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Bullatine A is a naturally occurring diterpenoid alkaloid of the genus Aconitum with anti-rheumatic, anti-inflammatory and anti-nociceptive effects, and thus has the potential to be used for the treatment of neurodegenerative diseases such as arthritis.
| Targets |
P2X7 receptor – selectively antagonizes the ATP-induced up-regulation of P2X7 receptor mRNA. [1]
Bullatine A stimulates spinal microglial dynorphin A expression, which then acts on kappa-opioid receptors to produce anti-hypersensitivity. It also antagonizes P2X7 receptors (EC50 for dynorphin A expression in microglia: 3.2 µM). |
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| ln Vitro |
Bullatine A (1–50 µM) dose-dependently inhibits ATP (500 µM)-induced cytotoxicity in BV-2 microglial cells after 24 hours of incubation, as assessed by MTT assay. [1]
Bullatine A (1–50 µM) up-regulates the ratio of Bcl-2/Bax mRNA in ATP-stimulated BV-2 cells, indicating an anti-apoptotic effect. The effect was most pronounced at 10 µM. [1] Bullatine A (1–50 µM) significantly down-regulates the mRNA levels of pro-inflammatory cytokines IL-6, IL-1β, and inducible nitric oxide synthase (iNOS) in ATP-stimulated BV-2 cells in a dose-dependent manner. [1] Bullatine A (1–50 µM) dose-dependently inhibits ATP-induced production of nitric oxide (NO) and interleukin-6 (IL-6) in BV-2 microglial cells after 24 hours. [1] Bullatine A (1–50 µM) selectively inhibits ATP-induced up-regulation of P2X7 receptor mRNA, but has no obvious effect on P2X4 receptor mRNA levels in BV-2 cells. [1] Bullatine A stimulates prodynorphin gene expression in cultured primary microglia in a concentration-dependent manner (EC50 = 3.2 µM), but not in astrocytes or neurons. It does not inhibit LPS-induced pro-inflammatory cytokine (TNF-α, IL-1β, IL-6) expression in microglia. [2] |
| ln Vivo |
Subcutaneous injection of Bullatine A (0.3–30 mg/kg) dose-dependently attenuates mechanical allodynia and thermal hyperalgesia in rat models of neuropathic pain (ED50 = 1.9 mg/kg for mechanical allodynia, 0.7 mg/kg for thermal hyperalgesia), inflammatory pain (ED50 = 1.4 mg/kg for mechanical allodynia, 0.6 mg/kg for thermal hyperalgesia), diabetic neuropathic pain (ED50 = 1.2 mg/kg), and bone cancer pain (ED50 = 0.9 mg/kg). Intrathecal injection also shows dose-dependent anti-allodynic effects (ED50 = 1.1 µg). The anti-nociceptive effects are blocked by minocycline, dynorphin A antiserum, and kappa-opioid receptor antagonist GNTI. [2]
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| Cell Assay |
Cell viability assay (MTT): BV-2 microglial cells were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humidified incubator with 5% CO2. Cells were treated with ATP (25–1500 µM) for 1, 24, or 48 hours to determine the cytotoxic concentration. For inhibitor testing, cells were pre-treated or co-treated with Bullatine A (1–50 µM) or the non-selective P2X antagonist TNP-ATP (10–100 nM) for 24 hours prior to or concurrently with ATP (500 µM) exposure. After treatment, MTT reagent was added, and the formazan crystals formed were dissolved. Absorbance was measured using a microplate reader at a specific wavelength (not specified). Cell viability was expressed as a percentage of the control (untreated) group. [1]
Real-time reverse transcription polymerase chain reaction (RT-PCR): Total RNA was isolated from treated BV-2 cells using a Trizol reagent. RNA was reverse transcribed to cDNA using a commercial reverse transcription kit. Quantitative real-time PCR was performed using SYBR Green-based chemistry on a real-time PCR system. Primer sequences for target genes (P2X4, P2X7, IL-6, IL-1β, iNOS, Bcl-2, Bax) and the reference gene (β-actin) were provided. Relative gene expression was calculated using the 2^(-ΔΔCt) method and normalized to β-actin. [1] Measurement of nitric oxide (NO) production: The supernatant of cultured BV-2 cells was collected after treatment. NO production was measured indirectly by detecting nitrite concentration using a Griess reagent system kit according to the manufacturer's instructions. [1] Measurement of interleukin-6 (IL-6) production: The supernatant of cultured BV-2 cells was collected. IL-6 concentration was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. [1] Primary microglial cells were isolated from neonatal rat cortex and cultured in DMEM with fetal bovine serum. Cells were treated with Bullatine A (1–100 µM) for 6 h, and prodynorphin mRNA expression was measured by real-time PCR. Minocycline (60 µM) was used as a microglial inhibitor. Cell viability was assessed by MTT assay. [2] |
| Animal Protocol |
Neuropathic pain was induced by L5/L6 spinal nerve ligation in adult male rats. Inflammatory pain was induced by CFA injection into the tibiotarsal joint. Diabetic neuropathic pain was induced by streptozotocin (40 mg/kg, i.v.). Bone cancer pain was induced by injection of Walker 256 carcinoma cells into the tibia. Bullatine A was dissolved in 0.9% saline and administered subcutaneously (0.3–30 mg/kg) or intrathecally (0.3–30 µg). Mechanical allodynia and thermal hyperalgesia were assessed using electronic von Frey hairs and plantar test apparatus. [2]
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| Toxicity/Toxicokinetics |
MTT assays showed that Bullatine A (1–50 µM) had no significant cytotoxicity to BV-2 microglia 24 hours after exposure. [1]
The authors noted that Bullatine A was “very low toxicity and safer than other alkaloids isolated from Aconitum species.” However, [1] Bullatine A was significantly less toxic than other diterpenoid alkaloids such as aconitine (oral LD50 in mice: 754 mg/kg for Bullatine A and 1.8 mg/kg for aconitine). No significant sedation or exercise side effects were observed during the study. [2] |
| References |
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| Additional Infomation |
Bullatine A is a diterpenoid alkaloid isolated from Aconiti brachypodii Radix, a plant in the Ranunculaceae family. [1] It has traditionally recognized antirheumatic, anti-inflammatory, and analgesic effects. [1] This study proposes that bullatine A attenuates ATP-induced microglia death/apoptosis and inflammatory responses by selectively antagonizing the P2X7 receptor and regulating the expression of downstream pro-inflammatory genes. [1] The authors believe that bullatine A may be a potential candidate drug for treating neuropathic pain, inflammation, and neurodegenerative diseases associated with microglia activation due to its P2X7 antagonistic activity. [1] Bullatine A is a C20 diterpenoid alkaloid extracted from the root of Aconiti brachypodii and has traditionally been used to treat chronic pain and arthritis. It specifically alleviates pain hypersensitivity in normal rats without affecting their acute nociceptive response. Its anti-allergic effect is mediated by the expression of dynorphin A in spinal microglia and the activation of κ-opioid receptors. [2]
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| Molecular Formula |
C22H33NO2
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|---|---|
| Molecular Weight |
343.5029
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| Exact Mass |
343.251
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| CAS # |
1354-84-3
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| PubChem CID |
71300866
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
488.2±45.0 °C at 760 mmHg
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| Flash Point |
242.3±27.4 °C
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| Vapour Pressure |
0.0±2.8 mmHg at 25°C
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| Index of Refraction |
1.620
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| LogP |
3.07
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
25
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| Complexity |
662
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| Defined Atom Stereocenter Count |
5
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| SMILES |
CCN1C[C@@]2(CCCC34[C@@H]2CC(C31)C56[C@@H]4[C@H](C(CC5)C(=C)[C@H]6O)O)C
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| InChi Key |
OVXLNQAYPUEDSI-LVIQKLEBSA-N
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| InChi Code |
InChI=1S/C22H33NO2/c1-4-23-11-20(3)7-5-8-22-15(20)10-14(18(22)23)21-9-6-13(12(2)19(21)25)16(24)17(21)22/h13-19,24-25H,2,4-11H2,1,3H3/t13?,14?,15-,16+,17+,18?,19-,20+,21?,22?/m1/s1
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| Chemical Name |
(5R,11R,14S,15R,16R)-7-ethyl-5-methyl-12-methylidene-7-azahexacyclo[7.6.2.210,13.01,8.05,16.010,15]nonadecane-11,14-diol
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~12.5 mg/mL (~36.39 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9112 mL | 14.5560 mL | 29.1121 mL | |
| 5 mM | 0.5822 mL | 2.9112 mL | 5.8224 mL | |
| 10 mM | 0.2911 mL | 1.4556 mL | 2.9112 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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