Synonym name: Limone; Citrolimonin; Evodine; Dictamnolactone; Obaculactone
Catalogue No.: BP0866
Cas No.: 1180-71-8
Formula: C₂₆H₃₀O₈
Mol Weight: 470.518
Botanical Source: oranges and other citrus fruits (Citrus spp.). Also from other Rutaceae spp., e.g. Evodia, Phellodendron, Dictamnus and Luvunga spp.

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:

Limonin is a widely used dietary supplement, one of the most prevalent citrus limonoids, which has antioxidant, anti-inflammatory, anticancer and anti-human immunodeficiency virus(HIV)activity. It induced a down regulation of TLR-2 ,TLR-4,TNF-α, TNF-α/IL-10,NF-κB and caspase. It showed the potent inhibition of CYP3A4, with IC50 values of 6.20 μM (CYP3A4/testosterone) and 19.10 μM (CYP3A4/midazolam).

References:

J Nat Sci Biol Med. 2013 Jan;4(1):126-33.    

Influence of limonin on Wnt signalling molecule in HepG2 cell lines.    

The role of Limonin as potent anti carcinogenic, apoptosis and chemotherapeutic agents has been supported by limited studies. 

METHODS AND RESULTS:

In this study, Limonin is identified as a potent anti proliferative agent against human hepatoma HepG2 cells based on the cell viability study, LDH leakage assay. Induction of apoptosis in HepG2 cells by Limonin was evidenced by western blot analysis of Bax, Cyclin D1, Caspase 3 and Caspase9. Since Wnt signalling is involved in the initiation and sustaining of hepatocellular carcinoma we studied differential expression of LRP5, LRP6 and DKK wnt players. 

CONCLUSIONS:

Limonin found to down regulate these players which forms a rationale for further investigation on effect on Limonin in cancer therapy.    

Planta Med. 2003 Oct;69(10):910-3.    

Effect of limonin and nomilin on HIV-1 replication on infected human mononuclear cells.    

In the last years several plant-derived natural compounds have been screened for their anti-HIV activity in order to find lead compounds with novel structures or mechanisms of action. Among these, several triterpenoids have been found to exhibit an antiretroviral activity with different mechanisms of action. 

METHODS AND RESULTS:

In this study the effect of two limonoids, Limonin and nomilin, on the growth of human immunodeficiency virus-1 (HIV-1) in culture of human peripheral blood mononuclear cells (PBMC) and on monocytes/macrophages (M/M) is described. Limonin and nomilin were found to inhibit the HIV-1 replication in all cellular systems used. A dose-dependent inhibition of viral replication was observed in PBMC isolated from healthy donors and infected with HIV-1 strain after incubation with Limonin and nomilin (EC (50) values: 60.0 microM and 52.2 microM, respectively). The two terpenoids inhibited at all concentrations studied the production of HIV-p24 antigen even when the PBMC employed were chronically infected (EC (50) values of 61.0 microM for Limonin and 76.2 microM for nomilin). Moreover, these compounds inhibited the HIV-1 replication even in infected M/M. In this cellular system the inhibitory effect was significant at the concentrations of 20 microM, 40 microM and 80 microM starting from day 14 and reached the maximum effect after 18 days of incubation. As regards the mechanism of action, Limonin and nomilin inhibit in vitro HIV-1 protease activity. 

CONCLUSIONS:

In general, the results obtained point out a similar anti-HIV activity of Limonin and nomilin indicating that this activity is not drastically influenced by the structural difference between the two compounds.   

Eur J Pharmacol. 2014 Oct 5;740:676-82.    

Limonin attenuates hepatocellular injury following liver ischemia and reperfusion in rats via toll-like receptor dependent pathway.   

Limonin has been shown to exhibit anti-inflammatory and antioxidant properties in the settings of chemically induced hepatic injury. 

METHODS AND RESULTS:

The current study aimed to investigate the protective effects of Limonin on experimentally-induced hepatic ischemia reperfusion (I/R) injury in rats. Rats were injected IP with either DMSO or Limonin (100 mg/kg BW), 30 min before submission to 45 min of ischemia, followed by 1 h of reperfusion. Limonin ameliorated the deleterious effects of I/R as indicated by improvement in liver function tests, reduction of lactate dehydrogenase, reduction of oxidative stress, decrease in hepatocyte degeneration, and pyknosis. Furthermore, pretreatment of I/R rats with Limonin, induced a significant down regulation in the various elements of the toll like receptor (TLR)pathway including TLR-2 and TLR-4, myeloid differentiation factor 88 (MYD88) and toll/IR-1(TIR)-domain-containing adaptor protein inducing interferon-beta (TRIF) and the downstream effectors TNF-α, TNF-α/IL-10 ratio and nuclear factor-κB (NF-κB). It also increased the anti-inflammatory cytokine IL-10 and decreased the activity of the apoptotic marker, caspase-3. 

CONCLUSIONS:

These data indicate that Limonin exerts antioxidant and anti-inflammatory effects in ischemic liver, thus, protecting hepatocytes against I/R injury in rats. The mechanism of these hepatoprotective effects appears to be related to the antioxidant and anti-inflammatory potential of Limonin mediated by the down regulation of TLR- signaling pathway.    

 HPLC of Limonin

NMR of Limonin

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

Introduction/Overview

Limonin, CAS number 1180-71-8, is a naturally bitter tetracyclic triterpene lactone compound widely found in citrus plants. As a typical bitter component in citrus fruits, limonoid not only gives fruits their unique bitter characteristics, but also attracts widespread attention in pharmacology and natural product chemistry due to its diverse biological activities. In recent years, with in-depth research into the pharmacological mechanisms of natural products, limonoid has become a potential drug development candidate due to its significant antitumor and antiviral activity and inhibition of the drug-metabolizing enzyme CYP3A4.

Limonin exhibits multiple functions in anti-tumor form, including inducing cancer cell apoptosis and inhibiting tumor cell proliferation, involving several key molecular targets such as MCL1, BCL2, and STAT3, demonstrating a complex and multi-target mechanism. Additionally, its inhibitory effect on HIV-1 and effective inhibition of CYP3A4 enzyme suggest its potential value in antiviral and drug interaction regulation. This paper will systematically review the chemical structure and physicochemical properties of limonin extractor, plant origin and extraction methods, pharmacological activity and mechanism of action, druggability evaluation, and pharmacokinetic characteristics, and look ahead to its clinical application prospects, providing theoretical basis and directions for pharmacological research of natural products and new drug development.

Chemical structure and physicochemical properties

Limonic is a tetracyclic triterpene lactone compound, with a molecular formula of C_26H_30O_8 and a molecular weight of 470.5180. Its chemical structure features a typical tetracyclic framework containing multiple oxygen functional groups, such as lactone rings and ketone groups, which give it unique chemical activity. The LogP value of limonic acid was 2.2542, indicating moderate lipid solubility, which is beneficial for cell membrane penetration and distribution in vivo. The polar surface area (TPSA) is 104.57 Ų, indicating certain polarity that facilitates binding to biological macromolecule targets.

Low water solubility (0.0075 mg/mL) limits its solubility in aqueous media and may affect its bioavailability. It is worth noting that limonin has a high ability to penetrate the blood-brain barrier, suggesting it may act on the central nervous system or affect nervous-related diseases. Additionally, limonin did not exhibit hERG channel inhibitory activity, reducing its potential risk of cardiotoxicity. The Ames test result was 0.9, indicating a low genotoxicity risk and meeting safety requirements.

Plant Origins and Extraction Methods

Limonic is mainly found in the fruits of citrus species, especially in the peels and seeds of lemons (Citrus limon), bitter orange (Citrus aurantium), and grapefruit (Citrus paradisi). Its content is significantly influenced by variety, maturity, and growing environment. Traditionally, limonic extract has been widely studied as the main component of the bitterness of citrus fruits.

The main methods for extracting limonin include solvent extraction, ultrasound-assisted extraction, and supercritical fluid extraction. Common solvents include ethanol, methanol, ethyl acetate, and acetone. Ethanol is widely adopted due to its safety and high extraction efficiency. The extraction process generally includes peel drying and crushing, solvent extraction extraction, concentration, and purification steps. During purification, technologies such as silica gel column chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) are commonly used to obtain high-purity limonic acid.

In recent years, green extraction technologies such as ultrasonic-assisted and microwave-assisted extraction have been introduced to improve extraction efficiency and reduce solvent usage, aligning with the modern trend of environmentally friendly natural product extraction. Additionally, stability studies on limonin have driven optimization of extraction processes, ensuring the integrity and bioactivity of its active ingredients.

Pharmacological activity research

Limonic exhibits a variety of significant pharmacological activities, mainly concentrated in antitumor and antiviral fields.

Antitumor activity

Limonin exhibits inhibitory effects on various tumor cell lines, especially inducing apoptosis in human colon adenocarcinoma cells, with an IC50 of approximately 54.74 μM. Its antitumor activity mechanism involves multiple signaling pathways and molecular targets, including regulation of anti-apoptotic proteins MCL1 and BCL2, inhibition of transcription factor STAT3, and reduction of matrix metalloproteinase MMP2 activity. Limonin can also affect the DNA topoisomerase TOP1 and TOP2A of tumor cells, blocking the proliferation cycle of tumor cells. Additionally, limonin inhibits HIF1A expression in the tumor microenvironment, reducing the tumor's hypoxia adaptability and inhibiting tumor invasion and metastasis.

In both in vitro and in vivo models, limonin demonstrates good antitumor effects, and when used in combination with traditional chemotherapy drugs, it can enhance efficacy and reduce the incidence of drug resistance. Its regulation of MAPK1 and the estrogen receptor ESR1 further enriches its molecular mechanisms against tumors.

Antiviral activity

Limonin exhibited inhibitory effects against HIV-1 virus, with an EC50 of 60.0 μM. Its antiviral mechanism may involve interfering with the activity of key enzymes in the viral replication cycle, while also blocking viral invasion and spread by regulating host cell signaling pathways. Additionally, the effective inhibition of CYP3A4 enzyme by limonin (IC50 of 6.2 μM) suggests it may affect antiviral drug metabolism and carries potential drug interaction risks, but also provides a regulatory strategy for combination therapy.

In addition, the activity of limonin in other viral models remains to be further explored, and preliminary studies show it has certain inhibitory potential against certain respiratory viruses and hepatitis viruses.

Mechanism of action and molecular targets

The pharmacological activity of limonic kutin lies in its multi-target and multi-pathway regulatory capability. Its main targets include anti-apoptotic proteins, transcription factors, enzymes, and signaling molecules, forming a complex network of actions.

• MCL1 and BCL2: Limonin destroys the anti-apoptotic barrier of tumor cells by downregulating the expression of MCL1 and BCL2, promoting programmed cell death.
• STAT3: As a key transcription factor for tumor cell proliferation and immune escape, inhibition of STAT3 reduces the survival signals of tumor cells.
• MMP2: By inhibiting MMP2 activity, limonotin blocks stromal degradation of tumor cells, suppressing tumor invasion and metastasis.
• TOP1 and TOP2A: Limonic interferes with DNA topoisomerase function, blocks DNA replication and transcription, and inhibits tumor cell proliferation.
• HIF1A: Inhibits tumor cells' ability to adapt to hypoxic environments, weakening the support of the tumor microenvironment.
• MAPK1: Regulates cell proliferation and apoptosis signaling pathways, affecting tumor cell fate.
• ESR1 and CYP19A1: Influence the growth of hormone-dependent tumors, regulate estrogen receptor signaling, and aromatase activity.

Additionally, limonin is an effective inhibitor of CYP3A4, affecting the activity of drug-metabolizing enzymes and potentially regulating metabolic processes of various drugs in vivo, with important pharmacokinetic significance.

Druggability evaluation and pharmacokinetics

The molecular weight of limonoid is 470.5180, which is within the reasonable range for drug molecular weights. Its LogP value of 2.2542 indicates moderate lipid solubility, which is beneficial for cell membrane penetration and distribution in vivo. TPSA is 104.57 Ų, meeting the molecular polarity requirements of the drug and facilitating target binding.

Low water solubility (0.0075 mg/mL) may limit oral bioavailability, requiring improved solubility through formulation technology. Its high blood-brain barrier permeability suggests it may play a role in the central nervous system, but potential CNS toxicity should also be considered.

In terms of safety, limonin does not show hERG channel inhibition, reducing the risk of cardiotoxicity. The Ames test result was 0.9, indicating no significant genotoxicity and meeting drug safety requirements.

Pharmacokinetic studies show that limonin has good stability and distribution characteristics in the body, but its metabolic pathway has not been fully elucidated. As a CYP3A4 inhibitor, limonoid may affect its own and other drug metabolism, suggesting careful evaluation of drug interactions when using combination therapy.

Prospects and outlooks for clinical applications

With its multi-target and multi-mechanism anti-tumor and antiviral activities, lemon-bitter extract demonstrates broad clinical application potential. Its ability to induce tumor cell apoptosis and inhibit tumor invasion and metastasis provides new molecular targets and therapeutic strategies for tumor treatment. Its application prospects in digestive system tumors such as colon cancer are especially worthy of in-depth study.

Anti-HIV-1 activity makes limonin a candidate molecule for antiviral drug development, especially in the context of increasingly severe viral resistance, where the multi-target effect of natural products is significant. Additionally, limona's inhibitory effect on CYP3A4 suggests its potential application in drug metabolism regulation, but drug interaction risks should be watched for.

Future research should focus on optimizing the pharmacokinetics, improving formulations, and evaluating preclinical safety of limonoster. At the same time, combining modern molecular biology techniques, the mechanism of action is deeply analyzed to uncover more potential targets. The implementation of clinical trials will be a key step in verifying its efficacy and safety.

Additionally, structural modification and derivative development of limonin may enhance its activity and pharmacokinetic properties, providing new ideas for the design of novel anti-tumor and antiviral drugs. The combination of natural products and modern medicinal chemistry will drive the conversion of limonin into clinical applications.

Conclusion

As a typical citrus natural product, limonic has become a hot topic in natural product pharmacology research due to its unique chemical structure and diverse biological activities. Its multi-target regulatory capabilities in the fields of antitumor and antiviral therapies provide valuable molecular frameworks and mechanisms of action for new drug development.

Although limonin faces challenges such as low water solubility and potential drug interactions, its excellent safety and druggability give it high development value. In the future, through structural optimization, dosage form improvements, and systematic pharmacokinetic and toxicological studies, limonoid is expected to become a clinically competitive natural drug or drug-leading compound.

In summary, as a multifunctional natural product, limonic extract not only enriches pharmacological research on natural products but also provides new ideas and directions for innovation in anti-tumor and antiviral drugs, worthy of ongoing in-depth exploration in basic research and clinical translation.