Caspase-8; Caspase-9; Apoptosis
Fas (CD95) death receptor (up-regulation mediated by p53) [1]
p53 tumor suppressor (stabilization) [1]
Mitochondrial apoptosis pathway (induction of BAX/BAK translocation and cytochrome c release) [1]

Ginsenoside Rh2 induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. Ginsenoside Rh2 is a promising candidate for the development of anti-tumor medications since it induces cancer cells to die via multiple pathways. Ginsenoside Rh2 initiates p53-dependent Fas expression, which leads to the activation of caspase-8 and p53-independent caspase-9-mediated intrinsic pathway, ultimately leading to cancer cell death. The human tumor cell lines HeLa, SK-HEP-1, SW480, and PC-3 are used to determine Ginsenoside Rh2's cytotoxic activity. SK-HEP-1 and SW480 cells are less sensitive to Ginsenoside Rh2, with IC50 values of 3.15 g/mL and 4.06 μg/mL, respectively, while HeLa cells are significantly inhibited in their ability to divide, with an IC50 value of 2.52 μg/mL. With an IC50 value of 7.85 μg/mL, 3-fold higher than HeLa cells, PC-3 cells are the least susceptible to Ginsenoside Rh2[1].

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Ginsenoside Rh2 (G-Rh2) exhibited anti-proliferative effects on human cancer cell lines HeLa (IC50 = 2.52 µg/mL), SK-HEP-1 (IC50 = 3.15 µg/mL), SW480 (IC50 = 4.06 µg/mL), and PC-3 (IC50 = 7.85 µg/mL) after 48 hours of treatment in serum-free medium, as determined by MTT assay [1]
In HeLa cells, G-Rh2 (7.5 µg/mL) simultaneously activated caspase-8, caspase-9, and caspase-3/7 in a time-dependent manner. Caspase-8 activity increased notably after 2 hours, followed by caspase-9 activation after 4 hours. Cleavage of caspases was confirmed by Western blot [1]
Co-treatment of HeLa cells with G-Rh2 and specific peptide inhibitors for caspase-8 or caspase-9 significantly attenuated apoptosis, as assessed by cell morphology changes and PARP cleavage [1]
G-Rh2 (7.5 µg/mL) up-regulated mRNA and protein expression of death receptors Fas and TNF-R1, and ligand TNF-α, in HeLa cells in a time-dependent manner [1]
Silencing of Fas using siRNA in HeLa cells significantly attenuated G-Rh2-induced caspase-8 and caspase-3 activation and PARP cleavage, whereas silencing TNF-R1 had no effect [1]
G-Rh2-induced Fas up-regulation and caspase-8 activation were dependent on wild-type p53 status, as observed in p53 non-mutated HeLa and SK-HEP-1 cells, but not in p53-mutated SW480 and PC-3 cells [1]
siRNA-mediated knockdown of p53 in HeLa cells diminished G-Rh2-induced Fas expression and caspase-8 activation, but did not significantly affect caspase-9 activation [1]
G-Rh2 (7.5 µg/mL) induced translocation of pro-apoptotic proteins BAX and BAK from the cytosol to mitochondria in HeLa cells, leading to cytochrome c release into the cytosol and subsequent caspase-9 activation [1]
In p53-mutated SW480 cells, G-Rh2 similarly induced BAX/BAK mitochondrial translocation, dissipation of mitochondrial membrane potential (ΔΨm), cytochrome c release, and caspase-9 activation, indicating a p53-independent intrinsic apoptotic pathway [1]
G-Rh2 was noted to interact with serum bovine serum albumin (BSA), and its apoptotic activity was attenuated in the presence of serum [1]

Tumor sizes from the three groups that are tumor-bearing are measured 15 days after B16-F10 cells were injected. In comparison to the tumor group, the tumor sizes in the G-L and G-H groups (where G-L and G-H denote a low or high dose of ginsenoside Rh2 injection, respectively) are smaller (P<0.05). The Ginsenoside Rh2 treated groups outlive the tumor group that is not receiving treatment, and the effect is dose-dependent (P<0.05), according to the survival analysis [2].

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Treatment with ginsenoside Rh2 inhibited tumor growth and improved survival in a melanoma mouse model. The antitumor effect was dose-dependent, with a high dose (0.5 mg/kg) showing greater efficacy than a low dose (0.2 mg/kg). [2]
Ginsenoside Rh2 increased infiltration of CD4⁺ and CD8a⁺ T-lymphocytes into tumor tissues. [2]
Spleen lymphocytes from ginsenoside Rh2-treated mice exhibited enhanced cytotoxicity against B16-F10 melanoma cells in a non-radioactive cytotoxicity assay. [2]
Adoptive transfer of spleen lymphocytes from ginsenoside Rh2-treated mice to naïve mice resulted in delayed tumor growth and improved survival in the recipient mice. [2]

Ginsenoside Rh2 (7.5 μg/mL) is applied to HeLa, SK-HEP-1, SW480, and PC-3 cells in serum-free media for the indicated times before the cells are harvested. 50 micrograms of cell lysates are incubated with 200 nM Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC for 1 hour at 37°C in a reaction buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM DTT, 0.1% CHAPS, and 10% sucrose. Fluorescence emission at 535 nm and excitation at 405 nm are used to monitor the reaction[1].

Determination of cell viability is performed by using MTT assay, which is used to calculate the growth inhibition induced by increasing concentrations of drug. In a 96-well plate, 1104 HeLa, SK-HEP-1, SW480, and PC-3 cells that are exponentially growing are seeded in triplicate. Cells are treated with increasing concentrations of ginsenoside Rh2 (1, 2.5, 5, 7.5, and 10 μg/mL) in serum-free media for 48 hours after 24 hours of incubation. Each well receives 20 μL of MTT (5 mg/mL) at the conclusion of the treatment, which is then incubated for an additional 4 hours. The DMSO is used to solubilize the formazan grains produced by viable cells, and an ELISA reader is used to measure the color intensity at 550 nm[1].

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MTT assay for cell viability: Exponentially growing cells were seeded in 96-well plates and treated with increasing concentrations of G-Rh2 in serum-free medium for 48 hours. MTT solution was added, incubated for 4 hours, formazan crystals were dissolved in DMSO, and absorbance was measured at 550 nm [1]
Caspase activity assay: Cell lysates from G-Rh2-treated cells were incubated with fluorogenic caspase substrates (Ac-DEVD-AFC for caspase-3, Ac-IETD-AFC for caspase-8, Ac-LEHD-AFC for caspase-9) in reaction buffer at 37°C for 1 hour. Fluorescence was measured at excitation 405 nm and emission 535 nm [1]
Western blot analysis: Cell lysates or fractionated samples were separated by SDS-PAGE, transferred to membranes, blocked, and probed with specific antibodies against targets such as caspases, PARP, Fas, TNF-R1, p53, BAX, BAK, cytochrome c, and loading controls (β-actin, α-tubulin, COX II). Detection was performed using HRP-conjugated secondary antibodies and enhanced chemiluminescence [1]
RNA purification and RT-PCR: Total RNA was isolated, reverse transcribed, and PCR was performed using gene-specific primers to analyze mRNA levels of death receptors and ligands. β-actin served as an internal control [1]
Mitochondrial and cytosolic fractionation: Cells were fractionated using a mitochondrial isolation kit. Mitochondrial and cytosolic extracts were prepared and analyzed by Western blot [1]
JC-1 staining for mitochondrial membrane potential (ΔΨm): SW480 cells treated with G-Rh2 were stained with JC-1 dye. Fluorescence was measured using a microplate reader; red fluorescence (aggregated form) indicates intact ΔΨm, green fluorescence (monomeric form) indicates depolarization [1]
Immunostaining for cytochrome c: Cells treated with G-Rh2 were fixed, permeabilized, blocked, incubated with anti-cytochrome c primary antibody, followed by FITC-conjugated secondary antibody, and visualized by fluorescence microscopy [1]
RNA interference: HeLa cells were transfected with siRNA duplexes targeting Fas, TNF-R1, or p53 using a lipid-based transfection reagent. After 24 hours, cells were treated with G-Rh2, and lysates were analyzed for protein expression and caspase activity [1]

Mice: In 4 groups of 80 mice each, the Tumor group, G-L group, G-H group, and Control group of male C57BL6 mice (3–4 weeks old) are randomly assigned. A low or high dose of ginsenoside Rh2 is referred to as G-L or G-H. The B16-F10 cell line is administered to the mice in the tumor group, G-L group, and G-H group. These 3 groups develop into cancer-bearing groups. The identical volume of PBS is injected in the control group in place of the drug. In the G-L and G-H groups of mice, ginsenoside Rh2 is injected into the left back. After day 5, the dose for the G-H group is 0.5 mg/kg or 0.2 mg/kg for the G-L group. PBS is injected into the tumor and control groups at the same time points.

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A melanoma mouse model was established by injecting B16-F10 cells subcutaneously into the left back of C57BL6 mice. Mice were randomly divided into four groups: Tumor group (injected with tumor cells), G-L group (low dose of ginsenoside Rh2, 0.2 mg/kg), G-H group (high dose of ginsenoside Rh2, 0.5 mg/kg), and Control group (injected with PBS). Ginsenoside Rh2 or PBS was administered every 2 days starting from day 5 after tumor inoculation. Tumor size was measured periodically, and mice were sacrificed for histological and immunological analysis. [2]
For adoptive transfer experiments, spleen lymphocytes were isolated from donor mice 15 days after tumor inoculation and injected intravenously into recipient naïve mice. Recipient mice were then challenged with B16-F10 cells, and tumor growth and survival were monitored. [2]

The study pointed out that ginsenoside Rh2 interacts with serum albumin (BSA), and its pro-apoptotic activity is weakened in the presence of serum, suggesting that it may bind to serum proteins, thereby affecting its bioavailability and efficacy [1].

[1]. p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells. Protein Cell. 2014 Mar;5(3):224-34.

[2]. Ginsenoside Rh2 enhances the antitumor immunological response of a melanoma mice model. Oncol Lett. 2017 Feb;13(2):681-685.

(20S)-Ginsenoside Rh2 is a ginsenoside found in plants of the genus Panax. Its structure is that of a dammarane-type ginsenoside, with hydroxyl groups substituted at positions 3β, 12β, and 20 (pro-S position). Specifically, the hydroxyl group at position 3 is converted to the corresponding β-D-glucopyranoside, and a double bond is introduced at positions 24-25. It possesses various effects as a plant metabolite, antitumor agent, apoptosis inducer, cardioprotective agent, bone mineral density maintainer, and liver protectant. It is a β-D-glucoside, 12β-hydroxysterol, ginsenoside, tetracyclic triterpenoid, and 20-hydroxysterol. It is derived from the hydride of dammarane. Ginsenoside Rh2 has been reported in Panax notoginseng and ginseng, and relevant data are available.

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Ginsenoside Rh2 (G-Rh2) is a ginsenoside belonging to the protopanaxadiol family and is isolated from ginseng root[1]
This study shows that G-Rh2 induces apoptosis in human cancer cells through multiple pathways: it activates the exogenous apoptosis pathway by upregulating Fas death receptor and activating caspase-8 in a p53-dependent manner, and activates the endogenous apoptosis pathway by p53-independent mitochondrial BAX/BAK translocation, cytochrome c release and activation of caspase-9[1]
Even in p53-mutant cancer cells, G-Rh2 can effectively induce apoptosis. Inducing apoptosis via endogenous pathways (e.g., SW480 cells) highlights its potential as a broad-spectrum pro-apoptotic agent [1]. This study suggests that G-Rh2 may stabilize p53 protein by inhibiting ubiquitin-mediated degradation of p53 protein [1]. One of the challenges in developing G-Rh2-based drugs is its interaction with serum components, which weakens its activity, indicating a need to develop formulations that can overcome serum interference [1].

C36H62O8

622.8727

622.444

C, 69.42; H, 10.03; O, 20.55

78214-33-2

78214-33-2

119307

White to off-white solid powder

1.2±0.1 g/cm3

726.4±60.0 °C at 760 mmHg

393.1±32.9 °C

0.0±5.4 mmHg at 25°C

1.572

5.62

6

8

7

44

1070

15

O([H])C1([H])C([H])([H])C2([H])[C@@]3(C([H])([H])[H])C([H])([H])C([H])([H])C([H])(C(C([H])([H])[H])(C([H])([H])[H])C3([H])C([H])([H])C([H])([H])[C@@]2(C([H])([H])[H])[C@]2(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([C@](C([H])([H])[H])(C([H])([H])C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H])O[H])C21[H])OC1([H])C([H])(C([H])(C([H])(C([H])(C([H])([H])O[H])O1)O[H])O[H])O[H]

CKUVNOCSBYYHIS-IRFFNABBSA-N

InChI=1S/C36H62O8/c1-20(2)10-9-14-36(8,42)21-11-16-35(7)27(21)22(38)18-25-33(5)15-13-26(32(3,4)24(33)12-17-34(25,35)6)44-31-30(41)29(40)28(39)23(19-37)43-31/h10,21-31,37-42H,9,11-19H2,1-8H3/t21-,22+,23+,24-,25+,26-,27-,28+,29-,30+,31-,33-,34+,35+,36-/m0/s1

(2R,3R,4S,5S,6R)-2-[[(3S,5R,8R,9R,10R,12R,13R,14R,17S)-12-hydroxy-17-[(2S)-2-hydroxy-6-methylhept-5-en-2-yl]-4,4,8,10,14-pentamethyl-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]oxy]-6-(hydroxymethyl)oxane-3,4,5-triol

20(S)-Ginsenoside Rh2; 20(S)-Rh2; Ginsenoside-Rh2;AR-1A4936; AR1A4936; AR 1A4936

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50~100 mg/mL (80.3~160.6 mM)
Ethanol: ~100 mg/mL (~160.6 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.01 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (2.01 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)