Eupafolin

• Anti-inflammatory
• Antioxidant
• Neuroprotective
• Anticancer

• Hepatoprotective
• Cardioprotective
• Antimicrobial
• Immunomodulatory
• Antiallergic

Eupafolin (6-methoxyluteolin or 5,7,3′,4′-tetrahydroxy-6-methoxyflavone) exerts its diverse biological effects through multiple molecular pathways. As a methoxyflavone with four hydroxyl groups and one methoxy group, eupafolin possesses a unique pharmacological profile that contributes to its various health benefits. One of eupafolin’s most extensively studied mechanisms is its anti-inflammatory activity. Eupafolin potently inhibits the nuclear factor-kappa B (NF-κB) signaling pathway, a master regulator of inflammatory responses.

It prevents the phosphorylation and degradation of inhibitor of kappa B (IκB), thereby blocking the nuclear translocation of NF-κB and subsequent transcription of pro-inflammatory genes. This leads to reduced expression of inflammatory mediators including tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Eupafolin also modulates the mitogen-activated protein kinase (MAPK) signaling pathways, including p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). By inhibiting the phosphorylation and activation of these kinases, eupafolin further suppresses the production of pro-inflammatory cytokines and chemokines.

A particularly significant anti-inflammatory mechanism of eupafolin is its ability to inhibit inducible nitric oxide synthase (iNOS) expression and activity, leading to reduced production of nitric oxide (NO), a key mediator in inflammation. This mechanism has been demonstrated in various cell types, including macrophages and microglia, and contributes to eupafolin’s potential benefits in conditions characterized by excessive inflammation. The antioxidant properties of eupafolin are mediated through both direct and indirect mechanisms. With its four hydroxyl groups, eupafolin can directly scavenge reactive oxygen species (ROS) and free radicals, neutralizing these harmful molecules before they can damage cellular components.

The catechol structure in the B-ring (3′,4′-dihydroxy) is particularly important for this activity, as it can donate hydrogen atoms to stabilize free radicals. Beyond direct scavenging, eupafolin activates the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, a master regulator of cellular antioxidant defenses. By promoting the nuclear translocation of Nrf2, eupafolin enhances the expression of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1). This dual approach to antioxidant protection—direct scavenging and enhancement of endogenous antioxidant systems—provides comprehensive defense against oxidative stress.

In the central nervous system, eupafolin exhibits neuroprotective effects through multiple mechanisms. It protects neurons from oxidative stress and excitotoxicity by reducing glutamate-induced calcium influx and maintaining mitochondrial function. Eupafolin also inhibits neuroinflammation by suppressing microglial activation and reducing the production of pro-inflammatory mediators in the brain. Additionally, it has been shown to protect against cerebral ischemia/reperfusion injury by preserving the integrity of the blood-brain barrier and reducing neuronal apoptosis.

In cancer cells, eupafolin demonstrates antiproliferative and pro-apoptotic effects. It induces cell cycle arrest primarily at the G2/M phase by modulating the expression and activity of cell cycle regulators, including cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors. Eupafolin also triggers apoptosis through both intrinsic (mitochondrial) and extrinsic (death receptor) pathways. It modulates the expression of Bcl-2 family proteins, decreasing anti-apoptotic proteins (Bcl-2, Bcl-xL) and increasing pro-apoptotic proteins (Bax, Bad), leading to mitochondrial membrane permeabilization, cytochrome c release, and activation of caspase cascades.

Furthermore, eupafolin has been shown to inhibit angiogenesis and metastasis, potentially limiting cancer progression and spread. In the cardiovascular system, eupafolin demonstrates protective effects by improving endothelial function, reducing inflammation, and preventing oxidative damage. It enhances nitric oxide (NO) production by endothelial cells through activation of endothelial nitric oxide synthase (eNOS), promoting vasodilation and improving blood flow. Eupafolin also inhibits platelet aggregation and thrombus formation, potentially reducing the risk of thrombotic events.

In the liver, eupafolin exhibits hepatoprotective effects by reducing oxidative stress, inflammation, and lipid accumulation. It activates AMP-activated protein kinase (AMPK), which enhances fatty acid oxidation and reduces lipogenesis, potentially benefiting conditions like non-alcoholic fatty liver disease (NAFLD). Eupafolin also induces phase II detoxification enzymes through Nrf2 activation, enhancing the liver’s capacity to metabolize and eliminate toxins. The balanced hydroxyl/methoxy structure of eupafolin (four hydroxyl groups and one methoxy group) contributes to its unique pharmacological profile.

The methoxy group at the 6-position enhances its lipophilicity and membrane permeability, while the hydroxyl groups maintain significant antioxidant capacity. This structural feature also influences its interaction with various molecular targets, contributing to its diverse biological activities.

Optimal dosage ranges for eupafolin in humans have not been well established through clinical trials. Most research has focused on eupafolin as a component of herbal extracts, particularly from Eupatorium perfoliatum (Boneset) and other plants, rather than as an isolated compound. Based on preclinical studies and limited human research with related compounds, estimated effective doses would range from 5-30 mg of eupafolin daily. For Eupatorium perfoliatum extracts, typical daily doses range from 1-3 grams of dried herb or 300-900 mg of standardized extract containing 0.1-1% eupafolin.

This would correspond to approximately 0.3-9 mg of eupafolin daily. It’s important to note that eupafolin’s bioactivity may be influenced by other compounds present in herbal extracts, potentially leading to synergistic effects that allow for lower effective doses compared to isolated eupafolin.

Eupafolin has moderate oral bioavailability, estimated at approximately 15-25% in animal studies. This is higher than many highly hydroxylated flavonoids but still limited by several factors. The presence of four hydroxyl groups provides good water solubility, while the single methoxy group at the 6-position enhances lipophilicity, creating a balanced structure that facilitates absorption. However, eupafolin’s bioavailability is limited by first-pass metabolism in the liver, efflux by P-glycoprotein transporters in the intestine, and phase II metabolism (primarily glucuronidation and sulfation).

The catechol structure in the B-ring (3′,4′-dihydroxy) makes eupafolin particularly susceptible to methylation by catechol-O-methyltransferase (COMT) and conjugation by UDP-glucuronosyltransferases (UGTs), which can significantly reduce the amount of free eupafolin in circulation. In animal studies, eupafolin has demonstrated tissue distribution to various organs, including the liver, lungs, and kidneys, with moderate penetration into the brain. The presence of other compounds in herbal extracts, particularly from Eupatorium perfoliatum, may influence eupafolin’s bioavailability through various mechanisms, including competitive inhibition of metabolic enzymes or transporters.

Nanoemulsion formulations – can increase bioavailability by 3-10 fold by improving solubility and enhancing intestinal permeability, Liposomal encapsulation – protects eupafolin from degradation and enhances cellular uptake, Self-emulsifying drug delivery systems (SEDDS) – improve dissolution and absorption in the gastrointestinal tract, Phospholipid complexes – enhance lipid solubility and membrane permeability, Microemulsions – provide a stable delivery system with enhanced solubility, Combination with piperine – inhibits P-glycoprotein efflux and intestinal metabolism, Cyclodextrin inclusion complexes – improve aqueous solubility while maintaining stability, Solid dispersion techniques – enhance dissolution rate and solubility, Co-administration with other flavonoids that may compete for metabolic enzymes, potentially extending eupafolin’s half-life, Nanoparticle formulations – improve stability and targeted delivery, particularly relevant for anticancer and neuroprotective applications

Eupafolin is best absorbed when taken with meals containing some fat, which can enhance solubility and stimulate bile secretion, improving dissolution and absorption. The presence of other flavonoids may enhance eupafolin’s bioavailability through competitive inhibition of metabolic enzymes or transporters. For anti-inflammatory and antioxidant effects, timing is less critical than consistency of use, though divided doses throughout the day may maintain more consistent blood levels due to eupafolin’s relatively short half-life (approximately 3-5 hours in animal studies). For neuroprotective effects, consistent daily dosing is important for maintaining protective mechanisms against oxidative stress and neuroinflammation.

For cardiovascular benefits, morning dosing may be beneficial due to potential effects on endothelial function and blood flow throughout the day. Enhanced delivery formulations like nanoemulsions or liposomes may have different optimal timing recommendations based on their specific pharmacokinetic profiles, but generally follow the same principles of taking with food for optimal absorption. Traditional use of Eupatorium perfoliatum containing eupafolin often involves preparing it as a tea or tincture, which may have different absorption characteristics compared to modern extract formulations. When consumed as a tea, the hot water extraction efficiently extracts eupafolin due to its good water solubility, but the absence of lipids may limit absorption compared to when taken with a meal.

• Gastrointestinal discomfort (mild to moderate)
• Nausea (uncommon)
• Diarrhea (uncommon)
• Headache (rare)
• Dizziness (rare)
• Allergic reactions (rare, but more common in individuals with allergies to plants in the Asteraceae family)
• Mild sedation (rare)
• Dry mouth (uncommon)

• Pregnancy and breastfeeding (due to insufficient safety data and potential effects on hormone levels)
• Individuals with known allergies to plants in the Asteraceae family (including ragweed, chrysanthemums, marigolds, and daisies)
• Scheduled surgery (discontinue 2 weeks before due to potential anticoagulant effects)
• Individuals taking medications metabolized by CYP enzymes (due to potential interactions)
• Individuals with severe liver or kidney disease (due to limited data on metabolism and excretion in these populations)
• Individuals with bleeding disorders (due to potential antiplatelet activity)
• Individuals with autoimmune conditions (due to immunomodulatory effects, which could potentially exacerbate or ameliorate symptoms depending on the condition)

• Anticoagulant and antiplatelet medications (may enhance bleeding risk due to potential antiplatelet effects)
• Cytochrome P450 substrates (eupafolin may affect the metabolism of drugs that are substrates for CYP enzymes, particularly CYP1A2, CYP2C9, and CYP3A4)
• P-glycoprotein substrates (may alter drug transport and absorption)
• Immunosuppressants (may interfere with therapeutic effects through immunomodulatory actions)
• Anti-inflammatory medications (potential for additive effects or interactions with NSAIDs and other anti-inflammatory drugs)
• Antioxidant medications (potential for additive effects with other antioxidants)
• Antihypertensive medications (may enhance blood pressure-lowering effects)
• Antidiabetic medications (may enhance blood glucose-lowering effects)
• Chemotherapeutic agents (potential interactions due to effects on cell cycle and apoptosis pathways, which could either enhance or reduce efficacy depending on the specific drug)

Due to limited human clinical data on isolated eupafolin, a definitive upper limit has not been established. Based on safety data for Eupatorium perfoliatum extracts (which contain eupafolin) and animal toxicity studies, doses up to 30 mg of eupafolin daily or 900 mg of standardized extract daily appear to be well-tolerated in most individuals. For general supplementation, doses exceeding these levels are not recommended without medical supervision due to potential drug interactions and limited long-term safety data at higher doses. It’s important to note that the presence of other bioactive compounds in herbal extracts may contribute to the overall safety profile, making it difficult to establish precise upper limits for isolated eupafolin.

Traditional use of Eupatorium perfoliatum (Boneset) in high doses has been associated with toxicity, including nausea, vomiting, diarrhea, and liver damage, though these effects are likely due to other compounds in the plant rather than eupafolin specifically.

Eupafolin itself is not approved as a drug by the FDA and is not commonly available as an isolated supplement. Plant extracts containing eupafolin, such as Eupatorium perfoliatum extracts, are regulated as dietary supplements under the Dietary Supplement Health and Education Act (DSHEA) of 1994. Manufacturers cannot make specific disease treatment claims but may make general structure/function claims with appropriate disclaimers. The FDA has not evaluated the safety or efficacy of eupafolin specifically.

Eupatorium perfoliatum is generally recognized as safe (GRAS) when used in traditional amounts as an herb or supplement, though it is not commonly used in food products due to its bitter taste.

Eu: In the European Union, eupafolin is not approved as a medicinal product. Eupatorium perfoliatum extracts containing eupafolin may be sold as food supplements, subject to the general food safety regulations. The European Food Safety Authority (EFSA) has not issued specific health claims for eupafolin or Eupatorium perfoliatum extracts. In some EU countries, particularly Germany, Eupatorium perfoliatum is recognized as a traditional herbal medicinal product for specific indications.

Germany: In Germany, Eupatorium perfoliatum is recognized by Commission E (the German regulatory authority for herbs) as a traditional herbal medicinal product for treating fever and common cold. It is available in various formulations, including teas, tinctures, and tablets.

Canada: Health Canada regulates Eupatorium perfoliatum extracts as Natural Health Products (NHPs). Several products containing these extracts have been issued Natural Product Numbers (NPNs), allowing them to be sold with specific health claims related to traditional use, such as ‘traditionally used to help relieve symptoms of colds and flu’ and ‘traditionally used to help reduce fever.’ Isolated eupafolin is not specifically approved as a standalone ingredient.

Australia: The Therapeutic Goods Administration (TGA) regulates Eupatorium perfoliatum extracts as complementary medicines. Several products containing these extracts are listed on the Australian Register of Therapeutic Goods (ARTG). Traditional use claims are permitted with appropriate evidence of traditional use. Eupafolin as an isolated compound is not specifically regulated.

China: Eupatorium perfoliatum is not officially recognized in the Chinese Pharmacopoeia, though some related Eupatorium species are used in Traditional Chinese Medicine. Eupafolin as an isolated compound is primarily used in research rather than as an approved therapeutic agent.

Japan: Eupatorium perfoliatum is not commonly used in Japanese traditional medicine (Kampo). Eupafolin as an isolated compound is not specifically regulated for therapeutic use.

Medium to high

Isolated eupafolin is rarely available commercially for supplementation and is primarily sold as a research chemical at prices ranging from $300-$800 per 10-25 mg, making

it prohibitively expensive for regular supplementation. Standardized Eupatorium perfoliatum extracts containing eupafolin along with other flavonoids typically cost $0.50-$2.00 per day for basic extracts and $2.00-$5.00 per day for premium, highly standardized formulations. Dried Eupatorium perfoliatum for tea preparation is the most cost-effective option, typically costing $0.20-$0.80 per day, though

it provides less consistent and potentially lower amounts of eupafolin.

The cost-effectiveness of eupafolin must be evaluated in the context of herbal extracts containing it, as isolated eupafolin is not practically available for regular supplementation due to its high cost and limited commercial availability. For anti-inflammatory effects, Eupatorium perfoliatum extracts containing eupafolin offer moderate value compared to other botanical anti-inflammatories. While not as potent as some pharmaceutical anti-inflammatories, they provide a broader spectrum of benefits at a lower cost and potentially fewer side effects for long-term use. For antioxidant benefits, there are likely more cost-effective options than eupafolin-containing extracts, as many other botanical antioxidants have stronger clinical evidence and lower costs.

For neuroprotective effects, particularly in the context of cerebral ischemia/reperfusion injury, the value proposition is promising based on preclinical studies, but clinical evidence is still lacking. The long-term benefits for neurodegenerative conditions would need to be substantial to justify ongoing supplementation costs. For anticancer applications, the current state of research does not support the use of eupafolin or Eupatorium perfoliatum extracts as standalone cancer treatments, regardless of cost considerations. They may have potential value as adjunctive therapy, but this should only be considered under medical supervision as part of a comprehensive treatment plan.

When comparing the cost-effectiveness of Eupatorium perfoliatum extracts containing eupafolin to other supplements with similar indications: For fever and cold symptoms (based on traditional use), they are comparably priced to other herbal remedies like elderberry or echinacea, but with less clinical evidence supporting their use. For anti-inflammatory effects, they are moderately priced compared to alternatives like curcumin or boswellia, offering good but not exceptional value. The most cost-effective way to consume eupafolin is through traditional Eupatorium perfoliatum tea, which can be prepared from dried herb at a fraction of the cost of processed extracts. However, the concentration of eupafolin and other active compounds may be lower and less consistent in tea preparations compared to standardized extracts.

Enhanced delivery systems such as nanoemulsions, liposomes, or SEDDS offer better bioavailability and potentially superior therapeutic outcomes, which may justify their higher cost for specific health conditions. For example, eupafolin nanoparticles have shown enhanced efficacy in protecting against particulate matter-induced inflammation and oxidative stress in skin cells, suggesting potential value for environmental protection applications.

Pure eupafolin is moderately stable, with a typical shelf life of 2-3 years when properly stored. The balanced hydroxyl/methoxy structure (four hydroxyl groups and one methoxy group) provides moderate stability, though the catechol structure in the B-ring (3′,4′-dihydroxy) makes it somewhat susceptible to oxidation. Standardized herbal extracts containing eupafolin, such as Eupatorium perfoliatum extracts, typically have a shelf life of 1-2 years from the date of manufacture. Dried herb material (e.g., Eupatorium perfoliatum) properly stored can maintain acceptable eupafolin content for 1-2 years.

Tea preparations have a much shorter shelf life, with optimal potency maintained for only a few hours after preparation. Enhanced delivery formulations such as nanoemulsions or liposomes generally have shorter shelf lives of 1-2 years, depending on the specific formulation and preservative system.

Store in a cool, dry place away from direct sunlight in airtight, opaque containers. Refrigeration is recommended for liquid formulations and can extend shelf life of extracts containing eupafolin. Protect from moisture, heat, oxygen, and light exposure, which can accelerate degradation. For research-grade pure eupafolin, storage under inert gas (nitrogen or argon) at -20°C is recommended for maximum stability.

For dried herb material (e.g., Eupatorium perfoliatum), store in airtight containers away from light and moisture to preserve the eupafolin content. The addition of antioxidants such as vitamin E or ascorbic acid to formulations can help prevent oxidation and extend shelf life. Enhanced delivery formulations may have specific storage requirements provided by the manufacturer, which should be followed carefully to maintain stability and potency. Avoid repeated freeze-thaw cycles, particularly for liquid formulations, as this can destabilize the product.

Exposure to UV light and sunlight – causes photodegradation, particularly affecting the catechol structure in the B-ring, High temperatures (above 30°C) – accelerates decomposition, Moisture – can promote hydrolysis and microbial growth, particularly in liquid formulations, Oxygen exposure – leads to oxidation, particularly of the catechol structure in the B-ring, pH extremes – eupafolin is most stable at slightly acidic to neutral pH (5-7), Metal ions (particularly iron and copper) – can catalyze oxidation reactions, with the catechol structure being particularly susceptible to metal-catalyzed oxidation, Enzymatic activity – may occur in improperly processed plant extracts, particularly polyphenol oxidases that can degrade eupafolin, Incompatible excipients in formulations – certain preservatives or other ingredients may interact negatively with eupafolin, Repeated freeze-thaw cycles – can destabilize enhanced delivery formulations such as nanoemulsions or liposomes

Synthesis Methods

Natural Sources

Quality Considerations

Eupafolin itself was not identified or isolated until the modern era, but it is a constituent of several plants that have been used in traditional medicine systems for centuries. While the specific contribution of eupafolin to the traditional uses of these plants was unknown to ancient practitioners, it is now recognized as one of the bioactive compounds in these historically important medicinal materials. Eupafolin is primarily found in Eupatorium perfoliatum (Boneset), which has a rich history in traditional North American medicine. Eupatorium perfoliatum was widely used by various Native American tribes, including the Cherokee, Iroquois, and Mohegan, long before European settlement.

The common name ‘Boneset’ is derived from its traditional use in treating dengue fever (also called ‘breakbone fever’ due to the severe bone and joint pain it causes) and other febrile illnesses that cause bone pain. Native Americans used the plant for treating fevers, colds, influenza, and rheumatic conditions. They typically prepared it as a hot tea or decoction from the leaves and flowering tops. When European settlers arrived in North America, they quickly adopted Boneset into their pharmacopeia, learning its uses from Native Americans.

By the 18th and 19th centuries, Boneset became one of the most widely used medicinal plants in American folk medicine and was included in the United States Pharmacopeia from 1820 to 1916. It was particularly valued during epidemics of influenza, dengue fever, and malaria. The famous American physician Dr. Benjamin Smith Barton wrote in 1798 that Boneset was ‘a medicine of great value’ and recommended it for treating fevers.

In traditional use, Boneset was primarily employed for its diaphoretic (sweat-inducing), antipyretic (fever-reducing), and anti-inflammatory properties. It was commonly used to treat influenza, fever, upper respiratory infections, rheumatism, and digestive disorders. The plant was typically prepared as a hot infusion or tea, which was consumed several times daily during acute illnesses. The intensely bitter taste of Boneset tea was considered a sign of its medicinal potency, though it made the remedy somewhat unpleasant to take.

In addition to Eupatorium perfoliatum, eupafolin is also found in other plants with traditional medicinal uses. Phagnalon rupestre has been used in Mediterranean traditional medicine for treating inflammatory conditions and wounds. Dittrichia viscosa (formerly Inula viscosa) has been used in traditional Mediterranean and Middle Eastern medicine for its anti-inflammatory, antimicrobial, and wound-healing properties. Various Artemisia species containing eupafolin have been used in traditional medicine systems worldwide for treating fevers, parasitic infections, and inflammatory conditions.

Eupafolin was first isolated and characterized in the mid-20th century as part of the scientific investigation into the active components of these traditional medicinal plants. Its structure was elucidated as 6-methoxyluteolin or 5,7,3′,4′-tetrahydroxy-6-methoxyflavone, identifying it as a partially methoxylated flavone. Modern scientific interest in eupafolin began to grow in the late 20th and early 21st centuries as research revealed its anti-inflammatory, antioxidant, and potential anticancer properties. The discovery of eupafolin’s effects on inflammatory signaling pathways, particularly its inhibition of iNOS expression and NF-κB activation, has provided scientific explanations for some of the traditional uses of Eupatorium perfoliatum, particularly its applications in inflammatory and febrile conditions.

Today, Eupatorium perfoliatum extracts containing eupafolin are used in various herbal formulations, particularly in North America and Europe, for treating inflammatory conditions, upper respiratory infections, and as an immunomodulator. However, the use of pure eupafolin as a supplement is still relatively uncommon, with most commercial products containing it as part of complex herbal extracts.

No meta-analyses specifically on eupafolin are currently available; most analyses focus on Eupatorium perfoliatum extracts or flavonoids as a group.

Limited ongoing trials specifically investigating eupafolin; most research remains at the preclinical stage, Several preclinical studies investigating eupafolin’s potential in neurodegenerative diseases, particularly focusing on its neuroprotective properties in cerebral ischemia/reperfusion injury, Research on eupafolin’s anti-inflammatory and antioxidant effects in various disease models, including respiratory and skin conditions, Investigations into eupafolin’s potential as an anticancer agent, particularly focusing on its effects on the STAT3 and PI3K/Akt/mTOR signaling pathways, Studies on novel delivery systems to enhance eupafolin’s bioavailability and targeted delivery