- Intrinsic apoptosis pathway (mitochondrial pathway): downregulates Bcl-2 and survivin, upregulates Bax, cytochrome C release, and activates caspase-9 and caspase-3. [1]
- ABCB1 (P-glycoprotein): inhibits DOX-induced P-gp upregulation, increases intra-tumor DOX accumulation, and shows synergistic antitumor activity with DOX. [3]

- 23-HBA inhibited the proliferation of K562 cells with an IC₅₀ of 18.7 ± 1.3 μM (48 h, MTT assay). It also showed cytotoxicity against B16 (39.9 ± 2.7 μM), HeLa (78.5 ± 6.3 μM), and HUVEC (94.8 ± 7.6 μM). [1]
- 23-HBA induced S phase arrest in K562 cells in a concentration-dependent manner (20–80 μM, 24 h). The percentage of cells in S phase increased from 26.35% (control) to 31.6%, 32.81%, and 52.34% at 20, 40, and 80 μM, respectively. [1]
- 23-HBA induced apoptosis in K562 cells as shown by Hoechst 33342/PI staining and Annexin V-FITC/PI flow cytometry. Total apoptotic cells increased from 3.8% (control) to 8.3%, 11.4%, and 18.2% at 20, 40, and 80 μM, respectively. [1]
- 23-HBA (20–80 μM, 24 h) disrupted mitochondrial membrane potential (MMP) in K562 cells, as shown by JC-1 staining. The red/green fluorescence ratio decreased from 12.5 (control) to 0.38 at 80 μM (p < 0.01). [1]
- Western blot analysis showed that 23-HBA (20–80 μM, 24 h) increased Bax expression, decreased Bcl-2 and survivin expression, increased cytochrome C release into cytosol, and activated cleaved caspase-9 and cleaved caspase-3 in K562 cells. [1]
- 23-HBA alone (0.2–20 μM) had minimal cytotoxicity but significantly enhanced DOX cytotoxicity in NCI-H460, SGC7901, HepG2, and sarcoma 180 cells in a concentration-dependent manner. [3]
- Median-effect analysis showed that 23-HBA combined with DOX produced synergistic effects (CI < 1) in all four cell lines at various effect levels (ED50–ED95). DRI values for DOX ranged from 1.48 to 29.74. [3]
- 23-HBA (0–80 μM, 24 h) did not affect ABCB1 mRNA or protein expression in HepG2/ADM cells, nor did it alter ABCB1 membrane localization. [2]
- 23-HBA (0.625–5 μM) increased intracellular accumulation of DOX and rhodamine123 in ABCB1-overexpressing HepG2/ADM and MCF-7/ADR cells in a dose-dependent manner, as shown by confocal microscopy and flow cytometry. It also inhibited rhodamine123 efflux. [2]
- 23-HBA at 5 μM did not reverse ABCC1-mediated resistance to vincristine or vinblastine in KB-CV60 and KB-MRP1 cells. [2]
- 23-HBA (0.625–5 μM) did not affect the viability of drug-sensitive parental cells (HepG2, MCF-7, KB-3-1) at the concentrations used. [2]
- BBA (a synthetic derivative of 23-HBA) at 5 μM significantly reversed ABCB1-mediated resistance to vincristine (fold reversal 132.29 in HepG2/ADM, 151.04 in MCF-7/ADR, 30.64 in HEK293/ABCB1), vinblastine, and paclitaxel. BBA was not a transported substrate of ABCB1. [2]

- In sarcoma 180 tumor-bearing ICR mice, co-administration of 23-HBA (20 or 100 mg/kg, i.g., daily for 7 days) with DOX (3 mg/kg, i.p., every other day for 4 times) significantly enhanced antitumor activity compared to DOX alone. Tumor weight inhibition increased from 43% (DOX alone) to 52% and 59% with 20 and 100 mg/kg 23-HBA, respectively (p < 0.05). 23-HBA alone had no significant effect. [3]
- 23-HBA (100 mg/kg, i.g.) given 1 h before DOX (5 mg/kg, i.p.) significantly increased DOX concentration in tumor tissue at 1 h post-dose (p < 0.05) but did not alter plasma pharmacokinetics of DOX. [3]
- 23-HBA (20 or 100 mg/kg) dose-dependently increased intra-tumor DOX concentration and inhibited DOX-induced upregulation of P-gp expression in sarcoma 180 tumors (Western blot). [3]
- In KB-C2 xenograft nude mice, the BBA derivative (15 mg/kg, po, q3d × 6) combined with paclitaxel (18 mg/kg, ip, q3d × 6) significantly reduced tumor volume and weight compared to paclitaxel alone (p < 0.05). No significant body weight loss was observed in the combination group. [2]
- In the KB-C2 xenograft model, BBA (15 mg/kg, po) alone had no effect on tumor growth. [2]

- ABCB1 ATPase activity assay: Membrane vesicles from ABCB1-expressing cells were incubated with increasing concentrations of BBA (0–30 μM) in the presence or absence of verapamil (5 μM). The inorganic phosphate released was measured. BBA stimulated basal ATPase activity at lower concentrations (peak at ~2.5 μM, 1.5-fold) and inhibited activity at higher concentrations. BBA inhibited verapamil-stimulated ATPase activity by up to 60% at 30 μM. [2]
- [¹²⁵I]IAAP photoaffinity labeling: Membrane vesicles were incubated with BBA (0–30 μM) and [¹²⁵I]IAAP, exposed to UV light, separated by SDS-PAGE, and visualized by autoradiography. BBA inhibited IAAP incorporation into ABCB1 in a concentration-dependent manner, with ~50% inhibition at 30 μM. [2]

- MTT assay for cytotoxicity: Cells (5 × 10³/well) were seeded in 96-well plates, treated with compounds for 48 h, incubated with MTT (0.5 mg/mL) for 4 h, dissolved in DMSO, and absorbance measured at 490 nm. IC₅₀ values were calculated from concentration-response curves. [1][2][3]
- Cell cycle analysis: K562 cells (1 × 10⁶) were treated with HBA (20–80 μM) for 24 h, fixed in 70% ethanol, stained with PI/RNase, and analyzed by flow cytometry. [1]
- Apoptosis detection (Annexin V-FITC/PI): K562 cells were treated with HBA (20–80 μM) for 24 h, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. [1]
- MMP measurement (JC-1): K562 cells were treated with HBA (20–80 μM) for 24 h, stained with JC-1 for 20 min, and analyzed by fluorescence spectrophotometry and fluorescence microscopy. [1]
- Western blotting: Cells were lysed in RIPA buffer with protease inhibitors, proteins separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with specific antibodies, and detected by ECL. [1][2][3]
- Intracellular DOX/rhodamine123 accumulation: ABCB1-overexpressing cells were treated with BBA (0.625–5 μM) for 2 h, then incubated with DOX (10 μM, 2 h) or rhodamine123 (5 μM, 1 h), and analyzed by confocal microscopy and flow cytometry. [2]
- Rhodamine123 efflux assay: Cells were loaded with rhodamine123 (5 μM) ± BBA or verapamil for 2 h, washed, and incubated in fresh medium ± compounds. Fluorescence was measured at 0, 10, 20, 40 min. [2]

- Sarcoma 180 xenograft model: Male ICR mice were inoculated subcutaneously with sarcoma 180 cells (0.2 mL of cell suspension). After 24 h, mice were randomized into 6 groups (n=10). DOX (3 mg/kg, i.p.) was administered on alternate days (qod × 4). 23-HBA (20 or 100 mg/kg, i.g.) was given daily 1 h before DOX. On day 8, tumors were excised and weighed. [3]
- KB-C2 xenograft model: Nude mice were implanted subcutaneously with KB-C2 cells (1 × 10⁷). When tumors reached ~0.5 cm in diameter, mice were randomized into 4 groups (n=6). Paclitaxel (18 mg/kg, i.p.) was given q3d × 6. BBA (15 mg/kg, po) was given q3d × 6, 2 h before paclitaxel. Tumor volumes and body weights were recorded every 3 days. [2]
- Pharmacokinetic study: Tumor-bearing mice received 23-HBA (100 mg/kg, i.g.) or vehicle, followed 0.5 h later by DOX (5 mg/kg, i.p.). Blood and tumor tissues were collected at various times (0.083–24 h). DOX concentrations were measured by HPLC. [3]

- In tumor-bearing mice, co-administration of 23-HBA (100 mg/kg, i.g.) 0.5 h before DOX (5 mg/kg, i.p.) did not significantly alter the plasma pharmacokinetics of DOX. However, it significantly increased DOX concentration in tumor tissue at 1 h post-dose (p < 0.05). [3]

- In sarcoma 180 tumor-bearing mice, 23-HBA alone (20 or 100 mg/kg, i.g., daily for 7 days) showed no significant effect on tumor weight, body weight, feeding behavior, or motor activity, indicating low toxicity. [3]
- Histopathological examination of heart sections showed that DOX treatment caused hemorrhagic spots, but combination with 23-HBA (20 or 100 mg/kg) appeared to alleviate DOX-induced cardiotoxicity (normal morphology). [3]
- In KB-C2 xenograft mice, combination treatment with BBA (15 mg/kg, po) and paclitaxel (18 mg/kg, ip) did not cause significant body weight loss, suggesting no additional toxicity. [2]
- In acute toxicity tests, BBA showed no significant toxicity at 5 g/kg/day in single dose (mentioned in discussion). [2]

[1]. Cytotoxicity of the compounds isolated from Pulsatilla chinensis saponins and apoptosis induced by 23-hydroxybetulinic acid. Pharm Biol. 2015 Jan;53(1):1-9.

[2]. BBA, a derivative of 23-hydroxybetulinic acid, potently reverses ABCB1-mediated drug resistance in vitro and in vivo. Mol Pharm. 2012 Nov 5;9(11):3147-59.

[3]. 23-Hydroxybetulinic acid from Pulsatilla chinensis (Bunge) Regel synergizes the antitumor activities of doxorubicin in vitro and in vivo. J Ethnopharmacol. 2010 Apr 21;128(3):615-22.

23-Hydroxybetulic acid is a triterpenoid compound, a metabolite derived from the hydrogenation of lupinane. It has been reported to be present in sage, peony, and other organisms with relevant data.

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- 23-HBA (23-hydroxybetulinic acid) is a pentacyclic triterpene and the major active constituent of Pulsatilla chinensis (Bunge) Regel, a traditional Chinese medicinal herb used as an adjuvant in cancer treatment. [1][3]
- Glycosylation at C3 and C28 of 23-HBA significantly reduces its cytotoxicity. Pulchinenoside A (PA, glycosylated at C3) had an IC₅₀ of 138.2 μM in K562 cells, while anemoside B4 (AB4, glycosylated at both C3 and C28) had IC₅₀ > 320 μM. [1]
- BBA (23-O-(1,4′-bipiperidine-1-carbonyl)betulinic acid) is a synthetic derivative of 23-HBA that exhibits potent ABCB1-mediated MDR reversal activity. BBA is not a transported substrate of ABCB1 and has persistent reversal activity. [2]
- 23-HBA synergizes with DOX by increasing intra-tumor DOX accumulation and inhibiting DOX-induced P-gp upregulation, without altering DOX plasma pharmacokinetics. [3]

C30H48O4

472.70

472.355

85999-40-2

21672692

White to off-white solid powder

1.1±0.1 g/cm3

585.8±25.0 °C at 760 mmHg

305 °C

322.1±19.7 °C

0.0±3.7 mmHg at 25°C

1.543

7.29

3

4

3

34

883

11

CC(=C)[C@@H]1CC[C@]2([C@H]1[C@H]3CC[C@@H]4[C@]5(CC[C@@H]([C@@]([C@@H]5CC[C@]4([C@@]3(CC2)C)C)(C)CO)O)C)C(=O)O

HXWLKAXCQLXHML-WGOZWDAUSA-N

InChI=1S/C30H48O4/c1-18(2)19-9-14-30(25(33)34)16-15-28(5)20(24(19)30)7-8-22-26(3)12-11-23(32)27(4,17-31)21(26)10-13-29(22,28)6/h19-24,31-32H,1,7-17H2,2-6H3,(H,33,34)/t19-,20+,21+,22+,23-,24+,26-,27-,28+,29+,30-/m0/s1

(1R,3aS,5aR,5bR,7aR,8R,9S,11aR,11bR,13aR,13bR)-9-hydroxy-8-(hydroxymethyl)-5a,5b,8,11a-tetramethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysene-3a-carboxylic acid

23-hydroxybetulinic acid; 23-HBA; RefChem:910332; 85999-40-2; Anemosapogenin; 23-Hydroxybetulinicacid;

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 (211.55 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.29 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.)