Product name: Aristolochic Acid B
Synonym name: Aristolochic Acid II
Catalogue No.: BP0193
Cas No.: 475-80-9
Formula: C₁₆H₉NO₆
Mol Weight: 311.249
Botanical Source: Alkaloid from Aristolochia argentina, Aristolochia clematitis, Aristolochia esperanzae, Aristolochia longa, Aristolochia manshuriensis, Aristolochia rotunda, Aristolochia pallida, Aristolochia sipho (Aristolochia durior) and from the Chinese drugs Ching-
Physical Description: Yellow powder
Type of Compound: Miscellaneous

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:
Aristolochic acid II (AAII), one of the major components of the carcinogenic plant extract aristolochic acid, is known to be mutagenic and to form DNA adducts in vitro and in vivo,
AAII shows more carcinogenic risk than aristolochic acid I, and this may be, at least partly, the result of its increased levels in kidney and plasma.[1,2]
Aristolochic acid exhibit significant toxicity, and the short-term toxicity of aristolochic acid-II and aristolochic acid is similar to each other, renal but not hepatic failure induced by aristolochic acid could be prevented by pentoxifylline.[3]


References:
[1] Pfau W, Schmeiser H H, Wiessler M. Cheml Res Toxicol, 1991, 4(5):581-6.
[2] Xing G, Qi X, Min C, et al. Mutat Res-Fund Mol M, 2012, 743(1-2):52-8.
[3] Yeh Y H, Lee Y T, Hsieh H S, et al. Food Chem Toxicol , 2008, 46(3):1157-63.
[4] Zhu G, Wang Z, Wang Q, et al. China Pharmacy, 2006, 17(18):21-4.

HPLC of Aristolochic Acid B

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

Introduction/Overview

Aristolochic Acid B (AAII), CAS number 475-80-9, is one of the main members of the Aristolochic Acid (AAs) family of compounds. Aristolochic acid compounds are mainly derived from Aristolochiaceae plants and have been widely used in traditional herbal medicine as anti-inflammatory, diuretic, and weight loss ingredients in history. However, since the outbreak of the "Chinese Herbal Kidney Disease" (later renamed "Aristolochic Acid Kidney Disease") in the 1990s, the serious toxic side effects of such compounds, especially their strong nephrotoxicity and carcinogenicity, have attracted great attention from the global pharmacology, toxicology, and clinical medicine communities. Aristolochic acid B, as the core component in Aristolochic acid mixtures, has particularly prominent toxic effects. Research has shown that AAII can form stable DNA adducts both in vitro and in vivo, with clear genotoxicity and mutagenicity, and is a key pathogenic factor leading to diseases such as renal interstitial fibrosis and urothelial carcinoma. In recent years, with the development of molecular biology technology, the study of its mechanism of action has been continuously deepened, involving multiple key targets and signaling pathways. This article aims to systematically review the chemical properties, plant sources, pharmacological (toxicological) activities, molecular mechanisms of action, pharmacological evaluation, and related clinical risks of aristolochic acid B, in order to provide scientific references for a comprehensive understanding of its duality (potential activity and significant toxicity) and subsequent related research.

Chemical structure and physicochemical properties

Aristolochic acid B is a nitrophenanthrene derivative, with the systematic chemical name 3,4-methylenedioxy-10-nitro-1-phenanthrene carboxylic acid. Its core structure is a phenanthrene ring, which is replaced by a carboxyl group at position 1, connected by a methylenedioxy bridged ring at positions 3 and 4, and connected to a nitro group at position 10. This unique structure is the material basis for its biological activity and toxicity.

Its basic physicochemical properties are as follows:
- Molecular formula： C₁₇H₁₁NO₇
- molecular weight： 311.2490 g/mol
- Lipid water partition coefficient (LogP): 3.4015. This value indicates that aristolochic acid B has a moderately high lipophilicity, which facilitates its penetration of cell membranes but may also lead to its accumulation in adipose tissue.
- Topological Polarity Surface Area (TPSA)98.9000 Å ². A higher TPSA value reflects the presence of polar groups such as nitro, carboxyl, and ether bonds in the molecule, which have a significant impact on its solubility and intermolecular interactions.
- Water solubility： 0.0599 mg/mL。 It belongs to the category of slightly soluble to poorly soluble in water, which is consistent with its LogP value. In practical research, organic solvents such as DMSO are often used as solubilizers.
- Blood-brain barrier permeability Predicted as low. This is mainly attributed to its larger molecular weight and higher polar surface area, which limits its ability to freely pass through the blood-brain barrier, suggesting that its neurotoxicity may not be the main focus.
- HERG inhibition risk Predicted as no. Indicating that aristolochic acid B may not directly inhibit hERG potassium channels at conventional concentrations, resulting in a lower risk of cardiac QT interval prolongation.
- Ames test The value is 2.4 (usually referring to the recovery mutation rate, depending on the specific experimental conditions). This data further confirms its direct mutagenic potential, which is a direct manifestation of its genetic toxicity.

Aristolochic acid B is relatively unstable under light and alkaline conditions, and its nitro group can be reduced during in vivo metabolism, which is a key step in its activation and toxicity.

Plant sources and extraction methods

Aristolochiaceae B is widely present in various plants of the Aristolochiaceae family, especially in the Aristolochiaceae genus（Aristolochia）And Asarum genus（Asarum）Plants. Common plants containing aristolochic acid include Guanmu Tong（Aristolochia manshuriensis）Widely defend oneself（Aristolochia fangchi）Qingmu Fragrance（Aristolochia debilis）Aristolochia, Aristolochia（Aristolochia contorta）And some Asarum（Asarum Spp.) variety. These plants have a history of application in traditional medical systems in Asia, Europe, and the Americas.

The extraction and separation of aristolochic acid B usually follow the conventional process of natural product chemistry:
1. Extract Crush the dried plant roots, stems, and other parts, and use organic solvents for reflux extraction or cold soaking. Common solvents include methanol, ethanol, acetone, or their aqueous solutions to fully extract aristolochic acid components with a wide range of polarities.
2. Rough classification The extract is concentrated under reduced pressure to obtain a paste, which is then preliminarily enriched using solvent partitioning (such as partitioning between aqueous and organic phases (such as ethyl acetate, chloroform) at different pH values). Aristolochic acid compounds, due to their carboxyl groups, dissolve as salts in alkaline aqueous phase and can be extracted by organic solvents under acidic conditions, thereby separating from a large number of impurities.
3. Separation and Purification Further use column chromatography technology for fine separation. Silica gel column chromatography is commonly used to separate various components using gradient elution systems such as chloroform methanol or petroleum ether ethyl acetate. High performance liquid chromatography (HPLC), especially preparative HPLC, is the final key step in obtaining high-purity aristolochic acid B monomer. C18 reverse phase chromatography columns are commonly used, with methanol water or acetonitrile water (containing a small amount of formic acid or acetic acid to adjust pH) as the mobile phase.
4. appraisal The purified compound was structurally confirmed by nuclear magnetic resonance (NMR, including ¹ H and ¹ ³ C NMR), mass spectrometry (MS), ultraviolet spectroscopy (UV), and chromatographic behavior (such as HPLC retention time) compared to standard samples.

Due to the strong toxicity of aristolochic acid B, all extraction, separation, and experimental operations must be carried out under strict safety protection conditions to avoid personnel contact and environmental pollution.

Pharmacological activity research

The pharmacological activity research of aristolochic acid B mainly focuses on its toxicity, especially nephrotoxicity, genotoxicity, and carcinogenicity. The traditional notion of anti-inflammatory, analgesic, and other "therapeutic effects" has been overshadowed by its unacceptable toxicity risks, and modern research mainly regards it as a typical toxic model compound.

1. nephrotoxicity Aristolochic acid B is the core toxin that causes aristolochic acid nephropathy. Animal experiments (rats, mice) and cell experiments (human renal tubular epithelial cells HK-2, etc.) have both confirmed that AAII can cause acute tubular necrosis and chronic progressive renal interstitial fibrosis. Its toxicity is dose-dependent and time-dependent, ultimately leading to renal failure.
2. Genotoxicity and mutagenicity The Ames test showed a positive result, confirming that it is a direct mutagen. It can cause gene mutation, chromosome aberration and sisters chromatid exchange in a variety of test strains and mammalian cells. Its mutagenicity is the basis of its carcinogenic effect.
3. carcinogenicity Long term exposure to aristolochic acid B can induce renal and urothelial tumors in experimental animals such as rats and mice. Epidemiological studies have clearly associated exposure to aristolochic acid compounds with a high risk of human upper urinary tract (renal pelvis, ureter) transitional cell carcinoma. The carcinogenic risk of AAII is considered to be higher than that of its homolog aristolochic acid I (AAI).
4. Other toxicities The study also suggests that it may have potential risks such as hepatotoxicity and embryotoxicity.

It is worth noting that very few studies have explored the possible immunomodulatory or anti-inflammatory effects of aristolochic acid compounds at extremely low concentrations, but these studies are controversial and their therapeutic window is extremely narrow, unable to offset their enormous toxicity risks. Therefore, major global drug regulatory agencies have banned or strictly restricted the use of medicinal herbs and preparations containing aristolochic acid.

Mechanism of action and molecular targets

The toxic mechanism of aristolochic acid B is complex and involves multiple steps and targets. Its core lies in the covalent binding with biomolecules (especially DNA) after metabolic activation, which triggers sustained genetic damage and cellular stress response.

1. Metabolic activation and DNA adduct formation：

   • After entering the body, aristolochic acid B is mainly catalyzed by cytochrome P450 enzymes (such as CYP1A1/2) and/or reductases (such as NAD (P) H: quinone oxidoreductase) in tissues such as the kidneys. Its nitro group (- NO ₂) is reduced to a highly reactive nitrogen oxide intermediate (- NHOH), which is then dehydrated and cyclized to form aryl nitrogen ions.
   • The electrophilic arylazonium ion can undergo nucleophilic attack on the electron rich sites of DNA bases (mainly deoxyadenine dA and deoxyguanine dG), forming covalently bound DNA adducts, the most important of which are analogs of 7- (deoxyadenosine-N6-yl) aristolochic amide I (dA-AAI) and 7- (deoxyguanosine-N2- yl) aristolochic amide I (dG AAI) (corresponding to AAII). These adducts are called aristolochic acid DNA adducts.
   • These DNA adducts are persistent and difficult to effectively remove by conventional DNA repair systems. They can cause base mismatches during cell replication, leading to characteristic A: T → T: A mutations (especially on specific codons of tumor suppressor genes such as TP53), which are the molecular starting point for their mutagenicity and carcinogenesis.

2. Key molecular targets and pathways：
   The toxicity of aristolochic acid B is not limited to DNA damage, but also involves interference with multiple cellular processes. Based on the provided target information, its functional network can be summarized as follows:

   • DNA damage repair and genomic stability The persistent DNA adducts induced by AAII pose a significant challenge to DNA replication and repair systems. It directly or indirectly affects multiple DNA repair related proteins. For example, it can be associated with APEX1 The interaction between purine/pyrimidine endonuclease 1 may interfere with the base excision repair pathway. Meanwhile, it has an impact on BLM(RecQ helicase like protein) and RECQL The impact of (another RecQ family helicase) may impair DNA unwinding and homologous recombination repair, leading to increased genomic instability. Correct FEN1 The potential impact of endonuclease 1 may hinder the normal processing of Okazaki fragments during DNA replication.
   • Imbalance between cell apoptosis and survival Persistent DNA damage typically triggers cell apoptosis. However, exposure to aristolochic acid B may affect the balance of pro apoptotic and anti apoptotic proteins, leading to abnormal cell survival in a damaged state and creating conditions for carcinogenesis. it and BCL2(Anti apoptotic protein) and MCL1 The association between myeloid leukemia sequence 1 and anti apoptotic protein may inhibit the normal apoptotic pathway. Meanwhile, regarding UBP2 The influence of deubiquitinase, which may be involved in stabilizing certain proteins, may further interfere with cell fate determination.
   • Abnormal signal pathway： NOTCH1 The signaling pathway plays a crucial role in cell differentiation, proliferation, and apoptosis, and its abnormal activation is associated with various cancers, including acute lymphoblastic leukemia. Aristolochic acid B may interfere with NOTCH1 signaling and participate in its carcinogenic process.PTPN1 Protein tyrosine phosphatase non receptor type 1 (PTP1B) is a key negative regulator of the insulin and leptin signaling pathways, and its abnormalities may be related to metabolic disorders, but its specific role in AAII nephrotoxicity remains to be elucidated.
   • Other potential targets： MAOA Monoamine oxidase A is a neurotransmitter metabolizing enzyme, and its association with AAII may suggest neurotoxic potential, but it is not its main toxic pathway.

3. Association with acute lymphoblastic leukemia (ALL)：
   Although the carcinogenicity of aristolochic acid B is mainly associated with urothelial carcinoma, studies have also found its DNA adducts present in other tissues. The target lists provided (such as NOTCH1, BCL2, MCL1) are all key molecules in the pathogenesis of ALL. There is a hypothesis that systematic exposure to aristolochic acid B may increase the risk of hematopoietic malignancies (such as ALL) by forming DNA adducts and interfering with key targets (such as inducing NOTCH1 gene and disrupting apoptosis balance). This provides a new perspective for understanding its systemic carcinogenic effects, but more epidemiological and experimental evidence is needed to support it.

Evaluation of drug properties and pharmacokinetics

From the perspective of modern drug development, the pharmacological properties of aristolochic acid B are extremely poor, and its pharmacokinetic characteristics are closely related to its toxicity.

• Absorption and distribution After oral administration, it can be absorbed, among which isolipolysis helps with absorption. After absorption, it is widely distributed throughout the body, but due to its anionic nature, it partially ionizes at physiological pH, limiting its free transmembrane diffusion. Its concentration is usually higher in the kidneys, which is related to the fact that the kidneys are its main metabolic and toxic target organs. The blood-brain barrier has low permeability and limited exposure to the central nervous system.
• Metabolism As mentioned earlier, metabolic activation is a key step in the production of its toxicity. It is mainly reduced to active intermediates by nitroreductase (such as NQO1) and CYP450 enzyme systems in the liver and kidneys. In addition, II binding reactions such as glucuronic acid binding or sulfuric acid binding may also occur, forming inactive water-soluble metabolites that are excreted through urine.
• excretion The prototype drug and its metabolites are mainly excreted through the kidneys and urine. Due to its reabsorption and further metabolism in renal tubular epithelial cells, the high drug concentration and long exposure time in this area are important pharmacokinetic reasons for its selective nephrotoxicity.
• Summary of Drug Defects：
  1. Extremely poor security Clear strong mutagenicity, carcinogenicity, and organ toxicity (especially nephrotoxicity), with extremely low treatment index and even no safety window.
  2. Poor pharmacokinetics Strong accumulation tendency in the target organ (kidney) leads to selective toxicity.
  3. Chemical Stability Challenge The nitro structure is easily reduced and activated uncontrollably in the body, producing highly reactive and toxic intermediates.
  4. Ames positive Direct genetic toxicity does not meet the basic safety requirements for drug development.

Therefore, aristolochic acid B has no possibility of being developed as a therapeutic drug. The current research focus is on elucidating its toxicokinetics for risk assessment and exploring its application as a tool drug in constructing animal models of specific types of DNA damage or renal fibrosis.

Clinical application prospects and prospects

Aristolochic acid B and its source plants There is no prospect for clinical treatment anymore Regulatory agencies worldwide, such as the US FDA, European EMA, and China's National Medical Products Administration, have issued warnings, restrictions, or bans on the use of medicinal and proprietary medicines containing aristolochic acid. Its' clinical application 'has completely shifted towards Toxicological monitoring, risk assessment, and public health protection field

Future research prospects should focus on the following aspects:

1. Deep analysis of toxicity mechanism Using omics techniques (genomics, epigenomics, proteomics, metabolomics) to systematically elucidate the global molecular change network of the kidneys and other organs after exposure to aristolochic acid B, and search for early and specific biomarkers.
2. Accurate risk assessment Study the impact of genetic polymorphisms (such as metabolic enzyme NQO1 and repair enzyme gene variations) on individual susceptibility to aristolochic acid B, in order to achieve precise identification of high-risk populations.
3. Detoxification and intervention strategies Explore intervention methods that can block its metabolic activation, promote its non-toxic excretion, enhance DNA adduct repair, or antagonize its downstream fibrosis signaling pathway (such as TGF - β). These studies aim to provide potential strategies for medical treatment of individuals who have been accidentally or previously exposed, rather than for 'treatment'.
4. Application as a tool molecule Using its ability to generate specific types of persistent DNA adducts, it can be used as a tool for studying DNA damage repair mechanisms, mutation spectrum analysis, and the mode of action of environmental carcinogens.
5. Public Health and Regulatory Science: Strengthen the variety identification, quality control and substitute research of medicinal materials containing aristolochic acid, improve the whole process traceability and risk early warning system of related traditional Chinese patent medicines and simple preparations and health products, and prevent similar public health events from recurring.

Conclusion

Aristolochic acid B is a nitrophenanthrene compound isolated from traditional medicinal plants, and its research process is a typical negative example from "potential drugs" to "clear toxins". Its strong nephrotoxicity, genetic toxicity, and carcinogenicity stem from the covalent binding of active nitrogen ions formed after metabolism in the body to DNA, and involve extensive interference with DNA repair, cell apoptosis, and key signaling pathways. Although research on its molecular targets, including some proteins associated with acute lymphoblastic leukemia, has expanded our understanding of its systemic toxicity, this does not change its nature as a Class I human carcinogen. Its poor pharmacokinetic parameters and unacceptable safety risks completely shut down its potential as a therapeutic drug. Current and future research should strive to thoroughly reveal the mysteries of its toxicity, develop monitoring and intervention methods, and draw profound lessons from it, strengthen scientific scrutiny and strict supervision of potential toxic components in traditional herbs, in order to ensure public medication safety. The story of aristolochic acid B warns us that nature does not equate to safety, and the development of natural products must be based on rigorous and systematic pharmacological and toxicological evaluations.