ACG-008-Risk-Seeds of annona squamosa-FR-2011

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From Champy, P. (2011). Acetogenins from the seeds of the Custard Apple (Annona squamosaL.) and their health outcomes. In V. R. Preedy, R. R. Watson, V. B. Patel (Editors), Nuts& Seeds in Health and Disease Prevention (1st ed.) (pp 429-437). London, Burlington,

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CHAPTER 51

Acetogenins from theSeeds of the Custard Apple(Annona squamosa L.) andtheir Health OutcomesPierre ChampyLaboratoire de Pharmacognosie, UMR CNRS 8076 BioCIS, Faculte de Pharmacie,Universite Paris-Sud 11, France

CHAPTER OUTLINE

Introduction 430Botanical Description 430Historical Cultivation andUsage 430Present-Day Cultivation andUsage 430

Applications to Health Promotionand Disease Prevention 431Adverse Effects and Reactions(Allergies and Toxicity) 434Summary Points 435References 435

LIST OF ABBREVIATIONS

ACG, annonaceous acetogenins

ATP, adenosine tri-phosphate

EC, effective concentration

FDA, Food and Drug Administration

HPLCeDAD, high performance liquid chromatographyediode array detection

IC, inhibitory concentration

NADH, nicotinamide adenine dinucleotide, reduced form

PSP, progressive supranuclear palsy

ROS, reactive oxygen species

SARs, structure activity relationships

THF, tetrahydrofuran

UQ, ubiquinone

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Nuts & Seeds in Health and Disease Prevention. DOI: 10.1016/B978-0-12-375688-6.10051-9

Copyright � 2011 Elsevier Inc. All rights reserved.


http://dx.doi.org/10.1016/B978-0-12-375688-6.10051-9

INTRODUCTIONAnnona squamosa L., a small tropical tree, is a famous cultivated Annonaceae. Its fruit is known

as the custard apple, sugar apple, or fruta do conde. Its seeds are poisonous, and have multiple,mainly traditional, uses. They contain high amounts of annonaceous acetogenins (ACGs), for

which a phytochemical update is proposed. This group of polyketides comprises the most

potent inhibitors of mitochondrial complex I (Bermejo et al., 2005). Recent biologicaloutcomes are presented, in regard to antitumoral and pesticidal potential. ACGs are being

proposed as environmental neurotoxins, toxicological data are summarized, along with

concerns about seed uses.

BOTANICAL DESCRIPTIONThe pseudosyncarpic fruits of A. squamosa are green, and display marked carpel protuberances.They are heart-shaped, measure approximately 7.5 cm in length, and weigh 100e400 g,

depending on the cultivar and cultivation conditions. Their whitish, custard-like sweet pulp

contains 35 to 50 black seeds of 1e1.5 cm in length and 0.5e0.8 cm in width, with a glossycuticle (Figure 51.1).

HISTORICAL CULTIVATION AND USAGEOriginating from Central America, like most Annona species, the tree is believed to have spread

to Mexico, South America, and the Caribbean in the 16th to 17th centuries, and is now

commonly found in domestic gardens in tropical America. It was brought to India by thePortuguese during the same period, then to South-east Asia, and was also introduced into

Africa and Oceania (Pinto et al., 2005). Alimentary use of the fruit appears mostly to be

a South-American and Asian habit. In tropical areas, various and convergent medicinal uses,mainly of bark and leaves, are reported.

PRESENT-DAY CULTIVATION AND USAGECustard apple grows at low altitudes (0e1500m), and is widely cultivated in tropical to semi-

arid regions, in orchards or on commercial farms. Considered to be aminor crop by the FAO, it

is the third most commercially cultivated Annonaceae in South and Central America (behindA. cherimolia and A. muricata), Brazil being one of the main producers (cultivation > 1200 ha,

and production > 11,000 tonnes, in 2000). The tree is also cultivated in India (44,000 ha in

the 1980s), Sri Lanka, Malaysia, Viet Nam, the Philippines, and Taiwan (2500 tonnes per year).

FIGURE 51.1A. squamosa fruits and seeds.

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PART 2Effects of Specific Nuts and Seeds


Smaller production areas are encountered in southern Florida, Australia (2 tonnes in 2003),tropical Africa, and Egypt (170 tonnes in 1997). Exportation to northern market is, however,

limited. The ripe fruit was being sold at US$ 0.56/kg in 2004, for direct consumption or for

industrial processing as juices or ice creams. For this use, prior peeling and removal of seeds isperformed (Pinto et al., 2005).

APPLICATIONS TO HEALTH PROMOTION ANDDISEASE PREVENTIONScarcity of traditional internal use of seeds, and convergence in topical treatment against

external parasites with crushed seeds or oil, are remarkable. Seeds are also often reported as

traditional pesticides, and, less frequently, as fish poison. Among various other bioactivesecondary metabolites (i.e., isoquinolines, ent-kauranes, cyclopeptides), ACGs appear to

support these uses. These white, waxy polyketides, specific to the Annonaceae family, havebeen encountered in all Annona species studied so far. Derived from long chain fatty acids, they

constitute 35 or 37 carbon atoms, with an alkyl chain bearing a central oxygenated system

(tetrahydrofuranic (THF) rings) and a terminal butyrolactone. Inner classification is based onthe structure of these moieties (Figure 51.2).

Extensive chemical studies of the seeds of A. squamosa led to isolation of 74 ACGs, most

bearing two adjacent THF rings (e.g., rolliniastatin-2 (1), squamocin (2)) or a single THFring (e.g., annonacin (3); Figure 51.3). ACGs are also reported in the bark (Bermejo et al.,

2005) and fruit pulp (Champy et al., 2008). Yang and colleagues, in a simultaneous HPLCe

DAD determination of eight ACGs from a supercritical CO2 extract of seeds from China,evidenced 1 and 2 as major representatives (0.58 and 0.37mg/g; total ACGs, 2.29mg/g)

FIGURE 51.2Structural features of ACGs from A. squamosaseeds. Structural characteristics that are the mostfavorable for complex I inhibition are underlined

(compare with structure of (1), Figure 51.3). Typesand sub-types are classified according to Cave

et al., 1997, and Bermejo et al., 2005 (in italics).

Percentages are calculated with respect to the total

number of ACGs isolated from seeds (Bermejo

et al., 2005; Souza et al., 2008, and references

cited in Figure 51.4). Length of 13 carbon atoms for

alkyl spacer between lactone and hydroxylated THF

system: ~50% of type A and type B ACGs. Note

that a type D (three adjacent THF groups), a type E

bearing three epoxides, a C-18, and a bis-lactonic

C-22 representative were also obtained.

CHAPTER 51Acetogenins of Annona Squamosa Seeds

431


(Yang et al., 2009a). In our experience, with several batches from Brazil, 2 was the main ACG(~60% of total ACGs), with a similar yield.

Since the last review on ACGs (Bermejo et al., 2005), eight articles have been published,describing isolation of 46 of these compounds from the seeds, of which 19 were obtained for

the first time in the species (annotemoyins-1 and -2, bullatencin, cis-bullatencin, corepox-

ylone, diepomuricanins A and B, dieporeticenin, glabranin, glabrencin B, probably narumicin-II (“Compound 1” in Sousa et al., 2008), reticulatains-1 and -2, solamin, erythro-solamin,

tripoxyrollin, and uvariamicins I, II, and III; “isosquamocin” is also mentioned (Grover et al.,

2009)); 17 display original structures (note that homonymies exist for squamostatin-C and forsquamocenin) (Figure 51.4).

ACGs are very strong inhibitors of mitochondrial complex I (NADH ubiquinone oxido-

reductase). Most act as uncompetitive semiquinone antagonists, with the lactonic ring asa probable inhibitory pharmacophore, and THF system allowing positioning in mitochondrial

internal membrane (Bermejo et al., 2005; Barrachina et al., 2007; see also Derbre et al., 2005

and references cited in Kojima and Tanaka, 2009). Rolliniastatin-2 (1) is the most activerepresentative, squamocin (2) displaying close potency (Table 51.1; see also structureeactivity

relationships (SARs) depicted in Figure 51.1).

ACGs show tremendous cytotoxicity, with IC50 values ranging from 10 mM to 10�4 nM.

However, striking discrepancies in SARs versus that for complex I appear in the literature.

Differential intracellular distribution of these amphiphilic, apolar compounds might beimplicated (Hollerhage et al., 2009). Fluorescent analogs of 2 showed mitochondrial tropism

in Jurkat cells (Derbre et al., 2005). Alternative targets are also proposed but their relevance is

unknown (Derbre et al., 2008; Liaw et al., 2008; Takahashi et al., 2008). In vitro studies invarious mammal cell lines or primary cultures reported death to be triggered by ROS

production or ATP deprivation, depending on the in vitro paradigm. Apoptotic mechanisms

consistent with a mitochondrial pathway were observed, notably with 2 as a pharmacologicaltool. The perspective of ACGs being anticancer agents has thus motivated most research on

these metabolites during the past three decades, with promising milestones being achieved.

Selectivity for cancerous cells in regard to normal ones was proposed, on the basis ofdiscrepancies in ATP requirements, but this issue remains under discussion (Garcia-Aguirre

et al., 2008). ACGs also proved cytotoxic in multi-drug resistant cell lines expressing the ATP-

dependent MDR efflux transporter (McLaughlin, 2008). Numerous semisynthesic analogsdesigned for activity enhancement or mechanistic studies were obtained, 2 being a lead

FIGURE 51.3Prototypical ACGs from A. squamosa seeds.


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compound (Kojima & Tanaka, 2009). Among other ACGs, 1 underwent promising antitumorassays, and was reported as being well tolerated in several animal species (McLaughlin, 2008;

see also Cuendet et al., 2008). However, to our knowledge, no ACG passed preclinical eval-

uation, and no clinical studies were published. According to McLaughlin (2008), dietarysupplements containing ACGs gave satisfactory results as oral adjuncts to chemotherapy in

cancer patients. Indeed, poorly evaluated Annonaceae dietary supplements are sold for cancer

treatment and prevention, on the Internet and in health stores. None contain A. squamosaseeds or seed extracts, but between 2007 and 2010 five patents related to the use of A. squamosa

seeds ACGs in cancer were deposited in China.

ACGs also proved molluscidal and anthelmintic (antibacterial, antifungal, immunosuppres-

sive, and antiparasitic activities have also been reported, Bermejo et al., 2005). They display

impressive acaricidal and insecticidal potency (McLaughlin, 2008): Among extracts containingACGs, those of A. squamosa seeds were extensively studied (Grover et al., 2009). Promising

FIGURE 51.4Original ACGs isolated from A. squamosa seeds between 2005 and 2009. Relative configurations: er, erythro; th, threo; c,cis; t, trans. *Undetermined absolute configurations. References: Compounds (8), Yu et al. (2005); (6,7, 11e16), Liaw et al.

(2008); (4,5,9,10), Bajin ba Ndob et al. (2009); (17, 18), Yang et al. (2009b); (19, 20), Yang et al. (2009c).


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semisynthesic ACGs were also obtained, including b-amino-(2), a dual complex I/complex III

inhibitor designed by Duval and colleagues (Kojima & Tanaka, 2009). A patent for an anti-head-lice shampoo containing a standardized extract of seeds of A. squamosa has been regis-

tered, alike Asimina triloba products containing similar ACGs (McLaughlin, 2008). Agro-

chemical valorization of A. squamosa seeds appears to be a potentially important outcome,with five publications in 2009 and three Indian patents on standardized apolar extracts since

2006. This is reminiscent of rotenoid-containing Fabaceae and of rotenone, a reference lipo-

philic complex I inhibitor sharing the enzyme binding domain of ACGs, with potency close tothat of 3 (Table 51.1).

ADVERSE EFFECTS AND REACTIONS (ALLERGIES AND TOXICITY)Seeds of A. squamosa are of notorious toxicity, and are thus barely used orally in traditional

medicine (except as an abortive in India, where aqueous extracts are used). They arereported to cause irritation to the eye and mucosa. Oral ingestion provokes vomiting, related

to the ACG content (McLaughlin, 2008). The plant is mentioned in the poisonous plants

database of the FDA (American Food and Drug Administration), and the AFSSA (AgenceFrancaise de Securite Sanitaire des Aliments: Saisine 2007-SA-0231, 2007/12/21; pp 3, 5;

Saisine 2008-SA-0171, 2010/04/28, 7 p.) has expressed safety concerns regarding its use in

dietary supplements.

In relation to pesticide use, safety evaluation of a defatted seed extract (MeOH/CH2Cl2 1:1) in

female Wistar Rats was proposed by Grover et al. (2009). Mortality was observed at 2 g/kg p.o.

At doses of 150 and 300mg/kg, genotoxicity was evidenced in leukocytes and bone marrow,from 4 to 72 h after ingestion, possibly due to ACGs (Garcia-Aguirre et al., 2008). Consistent

with complex I inhibition, involvement of ROS was suggested by significantly enhanced lipid

peroxidation, and decreased glutathione and glutathione S-transferase levels. However, anMeOH extract likely to contain ACGs did not increase oxidative markers in the livers of female

Swiss mice (dosage 200mg/kg, p.o., 10 days; Panda & Khar, 2007; see also Damasceno et al.,

2002; Pardhasaradhi et al., 2005). Histological examination of liver and kidney revealed nolesions. Authors have expressed concern about the use of A. squamosa seed extract as a pesticide

until more tests are carried out (Grover et al., 2009).

Nevertheless, complex I dysfunction has been reported in Parkinson’s disease (a movement

disorder with progressive degeneration of dopaminergic neurons in substantia nigra), as well as

in the tauopathy progressive supernuclear palsy (PSP), an atypical form of parkinsonism.Complex I inhibitors such as 1-methyl-4-phenylpyridinum, paraquat or rotenone are used to

establish animal models of neurodegeneration, and are linked to the occurrence of parkin-

sonism (Gibson et al., 2010). PSP-like syndromes were observed in genetically heterogeneouspopulations regularly consuming alimentary and medicinal Annonaceae products. Thus, in

TABLE 51.1 Complex I Inhibition Potential ACGs from A. squamosa Seeds

NADH OxidaseIC50 (nM)

NADH/DB OxidoreductaseIC50 (nM)

Ki (nM)

Rolliniastatin-2 (1) 0.85e1.2a 0.6e

Squamocin (2) 0.8b; 1.3c 2.0b 0.4e

Annonacin (3) 2.3� 0.3d 26.1� 3.2d

Rotenone 30c; 5.1� 0.9d 28.8� 1.5d 4.0e

IC50-Complex I, half-maximal concentration inhibiting NADH oxidase activity in the absence and presence of an exogenous

ubiquinone analog (decyl-UQ; NADH-DB oxido-reductase activity), in bovine heart sub-mitochondrial particles; data from:aMiyoshi et al.1998 and Fujita et al., 2005; cited in Kojima & Tanaka (2009);bDuval et al. (2005); cited in Kojima & Tanaka (2009);cDerbre et al., 2006, cited in Kojima & Tanaka (2009);dTormo et al. (2003); cited in Bermejo et al. (2005);eDegli-Espoti et al. (1994), cited in Bermejo et al. (2005).


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Guadeloupe (French West Indies), such patients account for two-thirds of all cases ofparkinsonism, compared to approximately 30% of atypical forms in European countries. They

display a combination of movement disorders and dementia, the disease being thoroughly

characterized. Autopsies performed in three patients revealed accumulation of neuronal Tau-fibrils (see references cited in Camuzat et al., 2008; Champy et al., 2009). ACGs were identified

as candidate toxins using PC12 cells (unpublished data), as confirmed for annonacin (3) in

mesencephalic primary cultures. In striatal primary cultures, ACGs induced ATP loss, Tauhyperphosphorylation and redistribution, microtubular disruption, and cell death at low

nanomolar concentrations (Table 51.2; Hollerhage et al., 2009).

Subchronic systemic intoxication of Lewis rats with 3 (continuous i.v., 3.8; 7.6 mg/kg per day,

28 days) did not cause locomotor dysfunction or signs of illness. However, 3 crossed the

bloodebrain barrier, reduced cerebral ATP levels, and caused neuronal cell loss and gliosis inthe brain stem and basal ganglia. These features are similar to those obtained with rotenone

(Hoglinger et al., 2006), and are reminiscent of the humandisease. ACGs are therefore proposed

as etiological agents for cases of sporadic atypical parkinsonism and tauopathies worldwide,upon chronic exposure. However, pharmacokinetic parameters remain to be determined, and

further epidemiological studies are needed before drawing firm conclusions. It is noteworthy

that rotenone, widely used as an organic pesticide with low environmental reminiscence, wasbanned in 2008 in the European Union. In the absence of a defined benefiterisk balance, these

facts challenge the various alternatives proposed for valorization of A. squamosa seeds.

SUMMARY POINTSl Annona squamosa is a cultivated pantropical fruit tree, and its seeds are by-products.

l Annona squamosa seeds constitute a major source of Annonaceous acetogenins (ACGs),which are potent lipophilic complex I inhibitors.

l Sources of ACGs are proposed as antitumoral dietary supplements.

l The seeds have major potential as an organic pesticide, with patents applied.l An extract of A. squamosa seeds was shown to be mildly genotoxic.

l An epidemiological link between Annonaceae and atypical parkinsonian syndromes was

evidenced.l ACGs are neurotoxic in vitro and in vivo.

l The benefiterisk balance of use of A. squamosa seeds remains undefined, and caution

should therefore prevail.

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TABLE 51.2 In vitro Neurotoxicity of ACGs (Striatal Primary Cultures)

IC50-Cpx I (nM) EC50-ATP (nM) EC50-ND (nM) EC5-Tau (nM)

Rolliniastatin-2 (1) 0.9 3.6* 1.1* 0.6*

Squamocin (2) 1.4 2.9 1.1 0.6Annonacin (3) 54.8 134.0 60.8 44.1Rotenone 6.8 7.3 8.1 7.2

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Health & Medicine

ACG-008-Risk-Seeds of annona squamosa-FR-2011