Protein prenylation (via geranylgeranyltransferase I, GGTase I) – involved in the promoting effect of Isobavachin on neuronal differentiation. [1]
Mitogen-activated protein kinase (MAPK) pathway components: ERK, p38, JNK – their phosphorylation states are modulated by Isobavachin. [1]

Isobavachin (10⁻⁷ mol/L) significantly increased the population of neuron‑like cells (axons ≥3 times longer than cell bodies) from mouse ES cells compared to solvent control. Neurons expressing β‑tubulin III and astrocytes expressing GFAP were more obvious after IBA treatment. [1]
Nestin (neural progenitor marker) was highly expressed in embryoid bodies (EBs) at d4 and d8+0 with or without IBA treatment, but decreased gradually during differentiation; Oct3/4 (pluripotency marker) disappeared at terminal differentiation. mRNA and protein levels of β‑tubulin III were expressed from EB stage to terminal differentiation; GFAP was upregulated in a time‑dependent manner (d8+5 and d8+10). NEFM (neurofilament marker) was upregulated by Isobavachin. [1]
Co‑treatment with GGTI‑298 (10⁻⁶ mol/L, a GGTase I inhibitor) selectively abolished the IBA‑induced neuronal and astrocyte differentiation, while GGTI‑298 had no effect on RA‑induced differentiation, indicating that the promoting effects of IBA and RA act via different pathways. [1]
In the MAPK pathway, p38 and JNK phosphorylation were down‑regulated, while ERK phosphorylation was up‑regulated after Isobavachin treatment at different neuronal differentiation passages (ES, EB d4, d8+0, d8+5, d8+10). ERK phosphorylation was high in ES cells, low in early EB (d4, d8+0), and increased greatly during late neuronal differentiation (d8+5, d8+10). p38 phosphorylation was detected in ES and EB stages and then down‑regulated on d8+5 and d8+10; little p38 phosphorylation was detected during the entire differentiation course with IBA. JNK phosphorylation was inhibited during neuronal differentiation compared to early stages. [1]

Mouse ES cells (D3 line) were routinely cultured on primary mouse embryonic fibroblasts in DMEM supplemented with 10% FBS, 0.1 mmol/L β‑mercaptoethanol, 1× non‑essential amino acids, and 1×10⁶ U/L LIF. A hanging drop method was used to initiate embryoid body formation. Drops (30 μL) containing approximately 900 ES cells were cultured for 2 days in hanging drops. After EB formation, they were transferred to agar‑coated Petri dishes and cultured in suspension for another 2 days. On day 4, Isobavachin (10⁻⁷ mol/L) was added, and EBs were cultured in suspension for another 4 days. GGTI‑298 (10⁻⁶ mol/L) was added into the medium with IBA during differentiation when indicated. On d8+0, EBs were plated on poly‑D‑lysine‑coated culture plates in differentiation medium (neural basal medium + 1% B27 supplement) with IBA to induce neuronal differentiation. 10⁻⁷ mol/L retinoic acid was used as positive control, and 0.1% DMSO as solvent control. [1]
Morphological evaluation: inverted phase‑contrast microscope; cells with axons ≥3 times longer than cell bodies were considered neuron‑like cells. [1]
Immunocytochemistry: cultures were fixed in ice‑cold methanol with 0.3% hydrogen peroxide, blocked with 10% FBS in PBS, incubated with primary antibodies (β‑tubulin III 1:50, GFAP 1:50) overnight at 4°C, then with fluorescent secondary antibodies (1:200) for 2 h, and nuclei stained with DAPI (2 μg/mL). [1]
Western blot: cells lysed in extraction buffer (Tris‑HCl pH7.5, 20 mmol/L NaCl 150 mmol/L, EDTA 1 mmol/L, Triton X‑100 1%, sodium deoxycholate 0.5%, PMSF 1 mmol/L, leupeptin 10 μg/mL, aprotinin 30 μg/mL). Protein concentration quantified by modified Lowry assay. 40 μg protein per lane were separated by SDS‑PAGE, transferred to nitrocellulose membrane, blocked with 5% nonfat milk in TBS/T, incubated with primary antibodies (NEFM 1:500, β‑tubulin III 1:1000, GFAP 1:1000, p38 1:500, phos‑p38 1:500, ERK 1:1000, phos‑ERK 1:500, JNK 1:500, phos‑JNK 1:500, GAPDH 1:10000) overnight at 4°C, then with HRP‑conjugated secondary antibodies (1:5000) for 1 h, and detected by chemiluminescence. [1]
Semi‑quantitative RT‑PCR: total RNA extracted with Trizol, first‑strand cDNA synthesized with oligo(dT)6 primer and M‑MuLV reverse transcriptase. PCR performed with specific primers for GAPDH, β‑tubulin III, GFAP, Oct3/4, Nestin (conditions: 35 cycles, annealing temperatures 59‑69°C, Mg²⁺ 3 mmol/L). Products analyzed on 1.5% agarose gel with ethidium bromide staining. [1]

[1]. Promoting effects of isobavachin on neurogenesis of mouse embryonic stem cells were associated with protein prenylation. Acta Pharmacol Sin. 2011 Apr;32(4):425-32.

[2]. Antioxidant properties of extracts and compounds from Psoralea morisiana. European Journal of Lipid Science and Technology. 2005.

Isopavonicaine belongs to the flavanone class of compounds. It has been reported to be found in licorice, ursaria, and other organisms with relevant data.

---

Isobavachin can facilitate mouse ES cell differentiation into multiple neuronal cell subtypes (neurons and astrocytes). The mechanism involves protein prenylation, subsequently leading to phos‑ERK activation and phos‑p38 off pathway. The prenyl group at position 8 of ring A of IBA is capable of protein prenylation during neurogenesis of mouse ES cells. The study suggests that IBA and its derivatives are promising candidates for developing methods to derive more neuronal or neural progenitor cells for cell replacement therapy. [1]

C20H20O4

324.3704

324.136

31524-62-6

193679

Off-white to light yellow solid

1.2±0.1 g/cm3

558.3±50.0 °C at 760 mmHg

187-188℃

202.1±23.6 °C

0.0±1.6 mmHg at 25°C

1.622

4.85

2

4

3

24

474

1

CC(=CCC1=C2C(=CC=C1O)C(=O)C[C@@H](C3=CC=C(C=C3)O)O2)C

KYFBXCHUXFKMGQ-IBGZPJMESA-N

InChI=1S/C20H20O4/c1-12(2)3-8-15-17(22)10-9-16-18(23)11-19(24-20(15)16)13-4-6-14(21)7-5-13/h3-7,9-10,19,21-22H,8,11H2,1-2H3/t19-/m0/s1

(2S)-7-hydroxy-2-(4-hydroxyphenyl)-8-(3-methylbut-2-enyl)-2,3-dihydrochromen-4-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)

DMSO : ~100 mg/mL (~308.29 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.71 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.71 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (7.71 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.

---

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