The study suggests licoricidin exerts its anti-metastatic effects through multiple pathways, including the inhibition of cancer cell migration, modulation of the tumor microenvironment (reduction of M2 macrophage infiltration), and suppression of tumor angiogenesis and lymphangiogenesis. It reduces the expression of proteins associated with inflammation (HIF-1α, iNOS, COX-2), angiogenesis (CD31, VEGF-A, VEGFR-2), and lymphangiogenesis (LYVE-1, VEGF-C, VEGFR-3) in tumor tissues. It also decreases the activation of NF-κB (as shown by reduced P-p65 expression) and its downstream targets. [2]

The vitality of colon cancer cell lines with different pathological and genetic characteristics (e.g., SW480, HCT116, SW620, and LoVo cells) is dose-dependently inhibited by lidocaine (LCD) (0–20 μM; 24 hours), with IC50 values of 7.2, 5.4, 4.5 are 5.1 μM and 5.1 μM, respectively [1]. Apoptosis is induced by lidocaine (LCD) (0–20 μM; 0–12 h), and in a dose- and time-dependent manner, this is followed by the cleaved activation of caspase-3 [1]. Licoricidin (LCD) (0-20 μM; 0-12 hours) stimulates autophagy in SW480 cells, leading to a time- and dose-dependent increase in p62 degradation and LC3-I to LC3-II cleavage [1]. In 4T1 cells, lidocidin (LCD) (0–5 μg/ml; 18 hours) decreases MMP-9 production, VCAM expression, and cell migration [2].

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Licoricidin inhibited the migration of 4T1 mouse mammary carcinoma cells in a dose-dependent manner at concentrations that did not decrease cell viability. [2]
At a concentration of 2.5 μg/mL, licoricidin reduced the secretion of matrix metalloproteinase-9 (MMP-9) and the expression of vascular cell adhesion molecule-1 (VCAM-1) in 4T1 cells, as determined by Western blot analysis of conditioned media and cell lysates. [2]
Licoricidin did not decrease the viability of MCF-10A normal mammary epithelial cells, as assessed by MTT assay. [2]

The growth of SW480 xenografts in nude mice was considerably decreased by lidocaine (LCD) (intraperitoneal injection; 5, 10 or 20 mg/kg; once daily; 15 days) with an inhibition rate of 43.5% [1]. Moreover, the protein expression of VEGF-R2, VEGF-C, VEGF-R3, and LYVE-1 in tumor tissues of mice treated with liquiritin decreased [2]. Liquiritin (LCD) (ip; 5, 10, or 20 mg/kg; once daily; 32 days) lowers lung metastasis and expression of CD45, CD31, HIF-1α, iNOS, COX-2, and VEGF-A.

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In a BALB/c mouse orthotopic model with 4T1 mammary carcinoma cells, intraperitoneal (i.p.) administration of licoricidin at 2 or 4 mg/kg body weight/day for 21 days significantly reduced the number of metastatic lung nodules and lung weight (at the 4 mg/kg dose), without significantly suppressing the primary tumor weight. [2]
Immunohistochemical (IHC) and immunofluorescence (IF) analysis of tumor tissues from licoricidin-treated mice revealed decreased expression of inflammation-related proteins (CD45, HIF-1α, COX-2, iNOS, and phosphorylated p65 NF-κB). [2]
Licoricidin treatment led to a decrease in the infiltration of macrophages, specifically M2 macrophages, as evidenced by reduced expression of F4/80 (a mature macrophage marker) and CD206 (an M2 macrophage marker), while the expression of the M1 macrophage marker CD11c remained unchanged. [2]
Tumor tissues from licoricidin-treated mice showed decreased expression of proteins related to angiogenesis (CD31, VEGF-A, VEGF-R2) and lymphangiogenesis (LYVE-1, VEGF-C, VEGF-R3). Additionally, hemoglobin concentration in tumor tissues was reduced in mice treated with 2 mg/kg licoricidin. [2]

Cell Viability Assay[1]
Cell Types: SW480, HCT116, SW620 and LoVo Cell
Tested Concentrations: 0-20 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Colon cancer cell line viability diminished.

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Western Blot Analysis [1]
Cell Types: SW480 cell
Tested Concentrations: 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM
Incubation Duration: 0 hrs (hours), 1 hour, 3 hrs (hours), 6 hrs (hours), 12 hrs (hours)
Experimental Results: Induction of apoptosis .

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Cell Viability Assay (MTT): 4T1 cells were treated with various concentrations of licoricidin for 12 and 24 hours. Following treatment, MTT solution was added, and the formazan crystals formed were dissolved. The absorbance was measured to determine cell viability relative to the control group. This assay was used to confirm that the concentrations used in migration assays (up to 2.5 μg/mL) did not affect cell viability. Similarly, MCF-10A normal mammary epithelial cells were treated with licoricidin (0.5, 1.0, 2.5 μg/mL) for 12 and 24 hours, and their viability was assessed using the MTT assay. [2]
Transwell Migration Assay: 4T1 cells were serum-starved for 24 hours. Type IV collagen was used to pre-coat the lower side of a transwell filter. Cells were added to the upper compartment and treated with various concentrations of licoricidin (0.5, 1.0, 2.5 μg/mL). The lower chamber contained DMEM with FBS and BSA as a chemoattractant. After a 12-hour incubation, cells that migrated to the lower side of the filter were stained with hematoxylin and eosin. The number of migrated cells was counted and expressed as a percentage of the control. [2]
Western Blot Analysis: 4T1 cells were plated, serum-starved for 24 hours, and then treated with or without licoricidin (2.5 μg/mL) for 18 hours. Conditioned media was collected and concentrated, and cell lysates were prepared. Proteins from both the concentrated conditioned media and total cell lysates were separated by electrophoresis, transferred to membranes, and probed with antibodies against MMP-9 and VCAM-1. Protein bands were detected using a chemiluminescence system and quantified relative to the control. [2]

Animal/Disease Models: SW480 nude mouse xenograft tumor growth [1]
Doses: 5, 10 or 20 mg/kg
Route of Administration: intraperitoneal (ip) injection; one time/day; 15-day
Experimental Results: tumor volume reduction.

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Animal/Disease Models: BALB/c mouse orthotopic model [2]
Doses: 5, 10 or 20 mg/kg
Route of Administration: intraperitoneal (ip) injection; 5, 10 or 20 mg/kg; one time/day; 32-day
Experimental Results: Inhibition of 4T1 mice Lung metastasis of breast cancer cells.

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Animal Model for Metastasis:** Female BALB/c mice (5 weeks old) were acclimated. 4T1 cells (5 × 10⁴ cells in 0.1 mL Matrigel) were injected into the inguinal mammary fat pads to establish an orthotopic tumor model. [2]
**Dosing and Administration:** Beginning 7 days after the 4T1 cell injection, mice received daily intraperitoneal (i.p.) injections for 21 days. The treatment groups were: (1) vehicle control, (2) licoricidin at 2 mg/kg body weight/day, and (3) licoricidin at 4 mg/kg body weight/day. Licoricidin was first dissolved in DMSO at 10 mg/mL to create a stock solution, which was then diluted with physiological saline to achieve the final desired concentration for administration. [2]
**Tissue Collection and Analysis:** At the end of the treatment period (28 days post-4T1 cell injection), animals were sacrificed. Primary tumors, lungs, and other organs were isolated and weighed. The lungs were fixed in Bouin's solution to visualize and quantify surface metastatic tumor nodules. Tumor tissues were also fixed in 4% paraformaldehyde for immunohistochemistry (IHC) or frozen for immunofluorescence (IF) staining. [2]

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Animal Model for Metastasis: Female BALB/c mice (5 weeks old) were acclimated. 4T1 cells (5 × 10⁴ cells in 0.1 mL Matrigel) were injected into the inguinal mammary fat pads to establish an orthotopic tumor model. [2]
Dosing and Administration: Beginning 7 days after the 4T1 cell injection, mice received daily intraperitoneal (i.p.) injections for 21 days. The treatment groups were: (1) vehicle control, (2) licoricidin at 2 mg/kg body weight/day, and (3) licoricidin at 4 mg/kg body weight/day. Licoricidin was first dissolved in DMSO at 10 mg/mL to create a stock solution, which was then diluted with physiological saline to achieve the final desired concentration for administration. [2]
Tissue Collection and Analysis: At the end of the treatment period (28 days post-4T1 cell injection), animals were sacrificed. Primary tumors, lungs, and other organs were isolated and weighed. The lungs were fixed in Bouin's solution to visualize and quantify surface metastatic tumor nodules. Tumor tissues were also fixed in 4% paraformaldehyde for immunohistochemistry (IHC) or frozen for immunofluorescence (IF) staining. [2]

The study notes that licoricidin did not decrease the viability of MCF-10A normal mammary epithelial cells in vitro. The primary safety concern addressed in the background is regarding the main licorice component glycyrrhizin, which induces hypertension and hypokalemia, whereas licoricidin is an active component in a glycyrrhizin-depleted extract. [2]

[1]. Licoricidin inhibits the growth of SW480 human colorectal adenocarcinoma cells in vitro and in vivo by inducing cycle arrest, apoptosis and autophagy. Toxicol Appl Pharmacol. 2017 Jul 1;326:25-33.

[2]. Licoricidin, an Active Compound in the Hexane/Ethanol Extract of Glycyrrhiza uralensis, Inhibits Lung Metastasis of 4T1 Murine Mammary Carcinoma Cells. Int J Mol Sci. 2016 Jun 14;17(6).

[3]. Licoricidin enhances gemcitabine-induced cytotoxicity in osteosarcoma cells by suppressing the Akt and NF-κB signal pathways. Chem Biol Interact. 2018 Jun 25;290:44-51.

[4]. Licoricidin, an isoflavonoid isolated from Glycyrrhiza uralensis Fisher, prevents UVA-induced photoaging of human dermal fibroblasts. Int J Cosmet Sci. 2017 Apr;39(2):133-140.

Licoricidin is a hydroxyisoflavone compound belonging to the R-isoflavone class, with hydroxyl groups at the 7', 2', and 4' positions, a methoxy group at the 5' position, and isopentenyl groups at the 6' and 3' positions. It is isolated from licorice (Glycyrrhiza uralensis) and possesses antibacterial activity. Licoricidin is both an antibacterial agent and a plant metabolite. It belongs to the hydroxyisoflavone class, aromatic ether class, and methoxyisoflavone class. Licoricidin has been reported to be found in licorice (Glycyrrhiza uralensis), coarse licorice (Glycyrrhiza aspera), and other organisms with relevant data. See also: Licorice (Glycyrrhiza uralensis) root (part).

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Licoricidin is a polyphenolic compound identified as one of the active anti-metastatic components in a hexane/ethanol extract of Glycyrrhiza uralensis (HEGU). Previous studies by the same research group showed that licoricidin inhibits the metastatic capacity of DU145 human prostate cancer cells in vitro. This study extends those findings, demonstrating its anti-metastatic efficacy in a murine breast cancer model. The authors propose that the anti-metastatic activity of licoricidin is mediated by multiple mechanisms, including direct inhibition of cancer cell migration, reduction of M2 macrophage infiltration, and suppression of both tumor angiogenesis and lymphangiogenesis. [2]

C26H32O5

424.5293

424.224

30508-27-1

480865

White to off-white solid powder

1.2±0.1 g/cm3

610.8±55.0 °C at 760 mmHg

161.0-162.5℃

323.2±31.5 °C

0.0±1.8 mmHg at 25°C

1.597

6.36

3

5

6

31

636

1

O1C2C([H])=C(C(C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H])=C(C=2C([H])([H])[C@]([H])(C2C([H])=C([H])C(=C(C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H])C=2O[H])O[H])C1([H])[H])OC([H])([H])[H])O[H]

GBRZTUJCDFSIHM-KRWDZBQOSA-N

InChI=1S/C26H32O5/c1-15(2)6-8-19-22(27)11-10-18(25(19)29)17-12-21-24(31-14-17)13-23(28)20(26(21)30-5)9-7-16(3)4/h6-7,10-11,13,17,27-29H,8-9,12,14H2,1-5H3/t17-/m0/s1

4-[(3R)-7-hydroxy-5-methoxy-6-(3-methylbut-2-enyl)-3,4-dihydro-2H-chromen-3-yl]-2-(3-methylbut-2-enyl)benzene-1,3-diol

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 : ~100 mg/mL (~235.55 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.)