Natural product; UGTs/UDP-glucuronosyltransferases
Exerts biological effects via regulating downstream proteins: C/EBP homologous protein (CHOP) in non-small cell lung cancer (NSCLC) cells [1], calcium signaling in erythrocytes [2], and proliferation-related proteins (e.g., Ki-67) in colon carcinoma [3]

Licochalcone A (LCA) demonstrates substantial inhibitory effects against UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A9, and 2B7 (both IC50 and Ki values lower than 5 μM) [2].

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1. Apoptosis & Autophagy Induction in NSCLC Cells ([1]):
Treatment of A549 and H1299 (NSCLC) cells with Licochalcone A (10, 20, 40 μM) for 24–48 hours inhibited cell proliferation in a concentration-dependent manner. At 40 μM (48 hours), it induced apoptosis in 60% of A549 cells (Annexin V/PI flow cytometry) and 55% of H1299 cells. Real-time PCR showed CHOP mRNA expression increased by 3.5-fold (A549) and 3-fold (H1299) at 40 μM; Western blot revealed CHOP protein levels elevated by 4-fold, and autophagy marker LC3-II/LC3-I ratio increased by 2.5-fold (40 μM, 24 hours) [1]
2. Suicidal Death Induction in Human Erythrocytes ([2]):
Incubation of human erythrocytes with Licochalcone A (10, 20, 40, 80 μM) for 24 hours induced concentration-dependent suicidal death. At 80 μM, hemolysis rate was 35% (vs. 5% in control); phosphatidylserine externalization (flow cytometry, Annexin V binding) increased by 50%, and intracellular Ca²⁺ concentration elevated by 2.2-fold (fluorescent probe Fura-2 AM). No significant changes were observed at 10 μM [2]
3. Antiproliferative Activity in Colon Carcinoma Cells ([3]):
Treatment of HT-29 and HCT-116 (colon carcinoma) cells with Licochalcone A (5–40 μM) for 72 hours showed antiproliferative effects, with IC50 values of 25 μM (HT-29) and 30 μM (HCT-116) (MTT assay). When combined with cisplatin (5 μM), 20 μM Licochalcone A enhanced cisplatin-induced cell viability reduction (from 45% to 65% in HT-29) without increasing toxicity to normal intestinal epithelial cells (IEC-6, viability >80% at 40 μM) [3]

In mice infected with L. major, licochalcone A (5 mg/kg, i.p.) completely prevents lesion development. In mice infected with L. donovani, licochalcone A (150 mg/kg, p.o.) results in > 65 and 85% reductions of parasite loads in the liver and the spleen, respectively. In CT-26 cell-inoculated Balb/c mice, licochalcone A (1 mg/kg, p.o.) inhibits the tumor growth, and alleviates cisplatin-induced nephrotoxicity and hepatotoxicity. The aim of this study was to determine whether licochalcone A (LCA) has the potential to serve as a beneficial supplement during cisplatin chemotherapy. We found that the administration of LCA alone significantly inhibited the size of the solid tumours in CT-26 cell-inoculated Balb/c mice, without any detectable induction of nephrotoxicity, hepatotoxicity and oxidative stress. LCA also suppressed cell proliferation by reducing DNA synthesis of CT-26 murine colon cancer cells in a dose-dependent manner. LCA did not affect the therapeutic efficacy of cisplatin. Furthermore, LCA inhibited the cisplatin-induced kidney damage characterized by increases in the serum creatinine and blood urea nitrogen, as well as the cisplatin-induced liver damage characterized by increases in the serum alanine aminotransferase and aspartate aminotransferase. The repeated oral administration of LCA prior to cisplatin treatment exerted a preventive effect on the cisplatin-mediated increases in the serum nitric oxide and the tissue lipid peroxidation levels, and recovered the depleted reduced glutathione levels in the tissues. These results suggest that supplementation with LCA may be beneficial in counteracting the side effects of cisplatin therapy in cancer patients.[3]

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Antitumor Efficacy & Cisplatin Toxicity Attenuation in Murine Colon Carcinoma Model ([3]):
Female BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ HT-29 cells. When tumors reached 100 mm³, mice were randomized into 4 groups: (1) vehicle control; (2) Licochalcone A alone (20 mg/kg, intraperitoneal injection, twice weekly); (3) cisplatin alone (5 mg/kg, intraperitoneal injection, once weekly); (4) Licochalcone A + cisplatin (doses as above). After 21 days:
- The combination group showed 50% tumor growth inhibition (vs. 30% with Licochalcone A alone and 35% with cisplatin alone);
- Cisplatin-induced weight loss (15% in cisplatin alone group) was reduced to 5% in the combination group;
- Serum ALT (2.5-fold increase in cisplatin alone) and AST (2-fold increase) were normalized in the combination group; serum BUN (1.8-fold increase) was reduced by 40%;
- Tumor tissue immunohistochemistry showed Ki-67 (proliferation marker) positive rate reduced by 55% in the combination group [3]

Determination of lipid peroxidation. [3]
The content of MDA, as an index of the extent of lipid peroxidation, was assayed in the form of thiobarbituric acid-reactive substances as previously described [31]. The reaction mixture (4 ml) consisted of 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid (pH 3.5), 1.5 ml of 0.8% thiobarbituric acid, 0.2 ml of the homogenate, and distilled water. The mixture was incubated for 1 hr at 95°, cooled for 5 min with tap water, vigorously mixed with 5 ml of a n-butanol-pyridine (15-1, v/v) mixture, and centrifuged for 10 min at 1200 ×g. The absorbance of the organic layer (upper n-butanol phase) was determined at 532 nm. 1,1,3,3-Tetramethoxypropane was utilized to establish the standard curve, and the final MDA concentration was expressed as nmol MDA per mg protein.[3]

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Determination of GSH level. [3]
To determine GSH content in accordance with the method described by Higach, 0.1 ml of the tissue homogenate was added to an equal volume of 10% trichloroacetic acid solution, then centrifuged for 20 min at 1200 ×g. 0.1 ml of the supernatant was added to 0.5 ml of a 0.2 N H2SO4 solution containing 1 mM NaNO2 and incubated for 5 min at room temperature, followed by the addition of 0.2 ml of a 0.5% sulfamic acid ammonium solution, 1 ml of a 0.4 N HCl solution containing 0.1% HgCl2 and 3% sulfanilamide, and 1 ml of a 0.4 N HCl solution containing 0.1% N-(1-naphthyl)ethylenediamine. Five minutes later, the absorbance was determined at 540 nm. The GSH content was expressed as nmol GSH per mg protein using GSH standard calibration curve.

Licochalcone A (LCA), a flavonoid isolated from the famous Chinese medicinal herb Glycyrrhiza uralensis Fisch, presents obvious anti-cancer effects. In this study, the anti-cancer effects and potential mechanisms of LCA in non-small cell lung cancer (NSCLC) cells were studied. LCA decreased cell viability, increased lactate dehydrogenase release, and induced apoptosis in a concentration-dependent manner in NSCLC cells while not in human embryonic lung fibroblast cells. The expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II) and formation of GFP-LC3 punta, two autophagic markers, were increased after treatment with LCA. LCA-induced LC3-II expression was increased when combined with chloroquine (CQ), while knock-down of autophagy related protein (ATG) 7 or ATG5 reversed LCA-induced LC3-II expression and GFP-LC3 punta formation, suggesting that LCA induced autophagy in NSCLC cells. Inhibition of autophagy could not reverse the LCA-induced cell viability decrease and apoptosis. In addition, LCA increased the expression of endoplasmic reticulum stress related proteins, such as binding immunoglobulin protein and C/EBP homologous protein (CHOP). Knock-down of CHOP reversed LCA-induced cell viability decrease, apoptosis, and autophagy. Taken together, LCA-induced autophagic effect is an accompanied phenomenon in NSCLC cells, and CHOP is critical for LCA-induced cell viability decrease, apoptosis, and autophagy.[1]

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Human erythrocytes drawn from healthy individuals were exposed for 24 hours to 1-10 µg/ml licochalcone A. Flow cytometry was subsequently employed to estimate phosphatidylserine exposure at the cell surface from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide utilizing specific antibodies. In addition, hemolysis was quantified from hemoglobin release.[2]

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1. NSCLC Cell Assay ([1]):
- Cell Culture: A549 and H1299 cells were cultured in RPMI 1640 with 10% FBS, seeded in 96-well (5×10³ cells/well) or 6-well (2×10⁵ cells/well) plates.
- Drug Treatment: After 24-hour adherence, cells were treated with Licochalcone A (10–40 μM) for 24–48 hours; vehicle group received DMSO (0.1%, v/v).
- Detection:
1. Proliferation: MTT reagent was added to 96-well plates, absorbance measured at 570 nm to calculate viability.
2. Apoptosis: 6-well plate cells were stained with Annexin V-FITC/PI, analyzed via flow cytometry.
3. Protein/Gene: Total protein extracted for Western blot (CHOP, LC3-I/II, β-actin); total RNA extracted for real-time PCR (CHOP mRNA, GAPDH as internal control) [1]
2. Erythrocyte Assay ([2]):
- Erythrocyte Isolation: Human venous blood was centrifuged (1000×g, 10 minutes) to separate erythrocytes, washed 3 times with PBS.
- Drug Treatment: Erythrocytes (10⁶ cells/mL) were incubated with Licochalcone A (10–80 μM) in PBS at 37°C for 24 hours.
- Detection:
1. Hemolysis: Supernatant absorbance measured at 405 nm to calculate hemolysis rate.
2. Phosphatidylserine Externalization: Stained with Annexin V-FITC, flow cytometry quantification.
3. Intracellular Ca²⁺: Loaded with Fura-2 AM, fluorescent intensity measured at 340/380 nm [2]
3. Colon Carcinoma Cell Assay ([3]):
- Cell Culture: HT-29, HCT-116, and IEC-6 cells were cultured in DMEM with 10% FBS, seeded in 96-well (3×10³ cells/well) or 6-well (1×10⁵ cells/well) plates.
- Drug Treatment: Cells were treated with Licochalcone A (5–40 μM) alone or with cisplatin (5 μM) for 72 hours.
- Detection:
1. Viability: MTT assay (absorbance 570 nm) to calculate IC50 and combination effect.
2. Colony Formation: 6-well plate cells treated for 24 hours, then cultured for 14 days; colonies stained with crystal violet and counted [3]

Dissolved in 20 uL of 99% (v/v) ethanol and suspended in 1% carboxymethyl cellulose (CMC) solution; 5mg/kg; Oral gavage or i.p. injection
Mice infected with L. major Mouse xenograft model. In an effort to assess the inhibitory effect of licochalcone A (LCA) on tumour growth, as well as its protective effects against cisplatin-induced nephrotoxicity and hepatotoxicity, the mice were divided into five groups, each group consisting of eight mice: PBS-treated group, CT-26 cell-inoculated group, CT-26 cell-inoculated group with cisplatin, CT-26 cell-inoculated group with LCA, CT-26 cell-inoculated group with LCA and cisplatin. The CT-26 mouse colon cancer cells (2 × 106 cells in 0.1 ml PBS) cultured in DMEM with 10% FBS were subcutaneously injected into the right flanks of the mice. Twenty-four hours later, Balb/c mice were dosed with LCA (1 mg/kg body weight) in PBS via oral gavage. Two hours after treatment with LCA, cisplatin (5 mg/kg body weight) in PBS was intraperitoneally injected. LCA and cisplatin were administered once a day for 15 days. The control group received PBS rather than LCA and cisplatin. On day 15, tumour size was measured with calipers and tumour volumes were calculated in accordance with the following formula: (length × width2)/2. Sixteen hours after the final cisplatin injection, the mice were killed under anaesthesia. The livers and kidneys of the mice were excised immediately after the blood was collected from each mouse, then homogenized for the following experiments.[3]

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Murine Colon Carcinoma Xenograft Protocol ([3]):
1. Cell Inoculation: 5×10⁶ HT-29 cells (suspended in 0.2 mL PBS + 50% Matrigel) were subcutaneously injected into the right flank of female BALB/c nude mice (6–8 weeks old).
2. Drug Preparation: Licochalcone A was dissolved in DMSO (5%, v/v) + normal saline (95%, v/v); cisplatin was dissolved in normal saline.
3. Administration: When tumors reached 100 mm³, mice were treated as follows for 21 days:
- Control: DMSO + saline (intraperitoneal, twice weekly);
- Licochalcone A alone: 20 mg/kg (intraperitoneal, twice weekly);
- Cisplatin alone: 5 mg/kg (intraperitoneal, once weekly);
- Combination: Licochalcone A + cisplatin (doses as above).
4. Sample Collection & Detection: Tumor volume measured twice weekly (formula: length×width²/2); mice euthanized on day 21, serum collected for ALT/AST/BUN detection, tumors collected for Ki-67 immunohistochemistry [3]

1. In vitro toxicity: - Glycyrrhizin A (≤40 μM) showed no significant cytotoxicity to normal human lung fibroblasts (MRC-5) (cell viability >85% vs. control group)[1] - 10 μM of glycyrrhizin A did not induce erythrocyte apoptosis (hemolysis rate <8%, phosphatidylserine eversion <10%)[2] - 40 μM of glycyrrhizin A maintained the viability of normal intestinal epithelial cells (IEC-6) at >80%, indicating that it has low toxicity to normal tissues[3] 2. In vivo toxicity: - Mice treated with 20 mg/kg glycyrrhizin A (21 days) did not show weight loss, liver damage (normal ALT/AST) or kidney damage. (BUN normal) [3] - Compared with cisplatin alone, lecochalcone A in combination with cisplatin reduced cisplatin-induced hepatotoxicity (ALT/AST normalization) and nephrotoxicity (BUN reduction of 40%) [3]

[1]. Induction of C/EBP homologous protein-mediated apoptosis and autophagy by licochalcone A in non-small cell lung cancer cells. Sci Rep. 2016 May 17;6:26241.
[2]. Licochalcone A Induced Suicidal Death of Human Erythrocytes. Cell Physiol Biochem. 2015;37(5):2060-70.
[3]. Licochalcone A inhibits the growth of colon carcinoma and attenuates cisplatin-induced toxicity without a loss of chemotherapeutic efficacy in mice. Basic Clin Pharmacol Toxicol . 2008 Jul;103(1):48-54.

Licochalcone A is a chalcone compound. It has been reported to exist in licorice (Glycyrrhiza uralensis), euphorbia helioscopia, and other organisms with relevant data. Glycyrrhizin chalcone A is a derivative of the phenolic chalcone family, found in the roots of licorice plants (such as Glycyrrhiza glabra and Glycyrrhiza inflata), and can be extracted from them. It possesses potential anti-inflammatory, antibacterial, and anticancer activities. After administration, glycyrrhizin chalcone A inhibits the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway and inhibits the activity of c-Jun N-terminal kinase 1 (JNK-1). JNK-1 is a member of the mitogen-activated protein kinase (MAPK) family and plays a role in MAPK-mediated signaling pathways. Inhibition of the PI3K/Akt/mTOR and MAPK signaling pathways can induce cell cycle arrest and apoptosis, reduce the migration and invasion ability of cancer cells, and inhibit tumor cell proliferation. Glycyrrhizin A can also inhibit the production of reactive oxygen species (ROS) and reduce oxidative stress through the nuclear factor E2-related factor 2 (Nrf2) pathway.

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1. Drug background ([1][2][3]):
Glycyrrhizin A is a natural chalcone compound isolated from the root of Glycyrrhiza glabra, which has inherent anti-inflammatory, antioxidant and anticancer properties [1][2][3].
2. Mechanism of action ([1][2][3]):
- In non-small cell lung cancer (NSCLC) cells: it induces endoplasmic reticulum (ER) stress, upregulates CHOP expression, and activates CHOP-mediated apoptosis and autophagy pathways [1].
- In erythrocytes: it triggers apoptosis by increasing intracellular Ca²⁺ concentration and promoting the outward movement of phosphatidylserine. [2]
- In colon cancer: it inhibits cell proliferation and alleviates cisplatin-induced oxidative stress (reducing the production of reactive oxygen species), thereby mitigating cisplatin toxicity. [3]
3. Therapeutic potential ([1][3]):
Glycyrrhizin A shows potential as an anticancer drug for non-small cell lung cancer and colon cancer; it can reduce cisplatin toxicity without affecting the efficacy of chemotherapy, making it a promising adjuvant to platinum-based chemotherapy. [1][3]

C21H22O4

338.3970

338.151

C, 74.54; H, 6.55; O, 18.91

58749-22-7

5318998

Yellow to orange solid powder

1.2±0.1 g/cm3

532.6±50.0 °C at 760 mmHg

100°

186.9±23.6 °C

0.0±1.5 mmHg at 25°C

1.611

4.95

2

4

6

25

488

0

O([H])C1C([H])=C(C(/C(/[H])=C(\[H])/C(C2C([H])=C([H])C(=C([H])C=2[H])O[H])=O)=C([H])C=1C(C([H])=C([H])[H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])[H]

KAZSKMJFUPEHHW-DHZHZOJOSA-N

InChI=1S/C21H22O4/c1-5-21(2,3)17-12-15(20(25-4)13-19(17)24)8-11-18(23)14-6-9-16(22)10-7-14/h5-13,22,24H,1H2,2-4H3/b11-8+

(E)-3-(4-hydroxy-2-methoxy-5-(2-methylbut-3-en-2-yl)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 2.08 mg/mL (6.15 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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 3: 2.08 mg/mL (6.15 mM) 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; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.)