IngredientsThe Annonaceous acetogenins are promising antitumor and pesticidal agents found only in the plant family Annonaceae. Chemically, they are derivatives of long-chain fatty acids. More precisely, the Annonaceous acetogenins are a series of apparently polyketide-derived fatty acid derivatives that possess tetrahydrofuran rings and a methylated gamma-lactone (sometimes rearranged to a methyl ketolactone) with various hydroxyl, acetoxyl, and/or ketoxyl groups along the hydrocarbon chain.
Annonaceous acetogenins are a unique class of C35 or C37 secondary metabolites derived from the polyketide pathway. Most acetogenins are white waxy derivatives of long-chain fatty acids (C32 or C34), and the terminal carboxylic acid is combined with a 2-propanol unit at the C-2 position to form a methyl-substituted α,β-unsaturated-γ-lactone. One of their interesting structural features is a single, adjacent, or nonadjacent tetrahydrofuran (THF) or tetrahydropyran (THP) system with one or two flanking hydroxyl group(s) at the center of a long hydrocarbon chain. Biogenetically, it has been suggested that the THF or THP cores are generated by polyepoxidation of an unconjugated polyene followed by domino cyclizations.
A new class of polyketides, the Annonaceous acetogenins, has been isolated from Annonaceous plants growing in tropical and subtropical regions. The Annonaceae is a large family encompassing a wide variety of commercially and ethnobotanically important species. The most studied sources include:
In 1982, Jolad et al. isolated uvaricin, a new antitumor agent, from the roots of Uvaria acuminata (Annonaceae), a bis-tetrahydrofuranoid fatty acid lactone related to polyketides or acetogenins. It contained a number of original structural characteristics, particularly a linear acetogenin, a bis-tetrahydrofuran pattern flanked by hydroxyls and a terminal unsaturated lactone. Subsequent work described new products presenting the same structural characteristics; because these products formed a new class of natural compounds, and are only found in species belonging to the family Annonaceae, they are commonly called acetogenins from Annonaceae.
Since the isolation of the first acetogenin, uvaricin, more than 400 members of the family have been found and characterized. Out of the 532 compounds reviewed in one comprehensive survey, 115 previously non-described annonaceous acetogenins were added to the list of isolated compounds from 2005 to May 2019.
Acetogenins are classified according to the number and arrangement of their central oxygenated ring systems. The relative potency of acetogenins can be categorized as adjacent bis-THF ACGs > nonadjacent bis-THF ACGs > mono-THF ACGs > linear-THF ACGs. Key named members of each structural class include:
Among adjacent bis-THF ACGs, asiminocin, asiminecin, asiminacin, and asimin are the most active compounds with in-vitro activity (ED50) in the range of 10−9 to 10−12 μg/mL.
The fruits of A. muricata are extensively used to prepare syrups, candies, beverages, ice creams and shakes. In the supplement market, acetogenin-containing preparations are sold as dried leaf powder (in capsule form), standardized twig extracts of Asimina triloba, liquid extracts, teas, and fruit juices. Products that contain twig extracts of pawpaw (Asimina triloba, Annonaceae) are widely consumed anticancer alternative medicines. In the agricultural sphere, various plant parts of the paw paw tree were extracted and partitioned to concentrate the mixture of acetogenins into a standardized pesticidal extract (F005). A bioassay with brine shrimp larvae was used to determine the relative potencies of the various extracts; the small twigs (0–0.5 cm diameter) yielded the most potent extract (LC50 = 0.04 ppm).
Annona muricata is a member of the Annonaceae family and is a fruit tree with a long history of traditional use. A wide array of ethnomedicinal activities is contributed to different parts of A. muricata, and indigenous communities in Africa and South America extensively use this plant in their folk medicine.
The plant produces edible fruit all year round and is widely used as a traditional medicine for skin disease, respiratory disease, fever, bacterial infections, diabetes, hypertension, and cancer. More specifically, the seeds combat parasitic infections; the fruit is used for the treatment of arthritis, nervous disorders, and diarrhea; and the leaves are used to treat cystitis, headaches, insomnia, and cancer.
Traditional use extends across multiple regions:
Numerous investigations have substantiated these activities, including anticancer, anticonvulsant, anti-arthritic, antiparasitic, antimalarial, hepatoprotective and antidiabetic activities. It is important to note that traditional use preceded any knowledge of the acetogenin class of constituents; the plants were used empirically for centuries, and the acetogenins themselves were only formally identified and named beginning in 1982.
A. muricata contains chemicals such as acetogenins (annomuricins and annonacin), alkaloids (coreximine and reticuline), flavonoids (quercetin), and vitamins, which are predicted to be responsible for the biological activity of A. muricata.
The acetogenin fraction itself contains numerous individual compounds. Key named acetogenins identified in the scientific literature include:
It is generally accepted that the mode of action of acetogenins is the inhibition of NADH–ubiquinone oxidoreductase (complex I) in mitochondria. Inhibition suppresses ATP production, especially for cancer cells with high metabolic levels, leading to apoptosis.
Biologically, acetogenins exhibit their potent bioactivities through depletion of ATP levels via inhibiting complex I of mitochondria and inhibiting the NADH oxidase of plasma membranes of tumor cells. Thus, they thwart ATP-driven resistance mechanisms.
Acetogenins were proposed to inhibit the terminal electron transfer step of mitochondrial complex I between the Fe-S cluster N2 and the ubiquinone pool. The γ-lactone moiety may bind at the quinone binding site of complex I.
The annonaceous acetogenins are the most potent of the known inhibitors of bovine heart mitochondrial complex I. These inhibitors act at the terminal electron transfer step of the enzyme, in a similar way to the usual complex I inhibitors, such as piericidin A and rotenone; however, structural similarities are not apparent between the acetogenins and these known complex I inhibitors.
Studies on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase), are useful for elucidating the structural and functional features of the terminal electron transfer step of this enzyme. Previous studies of the structure-activity relationship revealed that except for the alkyl spacer linking the two toxophores (i.e., the hydroxylated THF and the γ-lactone rings), none of the multiple functional groups of these inhibitors is essential for potent inhibition.
The functional group related to their antineoplastic activity is suggested to be the mono or bis tetrahydrofuran ring accompanied by two or more hydroxy groups.
Beyond complex I inhibition, the antitumor mechanisms of action were through apoptotic actions, negative regulation of the tumor growth factor and genotoxicity. Some acetogenins function as DNA topoisomerase I toxins, prevent cancer cells from entering their G1 phase, activate pathways linked to Bax-Bak and caspase-3, and block NADH-ubiquinone oxidoreductase in mitochondria.
Three tetrahydrofuran (THF)-containing acetogenins were found to have potent and selective antiproliferative activity against human nasopharyngeal carcinoma (NPC) cell lines and their methotrexate-resistant counterparts. The THF-containing acetogenins induced G2/M phase arrest, mitochondrial damage and apoptosis, and increased cytosolic and mitochondrial Ca2+ in NPCs. Microarray analysis of NPC cells treated with squamostatin A demonstrated an increased endoplasmic reticulum (ER)-stress response.
Pawpaw crude extract (CE) and purified acetogenins inhibited hypoxia-inducible factor-1 (HIF-1)-mediated hypoxic signaling pathways in tumor cells. In T47D cells, pawpaw CE and the acetogenins 10-hydroxyglaucanetin (1), annonacin (2), and annonacin A (3) inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.02 μg/mL, 12 nM, 13 nM, and 31 nM, respectively. This inhibition correlates with the suppression of the hypoxic induction of HIF-1 target genes VEGF and GLUT-1.
Some acetogenins show growth inhibitory activity against multidrug resistant (MDR) cancer cells. Several acetogenins like bullatacin, motrilin, asimicin, trilobacin, annonacin, gigantetronenin and squamocin are efficacious in suppressing the proliferation of the MDR MCF-7/Adr cells. This activity against MDR cells is attributed to the ATP-depletion mechanism, since cells with overexpressed ATP-dependent efflux pumps (P-glycoprotein) are preferentially vulnerable to agents that block ATP synthesis.
In vitro evidence (extensive; predominantly cell-line based):
Over 500 Annonaceous acetogenins have been described to date; these compounds are characterized by a long aliphatic chain with an α,β-unsaturated γ-lactone ring and 0–3 tetrahydrofuran (THF) rings, and have shown cytotoxic activity against different human cancer cell lines including lung, breast, colon, kidney, pancreas, prostate and liver.
A comprehensive review documented 133 acetogenin compounds for which anti-tumor activity has been documented in the literature, compiling and studying their chemical structure, in vitro as well as in vivo anticancer biological activity.
These acetogenins showed cytotoxicity against A-549 lung carcinoma, MCF-7 breast carcinoma, and HT-29 colon adenocarcinoma cell lines. Squamotacin, the acetogenin from the bark of sugar apple, has been reported to possess extremely high cytotoxicity against human prostate tumor cell line (PC-3). Two acetogenins from seeds, squadiolins A and B, showed high cytotoxicity against human hepatocellular carcinoma (HepG2) and human breast cancer (MDA-MB-231) cells.
In vivo evidence (animal models; no completed human trials):
In-vivo studies indicate that compounds like bullatacin, desacetyluvaricin, bullatalicin and annonacin have demonstrated significant activity in mouse models, thereby exhibiting potential for lead development as a potential anticancer agent/drug.
The chloroform fraction from the methanolic extract of Annona muricata seeds presented activity against triple-negative breast cancer, both in vitro and in vivo, which would be associated with the presence of acetogenins, mainly muricatacin.
As a result of a systematic literature search, it was found that the AAs are cytotoxic compounds that can induce apoptosis, cell cycle arrest, and autophagy in vitro, in addition to exhibiting tumor growth inhibition in vivo.
Limitation on clinical translation:
For the past three decades, numerous studies have elucidated the antiproliferative effects of acetogenins in hopes of developing a new class of clinical anticancer agents. However, clear and definitive action mechanisms of acetogenins were less clarified. Critically, acetogenins are thus unlikely to be effective therapeutic agents unless chemical alteration can preserve the apoptotic activity while reducing neurotoxicity. As of the available literature, no completed phase II or III human clinical trials of isolated acetogenins as anticancer agents have been published. The evidence base remains at the level of in vitro and animal studies.
Antimalarial: Acetogenins exhibit a broad range of potent biological activities including cytotoxicity, antitumor, antimalarial, antimicrobial, immunosuppressant, antifeedant, and pesticidal activity. Antimalarial effects have been demonstrated in vitro and in animal models but have not advanced to human clinical trials.
Antiparasitic: Asimina triloba contains annonaceous acetogenins which display antitumor, pesticidal, antimalarial, anthelmintic, piscicidal, antiviral, and antimicrobial effects, suggesting many potentially useful applications.
The evidence for anti-infective activity is predominantly from in vitro bioassays and limited in vivo models. No controlled human trials exist for any of these applications.
This is one of the best-documented application areas in terms of bioassay evidence. Various plant parts of the paw paw tree (Asimina triloba Dunal, Annonaceae) were extracted and partitioned to concentrate the mixture of acetogenins into a standardized pesticidal extract (F005). A bioassay with brine shrimp larvae was used to determine the relative potencies; the small twigs (0–0.5 cm diameter) yielded the most potent extract (LC50 = 0.04 ppm).
The unripe fruits, seeds, root wood, root bark, and stem bark were notably potent and, generally, yielded >2% of their dry weight as F005. Standardized pawpaw twig extracts have been investigated as botanical insecticides, including evaluation against several agricultural pest species, though formal registered biopesticide products have faced challenges relating to biomass availability and regulatory approval.
From 49 research articles that were obtained from 1981 to 2021, A. muricata's activities were shown to include anticancer (25%), antiulcer (17%), antidiabetic (14%), antiprotozoal (10%), antidiarrhea (8%), antibacterial (8%), antiviral (8%), antihypertensive (6%), and wound healing (4%).
These pharmacological activities are documented predominantly in preclinical (cell-based and animal) studies on whole plant extracts of A. muricata. The extent to which acetogenins specifically—as opposed to other constituents such as flavonoids and alkaloids—contribute to these effects has not been conclusively established in human studies.
The induction of secreted VEGF protein represents a key event in hypoxia-induced tumor angiogenesis. Both the pawpaw extract and the purified acetogenins blocked the angiogenesis-stimulating activity of hypoxic T47D cells in vitro. Pawpaw extract and acetogenins inhibited HIF-1 activation by blocking the hypoxic induction of nuclear HIF-1α protein. This is an in vitro finding; it has not been validated in clinical settings.
No standardized clinical dosage has been established for any isolated acetogenin compound, as no acetogenin has been approved as a pharmaceutical agent. Dosages referenced in the scientific literature are those used in experimental models or observed in dietary exposure studies.
Supplement products based on pawpaw twig extracts or soursop leaf do not have pharmacopeially established dosages. No published phase I dose-escalation study has defined a safe dose range for any purified acetogenin in humans.
The most extensively documented safety concern with acetogenins is their association with neurodegenerative disease. In Guadeloupe, epidemiological data have linked atypical parkinsonism with fruit and herbal teas from plants of the Annonaceae family, particularly Annona muricata.
In a key experimental study, annonacin inhibited complex I in brain homogenates in a concentration-dependent manner and, when administered systemically, entered the brain parenchyma and decreased brain ATP levels by 44%. In the absence of evident systemic toxicity, neuropathological abnormalities were observed in the basal ganglia and brainstem nuclei. Stereological cell counts showed significant loss of dopaminergic neurones in the substantia nigra (−31.7%), and cholinergic (−37.9%) and GABAergic neurones (−39.3%) in the striatum, accompanied by a significant increase in astrocytes (35.4%) and microglial cells (73.4%). The distribution of the lesions was similar to that in patients with atypical parkinsonism.
As an indication of its potential toxicity, an adult who consumes one fruit or can of nectar a day is estimated to ingest over 1 year the amount of annonacin that induced brain lesions in rats receiving purified annonacin by intravenous infusion.
ACGs could be involved in the pathogenesis of certain neurodegenerative disorders, such as in the case of the high prevalence of atypical parkinsonism that occurs in Guadeloupe, in some parts of the Afro-Caribbean region and among the Indian population residing in London and New Caledonia. This could be partially explained by the high consumption of dietary supplements and fruit products containing plant material from Annonaceae.
On Guadeloupe, atypical parkinsonism is abnormally frequent, and represents 75% of progressive parkinsonism while Parkinson's disease accounts for only 25%, which is an inversed percentage in comparison with Europe. Herbal tea made with Annonaceae leaves (containing benzyltetrahydroisoquinolines, tetrahydroprotoberberines, and acetogenins — potent mitochondrial complex I inhibitors) are commonly used on Guadeloupe.
In vitro and in vivo studies indicated annonacin can cause the development of signs associated with tauopathies, such as tau hyperphosphorylation, retrograde mitochondrial transport, tau redistribution from the axon to the neuronal body, and cell death. Acetogenin in A. muricata is recognized as a neurotoxin with the potential to cause neurodegenerative disorders. Acetogenin causes an increase in tau phosphorylation, which is associated with neurodegenerative tauopathy.
An in silico study found that almost all acetogenins are fat-soluble compounds that cross the blood-brain barrier and inhibit CYP2C19, CYP2C9 and CYP3A4; in addition to having acute and subchronic toxic potential. This lipophilicity and BBB penetration is consistent with the neurotoxic effects observed experimentally and epidemiologically.
In silico studies found that all examined acetogenins showed high cutaneous and intestinal absorption, moderate permeability in Madin-Darby canine kidney and Caco2 cells, strongly bound plasma proteins, freely crossed the blood-brain barrier, inhibited CYP2C19, CYP2C9 and CYP3A4, and have an affinity for CYP3A4, being metabolized by it. The inhibition of CYP2C19, CYP2C9, and CYP3A4 raises theoretical concern for interactions with drugs metabolized by these same enzymes (e.g., anticoagulants, immunosuppressants, many common pharmaceuticals), although these interactions have not been systematically characterized in vivo in humans.
In silico toxicity modeling found that all examined acetogenin compounds were toxic in at least one model. Acetogenins are thus unlikely to be effective therapeutic agents unless chemical alteration can preserve the apoptotic activity while reducing neurotoxicity. This observation summarizes the central challenge facing acetogenin drug development: the same Complex I inhibition that produces cytotoxicity against tumor cells also endangers non-cancerous neurons and other energy-dependent tissues.
No clinical data on acetogenin safety in pregnancy, lactation, or pediatric populations have been identified in the peer-reviewed literature. Given the documented neurotoxic profile in preclinical models, use of concentrated acetogenin preparations in these populations would carry uncharacterized risk.
In recent decades, annonaceous acetogenins have become highly studied plant secondary metabolites in terms of their isolation, structure elucidation, synthesis, biological evaluation, mechanism of action, and toxicity. The compounds are considered biologically in terms of their cytotoxicity for cancer cell lines, neurotoxicity, pesticidal effects, and miscellaneous activities.
Acetogenins have offered not only a challenging target for total synthesis, but they are also fascinating lead compounds for the development of novel antitumor agents. Despite decades of preclinical work, the transition to human clinical evidence remains incomplete. The evidence base for anticancer use consists almost entirely of cell-line and animal model data. No randomized controlled human trial of an isolated acetogenin compound for any indication has been published. The neurological safety concern—specifically the epidemiological and experimental linkage to atypical parkinsonism—represents a significant obstacle to therapeutic development that has not been resolved.
The versatility of the AA bioactivity renders them potential therapeutic agents for cancer treatment. It is therefore apparent that nature is worth further examination to aid in the discovery of more effective, accurate, and less harmful therapies in the fight against cancer.
Health conditions that Acetogenin may help support.
Acetogenins from Annona species (Graviola/soursop) potently inhibit parasite mitochondrial complex I in the nanomolar range against Leishmania, Trypanosoma, and helminths in vitro. Caringsunshine databases list acetogenin as a documented antiparasitic compound.
Body systems that Acetogenin may help support.
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