide (As
2O
3) [
71], capecitabine [
72], cisplatin [
73], gemcitabine [
74], mitomycin C and tegafur [
75]. ‘Shen-Yi capsule’ is a commercial product of ginsenoside Rg
3 distributed in the Chinese pharmaceutical market as an anticancer drug and used mostly in combination with several other chemotherapeutic agents [
76].
The ginsenoside Rh
2 could suppress proliferation in several cancer cells, including human breast cancer (MCF-7) [
77], lung cancer adenocarcinoma (A549 cells) [
78], human colorectal carcinoma (HCT116) [
79], prostate cancer (LNCaP and PC3) [
56]. Leukemia (HL-60) [
80], uterine leiomyoma [
81], neuroblastoma (SK-N-BE-2) [
82], glioblastoma multiforme (GBM) [
83], human gastric cancer (SGC-7901) [
84], hepatocellular carcinoma (HPEG-2) [
85], intestinal (Int-407 and Caco-2) [
86] and mouse melanoma (B16) [
87].
The antiproliferative effect of Rh
2 appears to be linked to its ability to induce apoptosis in cancer cells and arrest cell cycle progression. For instance, Rh
2 has been reported to affect the regulation of caspase enzymes, major proenzymes affecting apoptosis, in prostate cancer cells [
56], human neuroblastoma SK-N-BE(2) [
82] and human lung adenocarcinoma A549 cells [
15]. Rh
2 could induce calcium-dependent apoptosis and autophagy in HepG2 cells [
49] and cause cell cycle arrest at the G1 stage in human lung adenocarcinoma A549 cells [
88], MCF-7 human breast cancer cells and SK-HEP-1 hepatoma cells [
11].
Rh
2 was also able toinhibit cancer cells angiogenesis via the inhibition of vascular endothelial growth factor A (VEGF-A) protein in GBM cells [
83], induce cell detachment and modulate mitogen-activated protein (MAP) kinases in the prostate cancer cell lines, LNCaP and PC3 [
56], in addition to hepatic cellular carcinoma HepG2 cells [
10]. The inhibitory effects of Rh
2 against the growth of glioblastoma and hepatocellular carcinoma in vitro and in vivo in a mouse model have been outlined [
89,
90]; these effects were associated with a significant increase in apoptosis induction and decrease in proliferation of tumor cell through the inhibition of the epidermal growth factor receptor (EGFR) signaling pathway. In another in vivo study, Rh
2 could reduce lung tumor incidence in newborn mice after injection with benzo(a)pyrene [
64]. Likewise, Rh
2 showed anti-proliferative ability against prostatic cancer cells invasiveness in vivo and in vitro via the activation of transforming growth factor β (TGFβ) receptor signaling [
91].
The dose-dependent significant inhibition of Rg
5 against basic fibroblast growth factor (bFGF)-induced endothelial cell proliferation and migration has been revealed [
42]. Rg
5 also could arrest the cell cycle of human hepatoma SK-HEP-1 cells via the down-regulation of cyclin E-dependent kinase activity [
92] and of human breast cancer (MCF-7) in G0/G1 phase through the regulation of cell cycle-related proteins and the regulation of the expression of apoptosis-related proteins including Bax, PARP and cytochrome [
43]. In another experiment, the ability of Rg
5 to induce a significant increase in apoptosis induction and DNA damage in five human cervical cancer cell lines (HeLa, MS751, C33A, Me180 and HT-3) has been verified in a time and concentration-dependent manner [
93]. Through an in vitro and in vivo study, Rg
5 controlled chemotherapeutic multidrug resistance (MDR) mediated by ABCB1 transporter via the increase of the accumulation of ABCB1 substrates intracellularly [
93]. In addition, the combination of Rg
5 and docetaxel (TXT) in nude mice bearing an A549/T tumor could suppress the growth of drug-resistant tumors significantly through the suppression of AKT phosphorylation and Nrf2 expression [
94].
Compound K exhibited promising inhibitory activities against several types of cancer cell lines including lung carcinoma (B16-BL6 [
95] and 95-D [
96]), leukemia (HL-60 [
97], K562 [
96], Kasumi-1 and MV4-11 [
98]), hepatoma (HepG2 [
99], HGC-27 [
100] and SMMC7721 [
101]), colorectal cancer (colon 205 [
100], HCT-116, SW-480 and HT-29 [
102]), gastric carcinoma (MKN-45 [
98], BGC823 and SGC7901 [
103]), breast cancer (MCF-7 [
104]) pulmonary adenocarcinoma (PC-14 [
97]), nasopharyngeal carcinoma (HK-1 [
105]), prostate cancer (Du145 [
100]) and brain tumors (human astroglioma U87MG, CRT-MG and U373MG [
106]).
In addition, compound K significantly inhibited the growth and metastasis formation of glioblastoma U87MG and U373MG cell lines through several mechanisms, including cell cycle arrest at the G0/G1 phase, decreasing the expression levels of cyclin D1 and cyclin D3, apoptosis induction via nuclear condensation, increasing in ROS generation, mitochondrial membrane potential depolarization and activation of caspase-3, caspase-9 and poly(ADP-ribose) polymerase (PARP) enzymes [
107]. Furthermore, compound K showed promising in vivo anticancer activities against many cancer types; the ability of compound K to induce apoptosis through the loss of mitochondrial membrane potential and activation of caspase 3 in lung cancer of nude mice has been reported [
10]. In another study, compound K significantly inhibited metastasis induced by IV injection of B16-BL6 lung melanoma cells in syngeneic mice [
108]; it also exhibited inhibitory activity against colon cancer in an in vivo study via several pathways, including antiproliferation, apoptosis induction and cell cycle arrest in the G1 phase [
102].
The significant inhibition of the acidic polysaccharide (Ginsan) for benzo[a]pyrene-induced autochthonous lung tumors in mice through the induction of Th1 cell and macrophage cytokines has been outlined [
109]. PGP2a is an acidic protein–polysaccharide with a molecular weight of 3.2 × 10
4 Da and consists of galactose, arabinose, glucose and galacturonic acid in the molar ratio of 3.7:1.6:0.5:5.4, respectively. PGP2a inhibited the growth of human gastric cancer HGC-27 cells dose-dependently through apoptosis induction and cell cycle arrest in G2/M phase; PGP2a suppressed the protein expression of Twist and AKR1C2, with an increase of NF1 [
110].
4.2. Immunomodulatory and Anti-Inflammatory Effects
In a comparative study, black ginseng exhibited stronger anti-inflammatory and anti-nociceptive effects than red ginseng in xylene-induced ear edema model in mice and carrageenan-induced paw edema in rats; it inhibited the pro-inflammatory mediators, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines, IL-1β, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [
111]. In addition, the lipopolysaccharide-induced TNF-α release was significantly decreased after treatment with black ginseng extract [
41]. Furthermore, the dose-dependent recovering effect of black ginseng extract against the cisplatin-induced nephrotoxicity and the reduced (pig cell LLC-PK1) cells viability, has been indicated [
112]. In addition, in the same experiment, compound K could abrogate the elevated percentage of apoptotic LLC-PK1 cells significantly.
The black ginseng extract potently inhibited atopic dermatitis and asthma through suppression of the elevated IL-6 and IL-8 induced by
Dermatophagoides pteronissinus treatment in human acute monocytic leukemia (THP-1) and human eosinophilic leukemic (EoL-1) cell lines [
113]. Similarly, gamma-irradiated black ginseng extract could inhibit mast cell degranulation and suppress atopic dermatitis-like skin lesions in mice through different mechanisms, including the suppression of β-hexosaminidase and histamine in the stimulated mucosal mast cells [
114].
In another study, the significant inhibitory effect of pretreatment of dorsal skins of female ICR mice with Rg
3 against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase activity and 7,12-dimethylbenz[a]anthracene-initiated papilloma formation was declared [
15]. Rg
3 could inhibit COX-2 expression and eukaryotic transcription factor, NF-kappaB activation. Rg
3 could suppress the nitric oxide (NO), reactive oxygen species (ROS) and prostaglandin E2 (PGE2) productions induced by lipopolysaccharide (LPS) in RAW264.7 macrophage cells dose-dependently [
115].
The ginsenoside Rh
2 inhibited the production of NO, with an IC
50 value of 17 μM in LPS/interferon-γ-stimulated BV-2 microglial cells. This effect correlated with the inhibition of the expression of COX-2 and pro-inflammatory TNF-α, IL-1β, while it increased the expression of the anti-inflammatory cytokine IL-10 [
116]. Compound K exhibited anti-inflammatory activities in mice against several kinds of inflammations such as carrageenan-induced paw edema, colitic (DSS and TNBS-induced), sepsis (zymosan and LPS-induced) and xylene-induced ear edema through the inhibition of the activation of ROS, MAPKs and NF-κB/AP-1 with the enhancement of HO-1/ARE signaling [
108]. Compound K significantly increased the inflammatory pain threshold, reduced PGE2 level and decreased COX-2 expression in rats [
108]. In LPS-activated RAW264.7 cells, compound K inhibited the expression of the proinflammatory cytokines with a concentration of 5 μM; it also reduced the expression of other inflammatory mediators such as IL-1β, COX-2, iNOS and TNF-α through the mediation of NF-κB [
15]. Both of Rh
2 and compound K significantly inhibited the passive cutaneous anaphylaxis (PCA) reaction induced by IgE in rodents through membrane stabilizing effect [
11].
RGAP is an acidic polysaccharide of ginseng containing 56.9% acidic sugars and 28.3% neutral sugars. The intraperitoneal administration of RGAP exerted promising in vivo and in vitro immunomodulating activities through regulation of NO synthesis in female BALB/c mice [
117]. RGAP could also augment the humoral immune response of pidotimod in immunosuppressed mice in response to both lipopolysaccharide and sheep red blood cells through increasing the number of plaque-forming cells in the spleen [
118].
Ascorbic acid, cinnamic acid and esculetin were found to be the most abundant phenolics in
Panax ginseng; ascorbic acid and cinnamic acid effectively suppressed LPS-induced nitric oxide production in the RAW 264.7 cells; in addition, cinnamic acid could significantly inhibit the oxidative damage in the human neuroblastoma SH-SY5Y cells [
119].