Calycosin primarily targets the estrogen receptors (ERα and ERβ). It acts as a partial agonist, binding to these receptors and modulating their activity. The compound has been shown to promote the proliferation of estrogen receptor-positive cells via estrogen receptors and the ERK1/2 activation pathway. In addition to its direct interaction with estrogen receptors, calycosin also influences other signaling cascades, including the MAPK, PI3K/Akt, and NF-κB pathways. It also has calcium blocking activities.

In vitro, calycosin has demonstrated significant activity against various cancer cell lines. It induces apoptosis in human ovarian cancer SKOV3 cells by activating caspases and Bcl-2 family proteins. In breast cancer cells, it suppresses cell growth via ERβ-dependent regulation of IGF-1R, p38 MAPK, and PI3K/Akt pathways. Calycosin also promotes angiogenesis in human umbilical vein endothelial cell (HUVEC) cultures. Furthermore, it can reduce advanced glycation end products-induced macrophage migration and adhesion to endothelial cells, relieving local inflammation through the estrogen receptor-ERK1/2–NF-κB pathway.

In vivo, calycosin has shown promising therapeutic effects in several animal models. It induces angiogenesis in zebrafish embryos. In a rat model of cerebral ischemia/reperfusion, it has demonstrated neuroprotective effects, with mechanisms potentially involving the positive feedback between ER-α and miR-375. Its ability to promote angiogenesis in vivo is linked to the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1, and VEGFR2 mRNA expression. It also acts as a vasorelaxant in an endothelium-independent manner.

In vitro enzyme or receptor binding assays for calycosin typically involve radioligand competition binding studies to determine its affinity for estrogen receptors. Membrane preparations or purified ERα and ERβ proteins are incubated with a radiolabeled ligand, such as [³H]-17β-estradiol, in the presence of varying concentrations of calycosin. The amount of bound radioactivity is measured to calculate the IC50, quantifying the compound's ability to displace the natural ligand. Additionally, its effects on the activity of kinases like ERK1/2 can be assessed using purified enzyme assays with specific substrates.

In vitro cell-based assays for calycosin are performed using various cell lines, including estrogen receptor-positive breast cancer cells (e.g., MCF-7), ovarian cancer cells (SKOV3), and HUVECs. Cells are treated with the compound at various concentrations for different time points (e.g., 24-72 hours). Cell viability is measured using MTT or CCK-8 assays. Apoptosis is detected via Annexin V/PI staining and flow cytometry, or by measuring caspase activity. The expression of target genes and proteins like VEGF, ERK, and Bcl-2 is analyzed by qPCR and Western blotting.

In vivo animal experiments for calycosin are conducted using rodent models of cancer and angiogenesis. For tumor xenograft models, mice are subcutaneously injected with cancer cells (e.g., SKOV3) and then treated with calycosin (intraperitoneally or orally) to evaluate its anti-tumor efficacy. Tumor volume and weight are monitored. Angiogenesis is studied in zebrafish embryos, where the compound is added to the water, and the formation of new blood vessels is observed under a microscope. Neuroprotective effects are studied in a rat model of cerebral ischemia/reperfusion.

Metabolism / Metabolites
Astragalus contains two main bioactive isoflavone compounds: astragaloside and astragaloside-7-O-β-D-glucoside. To analyze the metabolites of astragaloside in a 9000 g rat liver supernatant culture system and the metabolites of astragaloside-7-O-β-D-glucoside in rat urine, this study employed high-performance liquid chromatography-diode array detector-electrospray ionization trap time-of-flight mass spectrometry (HPLC-DAD-ESI-IT-TOF-MSn). A total of 24 in vitro metabolites of astragaloside and 33 in vivo metabolites of astragaloside-7-O-β-D-glucoside were identified. In vitro metabolism revealed that monosaccharylation, pentosaccharylation, demethylation, dehydroxylation, dimerization, and trimerization are novel metabolic reactions of astragaloside; in vivo metabolism revealed that hydroxylation and hydrogenation are novel metabolic reactions of astragaloside-7-O-β-D-glucoside. In a 9000 g supernatant incubation system of rat liver, the main metabolic reactions of mangiferin were A-ring monohydroxylation, dimerization (CO coupling), dimerization (CC coupling), and dehydroxylation; in rats, the main phase I metabolic reactions of mangiferin-7-O-β-D-glucoside were deglycosylation, hydroxylation, demethylation, and dehydroxylation. Hydroxylation, dehydroxylation, and demethylation are common metabolic pathways for mangiferin and mangiferin-7-O-β-D-glucoside. Metabolites formed through these reactions, such as 8-hydroxymangiferin (S10, M10), pratercinone (5-hydroxymangiferin, S19, M27), mangiferin (S22, M28), daidzein (M22), 7,3',4'-trihydroxyisoflavone (aglycones of S13, M3, and M8), and equol (aglycones of M19 and M20), have been reported to possess numerous biological activities related to the pharmacological effects of mangiferin and mangiferin-7-O-β-D-glucoside. These findings will contribute to a deeper understanding of the metabolism and active forms of mangiferin and mangiferin-7-O-β-D-glucoside.
High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS(n)) was used, a method with high specificity and sensitivity, to identify the metabolites of mangiferin-7-O-β-D-glucopyranoside in vivo and in vitro in rats. Following oral administration of mangiferin-7-O-β-D-glucopyranoside, the parent compound and 12 metabolites were detected in rat urine. The parent compound and 6 metabolites were detected in rat plasma. In heart, liver, spleen, lung, and kidney samples, 6, 8, 7, 9, and 9 metabolites were identified, respectively, in addition to the parent compound. Three metabolites were found in rat intestinal flora cultures and feces, but the parent drug was not detected, indicating that the glycosidic bonds of the parent compound were broken in the intestine. In rats, the main phase I metabolic pathway of mangiferin-7-O-β-D-glucopyranoside includes deglycosylation, dehydroxylation, and demethylation; the phase II metabolic pathway includes sulfation, methylation, glucuronidation, and glycosylation (possibly). Furthermore, two metabolites commonly found in urine, plasma, and tissues were isolated from rat feces and characterized by nuclear magnetic resonance (NMR). The metabolite mangiferin showed significantly stronger antiviral activity against Coxsackievirus B3 (CVB3) and Human Immunodeficiency Virus (HIV) than mangiferin-7-O-β-D-glucopyranoside.
Pharmacokinetic (PK) properties of calycosin have been studied in various animal models. As an isoflavone, it is absorbed after oral administration but undergoes extensive first-pass metabolism. It is conjugated to glucuronide and sulfate metabolites, which are the predominant forms in the systemic circulation. The compound has a relatively short half-life, and its bioavailability is influenced by its metabolism. Calycosin is soluble in DMSO and is typically stored as a powder at -20°C.

Interactions
Dang Gui Bu Xue Tang (DBT) is a traditional Chinese medicine decoction containing Astragalus membranaceus (AR) and Angelica sinensis (ASR). It has a history of over 800 years in China and has long been used as a health supplement to treat menopausal discomfort in women. Multiple studies have shown that the synergistic effect of Astragalus membranaceus and Angelica sinensis in this decoction enhances the pharmacological efficacy of DBT. This study aimed to investigate the roles of different traditional Chinese medicines in the transport of DBT's active ingredients. The permeability of each component on Caco-2 cell monolayers was determined using a validated RRLC-QQQ-MS/MS method. Astragalus-derived chemicals, including astragaloside IV, gentianin, and gentianin, and Angelica sinensis-derived chemicals, including ferulic acid and ligustilide, were determined by RRLC-QQQ-MS/MS. Pharmacokinetic results showed that in the presence of ASR extract, the membrane permeability of gentianin and gentianin (two major flavonoids in AR) was significantly increased: this induction may be mediated by ASR-derived ferulic acid. Conversely, AR extracts had no effect on chemical permeability. Current findings suggest that components of ASR (such as ferulic acid) can enhance the membrane permeability of AR-derived formononetin and formononetin in cultured Caco-2 cells. This article proposes the possibility of a synergistic effect of traditional Chinese medicine in DBT.
Toxicology (toxicology) data for calycosin are limited, but as a natural phytoestrogen, it is generally considered safe at moderate doses. In vitro, it can promote the proliferation of estrogen receptor-positive cells, which raises a theoretical concern for hormone-sensitive cancers. However, its overall effect appears to be complex, as it has also been shown to induce apoptosis in certain cancer cell lines. Its safety profile requires further evaluation in long-term, high-dose studies.

[1]. Calycosin induces apoptosis in human ovarian cancer SKOV3 cells by activating caspases and Bcl-2 family proteins. Tumour Biol. 2015 Feb 12.

[2]. Calycosin and genistein induce apoptosis by inactivation of HOTAIR/p-Akt signaling pathway in human breast cancer MCF-7 cells. Cell Physiol Biochem. 2015;35(2):722-8.

[3].Calycosin suppresses breast cancer cell growth via ERβ-dependent regulation of IGF-1R, p38 MAPK and PI3K/Akt pathways. PLoS One. 2014 Mar 11;9(3):e91245.

[4]. Calycosin promotes proliferation of estrogen receptor-positive cells via estrogen receptors and ERK1/2 activation in vitro and in vivo. Cancer Lett. 2011 Sep 28;308(2):144-51.

Calycosin belongs to the 7-hydroxy isoflavone class of compounds, specifically, 7-hydroxy isoflavones with a hydroxyl group at the 3' position and a methoxy group at the 4' position. It possesses metabolic and antioxidant activities. Calycosin belongs to both the 7-hydroxy isoflavone and 4'-methoxy isoflavone classes. Functionally, it is related to isoflavones. It is the conjugate acid of Calycosin (1-). Calycosin has been reported to be found in Bowdichia virgilioides, Glycyrrhiza pallidiflora, and several other organisms with relevant data. Mechanism of Action…This study aimed to investigate the therapeutic effect of Calycosin (an active ingredient extracted from Bowdichia virgilioides) on macrophage infiltration of human umbilical vein endothelial cells (HUVECs) induced by advanced glycation end products (AGEs). A Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion. The expression of TGF-β1, ICAM-1, and RAGE proteins was detected by immunocytochemistry; the mRNA expression of TGF-β1, ICAM-1, and RAGE was detected by real-time quantitative PCR. The expression of estrogen receptor α, ICAM-1, and RAGE, as well as the phosphorylation status of ERK1/2 and NF-κB, were observed by immunofluorescence. Mangosteen significantly reduced AGEs-induced macrophage migration and adhesion to HUVECs. Mangosteen pretreatment significantly downregulated the protein and mRNA expression levels of TGF-β1, ICAM-1, and RAGE in HUVECs. Furthermore, mangosteen incubation significantly increased estrogen receptor expression and reversed AGEs-induced phosphorylation and nuclear translocation of ERK1/2 and NF-κB in HUVECs, while the estrogen receptor inhibitor ICI182780 inhibited this effect of mangosteen. These results indicate that gentianin can reduce AGEs-induced macrophage migration and adhesion to endothelial cells and alleviate local inflammation; moreover, this effect is achieved through the estrogen receptor-ERK1/2-NF-κB pathway.
Other information: Calycosin is a natural product derived from Radix Astragali and is used in traditional Chinese medicine. It has been studied for its potential in treating osteoporosis, cardiovascular diseases, and various cancers. Its role in angiogenesis and neuroprotection is also an active area of research. The compound is available as a research reagent for preclinical studies.

C16H12O5

284.2635

284.068

C, 67.60; H, 4.26; O, 28.14

20575-57-9

20575-57-9

5280448

White to off-white solid powder

1.4±0.1 g/cm3

536.8±50.0 °C at 760 mmHg

205.7±23.6 °C

0.0±1.5 mmHg at 25°C

1.669

2.41

2

5

2

21

432

0

O1C([H])=C(C(C2C([H])=C([H])C(=C([H])C1=2)O[H])=O)C1C([H])=C([H])C(=C(C=1[H])O[H])OC([H])([H])[H]

ZZAJQOPSWWVMBI-UHFFFAOYSA-N

InChI=1S/C16H12O5/c1-20-14-5-2-9(6-13(14)18)12-8-21-15-7-10(17)3-4-11(15)16(12)19/h2-8,17-18H,1H3

7-hydroxy-3-(3-hydroxy-4-methoxyphenyl)chromen-4-one

Calycosin

2934.99.03.00

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: ≥ 100 mg/mL (~351.8 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.79 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 25.0 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: ≥ 2.5 mg/mL (8.79 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), 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 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.79 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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 (Please use freshly prepared in vivo formulations for optimal results.)