Anti-inflammatory Activity: In LPS-stimulated RAW264.7 murine macrophages, 20(R)-Ginsenoside Rh2 inhibited NO production. Pretreatment with 10, 30, and 50 μM 20(R)-Ginsenoside Rh2 reduced nitrite levels to 97%, 86%, and 55% of LPS-only treated cells, respectively, with an IC50 > 50.0 μM.[1]
Anti-inflammatory Activity: In LPS-stimulated RAW264.7 murine macrophages, 20(R)-Ginsenoside Rh2 inhibited PGE2 production. A diminishing effect on the LPS-enhanced PGE2 level was clearly observed at a concentration of 50 μM.[1]
Antioxidative Activity: In LPS-stimulated RAW264.7 murine macrophages, 20(R)-Ginsenoside Rh2 reduced intracellular ROS levels. Pretreatment with 10, 30, and 50 μM 20(R)-Ginsenoside Rh2 reduced ROS levels to 98%, 96%, and 33% of LPS-only treated cells, respectively, with an IC50 of 34.8 μM.[1]
Antioxidative Activity: In unstimulated human HaCat keratinocytes, 20(R)-Ginsenoside Rh2 reduced intracellular ROS levels, notably at 5 and 25 μM. At 25 μM, it caused the ROS level to drop to 77% of that in untreated control cells.[1]
MMP Inhibitory Activity: In LPS-stimulated RAW264.7 murine macrophages, 20(R)-Ginsenoside Rh2 (10, 30, 50 μM) suppressed the enhanced gelatinolytic activity of pro-MMP-9 in a concentration-dependent manner, but did not modulate MMP-2 activity.[1]
MMP Inhibitory Activity: In unstimulated human HaCat keratinocytes, 20(R)-Ginsenoside Rh2 (0.2, 1, 5, 25 μM) inhibited the gelatinolytic activity of both MMP-9 and MMP-2, with a clear reduction for MMP-9 at 25 μM, but did not affect MMP-1 activity.[1]
MMP Inhibitory Activity: In TNF-α-stimulated human HaCat keratinocytes, 20(R)-Ginsenoside Rh2 inhibited the TNF-α-induced increase in MMP-9 gelatinolytic activity.[1]

Antiproliferative Activity: Assessed by MTT assay, 20(R)-Ginsenoside Rh2 exhibited cytotoxicity against various cancer cell lines, with IC50 values of 38.5 ± 2.4 μM for HL-60 human leukemia cells, 41.2 ± 1.5 μM for U937 cells, 49.6 ± 2.7 μM for P388 mouse lymphoblasts, 59.6 ± 3.2 μM for A549 human lung adenocarcinoma cells, >200 μM for HeLa human cervical carcinoma cells, >100 μM for HepG2 human hepatoblastoma cells, and 59.6 ± 3.2 μM for A431 human epidermoid carcinoma cells.[2]
Growth Inhibition: Trypan blue exclusion assay showed that 20(R)-Ginsenoside Rh2 (10-40 μM) inhibited the growth of HL-60 and U937 cells in a time- and concentration-dependent manner over 96 h. At 20 μM, it exhibited a cytostatic effect, while at 30 and 40 μM, it had a cytocidal effect.[2]
Cell Cycle Arrest: Flow cytometric analysis revealed that treatment with 20(R)-Ginsenoside Rh2 (20 μM) increased the proportion of cells in G1 phase in a time-dependent manner in HL-60 and U937 cells. After 96 h of treatment with 20 μM Rh2, the G1 phase population increased from 48.1% (control) to 67.7% in HL-60 cells and from 45.6% to 64.8% in U937 cells, with a concomitant decrease in S-phase cells.[2]
Expression of Cell Cycle-Related Proteins: Western blot analysis showed that 20(R)-Ginsenoside Rh2 (20 μM) treatment time-dependently upregulated protein and mRNA levels of p21^CIP1/WAF1 and p27^KIP1 in HL-60 and U937 cells, while downregulating protein levels of CDK4, CDK6, cyclin D1, cyclin D2, cyclin D3, and cyclin E; CDK2 levels remained unchanged.[2]
Binding of CDKs with CDKIs: Immunoprecipitation assays demonstrated that 20(R)-Ginsenoside Rh2 (20 μM) treatment for 96 h significantly enhanced the binding of p21^CIP1/WAF1 and p27^KIP1 to CDK2, CDK4, and CDK6 in HL-60 cells.[2]
CDK Kinase Activities: Kinase activity assays revealed that 20(R)-Ginsenoside Rh2 (20 μM) treatment for 96 h markedly reduced CDK2-, CDK4-, and CDK6-associated kinase activities in HL-60 cells (using histone H1 for CDK2 and GST-Rb fusion protein for CDK4/6 as substrates).[2]
Rb Phosphorylation and E2F1 Translocation: Western blot showed that 20(R)-Ginsenoside Rh2 (20 μM) treatment decreased Rb phosphorylation and reduced nuclear levels of E2F1 in HL-60 cells.[2]
Induction of Differentiation: NBT reduction assay indicated that 20(R)-Ginsenoside Rh2 (20 μM) treatment for 96 h stained approximately 54.5% of HL-60 cells (control 6.7%), compared to 46.2% with Vit D3 (20 nM). Esterase activity assays showed increases of 22.8% in AS-D chloroacetate esterase and 31.1% in α-naphthyl acetate esterase activities. Phagocytic activity was enhanced as evidenced by increased uptake of latex particles. Flow cytometry demonstrated significant upregulation of surface antigens CD11b, CD14, CD64, and CD66b, indicating differentiation toward monocyte/macrophage and granulocyte lineages.[2]
TGF-β1 Production: ELISA of culture supernatants showed that 20(R)-Ginsenoside Rh2 (20 μM) time-dependently increased TGF-β1 production in HL-60 cells. Real-time PCR confirmed upregulation of TGF-β1 mRNA.[2]
Reversal by TGF-β1 Neutralizing Antibody: Co-treatment with TGF-β1 neutralizing antibody reversed the Rh2-induced downregulation of CDK4/6, upregulation of p21/p27, and differentiation effects, indicating that Rh2's actions are dependent on TGF-β1 signaling.[2]

Zymographic Analysis of MMP Activity: To determine the gelatinolytic activity of MMP-1, -2, and -9, culture supernatant fractions were collected. Samples were electrophoresed on an 8% SDS-PAGE gel impregnated with 1 mg/ml gelatin under non-reducing conditions. After electrophoresis, proteins in the gel were renatured, followed by staining and destaining. The gel was stained with a solution of 0.1% Coomassie Brilliant Blue R-250, and gelatin-degrading enzymes were visualized as clear zones against a blue background. Identification of MMP-1, -2, and -9 activity bands was based on their molecular weights estimated using molecular mass markers.[1]
CDK Kinase Activity Assay Procedure: HL-60 cells treated with or without 20(R)-Ginsenoside Rh2 were harvested, and total lysates (500 μg protein) were prepared. Immunoprecipitation was performed using anti-CDK2, anti-CDK4, or anti-CDK6 polyclonal antibodies, followed by incubation with protein A-Sepharose CL-4B beads at 4°C for 18 h. The immunocomplexes were washed three times with lysis buffer and once with kinase assay buffer. Kinase reactions were carried out in kinase buffer containing [γ-³²P]ATP, with histone H1 (for CDK2) or GST-Rb fusion protein (for CDK4 and CDK6) as substrates. After incubation at 30°C for 30 min, reactions were separated by SDS-PAGE, and phosphorylated substrates were detected by autoradiography.[2]

Cell Culture and Treatment: Human HaCat keratinocytes and murine RAW264.7 macrophages were cultured in DMEM containing 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO₂ at 37°C. For RAW264.7 cells, 25 mM HEPES (pH 7.5) was added to the culture medium.[1]
Macrophage Experimental Procedure: RAW264.7 cells (5.0 × 10⁵) were seeded in 6-well plates and grown for 24 h. They were pre-incubated with 0, 10, 30, or 50 μM 20(R)-Ginsenoside Rh2 (dissolved in DMSO) for 1 h. After treatment with 1 μg/ml LPS, cells were further incubated for 24 h before analyses.[1]
Keratinocyte Experimental Procedure: HaCat cells (1.0 × 10⁵) were seeded in 6-well plates and grown for 24 h. They were then treated with 0, 0.2, 1, 5, or 25 μM 20(R)-Ginsenoside Rh2 in FBS-free medium and incubated for another 24 h. For TNF-α stimulation, cells were treated with 10 ng/ml TNF-α 1 h after 20(R)-Ginsenoside Rh2 treatment.[1]
Nitrite Measurement Procedure: Accumulated nitrite (NO₂⁻), generated from cell-released NO, in culture supernatants was determined using a colorimetric assay based on the Griess reaction. A standard curve was constructed using known concentrations (0-160 μM) of sodium nitrite.[1]
PGE2 Measurement Procedure: PGE2 in culture supernatants was determined using a commercial EIA kit according to the manufacturer's instruction. All experiments were performed in triplicate.[1]
Intracellular ROS Determination Procedure: For analysis of intracellular ROS, the redox-sensitive fluorescent probe DCFH-DA was used. RAW264.7 cells (3 × 10⁵) were pre-incubated with varying concentrations of 20(R)-Ginsenoside Rh2 for 1 h, then treated with LPS for 24 h. Cells were then incubated with 5 μM DCFH-DA for 30 min at 37°C and harvested. Intracellular ROS levels were immediately analyzed by flow cytometry.[1]
Cell Viability Assay Procedure: Cellular viability was quantified by the MTT assay. Trypsinized cells were incubated with 20 μl of 5 mg/ml MTT solution for 2 h. Cells were then lysed with isopropyl alcohol, and the amount of formazan generated from MTT reduction by mitochondria of living cells was determined by absorbance at 540 nm.[1]
Cell Culture and Drug Treatment: HL-60 (human promyelocytic leukemia), U937 (human histiocytic lymphoma), and other cell lines were cultured in RPMI 1640 or DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in 5% CO₂. 20(R)-Ginsenoside Rh2 was dissolved in DMSO (final concentration ≤0.05%) and added to cells at indicated concentrations (e.g., 10, 20, 30, 40 μM) for various times (24-96 h).[2]
MTT Cytotoxicity Assay: Cells (5×10⁴ cells/ml) were seeded in 96-well plates and incubated for 24 h, then treated with varying concentrations of 20(R)-Ginsenoside Rh2. After 96 h, 50 μl MTT (5 mg/ml) was added and incubated for 4 h. The medium was discarded, formazan crystals were dissolved in 100 μl DMSO, and absorbance was measured at 540 nm to calculate IC50.[2]
Trypan Blue Exclusion Assay: Cells (2×10⁵ cells/ml) were treated with 20(R)-Ginsenoside Rh2 for 24-96 h. Cell suspensions were mixed with trypan blue, and viable cells (excluding dye) were counted using a hemocytometer.[2]
Cell Cycle Analysis: Treated cells were fixed in 70% cold ethanol at 4°C for 1 h, washed with PBS, and resuspended in PBS containing 2.5 μg/ml RNase and 50 μg/ml propidium iodide. After 30 min incubation in the dark at room temperature, DNA content was analyzed by flow cytometry (10,000 events acquired) using Cell Quest software.[2]
Western Blotting: Cells were lysed in lysis buffer to obtain total protein or nuclear extracts (nuclei isolated by hypotonic buffer lysis and extracted with high-salt buffer). Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, blocked for 1 h, incubated with primary antibodies (1:1000) overnight at 4°C, washed, incubated with HRP-conjugated secondary antibodies (1:2000) for 1 h at RT, and detected by ECL.[2]
Immunoprecipitation: Cell lysates (100 μg protein) were incubated with anti-CDK2, anti-CDK4, or anti-CDK6 polyclonal antibodies at 4°C for 12 h, followed by incubation with protein A-Sepharose beads for 4 h. Immune complexes were washed, boiled in sample buffer, resolved by SDS-PAGE, transferred, and immunoblotted with anti-p21 or anti-p27 antibodies.[2]
RNA Extraction and Real-time PCR: Total RNA was isolated using Easy Blue kits, and cDNA was synthesized by reverse transcription. Real-time PCR for TGF-β1 mRNA was performed using SYBR Green with GAPDH as internal control. Primers: TGF-β1 forward 5'-gtg tgacat agg gtc tct gc-3', reverse 5'-gag ggt gca cat aca aca gcg-3'; GAPDH forward 5'-ggg gct ctc cac aga acat ac-3', reverse 5'-cag gtc agg tcc acc act ga-3'. Cycling conditions: 95°C for 5 s denaturation, 57°C for 10 s annealing, 72°C for 20 s extension, 40 cycles.[2]
NBT Reduction Assay: Cells were incubated with 0.1% NBT and 100 ng/ml PMA at 37°C for 25 min, smeared, and microscopically scored for cells containing blue-black formazan deposits (positive cells).[2]
Esterase Activity Assay: AS-D chloroacetate esterase and α-naphthyl acetate esterase kits were used according to manufacturer's instructions to stain cells, and the percentage of positive cells was counted.[2]
Phagocytic Activity Assay: Cells were incubated with fluorescent latex beads (1 μm diameter) at 37°C for 4 h, washed, and microscopically counted for cells containing ≥10 beads.[2]
Flow Cytometry for Surface Antigens: Cells were collected, incubated with FITC-labeled anti-CD11b, anti-CD14, anti-CD64, or anti-CD66b antibodies on ice for 30 min, washed, and analyzed by flow cytometry for fluorescence intensity.[2]
TGF-β1 ELISA: Culture supernatants were collected and assayed using Quantikine ELISA kit per manufacturer's protocol. Samples were acid-activated (1N HCl for 10 min, neutralized with 1.2N NaOH/0.5M HEPES) and added to microplates pre-coated with polyclonal antibody to measure TGF-β1 protein.[2]

[1]. Anti-inflammatory, antioxidative and matrix metalloproteinase inhibitory properties of 20(R)-ginsenoside Rh2 in cultured macrophages and keratinocytes. J Pharm Pharmacol. 2013 Feb;65(2):310-6.

[2]. Ginsenoside Rh2 induces cell cycle arrest and differentiation in human leukemia cells by upregulating TGF-β expression. Carcinogenesis. 2013 Feb;34(2):331-40.

[3]. Ginsenoside Rh2-mediated G1 phase cell cycle arrest in human breast cancer cells is caused by p15 Ink4B and p27 Kip1-dependent inhibition of cyclin-dependent kinases. Pharm Res. 2009 Oct;26(10):2280-8.

[4]. Antiviral activity of 20(R)-ginsenoside Rh2 against murine gammaherpesvirus. J Ginseng Res. 2017 Oct;41(4):496-502.

(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 functions 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 to exist in Panax notoginseng and ginseng, and relevant data are available for reference.

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Background: Ginsenoside Rh2 is a triterpene saponin and one of the major bioactive constituents of Panax ginseng, classified as a 20(S)-protopanaxadiol. Previous studies have shown that Rh2 inhibits proliferation of various cancer cells by inducing cell cycle arrest and apoptosis. This study is the first to investigate the molecular mechanism of Rh2-induced cell cycle arrest and differentiation in human leukemia HL-60 cells. It demonstrates that Rh2 upregulates TGF-β1 expression, leading to increased p21^CIP1/WAF1 and p27^KIP1, inhibition of CDK2/4/6 activities, reduced Rb phosphorylation and E2F1 nuclear translocation, resulting in G1 phase arrest and differentiation toward monocyte/macrophage and granulocyte lineages. These findings suggest the potential of Rh2 for leukemia treatment.[2]

C36H62O8

622.8727

622.444

112246-15-8

119307

White to off-white solid powder

1.19

726.4±60.0 °C at 760 mmHg

225 °C

393.1±32.9 °C

0.0±5.4 mmHg at 25°C

1.572

5.62

6

8

7

44

1070

15

CC(=CCC[C@@](C)([C@H]1CC[C@@]2([C@@H]1[C@@H](C[C@H]3[C@]2(CC[C@@H]4[C@@]3(CC[C@@H](C4(C)C)O[C@H]5[C@@H]([C@H]([C@@H]([C@H](O5)CO)O)O)O)C)C)O)C)O)C

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~125 mg/mL (~200.68 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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