Topoisomerase I ( IC50 = 5 μM ); HIV-1 ( IC50 = 1.4 μM ); NF-κB
DNA topoisomerase I (IC50 = 5 μM in enzyme activity assay) [1]
NF-κB (as an activator) [5]

Betulinic acid is a eukaryotic topoisomerase I inhibitor that inhibits topoisomerase I-DNA interaction and has an IC50 of 5 μM[1]. MDA-MB-231 cell viability is significantly suppressed by betulinic acid (10, 20, 40, 80, and 160 μM) in a time- and dose-dependent manner following treatment for 24 or 48 hours. MDA-MB-231 cells express less Bcl-2 when exposed to betulinic acid (20, 40 μM). Additionally, betulinic acid alters the morphology of MDA-MB-231 cells at 20 μM and the ultrastructure of MDA-MB-231 cells at 40 μM[2]. With an EC50 of 1.4 μM in acutely infected H9 lymphocytes, betulinic acid exhibits anti-HIV activities[4].

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In eukaryotic DNA topoisomerase I assay, Betulinic acid (ALS357; Lupatic acid) exhibited potent inhibitory activity with an IC50 of 5 μM, targeting the enzyme-DNA cleavage complex formation step. The 3-hydroxy group of the compound was identified as the major functional group responsible for the inhibitory effect [1]
- In human breast cancer MDA-MB-231 cells, Betulinic acid (ALS357; Lupatic acid) induced concentration-dependent apoptosis, characterized by chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Ultrastructural changes included mitochondrial swelling, endoplasmic reticulum dilation, and loss of cell membrane integrity [2]
- In HIV-infected cells, Betulinic acid (ALS357; Lupatic acid) showed anti-HIV activity by inhibiting viral replication, with moderate potency against HIV-1 strains [4]
- In various cancer cell lines (HeLa, MCF-7, A549), Betulinic acid (ALS357; Lupatic acid) activated the NF-κB signaling pathway, leading to upregulation of NF-κB target genes (e.g., IL-6, TNF-α) and enhanced cell survival. The activation was mediated via IκBα phosphorylation and degradation, followed by p65 nuclear translocation [5]

Betulinic acid (10 and 30 mg/kg, p.o.) blocks colon shortening and lowers lipid hydroperoxide and malondialdehyde (MDA) levels in mice with colitis brought on by dextran sulfate sodium (DSS). In mice with DSS-induced colitis, betulinic acid (30 mg/kg, p.o.) restores glutathione (GSH) levels, catalase activity, and superoxide dismutase (SOD) levels to normal. Additionally, betulinic acid (30 mg/kg, p.o.) prevents the rise in inflammatory markers brought on by DSS. Mice exposed to acetic acid (3, 10, 30 mg/kg, p.o.) or mustard oil (MO) do not exhibit writhing responses or visceral nociception.

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In dextran sulfate sodium (DSS)-induced colitis mice, oral administration of Betulinic acid (ALS357; Lupatic acid) at 20 and 40 mg/kg/day for 7 days alleviated colitis symptoms, including reduced body weight loss, colon shortening, and mucosal inflammation. It also attenuated visceral pain by inhibiting the expression of pro-inflammatory cytokines (IL-1β, TNF-α) and nociceptive mediators (TRPV1) in colon tissues [3]

The 25-mer annealed duplex consisting of oligonucleotide-1 and oligonicleotide-2 is subjected to an 8°C incubation period with 25 or 50 units of rat liver topoisomerase I, either in the presence or absence of betulinic acid in the binding buffer. Following incubation, the reaction mixtures are electrophoresed in a 7% non-denaturing polyacrylamide gel in 0.167 × TBE buffer at 4 °C. Ethidium bromide is then used to stain the DNA bands.

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DNA topoisomerase I activity assay: Purified eukaryotic DNA topoisomerase I was incubated with supercoiled pUC19 DNA in reaction buffer at 37°C. Betulinic acid (ALS357; Lupatic acid) was added at serial concentrations (0.1-20 μM), and the mixture was incubated for 45 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 55°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The intensity of relaxed DNA bands was quantified to assess enzyme inhibition. The results confirmed that the compound inhibited topoisomerase I by stabilizing the enzyme-DNA cleavage complex [1]

The assay makes use of CCK-8. After being grown at a density of 2 × 10 3 cells/well in 96-well plates, MDA-MB-231 cells are treated with DMSO vehicle or different concentrations of betulinic acid (5 µM to 160 µM) in 100 µL of medium for the specified durations. Following the treatment phase, 110 µL of medium containing 10 µL of the CCK-8 mixture is added to each well, and the plates are incubated at 37°C for one hour and thirty minutes. With the aid of a microplate reader, the absorbance at 450 nm wavelength is determined[2].

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Breast cancer cell apoptosis assay: MDA-MB-231 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with Betulinic acid (ALS357; Lupatic acid) at concentrations of 10, 20, and 40 μM for 24 and 48 hours. Cells were harvested, fixed with glutaraldehyde, and processed for transmission electron microscopy to observe ultrastructural changes. Apoptotic cells were quantified by DAPI staining and flow cytometry (annexin V-FITC/PI double staining) [2]
- Anti-HIV activity assay: HIV-1-infected T lymphoblastoid cells were cultured in the presence of Betulinic acid (ALS357; Lupatic acid) at 1-50 μM for 72 hours. Viral replication was measured by detecting p24 antigen levels in the culture supernatant using an enzyme-linked immunosorbent assay (ELISA) [4]
- NF-κB activation assay: Cancer cells (HeLa, MCF-7) were treated with Betulinic acid (ALS357; Lupatic acid) at 5-25 μM for 1-6 hours. Cytoplasmic and nuclear proteins were extracted, and western blot analysis was performed to detect IκBα phosphorylation/degradation and p65 nuclear translocation. NF-κB transcriptional activity was measured using a luciferase reporter assay with an NF-κB-responsive promoter [5]
- Topoisomerase I inhibition cell-based assay: HeLa cells were treated with Betulinic acid (ALS357; Lupatic acid) at 2-10 μM for 24 hours. DNA strand breaks were detected by comet assay, and the expression of DNA damage response proteins (γ-H2AX) was analyzed by immunofluorescence [1]

Oral betulinic acid (3, 10, or 30 mg/kg) or vehicle (5% v/v DMSO in peanut oil) is given to female Swiss albino mice. An observer who is blind to the treatments counts the number of writhing responses from each animal for 20 minutes after acetic acid (300 mg/kg) is given intraperitoneally after an hour. An animal exhibits a whining response when it rubs its belly on a table or floor while extending its hind limbs and body[3].

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Colitis and visceral pain model: C57BL/6 mice were randomly divided into control, DSS-induced colitis, and betulinic acid-treated groups (n=8 per group). Colitis was induced by administering 3% DSS in drinking water for 7 days. Betulinic acid (ALS357; Lupatic acid) was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) and administered orally at doses of 20 and 40 mg/kg/day for 7 days, starting from the first day of DSS administration. Body weight was recorded daily, and colon length was measured after sacrifice. Colon tissues were collected for histological analysis, cytokine detection, and TRPV1 expression measurement [3]

[1]. Betulinic acid, a potent inhibitor of eukaryotic topoisomerase I: identification of the inhibitory step, the major functional group responsible and development of more potent derivatives. Med Sci Monit. 2002 Jul;8(7):BR254-65.

[2]. Betulinic acid induces apoptosis and ultrastructural changes in MDA-MB-231 breast cancer cells. Ultrastruct Pathol. 2018 Jan-Feb;42(1):49-54.

[3]. Betulinic acid alleviates dextran sulfate sodium-induced colitis and visceral pain in mice. Naunyn Schmiedebergs Arch Pharmacol. 2017 Dec 26.

[4]. Anti-AIDS agents--XXVII. Synthesis and anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives. Bioorg Med Chem. 1997 Dec;5(12):2133-43.

[5]. Betulinic acid as new activator of NF-kappaB: molecular mechanisms and implications for cancer therapy. Oncogene. 2005 Oct 20;24(46):6945-56.

Betulinic acid is a pentacyclic triterpenoid compound belonging to lupinane, with a double bond at position 20(29) and a 3β-hydroxyl group and a 28-carboxyl substituent. It is found in the bark and other plant parts of various plants, including Syzygium claviflorum. Betulinic acid has anti-HIV, antimalarial, antitumor, and anti-inflammatory properties. It can be used as an EC 5.99.1.3 [DNA topoisomerase (ATP hydrolysis)] inhibitor, anti-HIV drug, antimalarial drug, anti-inflammatory drug, antitumor drug, and plant metabolite. It is a pentacyclic triterpenoid compound and also a hydroxy monocarboxylic acid. It is derived from the hydride of lupinane.
Betulinic acid has been used in clinical trials to study the treatment of dysplastic nevus syndrome.
Betulinic acid has been reported in Paeonia emodi, Bowdichia virgilioides, and other organisms with relevant data. Betulinic acid is a pentacyclic lupinane-type triterpenoid derivative of betulin (isolated from birch bark), possessing anti-inflammatory, anti-HIV, and antitumor activities. Betulinic acid induces apoptosis by inducing changes in mitochondrial membrane potential, generating reactive oxygen species, and opening mitochondrial permeability transition pores, leading to the release of mitochondrial apoptosis factors, activation of caspases, and DNA fragmentation. Although initially thought to have specific cytotoxicity against melanoma cells, it was later found to be cytotoxic to non-melanoma tumor cell types, including neuroectodermal tumor cells and brain tumor cells. Betulin is a lupinane-type triterpenoid derivative, originally isolated from birch (BETULA). It possesses anti-inflammatory, anti-HIV, and antitumor activities. See also: peony root (part); jujube fruit (part).

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strong>Betulic acid (ALS357; Lupaic acid) is a naturally occurring pentacyclic triterpenoid compound isolated from various plants and possesses a variety of biological activities[1][4]
- Mechanism of action: This compound exerts a variety of biological effects, including inhibiting DNA topoisomerase I (stabilizing the enzyme-DNA cleavage complex), inducing apoptosis in cancer cells (through mitochondria and endoplasmic reticulum pathways), anti-HIV replication, and activating the NF-κB signaling pathway[1][2][4][5]
- Structural modification: By modifying the 3-hydroxyl and 28-carboxyl groups, betulic acid (ALS357; Lupaic acid) derivatives with enhanced topoisomerase I inhibitory activity and bioavailability have been developed[1]
- Therapeutic potential: It has potential applications in cancer treatment (breast cancer, etc.), HIV treatment, and management of inflammatory bowel disease[2][3][4][5]
- NF-κB activation paradox: Although betulic acid (ALS357; Lupaic acid) In some cancer cells, NF-κB (a pro-survival pathway) is activated, but in breast cancer cells it mainly plays a pro-apoptotic role, suggesting a cell type-specific mechanism [2][5].

C30H48O3

456.7

456.36

C, 78.90; H, 10.59; O, 10.51

472-15-1

64971

White to off-white solid powder

1.1±0.1 g/cm3

550.0±33.0 °C at 760 mmHg

295-298 °C (dec.)(lit.)

300.5±21.9 °C

0.0±3.4 mmHg at 25°C

1.533

8.94

2

3

2

33

861

10

O([H])[C@@]1([H])C([H])([H])C([H])([H])[C@@]2(C([H])([H])[H])[C@]([H])(C1(C([H])([H])[H])C([H])([H])[H])C([H])([H])C([H])([H])[C@]1(C([H])([H])[H])[C@]2([H])C([H])([H])C([H])([H])[C@]2([H])[C@@]3([H])[C@]([H])(C(=C([H])[H])C([H])([H])[H])C([H])([H])C([H])([H])[C@]3(C(=O)O[H])C([H])([H])C([H])([H])[C@@]12C([H])([H])[H]

QGJZLNKBHJESQX-FZFNOLFKSA-N

InChI=1S/C30H48O3/c1-18(2)19-10-15-30(25(32)33)17-16-28(6)20(24(19)30)8-9-22-27(5)13-12-23(31)26(3,4)21(27)11-14-29(22,28)7/h19-24,31H,1,8-17H2,2-7H3,(H,32,33)/t19-,20+,21-,22+,23-,24+,27-,28+,29+,30-/m0/s1

(1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-hydroxy-5a,5b,8,8,11a-pentamethyl-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

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: 20~35.7 mg/mL (43.8~78.2 mM)
Ethanol: 10 mg/mL (~21.9 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.55 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 4 mg/mL (8.76 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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