Synonym name: Arenobufogenin
Catalogue No.: BP0190
Cas No.: 464-74-4
Formula: C₂₄H₃₂O₆
Mol Weight: 416.514
Botanical Source: secretion of Bufo arenarum

Purity: 95%~99%
Analysis Method: HPLC-DAD or/and HPLC-ELSD
Identification Method: Mass, NMR
Packing: Brown vial or HDPE plastic bottle
Can be supplied from milligrams to grams.

For Reference Standard and R&D, Not for Human Use Directly.
Inquire for bulk scale.

Description:

Arenobufagin is a potent Na + /K + pump inhibitor, and is also a specific inhibitor of VEGF-mediated angiogenesis. Arenobufagin has antineoplastic effect that involves cross talk between apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathway.

References:

Carcinogenesis. 2013 Jun;34(6):1331-42.    

Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway.    

Hepatocellular carcinoma (HCC) is a deadly form of cancer without effective chemotherapy so far. Currently, only sorafenib, a multitargeted tyrosine kinase inhibitor, slightly improves survival in HCC patients. 

METHODS AND RESULTS:

In searching for natural anti-HCC components from toad venom, which is frequently used in the treatment of liver cancer in traditional Chinese medicine, we discovered that Arenobufagin, a bufadienolide from toad venom, had potent antineoplastic activity against HCC HepG2 cells as well as corresponding multidrug-resistant HepG2/ADM cells. We found that Arenobufagin induced mitochondria-mediated apoptosis in HCC cells, with decreasing mitochondrial potential, as well as increasing Bax/Bcl-2 expression ratio, Bax translocation from cytosol to mitochondria. Arenobufagin also induced autophagy in HepG2/ADM cells. Autophagy-specific inhibitors (3-methyladenine, chloroquine and bafilomycin A1) or Beclin1 and Atg 5 small interfering RNAs (siRNAs) enhanced Arenobufagin-induced apoptosis, indicating that Arenobufagin-mediated autophagy may protect HepG2/ADM cells from undergoing apoptotic cell death. In addition, we observed the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway by Arenobufagin. Interestingly, inhibition of mTOR by rapamycin or siRNA duplexes augmented Arenobufagin-induced apoptosis and autophagy. Finally, Arenobufagin inhibited the growth of HepG2/ADM xenograft tumors, which were associated with poly (ADP-ribose) polymerase cleavage, light chain 3-II activation and mTOR inhibition. 

CONCLUSIONS:

In summary, we first demonstrated the antineoplastic effect of Arenobufagin on HCC cells both in vitro and in vivo. We elucidated the underlying antineoplastic mechanisms of Arenobufagin that involve cross talk between apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathway. This study may provide a rationale for future clinical application using Arenobufagin as a chemotherapeutic agent for HCC.    

J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Nov 15;939:86-91.    

Quantitative determination of arenobufagin in rat plasma by ultra fast liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.   

A rapid, sensitive, and selective ultra fast liquid chromatography-tandem mass spectrometry method was developed for quantitative determination of Arenobufagin in rat plasma. 

METHODS AND RESULTS:

Sample pretreatment involved a one-step protein precipitation with methanol using 0.1mL rat plasma. The separation was carried out on a Shim-pack XR-ODS II (75mm×2.0mm, i.d. 2.1μm) column with gradient elution at a flow rate of 0.30mLmin(-1). The mobile phase was acetonitrile and 0.1% formic acid in water. A post-column switching valve was applied to reduce the matrix effect. The detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode after electrospray ionization. Linear calibration curves for Arenobufagin were obtained over the concentration range 1.056-1056ngmL(-1), with a lower limit of quantification of 1.056ngmL(-1). The intra-day and inter-day precision values were lower than 15% and the accuracy ranged from 5.4% to 9.8% at all quality control levels. 

CONCLUSIONS:

The method was successfully applied to the determination and pharmacokinetic study of Arenobufagin in rat plasma following intraperitoneal administration.    

HPLC of Arenobufagin

Storage conditions：Short-term storage at 2~8℃, long-term storage at -20 ~ -80℃

Introduction/Overview

Arenobufagin (CAS No.: 464-74-4) is a typical natural product of the toad dienolidone family, belonging to the steroid lactone family. Its structure is closely related to toad lactone and exhibits significant biological activity, especially showing great research value in tumor treatment. In recent years, with the deepening development of natural product pharmacology, Shachan Dujing has become one of the hotspots in anti-tumor drug development due to its unique pharmacological mechanism and multi-target regulatory capabilities. This paper systematically reviews the latest research progress on the chemical structure and physicochemical properties, sources and extraction methods, pharmacological activity and mechanism of action, druggability evaluation, and clinical application prospects of Shachan Venom Essence, aiming to provide a theoretical basis and reference for further research and development of this compound.

Chemical structure and physicochemical properties

Shachan Venom Essence is a typical steroid lactone with a molecular formula of C24H34O6 and a molecular weight of 416.51. Its structural core is a typical tetracyclic steroid backbone, containing an lactone ring and multiple hydroxyl groups, giving it high stereoselectivity and bioactivity. Its LogP value is 1.76, indicating moderate lipid solubility and facilitating membrane penetration. The topological pole surface area (TPSA) is 107.28 Ų, and the number of hydrogen bond acceptors is 6, indicating strong hydrophilicity and binding potential in intermolecular interactions. Shachan venom is difficult to cross the blood-brain barrier (BBB), which to some extent reduces the risk of central nervous system toxicity, but also limits its application in central nervous system diseases.

It is worth noting that Shachan Dujing carries cardiotoxicity risks, manifesting as hERG channel inhibitory activity, indicating that it should focus on cardiac safety assessment during drug development. In addition, hepatotoxicity and genotoxicity (Ames test results) are still unclear and require further systematic research.

Plant Origins and Extraction Methods

The sand toad venom mainly comes from the secretions of the toad glands (Bufo spp.), especially in the skin venom of species such as the Chinese toad (Bufo gargarizans) and the black-spotted toad (Bufo melanostictus). Toad venom, as an important component of the traditional Chinese medicinal material "toad venom," has a long history and broad medicinal value.

The extraction method usually includes the following steps: first, collect fresh toad venom, dry it, and extract it with organic solvents (such as methanol, ethanol); Subsequently, liquid-liquid partitioning, column chromatography (silica gel, inverted C18 column) and other techniques were used for separation and purification; Finally, purity and structure are confirmed using modern analytical methods such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS). In recent years, the application of ultrasound-assisted extraction and microwave-assisted extraction technologies has improved extraction efficiency and purity, reduced production costs, and provided technical support for the large-scale preparation of Shachan poison essence.

Pharmacological activity research

Antitumor activity

As a natural toad dienolactone, Shachan Toad Toxic Essence exhibits significant antitumor activity. Numerous in vitro cell experiments and in vivo animal model studies have shown that Shachan toxin has inhibitory effects on various tumor cells, especially hepatocellular carcinoma (HepG2) and its multidrug-resistant strains (HepG2/ADM), which exhibit high sensitivity. Its antitumor effects include:

1. Induction of apoptosis: Shachan toxin activates endogenous apoptosis pathways to promote programmed tumor cell death, manifested as loss of mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and upregulation of apoptosis-related proteins (such as Caspase-3, Bax).

2. Regulating autophagy: Shachan venom can induce autophagy in tumor cells, regulate the expression of autophagy-related proteins (LC3-II, Beclin-1), promote the clearance of metabolic waste within cells, and enhance cell mortality.

3. Inhibition of cell proliferation and migration: By blocking key cell cycle proteins, it suppresses tumor cell proliferation and reduces cell migration and invasion capabilities.

Anti-angiogenic activity

Angiogenesis is a key process for tumor growth and metastasis. As a VEGF-mediated angiogenesis-specific inhibitor, Shachan Toxin Essence can significantly inhibit the proliferation, migration, and lumen formation of vascular endothelial cells. Its mechanism of action mainly involves inhibiting the VEGFR-2 signaling pathway, blocking the downstream PI3K/Akt/mTOR pathway, reducing the expression of angiogenesis-related factors, restricting angiogenesis in the tumor microenvironment, and inhibiting tumor growth.

Other pharmacological activities

Although Shachan Toxin is mainly known for its antitumor activity, it has potential regulatory effects on hyperglycemia-related targets (such as EHMT2, AMPK, SGLT2, etc.), suggesting its potential for application in metabolic diseases. However, current related research is relatively limited and requires further in-depth exploration.

Mechanism of action and molecular targets

The pharmacological mechanism of Shachan Toxin Essence is complex, involving multiple signaling pathways and molecular targets, mainly including:

1. Na+/K+ ATPase inhibition
   Shachan Venom is a potent Na+/K+ pump inhibitor that can bind to the α subunit of Na+/K+ ATPase, inhibiting its activity, leading to increased intracellular sodium concentration, which in turn affects calcium homeostasis and induces apoptosis and necrosis. This mechanism is an important basis for its cytotoxicity and cardiotoxicity.

2. PI3K/Akt/mTOR signaling pathway suppression
   This pathway plays a central regulatory role in cell growth, proliferation, autophagy, and apoptosis. Shachan venom essence inhibits PI3K kinase activity, blocks phosphorylation of Akt and mTOR, regulates downstream effector molecules, promotes tumor cell apoptosis and autophagy, and inhibits tumor growth.

3. VEGFR-2 signaling pathway is blocked
   Shachan toxin specifically inhibits the activation of vascular endothelial growth factor receptor 2 (VEGFR-2), blocks its downstream signal transduction, inhibits angiogenesis, reduces tumor blood supply, and limits tumor metastasis.

4. Multi-target regulation
   In addition to the main targets mentioned above, Shachan Toxin may also affect various proteins and enzymes related to metabolism, inflammation, and cell cycle, such as EHMT2, UBP2, PAI1, AMPK, SGLT2, etc., demonstrating its multi-target and multi-pathway pharmacological properties.

Druggability evaluation and pharmacokinetics

The drug-making evaluation of Shachan Poison Essence shows certain advantages and challenges:

• Physicochemical properties of the drug: moderate molecular weight, suitable LogP value for cell membrane penetration, reasonable TPSA and hydrogen bond receptor counts, and good bioavailability potential.
• Poor blood-brain barrier permeability: This limits its application in central nervous system diseases but reduces the risk of neurotoxicity.
• Toxicological risk: Cardiotoxicity (hERG channel inhibition) is a major safety bottleneck and requires special attention in drug design and clinical trials. Hepatotoxicity and genotoxicity remain unclear and require further systematic evaluation.
• Pharmacokinetic characteristics: Currently, research on the absorption, distribution, metabolism, and excretion (ADME) of Shachan toxin essence is relatively limited. Existing studies have shown that its oral bioavailability is low, its half-life in the body is moderate, and it is mainly metabolized through the hepatic enzyme system. The activity and toxicity of these metabolites require further investigation.
• Overcoming multidrug resistance: Shachan venom shows good activity against multidrug-resistant tumor cell lines, suggesting it may act by bypassing traditional resistance mechanisms and has important clinical translational value.

Prospects and outlooks for clinical applications

As a natural steroid lactone, Shachan Toxic Essence, with its remarkable antitumor activity and multi-target regulatory capabilities, shows broad clinical application prospects. Currently, research on Shachan Toxin Essence mainly focuses on the treatment of solid tumors such as hepatocellular carcinoma, and has shown outstanding results in overcoming multidrug-resistant tumors, showing potential as a novel anticancer drug.

Future clinical development should focus on the following aspects:

1. Security optimization
   To address cardiotoxicity issues, structural modification, dosage form optimization, or combination therapy strategies can reduce the risk of cardiotoxicity and improve safety.

2. Development of drug delivery systems
   By utilizing advanced drug delivery technologies such as nanocarriers and liposomes, the bioavailability and targeting of Shachan venom essence are improved, enhancing anti-tumor effects and reducing systemic toxic side effects.

3. Multi-indication development
   Beyond tumors, explore its potential applications in metabolic diseases (such as hyperglycemia) and vascular-related diseases, broadening the scope of clinical indications.

4. Clinical trials are advancing
   Accelerate the transition from preclinical research to clinical trials, systematically evaluate their efficacy, safety, and pharmacokinetic characteristics, and promote their development into clinically usable anti-tumor drugs.

Conclusion

As a unique natural product of the toad dienolactone class, Shachan Venom Essence demonstrates significant antitumor activity and anti-angiogenesis effects due to its potent Na+/K+ pump inhibition and multi-target regulation of the PI3K/Akt/mTOR and VEGFR-2 signaling pathways. Its potential in treating hepatocellular carcinoma and multidrug-resistant tumors gives it significant value as a next-generation anticancer drug. However, cardiotoxicity and pharmacokinetic properties remain the main challenges for its clinical application. In the future, through structural optimization, improvements in delivery systems, and multidisciplinary collaborative research, it is expected to promote the clinical translation of Shachan Venom Essence, benefiting more cancer patients. In summary, Shachan Toxin Essence, as a model for pharmacological research of natural products, deserves ongoing in-depth exploration and development.