Product name: Telocinobufagin
Synonym name:
Catalogue No.: BP1370
Cas No.: 472-26-4
Formula: C₂₄H₃₄O₅
Mol Weight: 402.531
Botanical Source: Ch'an Su and other toad poisons
Physical Description: Powder
Type of Compound: Steroids

Purity: 95%~99%
Analysis Method: HPLC-DAD or/and HPLC-ELSD
Identification Method: Mass, NMR
Packing: Brown vial or HDPE plastic bottle

Storage: Store in a well closed container, protected from air and light. Put into refrigerate or freeze for long term storage.
Whenever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20℃. Generally, these will be useable for up to two weeks.

The product could be supplied from milligrams to grams, up to kilograms
Inquire for bulk scale.

Descriptions:
Telocinobufagin (TCB), a bufadienolide extracted from Bufo paracnemis parotoid glands, shows a reversible local anesthetic action, similar to BUPI, however, without cardiac toxicity in vitro. [1]
Telocinobufagin is a novel endogenous digitalis, exhibits elevated plasma levels in patients with terminal renal failure.[2]
Telocinobufagin and marinobufagin have antimicrobial activity, the minimum inhibitory concentrations of telocinobufagin and marinobufagin were, respectively, 64.0 and 16.0 microg/mL for E. coli and both 128 microg/mL for S. aureus; they also promote an increase of the contraction force in isolated frog ventricle strips.[3]
Telocinobufagin has potential immune system regulatory effects, suggests that it could be developed as a novel immunotherapeutic agent to treat and other immune-mediated diseases, and it may become a new immunomodulatory agent in many regions.[4]
Telocinobufagin can enhance the Th1 immune response and protect against Salmonella typhimurium infection .[5]
Telocinobufagin can induce colon cancer cell apoptosis by oxidative stress and apoptosis pathway.[6]

References:
[1] Patrocínio B A, Claúdio M. Universidade Federal do Ceará, 2004.5.26.
[2] Komiyama Y, Dong X N, Masaki H, et al. Clin Biochem, 2005, 38(1):36-45.
[3] Cunha Filho G A, Schwartz C A, Resck I S, et al. Toxicon, 2005, 45(6):777-82.
[4] Cao Y, Song Y, An N, et al. Fund Clin Pharmacol, 2009, 23(4):457-64.
[5] Wu S C, Fu B D, Shen H Q, et al. Int Immunopharmacol, 2015, 25(2):353-62.
[6] Liang S T, Li X W, et al. Nan Fang Yi Ke Da Xue Xue Bao 2016, 36(7):921-6.
[7] Ma X C, Zhang B J, Xin X L, et al. Nat Prod Co, 2009, 4(4):179-84.

HPLC of Telocinobufagin

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

Introduction/Overview

Natural products, as a treasure trove of drug discovery, play an irreplaceable role in the history of human disease treatment. Among them, bufadienolide compounds have attracted much attention due to their unique structure and wide range of biological activities. Telocinobufagin (also known as Telobufotoxin or Telocinobufogenin, CAS number: 472-26-4) is an outstanding representative of this type of compound. As an orally effective steroid compound, Far Eastern Toad Venom was initially recognized for its cardiotonic effect, and its mechanism of action is similar to classical cardiac glycosides, which enhances myocardial contractility by inhibiting Na+/K+- ATPase on the myocardial cell membrane. However, with the deepening of modern pharmacological research, its more remarkable anti-tumor activity has gradually become a research focus. Research has shown that Yuanhua Bufalin can exert significant anti proliferative and pro apoptotic effects in various malignant tumor models by intervening in multiple key signaling pathways such as STAT3, PI3K/Akt, mTOR, etc. In addition, its potential in immune regulation, anti infection, and non opioid analgesia is also beginning to emerge. This article aims to systematically review the chemical structure, plant origin, pharmacological activity, molecular mechanism of action, pharmacological characteristics, and clinical application prospects of Yuanhua Bufotalin, in order to provide comprehensive academic references for the deep development and transformation research of this natural product.

Chemical structure and physicochemical properties

Yuanhua Bufotoxin is a C24 steroidal bufadienolide, whose basic skeleton consists of a steroid nucleus and an unsaturated hexagonal lactone ring (α - pyranone ring) connected at the C17 position. Its molecular formula is C24H34O5 and its molecular weight is 402.5310. Compared with many cardiac glycosides, their steroid nuclei usually contain multiple hydroxyl groups (such as C3, C5, C14 positions), which have important effects on their water solubility and biological activity. The unsaturated lactone ring in its structure is one of the key pharmacophores that exert Na+/K+- ATPase inhibitory activity.

From the analysis of parameters related to drug properties, the lipid water partition coefficient (LogP) of Yuanhua Bufotalin is 2.5404, indicating its moderate lipophilicity, which is beneficial for its penetration of cell membranes but may also affect its water solubility. Its topological polar surface area (TPSA) is 90.9000 Å ², which is relatively high, indicating the presence of more hydrogen bond donors and acceptors (mainly from hydroxyl and carbonyl groups) in the molecule. Its water solubility value is relatively low (about 0.0126 mg/mL), making it a poorly soluble compound, which may be one of the main challenges facing its oral absorption and formulation development. These physical and chemical properties collectively determine its lower blood-brain barrier permeability, limiting its potential applications in central nervous system related diseases, but may also reduce the risk of central side effects. In addition, preliminary pharmacological screening showed no significant inhibitory effect on hERG potassium channels (hERG inhibition: no), indicating a relatively low potential risk of arrhythmia; The Ames test result was 0.0, indicating that no mutagenicity was observed in this testing system, providing preliminary positive signals for its safety.

Plant sources and extraction methods

The poison essence of Yuanhua toad mainly comes from animals in the family Bufonidae, such as the Chinese toad（Bufo gargarizans）Or black eyed toad（Bufo melanostictus）In the dried secretion of the ear gland and skin (i.e. traditional Chinese medicine "toad venom"). Chansu is a mixture of various bufadienolactones. In addition to Yuanhua Bufalin, it also contains various active ingredients such as Huahua Bufalin, Bufalin, and Lipobufalin.

Its extraction and separation usually use organic solvent extraction combined with modern chromatographic techniques. The classic process is as follows: first, the dried toad venom powder is subjected to cold soaking or reflux extraction with polar organic solvents such as methanol or ethanol, and then concentrated to obtain the total toad venom extract. Subsequently, liquid-liquid extraction methods were used, such as segmented extraction with solvents of different polarities such as petroleum ether, chloroform, and ethyl acetate. Far Eastern toad venom was mostly concentrated in the chloroform or ethyl acetate parts. Further purification relies on column chromatography technology, often using silica gel column chromatography with gradient elution using chloroform methanol or petroleum ether ethyl acetate mixed solvents in different ratios. High performance liquid chromatography (HPLC), especially preparative HPLC, is the final key step in obtaining high-purity monomers of Far Eastern Toad Venom. A reverse phase C18 chromatographic column is often used with methanol water or acetonitrile water as the mobile phase. In recent years, new separation technologies such as high-speed countercurrent chromatography have also been applied to the efficient preparation of such compounds. During the extraction process, attention should be paid to the instability of such compounds, and strong light, high temperature, and acid-base environments should be avoided to maintain their activity.

Pharmacological activity research

Yuanhua Toad Venom Essence exhibits diverse and powerful pharmacological activities, and its research has expanded from traditional cardiovascular fields to multiple modern medical fields such as oncology, immunology, anti infection, and analgesia.

1. Antitumor activity This is the most highly regarded activity of Yuanhua Toad Venom Essence. Studies have confirmed that it has significant effects on inhibiting proliferation and inducing apoptosis of many human cancer cell lines, including non-small cell lung cancer, osteosarcoma, thyroid cancer, breast cancer, head and neck squamous cell carcinoma, etc. Its anti-tumor effect is concentration - and time-dependent, and it also shows potential for certain drug-resistant tumor cells.
2. Heart strengthening effect As a fundamental property of bufadienolactone, Yuanhua Bufotoxin is proficient in inhibiting Na+/K+- ATPase on the myocardial cell membrane, leading to an increase in intracellular Na+concentration. This, in turn, increases intracellular Ca2+concentration through Na+/Ca2+exchange, thereby enhancing myocardial contractility and producing positive inotropic effects. However, it should be noted that this effect has a narrow window of potential toxicity (such as arrhythmia).
3. Immune regulation and anti infection Research has found that Far Eastern Toad Venom can enhance Th1 type immune response and promote the production of cytokines such as interferon - γ (IFN - γ), which helps the body fight against intracellular pathogens. In the Salmonella typhimurium infection model, Far Eastern Toad Venom showed a preventive protective effect, which may be related to immune enhancement.
4. Analgesic effect In various animal models of acute pain, such as acetic acid writhing test and formalin test, Far Eastern Toad Venom has shown clear analgesic effects. Importantly, its analgesic effect is not blocked by the opioid receptor antagonist naloxone, indicating that it belongs to a non opioid analgesic mechanism, which provides new ideas for the development of non addictive analgesics.
5. Other activities Some studies have pointed out that Far Eastern Toad Venom may promote renal fibrosis, indicating that there may be organ toxicity risks when used in long-term or high-dose settings, which is a direction that needs to be closely monitored in future safety evaluations.

Mechanism of action and molecular targets

The multiple pharmacological activities of Yuanhua Bufotalin stem from its precise intervention in multiple key signaling pathways and molecular targets within cells.

1. The core mechanism of anti-tumor effect：

   • Inhibition of JAK2/STAT3 pathway STAT3 is an important oncogenic transcription factor. Yuanhua Bufalin can effectively inhibit the phosphorylation of JAK2, thereby blocking the activation, nuclear translocation, and transcription of downstream target genes (such as MCL1, BCL2, MMP2, HIF1A, etc.) of STAT3. These target genes regulate cell apoptosis resistance, angiogenesis, and invasion and metastasis, and their downregulation is key to inducing tumor cell apoptosis, inhibiting growth, and metastasis.
   • Interference with PI3K/Akt/mTOR signaling axis: Yuanhuabufagin can inhibit the activation of PI3K/Akt, and then down regulate its downstream mTOR signal and the key transcription factor Snail of epithelial mesenchymal transformation (EMT), thereby inhibiting the progress and metastasis of breast cancer and other tumors. Meanwhile, it can also inhibit this pathway by affecting the interaction between LARP1 (La related protein 1) and mTOR.
   • Affects cell cycle and DNA topoisomerase Yuanhua Bufalin has been reported to inhibit PLK1 (Polo like kinase 1), a key kinase that regulates cell mitosis, and its inhibition leads to cell cycle arrest. In addition, it may interact with topoisomerases (TOP1, TOP2A), interfering with DNA replication and repair.
   • Regulating apoptosis related proteins Directly or indirectly downregulate the expression of anti apoptotic proteins MCL1 and BCL2, disrupt the pro/anti apoptotic balance, activate the Caspase cascade reaction, and lead to cell apoptosis.
   • Affects estrogen signaling: It may interfere with the growth of estrogen dependent tumors (such as some breast cancer) by acting on ESR1 (estrogen receptor α) or inhibiting CYP19A1 (aromatase).

2. Heart strengthening mechanism Its classic target is Na+/K+- ATPase (sodium pump). Inhibition of enzyme activity is the molecular basis of its positive inotropic effect.

3. Mechanism of analgesic effect The specific molecular targets of non opioid analgesia are still under further investigation and may involve inhibiting the release of inflammatory mediators, regulating ion channels (such as sodium and calcium channels), or affecting other pain perception related signaling pathways (such as the MAPK1/ERK pathway).

4. Other Its promoting effect on renal fibrosis may be related to the activation of fibrogenic signals (such as TGF - β) after long-term inhibition of Na+/K+- ATPase in renal cells.

Evaluation of drug properties and pharmacokinetics

Despite the significant in vitro activity of Yuanhua toad venom essence, it faces many challenges on its path to commercialization.

• Absorption, distribution, metabolism, excretion (ADME)As a compound with low water solubility and moderate LogP value, its oral bioavailability may be limited. Limited pharmacokinetic studies suggest that it is widely distributed after oral absorption in rats, but its metabolism may be fast, mainly through liver metabolism, possibly involving hydroxylation, binding reactions, etc., and ultimately excreted through bile and urine. Its low blood-brain barrier permeability limits central application, but is beneficial for avoiding neurotoxicity.
• Formulation Challenge Improving its water solubility and oral absorption is the core of formulation development. Possible strategies include the production of phospholipid complexes, cyclodextrin inclusion complexes, nanocrystals, liposomes, or self microemulsion delivery systems.
• Security Window Similar to all cardiac glycosides, the therapeutic window of Yuanhua Bufalin is relatively narrow. The effective anti-tumor dose may not differ significantly from the dose that produces cardiac toxicity (arrhythmia) and potential nephrotoxicity. Therefore, precise dose control, therapeutic drug monitoring, and the development of targeted delivery systems to reduce exposure to normal tissues, especially myocardium and kidneys, are crucial.
• Drug interactions As a substance that may be metabolized by CYP450 enzyme, it may interact with other drugs metabolized by the same enzyme system.

Clinical application prospects and prospects

The clinical application prospects of Yuanhua Toad Venom Essence are broad, but the transformation path needs to be cautious and strategic.

1. Antitumor therapy: is the main development direction. Given its multi-target mechanism of action, especially its effective inhibition of difficult to drug targets such as STAT3, it has unique advantages in overcoming tumor drug resistance and inhibiting metastasis. Possible future development paths include:

   • combination therapy: Used in combination with existing chemotherapy drugs, targeted drugs, or immune checkpoint inhibitors to enhance efficacy, reduce respective doses, and toxicity.
   • Structural modification By using semi synthetic methods to modify its chemical structure, the aim is to improve efficacy, reduce toxicity, and enhance pharmacokinetic properties. For example, modifying its lactone ring or hydroxyl group may result in derivatives with higher selectivity.
   • Targeted delivery system Develop targeted drug delivery systems based on nanotechnology, such as folate and antibody modified nanoparticles, to deliver drugs specifically to tumor tissues, maximizing therapeutic efficacy while reducing cardiac and renal toxicity.

2. Analgesic application Its non opioid analgesic properties have important social value and clinical significance in the context of the global spread of the opioid crisis. Further clarification of its precise analgesic targets and mechanisms is needed to evaluate its efficacy and long-term safety for chronic pain.

3. As a pharmacological tool molecule Yuanhua Bufotalin is an excellent tool compound for studying the biological functions of Na+/K+- ATPase, STAT3 signaling pathway, and its role in diseases.

4. Challenges and Future Research Focus：

   • In depth security evaluation Systematic preclinical toxicology studies are required to clarify its dose limiting toxicity, target organ toxicity, and reversibility.
   • Pharmacokinetic optimization Comprehensively elucidate its ADME process in vivo, providing a basis for dosage form design and administration regimen.
   • Refined mechanism of action Using chemical biology methods such as affinity fishing and proteomics to identify its direct target of action and create a more comprehensive pharmacological network map.
   • Conduct high-quality clinical research Based on sufficient preclinical research, gradually advance phase I/II clinical trials to explore their human safety, pharmacokinetic characteristics, and preliminary efficacy.

Conclusion

Yuanhua Bufotoxin, as a natural bufadienolide derived from traditional Chinese medicine, has shown new vitality in modern pharmacological research due to its unique chemical structure and multi-target, multi pathway pharmacological mechanism of action. Its outstanding activities in anti-tumor, immune regulation, and non opioid analgesia demonstrate its enormous potential for transformation from a "traditional toxin" to a "modern medicine". However, its inherent drug development bottlenecks such as poor water solubility, narrow treatment window, and potential organ toxicity are also scientific challenges that must be faced and overcome during the research and development process. In the future, through interdisciplinary cooperation and the comprehensive application of research methods in medicinal chemistry, pharmacy, pharmacology, and clinical medicine, the deep development of Yuanhua Bufotalin is expected to be developed into an innovative drug derived from nature and used in clinical practice, providing new weapons for the treatment of major diseases such as cancer and pain. The research process has once again confirmed that in-depth exploration of the treasure trove of natural products, combined with modern science and technology for innovative development, is an important source of inexhaustible drug discovery.