IngredientsPolymethoxylated flavones (PMFs) are a unique class of flavonoids with two or more –OCH3 groups on their chemical flavone skeletons, and they are widely distributed in the Rutaceae, Compositae, Labiatae, Verbenaceae, and Zingiberaceae plant families. More precisely, PMF is a general term for flavones bearing from two to seven methoxy groups in different positions on their basic benzo-γ-pyrone (15-carbon) skeleton, consisting of two benzene rings joined by a linear three-carbon chain with a carbonyl group at the C4-position.
PMFs have a low polarity and are created when two or more hydroxyl groups on the A and B rings of the flavone nucleus are replaced with two or more methoxy groups. Depending on the location and number of methoxy substitutions, PMFs can be categorized into different types. Methoxylation renders the molecule more lipophilic while hydroxylation makes the molecule more hydrophilic; this change in hydrophobicity markedly affects their biological activities.
Compared with other flavonoids, PMFs with more –OCH3 groups tend to be less polar and show approximately planar structures, resulting in stronger bioactivity in localizing to and penetrating cancer cells due to higher hydrophobicity. This enhanced lipophilicity also improves their ability to cross biological membranes relative to unmethylated flavones.
Polymethoxyflavones (PMFs) are a group of flavonoids found almost exclusively in the genus Citrus, especially in the peels. Citrus fruits are the main dietary source of PMFs with significant effects on consumer health. PMFs occur naturally in citrus peels and citrus-derived foods as well as in other plants.
The most extensively studied dietary species include sweet orange (Citrus sinensis), sour orange (Citrus aurantium), mandarin (Citrus reticulata), clementine (Citrus clementina), lime (Citrus aurantifolia), grapefruit (Citrus paradisi), tangelo, and pummelo (Citrus maxima). Nobiletin is abundant in citrus fruits such as Citrus reticulata and Citrus depressa, and is particularly abundant in the peel (outer pericarp) and albedo (middle pericarp), which are the pericarp parts.
Thirty-five PMFs have been identified from citrus species so far. Among them, nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) and tangeretin (5,6,7,8,4′-pentamethoxyflavone) are the most abundant. Other identified PMFs include sinensetin, heptamethoxyflavone (3,3′,4′,5,6,7,8-heptamethoxyflavone, HMF), isosinensetin, 5′-demethylnobiletin, tetramethyl-O-scutellarein, pentamethoxyflavone, tetramethyl-O-isoscutellarein, and gardenin forms. A total of 42 PMFs have been detected in C. reticulata and C. sinensis peels.
The flavonoid biosynthesis location is in the cytoplasm of the flavedo cells and then they are transferred to the vacuoles. A previous study showed that five O-methyl transferase (OMT) genes isolated from Citrus depressa promoted the accumulation of nobiletin in the flavedo; therefore, PMFs might be synthesized from flavone aglycones by OMTs. Citrus PMFs act as essential barriers against pathogen attack and can be induced to accumulate in fruits.
In the dietary supplement market, PMFs are available primarily as standardized extracts of citrus peel, typically standardized to defined concentrations of nobiletin and tangeretin. Dried citrus peel derived from Citrus reticulata, also called "chenpi," possesses a complex mixture of flavonoids. Sources of conventional chenpi include geographic varieties from California, Guangxi, and Zhejiang; Xinhui orange peel extract had the highest content of polymethoxylated flavones, along with the greatest capacity to scavenge free radicals.
Supplement preparations include capsules, tablets, and blended nutraceutical formulations. Hot water extraction of citrus peels efficiently extracts phenolic compounds including flavanone glycosides, PMFs, and phenolic acids. Among citrus flavonoids, nobiletin and tangeretin are reported to be easier to extract by hot water than hesperidin. PMFs are highly lipophilic; absorption is enhanced by dietary fat.
In China, the dried mandarin peel called "Chenpi" (Citri Reticulatae Pericarpium), has been used for disease treatment for two thousand years, dating back to the Han dynasty, according to the earliest work on Chinese medicine, the Shennong Bencao Jing.
Citri Reticulatae Pericarpium (CRP), commonly referred to as Chenpi (陈皮) in Chinese, is an orange-colored Citrus reticulata Blanco fruit peel. Since thousands of years in China, CRP has been most frequently used as a qi-regulating drug in traditional Chinese medicine (TCM) prescriptions for clinical treatment. The "Xing qi" (行气) effect of CRP promotes the circulation of qi (energy) throughout the body and is widely used in a variety of "stagnation" characteristics of qi, such as food stagnation with pain and distention symptoms. Furthermore, as described in the books on TCM, CRP is also regarded as drying dampness and resolving phlegm.
The dried mature peels of Citrus reticulata cv. Chachiensis, named "Guangchenpi," have been used as a traditional Chinese medicine to treat cough, indigestion, and lung diseases for several hundred years. Flavonoids are one of the main medicinal ingredients of "Guangchenpi," and PMFs with four or more methoxy groups are the key component that can distinguish "Guangchenpi" significantly from ordinary "Chenpi."
In traditional Chinese medicine, the dried ripe pericarps of Citrus reticulata or its cultivars, namely Pericarpium Citri Reticulatae (PCR, Chenpi), were used to treat chronic diseases such as coughing, stomach upset, and skin inflammation. Throughout history, PMF-rich citrus peels were combined with other botanicals in herbal formulations to enhance their effectiveness. For example, Chenpi is frequently blended with licorice root, ginger, or ginseng in classic Chinese formulas designed to harmonize the stomach, promote energy, and relieve respiratory discomforts.
Citrus peels are a rich source of polymethoxylated flavones and have been widely used as a crude drug in traditional herbal medicines. Citrus fruits are a rich source of PMFs and hydroxylated PMFs, particularly in their peels, which have been used as commonly available herbal medicines for thousands of years.
Nobiletin-rich Citrus reticulata peel preparations have also been used in the Japanese Kampo tradition, with case studies exploring their use in the context of cognitive decline. In the course of a survey of substances from natural resources having anti-dementia and neuroprotective activity, nobiletin was found in the peel of Citrus depressa.
The most prominent citrus PMFs with proven pharmacological effects are tangeretin, nobiletin, 5′-demethylnobiletin, tetramethyl-O-scutellarein, pentamethoxyflavone, tetramethyl-O-isoscutellarein, and sinensetin.
Approximately 140 chemical compounds in total have been isolated and identified from Citri Reticulatae Pericarpium.
The beneficial anti-inflammatory effects of nobiletin in muscle cells are accompanied by suppression of ROS production and reduction of MAPK activity (especially JNK) and inhibition of the NF-κB signaling pathway. Results from cell and animal studies indicate that nobiletin exhibits a therapeutic anti-inflammatory effect through active inhibition of the NF-κB signaling pathway.
Research supports the view that tangeretin can effectively interfere with TNF-α-driven inflammatory signaling and suppress NF-κB/MAPK activity under pathological conditions. Molecular dynamics simulations and in vitro cellular experiments of tangeretin have shown that tangeretin stably binds to NLRP3 protein and inhibits the activation of NLRP3 inflammasome by regulating the MAPK/NF-κB/NLRP3 signaling pathway.
PMFs have shown many biological activities such as anticancer, anti-inflammatory, antiallergic, antimutagenic, antiatherosclerosis, hypoglycemic, antioxidant, and neuroprotective effects. The methoxylation of hydroxyl groups increases metabolic stability and favors passive diffusion through biological membranes, improving the delivery of antioxidant capacity to target tissues compared to unmethylated flavonoids.
Transcriptome and gene ontology analyses showed that nobiletin significantly downregulated genes associated with the regulation of lipid metabolic processes. Among these genes, angiopoietin-like 3 (ANGPTL3), an inhibitor of lipoprotein lipase (LPL) activity that regulates triglyceride-rich lipoprotein metabolism in circulation, was the protein most markedly downregulated by nobiletin.
In adipocyte studies, both nobiletin and tangeretin decreased triglyceride accumulation, and the inhibitory efficacy followed the order nobiletin > tangeretin > hesperetin, indicating that the number of methoxyl groups on the benzene ring plays an important role in reducing lipid accumulation. These results imply that polymethoxyflavones with a high degree of methoxylation are more easily permeable to cell membranes and demonstrate greater ability to regulate adipogenesis.
At the cancer initiation stage, bioactivation of polyaromatic hydrocarbon carcinogens and binding to DNA are markedly diminished through effects on CYP1A1/1B1 transcription but also through direct interactions with the proteins. At the cancer promotion stage, the proliferation of cancer cells, but not normal cells, is inhibited with greater potency than with unmethylated flavones. The anticancer mechanisms of PMFs include estrogen activity modulation, cytochrome P-450 enzyme system interaction, and arylhydrocarbon receptor (AhR) inhibition, along with various molecular targets.
The neuroprotective action mechanisms of PMFs involve diverse functions, such as antioxidant effects, anti-inflammatory effects, inhibition of Aβ pathology, suppression of neurodegeneration and neuronal cell death, and regulation of neurotrophic signals and synaptic plasticity. PMFs, the key lipid-soluble constituents in Citri Reticulatae Pericarpium, exhibit excellent blood–brain barrier permeability and anti-inflammatory properties, holding therapeutic potential for central nervous system disorders.
Methoxyflavones represent a flavonoid subclass in which all hydroxyl groups are capped by methylation; this results in dramatically increased metabolic stability and membrane transport in the intestine/liver, thus improving oral bioavailability. The methoxyflavones also show increased cancer chemopreventive properties. Most unmethylated dietary flavonoids have low bioavailability, but methylated dietary flavonoids not only retain the anticancer activity of unmethylated dietary flavonoids, but also have higher water solubility, metabolic stability, bioavailability, and lower toxicity.
Preclinical evidence: Several preclinical studies, including in vitro and animal models, have shown that PMFs can help lower cholesterol levels, reduce inflammation, and improve endothelial function, all of which are factors in cardiovascular health. In macrophage studies, nobiletin inhibited (50–72%) acetylated LDL metabolism as measured by cellular cholesterol ester mass. Among differentially expressed genes in hepatic cells treated with nobiletin, angiopoietin-like 3 (ANGPTL3), an inhibitor of lipoprotein lipase activity, was the protein most markedly downregulated.
Both nobiletin and tangeretin decreased triglyceride accumulation, decreased the secretion of monocyte chemoattractant protein-1 (MCP-1), and increased adiponectin in 3T3-L1 adipocytes. In addition, nobiletin prevented resistin secretion from mature 3T3-L1 adipocytes.
Human/clinical evidence — limited and largely indirect: A human clinical trial (the NIRVANA study) evaluated the effect of a nutraceutical preparation on lipid profile, endothelial function, and oxidative stress. Each capsule consisted of red yeast rice containing monacolin K, PMFs from a tangerine extract (mainly nobiletin and tangeretin), hydroxytyrosol from olive fruit extract, phenolic acids and flavonoids from an Ipomoea batatas extract, vitamin E, and coenzyme Q10. However, this clinical study cannot be considered as an effect of PMFs on human health in isolation, as they are constituents of a mixture of nutrients at very low concentration among other compounds, and the beneficial effect might be due to other bioactives or synergy among all of them.
Strength of evidence: Preclinical (in vitro and animal) evidence for cardiovascular and lipid-lowering effects is substantial. Isolated human clinical evidence for PMFs is extremely limited; no large, well-controlled human trials demonstrating cardiovascular benefit specifically attributable to PMFs have been published as of the current literature.
Preclinical evidence: A systematic review of the effect of PMFs on obesity identified 16 studies meeting the criteria. The range of dosages varied from 10 to 200 mg/kg over 5–26 weeks in animal studies, and 1–100 μmol over 2 hours to 8 days in in vitro studies. Nobiletin (10 to 100 μmol) administered to 3T3-L1 cells for 48 hours significantly inhibited differentiation of 3T3-L1 preadipocytes into adipocytes in a dose-dependent manner.
Studies have shown that nobiletin exerts anti-adipogenic effects through modification of the AMPK signaling pathway and prompts brown adipocyte-like phenotype in 3T3-L1 cells. In high-fat diet–induced obese mice, nobiletin decreased body weight gain, white adipose tissue weight, and plasma triglyceride; plasma glucose levels tended to decrease compared with the high-fat diet group, with improved plasma adiponectin levels and glucose tolerance.
Human/clinical evidence: The intake of PMFs at 12 mg/day reduced visceral fat in overweight Japanese individuals, indicating that dietary PMFs are absorbed in the intestines and circulate through the body in the bloodstream. This represents one of the few directly relevant human data points for PMFs in metabolic outcomes, but data from this single study are insufficient to draw broad clinical conclusions.
Strength of evidence: Evidence for anti-obesity and metabolic effects is predominantly from animal and in vitro studies, with one small human observation supporting bioavailability and visceral fat reduction. Larger, well-powered human trials are needed.
Preclinical evidence: Nobiletin improved cognitive deficits and the pathological features of AD, such as Aβ pathology, hyperphosphorylation of tau, and oxidative stress, in animal models. In addition, nobiletin improved motor and cognitive deficits in Parkinson's disease animal models. These observations suggest that nobiletin has the potential to become a novel candidate for the treatment and prevention of neurodegenerative diseases such as AD and Parkinson's disease.
Results from mechanistic studies suggested that PMFs have potential therapeutic value for Alzheimer's disease (AD)-like mice, with the inhibition of neuroinflammation likely being a key mechanism of their anti-AD effects. Sinensetin, tangeretin, nobiletin, and 3,5,6,7,8,3′,4′-heptamethoxyflavone were identified as potent neuroinflammatory inhibitors.
In a middle cerebral artery occlusion (MCAO) rat model, nobiletin treatment notably improved neurological deficits, brain water content, and brain index, and was accompanied by a decreased infarct area. Apoptosis induced by cerebral ischemia-reperfusion was decreased by nobiletin administration via upregulating Bcl-2 and downregulating Bax and caspase-3. The levels of pro-inflammatory mediators TNF-α and IL-6 were reduced and anti-inflammatory cytokine IL-10 was increased by nobiletin treatment. Furthermore, the expression of p-p38 and MAPKAP-2 was reduced by nobiletin treatment.
Human/clinical evidence: Basic preclinical studies prompted several clinical trials on the intake of citrus components and human neurological function; interestingly, clinical and epidemiological studies showed improved cognitive function and reduced disease risk in patients and healthy subjects, broadly consistent with preclinical studies. Clinical and epidemiological studies appear to support preclinical evidence and show improved cognitive function and reduced associated disease risk in healthy individuals and/or patients. However, the available clinical trials are small in number, and many involve citrus peel extracts rather than isolated PMFs, making it difficult to attribute effects specifically to PMFs.
Strength of evidence: Strong animal and mechanistic in vitro evidence; early supportive human epidemiological and limited clinical data. Dedicated large-scale randomized controlled trials of isolated PMFs in humans with neurodegenerative diseases are lacking.
Preclinical evidence: The anti-cancer activity of PMFs has been demonstrated in colon carcinogenesis animal models: PMFs inhibit the proliferation of cells, induce apoptosis, and reduce the formation of tumors. The anticancer mechanisms of PMFs include estrogen activity modulation, cytochrome P-450 enzyme system inhibition, and arylhydrocarbon receptor (AhR) inhibition, along with various molecular targets. These include estrogen activity, arylhydrocarbon receptor inhibition, multidrug resistance protein inhibition, and chemical sensitization.
Tangeretin has been studied specifically across multiple cancer cell types. Tangeretin is known to exhibit considerable selective toxicity to many types of cancer cell proliferation such as ovarian, brain, blood, and other cancer cell lines.
Human/clinical evidence: Although PMFs may be advantageous in the prevention and treatment of breast cancer, there is a lack of clinical evidence and data to support their efficacy. This assessment applies broadly across cancer types: the field remains substantially in preclinical stages.
Strength of evidence: Substantial in vitro and animal evidence; essentially no direct human clinical trial evidence for anticancer outcomes. Research remains preliminary for all cancer applications.
In a mouse model of osteoarthritis, injection of nobiletin (20 mg/kg) every 2 days for 8 weeks after surgery inhibited cartilage destruction and synovitis. Results indicated that nobiletin exhibited a therapeutic effect through active inhibition of the NF-κB signaling pathway. These findings are from in vivo animal and cell models; no dedicated human clinical trials in arthritis with isolated PMFs have been identified.
Several studies have shown that PMFs have a protective effect on bone resorption in mouse models of diseases including osteoporosis, rheumatoid arthritis, and periodontal disease. PMFs significantly suppressed the differentiation of osteoclasts (bone resorptive cells) through indirect and direct mechanisms.
Strength of evidence: Preclinical animal and cell evidence only; no human trials identified.
The difference of PMFs from other flavonoids is that the former possesses more than one methoxy group (–CH3O), which has been suggested to significantly influence the bioactivities of PMFs. Most PMFs are hydrophobic, leading to their poor bioavailability when consumed orally.
The poor solubility of PMFs limits their bioaccessibility in the human gut system. In addition, a high metabolic rate influences the amount of PMFs entering the human circulatory system. This ultimately leads to poor bioavailability and reduces the therapeutic dosages they can achieve in the target organs.
The understanding of PMF metabolism in humans and their direct effects on human health remains limited. The main sources of information on these health effects in humans are derived from two recent clinical trials, an observational study, and a pilot study. However, no data on bioavailability and metabolism (pharmacokinetics, human phase I and II metabolism in biological fluids and tissues, gut microbiota metabolism) were reported.
The absorption and metabolism of PMFs in the body involves methylation, demethylation, and hydroxylation processes. Tangeretin, for example, undergoes phase I demethylation in the liver and intestine, producing hydroxylated metabolites. The 3′-hydroxylated tangeretin (T3) was detected among tangeretin metabolites for the first time, suggesting that the 4′-demethylated and 3′-hydroxylated derivative of tangeretin is not only derived from 4′-demethylated tangeretin as previously reported, but also from T3.
Despite the general low oral bioavailability of PMFs, the methoxylated structure is significantly advantageous compared with unmethylated flavones. In methoxyflavones, all hydroxyl groups are capped by methylation, resulting in dramatically increased metabolic stability and membrane transport in the intestine and liver, thus improving oral bioavailability.
The following dosages reflect those specifically reported in the peer-reviewed literature cited, not recommendations.
Human pharmacokinetic data remain sparse. Many in vitro and some in vivo studies have shown potentially relevant biological effects of PMFs; these promising biological effects still require further research to establish their impact on human health.
PMFs have shown biological activities associated with the following areas: anticancer, anti-inflammatory, antiallergic, antimutagenic, antiatherosclerosis, hypoglycemic, antioxidant, and neuroprotective effects. In vivo and in vitro preclinical studies of PMFs have highlighted molecular mechanisms and signaling pathways for anti-inflammatory, anti-cancer, chemopreventive, antidiabetic, anti-obesity, hepatoprotective, and neuroprotective effects in neurodegenerative diseases.
PMFs are generally quite safe, which is a major advantage. In vitro genotoxicity testing: observed results indicate the safety profile of PMF mixture and excluded any possibility of genotoxicity from in vitro assay systems. One study found a statistically insignificant positive relationship between increasing PMF concentrations and spleen weight in sheep red blood cell (SRBC) immunized mice. In mice without immunization, there was no evidence for spleen weight changes.
Excessive long-term intake of PMFs may indicate some toxicity, since a high concentration of PMFs may lead to inhibited cell growth or cell death in various cell types, including osteoblasts, endothelial cells, and cancer cell lines. This concern is derived from cell culture experiments and may not reflect physiologically relevant concentrations from dietary or supplemental intake.
In P-glycoprotein transport studies using Caco-2 cells, PMFs significantly decreased talinolol transport from the basolateral to apical side, where tangeretin had the lowest IC50 of 3.2 μmol/L, followed by nobiletin, heptamethoxyflavone, and sinensetin with IC50 values of 3.5, 3.8, and 3.9 μmol/L, respectively. This study suggests that PMFs have a high potential to interact with P-glycoprotein–mediated transport, but based on their relatively low concentrations in citrus foods, the clinical relevance of these interactions needs to be further elucidated in in vivo studies.
Tangerine fruit juice was reported to upregulate CYP3A4 activity and inhibit P-glycoprotein due to the high content of flavonoid tangeretin. Some data reported the opposite effect—that tangeretin inhibits CYP3A4 and CYP1A2 in human liver microsomes. This discrepancy likely reflects differences in concentration, model system, and experimental conditions.
Citrus juice has been demonstrated to affect the pharmacokinetics of various kinds of drugs by modulating drug transporters and drug-metabolizing enzymes. For instance, the flavonoid fraction of clementine juice can induce or inhibit a number of human cytochrome enzymes such as CYP3A4 and CYP1A2. In the last two decades, many studies have discussed citrus fruit–drug interactions. The simultaneous intake of tangeretin and drugs was reported to have a strong influence on pharmacokinetics.
It has been reported that the concentration of flavonoids in fruits or juices is greater than 100 μM (up to 20 mM), which is usually much higher than the effective concentration for inhibiting OATP2B1 (IC50 = 8.3 μM); when taken as dietary supplements or drugs, the intestinal concentration of these flavonoids may be higher, so the risk of interaction with drugs that are substrates of OATP2B1 must be considered.
The effects of PMFs have been mainly evidenced through in vitro assays and in some cases using preclinical studies with animal models. However, the understanding of PMF metabolism in humans and their direct effects on human health remains limited. Though further research is needed in mutual effects, safety, and clinical trials, citrus PMFs still have great potential to be developed as therapeutic agents or dietary supplements.
Health conditions that Polymethoxylated flavones may help support.
Body systems that Polymethoxylated flavones may help support.
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