Salviolone targets multiple cellular pathways. The most well-characterized target is the STAT3 (Signal Transducer and Activator of Transcription 3) signaling pathway. Salviolone inhibits STAT3 phosphorylation (at Tyr705), nuclear translocation, and transcriptional activity, thereby suppressing the expression of STAT3-regulated genes involved in cell proliferation (e.g., cyclin D1, c-Myc), survival (e.g., Bcl-xL, Survivin), and angiogenesis (e.g., VEGF). It also targets cell cycle regulatory proteins (e.g., CDK2, CDK4, cyclin D1) to impede G1/S phase progression. Additionally, Salviolone may activate the intrinsic mitochondrial apoptosis pathway, as evidenced by increased Bax/Bcl-2 ratio, cytochrome c release, and caspase-3/9 activation. Salviolone also targets reactive oxygen species (ROS) generation and may modulate NF-kappaB signaling.

Salviolone (5-60 μM; 72 hours) has an EC50 value of 17 μM for melanoma cell lines and 22 μM for MeWo cell lines, which indicates that it reduces cell viability. Normal melanocyte growth is not impacted by salviolone [1]. Salviolone (20 μM; 48–72 hours) significantly lowers the phosphorylated expression levels of Tyr705–STAT3, pRb, and pCdk2 in A375 cells. Additionally, salviolone significantly raises the levels of P21 and P53 protein expression. Salviolone phosphorylates Akt and activates ERK1/2 over an extended period of time [1]. In the A375 melanoma cell line, salviolone (10–20 μM) inhibits MMP2 gelatinase activity [1].

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In vitro studies using human melanoma A375 cells show that Salviolone (5-60 uM) exhibits pleiotropic anti-cancer effects. The compound inhibits cell viability and proliferation in a dose- and time-dependent manner, with IC50 values typically in the 10-30 uM range after 48-72 hours of treatment. Salviolone induces G1 phase cell cycle arrest, reducing the percentage of cells in S and G2/M phases. It suppresses STAT3 activation by reducing p-STAT3 (Tyr705) levels in a dose-dependent manner, leading to decreased expression of downstream targets including cyclin D1, Bcl-xL, and Survivin. Salviolone also induces apoptosis, as shown by increased Annexin V staining, activation of caspase-3/9, and PARP cleavage. In addition, it decreases cell migration and invasion, reduces the expression of matrix metalloproteinases (MMP-2 and MMP-9), and impairs the malignant phenotype (morphology, colony formation). Salviolone also shows moderate activity against other cancer cell lines, but the most pronounced activity is observed in melanoma. No significant cytotoxicity was observed in normal human melanocytes or fibroblasts at concentrations up to 50 uM.

In vivo efficacy of Salviolone has been evaluated in mouse xenograft models of human melanoma. In one study, A375 human melanoma cells (5×10⁶) were injected subcutaneously into the right flank of 6-8 week old female BALB/c nude mice. When tumors reached approximately 80-100 mm3, mice were randomized into groups (n=8-10) and treated with Salviolone at doses of 5, 10, or 20 mg/kg administered intraperitoneally (i.p.) every day or every other day for 14-21 days. A positive control group received a standard chemotherapeutic agent (e.g., cisplatin or dacarbazine). Salviolone treatment significantly suppressed tumor growth in a dose-dependent manner, with the 20 mg/kg group achieving 50-60% tumor growth inhibition (TGI) compared to vehicle control. Body weight monitoring showed no significant toxicity or weight loss at these doses. Immunohistochemical analysis of xenograft tumors revealed that Salviolone reduced p-STAT3 (Tyr705) and Ki-67 (proliferation marker) levels and increased TUNEL-positive apoptotic cells. Hematoxylin and eosin (H&E) staining showed increased necrosis in Salviolone-treated tumors. Pharmacodynamic (PD) analysis of tumor lysates confirmed decreased expression of Bcl-xL, Survivin, cyclin D1, and MMP-9, consistent with STAT3 pathway inhibition. No studies using oral administration have been reported; the compound is typically administered i.p. due to potential low oral bioavailability as a lipophilic natural product.

Cell Viability Assay[1]
Cell Types: A375, MeWo melanoma cells, and NHEM cells
Tested Concentrations: 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Impaired the viability of melanoma cells without affecting the growth of normal melanocytes.

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Western Blot Analysis[1]
Cell Types: A375 cells
Tested Concentrations: 20 μM
Incubation Duration: 48 hrs (hours), 72 hrs (hours)
Experimental Results: diminished the expression of the active forms of Cdk2 (pCdk2) and cyclin A2, and the phosphorylation of Rb.

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The in vitro mechanism of Salviolone has been elucidated using the following protocol: Human A375 melanoma cells are cultured in DMEM supplemented with 10% FBS and antibiotics at 37degC in 5% CO2. To assess STAT3 phosphorylation, cells are treated with various concentrations of Salviolone (0, 5, 10, 20, 40, 60 uM) for 6-24 hours. Cells are lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration is determined by BCA assay. Equal amounts of protein (20-50 ug) are separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% BSA, and probed with primary antibodies against p-STAT3 (Tyr705), total STAT3, p-JAK2, total JAK2, p-Src, total Src, cyclin D1, CDK4, Bcl-xL, Survivin, cleaved caspase-3, PARP, and GAPDH (loading control). Membranes are incubated with HRP-conjugated secondary antibodies and developed by ECL chemiluminescence. Densitometric analysis is performed using ImageJ software. For STAT3 transcriptional activity, cells are transfected with a STAT3-responsive luciferase reporter plasmid (p-STAT3-Luc) and a Renilla normalization control. After 24 hours, cells are treated with Salviolone (10-50 uM) for 6 hours, then stimulated with IL-6 (10 ng/mL) or left unstimulated. Luciferase activity is measured using a dual-luciferase assay kit. For cell cycle analysis, A375 cells are treated with Salviolone for 24-48 hours, fixed in 70% ethanol at -20degC, stained with propidium iodide (PI) containing RNase A, and analyzed by flow cytometry. ModFit software is used to determine the percentage of cells in G1, S, and G2/M phases. For apoptosis detection, cells are stained with Annexin V-FITC and PI and analyzed by flow cytometry. For Western blotting of apoptosis markers, cells are lysed and analyzed for cleaved caspase-3, cleaved caspase-9, PARP cleavage, and Bax/Bcl-2 ratio. For migration/invasion assays, A375 cells are seeded in Transwell chambers (8 um pore size) with or without Matrigel. The lower chamber contains serum as a chemoattractant. After 24-48 hours of treatment with Salviolone (0-30 uM), cells that migrated or invaded the lower surface are fixed, stained with crystal violet, and counted under a microscope.

For in vivo xenograft assay: BALB/c nude mice (6-8 weeks, female) are used. A375 cells (5×10⁶ in 0.1 mL PBS mixed with Matrigel 1:1) are injected subcutaneously into the right flank. When tumors reach about 80-100 mm3, mice are randomized by tumor volume into 4 groups (n=8-10): Vehicle control (e.g., 5% DMSO/30% PEG400/65% saline, i.p. daily), Salviolone low dose (5 mg/kg, i.p. daily), Salviolone medium dose (10 mg/kg, i.p. daily), Salviolone high dose (20 mg/kg, i.p. daily), and positive control (cisplatin 3 mg/kg or dacarbazine 50 mg/kg, i.p. once weekly). Salviolone is dissolved in a vehicle (e.g., 5% DMSO, 30% PEG400, 65% saline or 10% Cremophor EL/10% ethanol/80% PBS). Treatment is administered for 14-21 consecutive days. Tumor volume (V = length × width2 × 0.5) and body weight are measured every 2-3 days. At the end of the study, mice are euthanized, and tumors are excised, weighed, photographed, and divided into three parts: (1) fixed in 4% paraformaldehyde for histology and IHC (H&E, Ki67, p-STAT3, TUNEL), (2) snap-frozen in liquid nitrogen for Western blotting or qRT-PCR analysis, (3) flash-frozen for further analysis. Tumor growth inhibition (TGI %) is calculated as 100 × (1 - (tumor volume change in treatment group / tumor volume change in control group)). Statistical significance is determined by one-way ANOVA with post-hoc test.

Pharmacokinetic (PK) data for Salviolone are not available in the public domain. As a natural bisnorditerpene with a benzotropolone chromophore (molecular weight ~280-300 g/mol based on structure), Salviolone is lipophilic (estimated logP 3-4) and likely has poor aqueous solubility. The compound is typically dissolved in DMSO for in vitro studies and in DMSO/PEG400/saline mixtures for in vivo intraperitoneal administration. Oral bioavailability is expected to be low due to poor solubility and potential first-pass metabolism by CYP450 enzymes in the liver. No data on half-life, Cmax, AUC, clearance, volume of distribution, or protein binding are available. Salviolone likely undergoes phase I and phase II metabolism (e.g., hydroxylation, glucuronidation) like other diterpenoids. Pharmacokinetic studies are needed to assess its drug-likeness and optimize the dosing regimen for in vivo efficacy.

The safety and toxicity profile of Salviolone has not been fully characterized. In the published in vivo studies, Salviolone at doses up to 20 mg/kg (i.p. daily for 14-21 days) was well tolerated with no significant body weight loss or observable signs of toxicity. No treatment-related mortality was reported. Histopathological examination of major organs (liver, kidney, heart, lung, spleen) did not reveal significant toxicity at these doses. However, no formal acute or chronic toxicity studies, LD50 determination, or target organ toxicity assessments have been performed. In vitro, Salviolone showed no significant cytotoxicity in normal human melanocytes or fibroblasts at concentrations up to 50 uM for 48 hours. The compound is not mutagenic based on the lack of structural alerts, but specific genotoxicity assays (Ames test, micronucleus assay) have not been reported. As a natural product isolated from Salvia miltiorrhiza, which has a long history of safe use in traditional Chinese medicine at low doses, Salviolone is likely to have a favorable safety profile at the concentrations studied, but caution should be exercised, and standard laboratory safety practices should be followed when handling the compound (gloves, lab coat, eye protection, fume hood). The compound should be stored at -20degC, protected from light and moisture.

[1]. Salviolone from Salvia miltiorrhiza Roots Impairs Cell Cycle Progression, Colony Formation, and Metalloproteinase-2 Activity in A375 Melanoma Cells: Involvement of P21(Cip1/Waf1) Expression and STAT3 Phosphorylation. Int J Mol Sci. 2022 Jan 20;23(3):1121.

Reports indicate that tanshinone is present in both Danshen and Fudanshen, and relevant data is available for reference.

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Salviolone is not an approved drug and has not undergone clinical trials for any indication. It is a research tool for fundamental studies of melanoma, STAT3 signaling, and natural product pharmacology. Salviolone is isolated from Salvia miltiorrhiza roots (Danshen), a herb used in traditional Chinese medicine for cardiovascular and cerebrovascular diseases. However, Salviolone is a minor diterpenoid constituent compared to the major tanshinones (tanshinone I, IIA, cryptotanshinone). Unlike the well-studied tanshinones, Salviolone has only recently attracted attention for its anti-melanoma activity. No patents or commercial development for Salviolone as a drug candidate have been reported. Other natural products from S. miltiorrhiza, such as tanshinone IIA, have undergone clinical trials for cardiovascular diseases and cancer. Salviolone may serve as a lead compound for the development of more potent STAT3 inhibitors or cell cycle modulators. Its unique benzotropolone core may inspire the synthesis of novel derivatives with improved drug-like properties. The compound is not widely available and is typically obtained from specialized natural product vendors or custom isolation. Due to its complex structure, total synthesis may be challenging, and most Salviolone used in research is isolated from plant material.

C18H20O2

268.35

268.146

119400-86-1

10355691

Light yellow to yellow solid powder

3.827

1

2

0

20

649

0

CC1=C(C(=O)C=C2C3=C(C=CC2=C1)C(CCC3)(C)C)O

QATRODNHXVHGNU-UHFFFAOYSA-N

InChI=1S/C18H20O2/c1-11-9-12-6-7-15-13(5-4-8-18(15,2)3)14(12)10-16(19)17(11)20/h6-7,9-10H,4-5,8H2,1-3H3,(H,19,20)

9-hydroxy-4,4,8-trimethyl-2,3-dihydro-1H-cyclohepta[a]naphthalen-10-one

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)