Smith RE; Tran K; Shejwalkar P; Hara K

Research Article

Austin Neurol & Neurosci. 2016; 1(1): 1005.

Neurotoxicity of Fruits, Seeds and Leaves of Plants in the Annonaceae Family

Smith RE¹*, Tran K¹, Shejwalkar P¹ and Hara K²

¹U.S. Food and Drug Administration, Total Diet and Pesticide Research Center, Lenexa, USA

²School of Engineering, Tokyo University of Technology, Japan

*Corresponding author: Smith RE, U.S. Food and Drug Administration, Total Diet and Pesticide Research Center, Lenexa, USA

Received: April 07, 2016; Accepted: May 31, 2016; Published: June 07, 2016

Abstract

Fruits, seeds, twigs and leaves of several plants in the Annonaceae family contain neurotoxic compounds called acetogenins. Overconsumption of at least one of these fruits, graviola (Annona Muricata), caused an atypical form of Parkinson’s disease on the islands of Guadeloupe, Guam and New Caledonia. It does not respond to the standard treatment with L-Dihydroxyphenylalanine (L-DOPA). This type of atypical Parkinsonism is similar to progressive supranuclear palsy, but with important differences. It is characterized by L-DOPA-resistant Parkinsonism, tremor, subcortical dementia and abnormal eye movements suggestive of Progressive Supranuclear Palsy (PSP). Patients also have hallucinations and dysautonomia, which are not characteristic of PSP. Furthermore, the oculomotor abnormalities and the tremor, which is jerky, differ from what is observed in classical PSP patients. The neurotoxicity is caused by mitochondrial dysfunction. A class of compounds called Acetogenins (ACGs) inhibits the mitochondrial NADH: Ubiquinone Oxidoreductase (complex-I of the respiratory chain). These compounds are lipophilic polyketides that are found in plants in the Annonaceae family. They have two toxicophores: a γ-Butyrolactone and one or more Tetrahydrofuran (THF) or Tetrahydropyran (THP) rings. There is also a long chain alkyl group on the other end of the molecule. Even though it strongly affects the physical-chemical properties of ACGs, it is not a toxicophores. Over 400 ACGs have been found in different plants in the Annonaceae family. They can be identified by Nuclear Magnetic Resonance (NMR) and liquid chromatography coupled to mass spectrometry (LC-MS). They form adducts with Ca2+ and Mg2+, which helps to confirm their presence in samples.

Keywords: Parkinson’s disease; Acetogenins; NADH: Ubiquinone Oxidoreductase; Graviola; Annona Muricata

Introduction

Fruits, seeds, twigs and leaves of several plants in the Annonaceae family contain neurotoxic compounds called acetogenins [1-3]. Epidemiological studies showed a link between the overconsumption of graviola (Annona Muricata) fruit and aqueous infusions of leaves (tea), and an atypical form of Parkinson’s disease on the islands of Guadeloupe, Guam and New Caledonia, as well as the Afro- Caribbean and Indian population in London [4-15]. It does not respond to the standard treatment with L-Dihydroxyphenylalanine (L-DOPA) [4]. This atypical form of Parkinsonism is similar to progressive supranuclear palsy, but with important differences [4]. It is characterized by L-DOPA-resistant Parkinsonism, tremor, subcortical dementia and abnormal eye movements suggestive of Progressive Supranuclear Palsy (PSP) [11]. However, Patients also have hallucinations and dysautonomia, which are not characteristic of PSP. Furthermore, the oculomotor abnormalities and the tremor, which is jerky, differ from what is observed in classical PSP patients [11]. The neurotoxicity is caused (at least in part) by mitochondrial dysfunction [3,8,12-15]. Patients in Guadeloupe were separated into three different categories. Only one-third of them presented the classical features of idiopathic Parkinson disease. Another one-third had a syndrome resembling Progressive Supranuclear Palsy (PSP).

The others were unclassifiable, according to criteria existing in 2007 [8].

A class of compounds called Acetogenins (ACGs) inhibits the mitochondrial NADH: Ubiquinone Oxidoreductase (complex I of the respiratory chain), which decreases ATP levels. This leads to neuronal cell loss and gliosis in the brain stem and basal ganglia [18-20]. However, other mechanisms of toxicity may be involved. This includes modulating histone H3 phosphorylation [16], complexation with Ca2+ ions [17] and somatodendritic redistribution of phosphorylated tau protein [12]. NADH: Ubiquinone Oxidoreductase is a mitochondrial enzyme present in not just neurons, but also cancer cells. As a result, there is a substantial body of literature on the anti-cancer properties of graviola, the North American pawpaw (Asimina triloba), acetogenins and acetogenin mimetics [19,21-25]. One recent article told how drinking a tea made from graviola leaves helped cure a patient who had metastatic negative breast cancer [23]. However, data on single individuals seldom means much to most oncologists. Still, it is worth noting that the dose of the major acetogenin in tea made graviola leaves (annonacin) is only 0.213% of the dose that one would receive from consuming the entire leaves that are sold as a dietary supplement [26]. That is, there are dietary supplements made from different parts of plants in the Annonaceae family that are sold as cures for cancer, even though they may be neurotoxic [1]. For graviola, “The estimated amounts of annonacin ingested in a year by eating one fruit or can of nectar a day were comparable to the dose (3.8 mg/kg/day) that induced widespread neurodegeneration in the basal ganglia and mesencephalon when infused intravenously into rats for 28 days (106 mg/kg)” [27]. More recently, it has been shown that annonacin can cross the blood-brain barrier and its bioavailability was about 3.2% of the orally ingested dose in rats [28].

The goals of this report are to review the literature on the neurotoxicities of the fruits, seeds and leaves of plants in the Annonaceae family and the biochemical toxins that are in them (acetogenins), as well as use Mass Spectrometry (MS) to find as many acetogenins as possible in the fruits of the North American pawpaw (Asimina triloba).

Plants in the Annonaceae Family

Acetogenins (ACGs) have been isolated from the fruit pulp, seeds, twigs, roots, stems, leaves and bark of many plants in the Annonaceae family, which contains about 2400 species in 108 genera, based on the sequences of nucleotides on multiple plastid DNA loci [29]. This is better than previous classifications based mostly on morphology. The new classification consists of four major clades, which were given the taxonomic rank of subfamily [29]. Some of the genera in the Annonoideae subfamily have acetogenins. They include Annona, Anonidium, Asimina, Boutiquea, Dasymaschalon, Diclinanona, Disepalum, Goniothalamus, and Neostenanthera. Even though many previous articles used the name Asimina triloba for the North American pawpaw, it is more properly called Annona triloba [29]. A partial list of plants in the Annonaceae family that have been sources of purified acetogenins is shown in Table 1 [16,17,30- 32]. Note that some of them share the same common names. Also, many of them have uses in folk remedies and traditional medicine, including treating cancer. A book about the Annonaceae family was even published recently [33].

Table 1: Laboratory values at hospital admission.




  
    Genus and Species  
    Popular Names  
  
  
    Annona x A.    atemoya (hybrid)  
    Atemoya, atemoia, A. squamosa x A.    cherimola  
  
  
    Annona cherimolia  
    Custard apple, Chirimuya, cherimoya  
  
  
    Annona coriacea  
    Soursop, fruta-de-conde  
  
  
    Annona crassiflora  
    Marolo, Araticum do cerrado  
  
  
    Annona glabra  
    Pond apple alligator apple, cancer    therapy  
  
  
    Annona jahnii  
    Manirito  
  
  
    Annona Montana  
    Mountain soursop  
  
  
    Annona muricata  
    Soursop, graviola, Brazilian pawpaw,    guanábana  
  
  
    Annona nutans  
    Yaguá-nambí  
  
  
    Annona purpurea  
    Custard apple, soncoya, ox-heart  
  
  
    Annona reticulata  
    Wild sweetsop,  
  
  
    Annona senegalensis  
    African custard apple  
  
  
    Annona spraguei  
    Chirimoya; Negrito; Nonita de mono  
  
  
    Annona squamosa  
    Sugar apple, custard apple, sweetsop,    fruta-do-conde, ata, pinha  
  
  
    Annona triloba  
    Pawpaw  
  
  
    Disepalum anomalum  
    Disepalum grandiflorum  
  
  
    Goniothalamus    donnaiensis  
    Tian fang gu  
  
  
    Rollinia emarginata  
    Aratiku (fruit of the sky)  
  
  
    Rollinia mucosa  
    Biribá, wild sweetsop, anón cimarrón,    cachiman  
  
  
    Rollinia sylvatica  
    Annona crassiflora Mart., Araticum do    Mato  
  
  
    Uvaria boniana  
    Guang ye zi yu pan  
  
  
    Uvaria calamistrata  
    Ci guo zi yu pan  
  
  
    Uvaria grandiflora  
    Calabao  
  
  
    Uvaria narum  
    Neelavalli, Valeeshakhota (Ayurvedic    medicine)  
  
  
    Uvaria tonkinensis  
    Dong jing zi yu pan  
  
  
    Xylopia aromitaca  
    Hemlock anon, malageuto, malageuto macho



Table 1:  Laboratory values at hospital admission.

The most popular plant in the Annonaceae family is A. Muricata, also known as soursop in English and Guanabana in Spanish [15]. Soursop, Cherimoya (A. Cherimola), Sugar Apple (A. Squamosa) And Atemoya (A. Squamosa X A. Cherimola) are the most important in Brazil, where they are known as graviola, cherimólia, pinha and atemoia [34]. There is even a Worker’s Nutrition program in Brazil, in which workers are encouraged to eat supposedly healthy fruits, including graviola [35]. Graviola is also recommended for oral rehydration therapy [36].

Acetogenins

Acetogenins from plants in the Annonaceae family are lipophilic polyketides [30]. Even though there is a review article that said that acetogenins are only found in this family [19], there is a report of acetogenins being found in the roots of a small tree in the genus Ampelocissus and family Vitaceae [37], and a different set of compounds also called acetogenins exist in red algae of the genus Laurencia as well as the sea hares (Dolabella Auricularia) that feed on them [38]. Also, some reports describe a set of compounds that are also called acetogenins, which are found in members of the Lauraceae family, including the popular avocado (Persea Americana) [39-41]. The so-called acetogenins from other families have very different structures and biological properties than those from the Annonaceae family [30]. Only the acetogenins from the Annonaceae family have been linked to atypical Parkinsonism [30].

Annonaceous Acetogenins (ACGs) have two pharmacophores: a γ-Butyrolactone and one or more Tetrahydrofuran (THF) or Tetrahydropyran (THP) rings. Like the acetogenins in avocados, there is also a long chain alkyl group on one end of the ACGs [42]. According to a recent review, over 400 ACGs have been found [42]. Figure 1 shows the generalized structures of the different classes of ACGs and Figure 2 shows some specific examples of ACGs [30]. They are derivatives of fatty acids and have 32 or 34 carbons, including a long chain alkyl group on one end and a monounsaturated or unsaturated lactones on the other end [30,42].

Figure 1:

    
    
    Figure 1:

Figure 2:

    
    
    Figure 2:

Figure 1 Structures of different types of anonaceous Acetogenins (ACGs), as described by others [19,30]. They have 32 or 34 carbons, including a long chain alkyl group on one end and a monounsaturated or unsaturated lactones on the other end. The R groups in LA, LB and LC can have 1-3 Tetrahydrofuran (THF) rings or a Tetrahydropyran (THP) ring. They also can contain different amounts of hydroxyl groups. There are many different types of R groups in the non-classical, ketolactone, lactone and the hydroxylated and reduced lactones, but one of them ends in a long chain alkyl group. For example, the R₁and R₂ in EA, EB and EC are aliphatic chains that end in a methyl on one end and a lactone on the other end

Figure 2 Structures of some of the known neurotoxic ACGs: annonacin (a mono THF ACG), annonacinone (a ketone), Squamocin, Bullatacin (di-THF ACGs), sootepensins A and sootepensins B (mono THF ACG) [22]. IC₅₀, EC₅₀ death and EC₅ tau are the concentrations needed to cause 50% inhibition of the mitochondrial complex I, death in 50% of the cultured neurons and redistribution of the tau protein in 5% of the neurons, which is a standardized measure of the minimum concentration needed to cause tau pathologies [12]. Bullatacin and the sootepensins were not tested for neurotoxicity, but Bullatacin was reported to have an IC₅₀ against MDR MCF-7/Adr breast cancer cells (0.0108 mg/mL) that was much lower than that of Adriamycin, which has been used for decades to help treat and cure breast cancer [45].

Several different parts of plants in the Annonaceae family have been analyzed for ACG content [1, 26-28, 43-51]. The methods used include Time of Flight Mass Spectrometry (TOF-MS), liquid chromatography coupled to mass spectrometry (LC-MS) and Nuclear Magnetic Resonance Spectrometry (NMR). Of special note is the ability to take the mass spectrum of a mass spectrum (MS/MS), in which a primary ion is generated in one quadrupole of the MS and then fragmented in another. In TOF-MS, analytes (such as acetogenins) are separated based on the time it takes for them to traverse the flight tube and reach the MS detector. Originally, TOF-MS was used to analyze mixtures of gases, such as N₂, O₂, CO and CO₂. However, N₂ and CO have the same molecular weights (28 atomic mass units, or amu) when measured with older TOF-MS instruments. On the other hand, modern TOF-MS instruments often have relatively high resolution and can be used to identify acetogenins [44]. However, a chemical matrix must be added to enable the production of ions when the sample is irradiated with a laser. This is called Matrix Assisted Laser Desorption and Ionization (MALDI). For example, a MALDITOF- TOF MS was used to produce specific patterns of fragments that unambiguous structural information on several acetogenins [44].

There are also ion trap MS instruments. For example, an LQT XL Orbitrap® was used to detect ACGs with mass accuracy of ±5 parts per million (ppm) [44]. This technology enables the determination of molecular formula. For example, the atomic mass of 12C is 12.0000, 16O is 15.9999 and 14N is 14.0031. Thus, the compounds C₆H₁₂, C₅H₈O and C₄H₈N₂ have molecular weights of 84.0939, 84.0575 and 84.0688, respectively and can be distinguished by high resolution MS. So, an LQT XL Orbitrap® was used to analyze the neurotoxic ACG, annonacin. In the positive ion mode, it produced ions with Na+ as an adduct, with sequential losses of five waters, four from the four –OH groups and one from the methyl lactone that opened in the acidic mobile phase. The Limit of Detection (LOD) was 0.3 nM in the MS mode and 0.5 μM in the MS/MS mode. In the negative ion mode, it produced a fragment that showed that C-20 was hydroxylated [44].

The same study also reported that MALDI-TOF-TOF produced more fragments at relatively high collision energies [44]. When lithium was added to the sample, it had a limit of detection of 10 nM in the MS mode and 10 μM in the MS/MS mode when Chemically Induced Ionization (CID) was used. When other ACGs were analyzed, it was found that fragment ions with an m/z=118 and/or 119 indicate the presence of a lactone of subtype-1, whereas their absence is correlated to the sub-type 2. Also, the loss of 112 amu from the lithium adducts indicates the presence of a hydroxyl group at C-4 [44].

Even though MS can identify acetogenins, pure analytical standards are needed to quantify them. Two of them, annonacin and squamocin, have been purified [43] and shared with other laboratories [1,26,50]. The concentrations of annonacin in lyophilized pawpaw (A. Triloba), graviola, atemoya, ata (A. Squamosa) and biriba (R. Mucosa) fruits were found to be 7720, 768, 3.8, 2.2 and 1.8 μg/g (dry weight), respectively [1,50]. The concentrations of squamocin in lyophilized graviola, atemoya, ata and biriba (R. mucosa) fruits were 162, 4.5, 68, 0.7 and 4.2 μg/g (dry weight), respectively [1,50]. We only had seeds from atemoya, but they had 454 and 14200 μg/g (0.454 and 14.2 mg/g) annonacin and squamocin, respectively [1]. Lyophilized graviola leaves had 306 and 17 μg/g annonacin and squamocin, respectively, but a tea made from the leaves had only 0.213% of the annonacin that was in the entire leaves and too little of the squamocin to detect [26]. That is, to analyze dried fruits, leaves or seeds for acetogenins, they must be completely extracted from the plant cells that contain them. Water (even at 100 0C) does not dissolve much of the hydrophobic (lipophilic) acetogenins. Instead, dry methanol at 100 0C and 10 MPa (100 atm) pressure in a sealed container can [1,26,50]. So, the dose of annonacin that one would consume in a tea made from dried graviola leaves would be only 0.213% of the dose that would be consumed if the entire leaves were ingested in a dietary supplement [26]. Despite the large differences in concentrations of annonacin and squamocin in graviola, atemoya, ata and biriba, they were all toxic to Lund Human Mesencephalic Neurons (LUHMES) at about the same doses [1]. So, it is quite likely that there are many other neurotoxic acetogenins in these and other fruits in the Annonaceae family.

Still, the pawpaw (Asimina Triloba) fruit might be of special concern in North America. Even though we know of no epidemiological studies on the fruit, it has been suggested that it may also be neurotoxic [49]. Not only does it contain much more annonacin than graviola, it contains many other acetogenins [52]. However, hot, pressurized methanol was not used to extract pawpaw samples when that review article was written, so there could be some that were not seen.

So, the same methanolic extract of lyophilized pawpaw fruit that was analyzed for its annonacin and squamocin content [50] was redissolved in CH₃OH containing sodium format and directly infused at a rate of 0.6 ml/min into a Bruker micro TOF-II MS in the current study. Positive ion electro spray (ESI+) was used and a mass range of 50-3000 amu was monitored. Then, Ca²⁺ was added to a portion of the extract and mass spectra were acquired. Subsequently, Mg²⁺ was added to a different portion of the extract and more mass spectra were acquired. The list of suspect acetogenins that was observed is shown in Table 2. This is based on the m/z values of the [M+H] ⁺ ion being within ±0.1 amu of that expected, based on the molecular formulas of known acetogenins. This includes annonacin and squamocin, which had been identified and quantified by Liquid Chromatography Coupled to High Resolution Mass Spectrometry (LC-HRMS) in a previous study [50]. They were the only two acetogenins that were available as analytical standards. At this time, the presence of the other acetogenins has not confirmed. This will require separation of the suspect acetogenins by LC.

Table 2: Laboratory values at hospital admission.




  
    [M+H]+  
    Molecular    Formula  
    [M+Ca]2+  
    [M+Mg]2+  
  
  
    295.1766  
    C18H30O3  
    167.0631  
    159.0273  
  
  
    327.1414  
    C21H26O3  
    183.0867  
    175.0459  
  
  
    381.3372  
    C22H36O5  
    210.0870  
    202.1596  
  
  
    385.2572  
    C25H36O3  
    212.0952  
    204.1028  
  
  
    531.4208  
    C35H62O3  
    285.2541  
    277.1915  
  
  
    533.4326  
    C35H64O3  
    286.2241  
    278.2161  
  
  
    535.4532  
    C35H66O3  
    287.1956  
    279.2199  
  
  
    543.4162  
    C37H66O2  
    291.1863  
    283.2520  
  
  
    545.4333  
    C35H60O4  
    292.1961  
    284.2578  
  
  
    559.4492  
    C37H66O3  
    299.1322  
    291.2193  
  
  
    561.4617  
    C35H60O5  
    300.1966  
    292.2272  
  
  
    563.4817  
    C35H62O5  
    301.1089  
    293.2359  
  
  
    565.4825  
    C35H64O5  
    302.2122  
    294.2376  
  
  
    569.4402  
    C33H60O7  
    304.2274  
    296.2301  
  
  
    573.4573  
    C37H64O4  
    306.2313  
    298.228  
  
  
    579.4121  
    C35H62O.  
    309.1774  
    301.2256  
  
  
    581.4306  
    C35H64O6  
    310.1995  
    302.2223  
  
  
    589.4555  
    C37H64O5  
    314.2260  
    306.2313  
  
  
    591.4020  
    C37H66O5  
    315.2041  
    307.2224  
  
  
    593.4023  
    C35H60O7  
    316.1911  
    308.2196  
  
  
    595.3589  
    C35H62O7  
    317.1945  
    309.2162  
  
  
    597.4213  
    C35H64O7  
    318.2056  
    310.212  
  
  
    605.3959  
    C37H64O6  
    322.2197  
    314.1795  
  
  
    607.3959  
    C37H66O6  
    323.2040  
    315.2045  
  
  
    609.4518  
    C37H68O6  
    324.1912  
    316.2069  
  
  
    611.4704  
    C35H62O8  
    325.1922  
    317.2171  
  
  
    613.4858  
    C35H64O8  
    326.1939  
    318.2103  
  
  
    621.3874  
    C37H64O7  
    330.1795  
    322.2137  
  
  
    623.3966  
    C36H62O8  
    331.1812  
    323.2099  
  
  
    627.3835  
    C35H62O9  
    333.1871  
    325.2097  
  
  
    629.3981  
    C35H64O9  
    334.1963  
    326.1835  
  
  
    631.3827  
    C35H66O9  
    335.1992  
    327.1882  
  
  
    637.3700  
    C37H64O8  
    338.2281  
    330.2037  
  
  
    639.4128  
    C37H66O8  
    339.2298  
    331.1953  
  
  
    645.3946  
    C35H64O10  
    342.1922  
    334.1986  
  
  
    663.3932  
    C39H66O8  
    351.1933  
    343.1974  
  
  
    665.3988  
    C39H68O8  
    352.2048  
    344.1937  
  
  
    673.3888  
    C37H68O10  
    356.1930  
    348.1985  
  
  
    681.3855  
    C39H68O9  
    360.2811  
    352.2079



Table 2:  Laboratory values at hospital admission.

Toxicity Studies

In addition to the epidemiological studies [4-11], several groups have investigated the in vitro neurotoxicity. The epidemiological studies showed that a particular variant of taupathy on the island of Guadeloupe was linked in case control studies to overconsumption of graviola fruit and tea made from the leaves [12]. As in other taupathies, there was an accumulation of the tau protein in the somatodendritic compartment of neurons. Acetogenins in graviola fruit and leaves are potent lipophilic inhibitors of mitochondrial NADH: Ubiquinone Oxidoreductase. The major acetogenin in graviola, annonacin, reduced cerebral ATP levels and causes neuronal cell loss and gliosis in the brain stem and basal ganglia [3,12]. It and several other acetogenins were found to not just reduce the ATP concentration in neurons, but also cause phosphorylated tau to be redistributed from the axon to the cell soma [12]. Annonacin has been shown to be toxic to rat primary mesencephalic [2] and striatal cells [12,13] as well as on human neuronal cells. Moreover, several acetogenins increased tau phosphorylation [12,13], changed the isoform of tau from 3R-tau to 4R-tau [54], both contributing to the pathophysiology of neurodegenerative tauopathies.

It was also shown that there is some synergy between the effects of annonacin and the expression of a mutated form of the tau protein (R406W, in which arginine 406 is mutated into tryptophan) [55]. There is much variability in the way that this mutation can affect patients. This suggests that environmental factors and diet being may also affect their health. So, it was quite important that annonacin increased the number of neurons that had phosphorylated tau in the somatodendritic compartment in several brain areas in R406W+/+ mice as opposed to mice that had only the endogenous mouse tau (R406W^-/-). It caused degradation of the neuronal tau protein, as well as an increase in its phosphorylation and redistribution. It also activated the tau kinase Cdk5 [55].

In a more recent study, dietary supplements that contained graviola stems and leaves or pawpaw twigs were found to toxic to LUHMES cells that were derived from human mesencephalic tissue [1]. These dietary supplements were sold over the internet and advertised as being cures for cancer. As a positive control, lyophilized graviola fruit was also tested and found to be neurotoxic. In addition, pawpaw, atemoya, ata and biriba fruits as well as atemoya seeds were toxic to LUHMES cells. Despite the large differences in concentrations of annonacin and squamocin in graviola and the other four fruits, they were all toxic at about the same doses [1]. This could be due, in part, to the presence of many other neurotoxic acetogenins that were not quantified. Also, the bioavailabilities and neurotoxicities of acetogenins may be quite different when consumed by themselves or as part of the fruits. That is, acetogenins are lipophilic and may not form homogeneous doses in aqueous growth media used for the LUHMES cells. Since previous studies of in vitro neurotoxicities were done on ethyl acetate extracts of graviola, this solvent was used to extract the dietary supplements, the five lyophilized fruits and the atemoya seeds. However, ethyl acetate also extracts triacylglycerides, which are abundant in twigs, stems and especially seeds [51]. After the ethyl acetate was evaporated off, the oily residue that remained was made up of primarily hydrophobic triacylglycerides. It is quite likely that most of the acetogenins were trapped inside the oil and were not completely solubilized when mixed with the growth medium. So, it is hoped that hot, pressurized methanolic extracts of different parts of plants from the Annonaceae family will be evaluated for possible neurotoxicities in the future. Such extracts do not contain triacylglycerides, but do contain amphiphilic fatty acid glycosides that can form micelles that can help form a nearly homogeneous suspension of acetogenins [56]. It would be best if doses of such extracts that are dissolved in a suitable vehicle were analyzed to ensure that they are stable and the acetogenins are distributed homogeneously. This is seldom done in academic studies that have limited funding, but it is always done by pharmaceutical companies and the U.S. Environmental Toxicology Program in toxicity studies performed in accordance with Good Laboratory Practices (GLP) [57].

Another recent study showed that an aqueous extract of graviola leaves caused neuronal degeneration in the substantia nigra and neuronal vacuolization in the hippocampus of rats [58]. The neuronal damage led to an increase in glial cells in the nucleus accumbens and cerebral cortex. That is, necrotic neurons became surrounded by glia. Moreover, cytoplasmic vacuolization, clear cell foci, and eosinophilic hepatocytes emerged in the livers of the rats [58]. These effects are especially noteworthy since a tea made from 2.5 g of graviola leaves that were added to 237 ml (one cup) of water at 90 0C for 10 min had only 0.213% of the annonacin that was present in the entire leaves [26]. There is even more annonacin in graviola fruit (768 μg/g dry weight) than in the leaves (306 μg/g dry weight) [1,26]. Pawpaw fruit had 7720 μg/g dry weight [50]. Atemoya seeds had 454 and 14200 μg/g (0.454 and 14.2 mg/g) annonacin and squamocin, respectively [1]. So, if a tea made from graviola leaves can cause damage to the brain and liver of rats, the fruits and seeds of this and other plants in the Annonaceae family probably can, too, but at much lower doses. Similarly, the entire leaves that are sold as dietary supplements could be toxic, too.

Conclusion

Overconsumption of graviola has been linked to an atypical form of Parkinson’s disease on the islands of Guadeloupe, Guam and New Caledonia. It does not respond to the standard treatment with L-Dihydroxyphenylalanine (L-DOPA). The neurotoxicity is caused by mitochondrial dysfunction. A class of compounds called Acetogenins (ACGs) inhibits the mitochondrial NADH: Ubiquinone Oxidoreductase (complex-I of the respiratory chain). These compounds are lipophilic polyketides that are found in plants in the Annonaceae family. Over 400 ACGs have been found in different plants in the Annonaceae family. They can be identified by NMR and LC-MS. They form adducts with Ca2+ and Mg2+, which helps to confirm their presence in samples. Annonacin has been shown to be toxic to rat primary mesencephalic [2] and striatal cells [12,13] as well as on human neuronal cells. Moreover, several acetogenins increased tau phosphorylation [12,13] and changed the isoform of tau from 3R-tau to 4R-tau [54], both contributing to the pathophysiology. It was also shown that there is some synergy between the effects of annonacin and the expression of a mutated form of the tau protein (R406W) [55]. Also, dietary supplements that contained graviola stems and leaves or pawpaw twigs were found to toxic to LUHMES cells that were derived from human mesencephalic tissue [1]. These dietary supplements were sold over the internet and advertised as being cures for cancer. As a positive control, lyophilized graviola fruit was also tested and found to be neurotoxic. In addition, pawpaw, atemoya, ata and biriba fruits as well as atemoya seeds were toxic to LUHMES cells [1]. Still, it is hoped that hot, pressurized methanolic extracts can be tested for neurotoxicity, since they are more likely to form nearly homogeneous doses of acetogenins in micelles made from fatty acid glycosides that are in all five fruits in the Annonaceae family that were tested [1,56].

References

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Citation: Smith RE, Tran K, Shejwalkar P and Hara K. Neurotoxicity of Fruits, Seeds and Leaves of Plants in the Annonaceae Family. Austin Neurol & Neurosci. 2016; 1(1): 1005.

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Genus and Species	Popular Names
Annona x A. atemoya (hybrid)	Atemoya, atemoia, A. squamosa x A. cherimola
Annona cherimolia	Custard apple, Chirimuya, cherimoya
Annona coriacea	Soursop, fruta-de-conde
Annona crassiflora	Marolo, Araticum do cerrado
Annona glabra	Pond apple alligator apple, cancer therapy
Annona jahnii	Manirito
Annona Montana	Mountain soursop
Annona muricata	Soursop, graviola, Brazilian pawpaw, guanábana
Annona nutans	Yaguá-nambí
Annona purpurea	Custard apple, soncoya, ox-heart
Annona reticulata	Wild sweetsop,
Annona senegalensis	African custard apple
Annona spraguei	Chirimoya; Negrito; Nonita de mono
Annona squamosa	Sugar apple, custard apple, sweetsop, fruta-do-conde, ata, pinha
Annona triloba	Pawpaw
Disepalum anomalum	Disepalum grandiflorum
Goniothalamus donnaiensis	Tian fang gu
Rollinia emarginata	Aratiku (fruit of the sky)
Rollinia mucosa	Biribá, wild sweetsop, anón cimarrón, cachiman
Rollinia sylvatica	Annona crassiflora Mart., Araticum do Mato
Uvaria boniana	Guang ye zi yu pan
Uvaria calamistrata	Ci guo zi yu pan
Uvaria grandiflora	Calabao
Uvaria narum	Neelavalli, Valeeshakhota (Ayurvedic medicine)
Uvaria tonkinensis	Dong jing zi yu pan
Xylopia aromitaca	Hemlock anon, malageuto, malageuto macho