Traditional Uses, Phytochemistry and Pharmacological Activities of Annonacae

In 1789, the Annonaceae family was catalogued by de Jussieu. It encompasses tropical and subtropical plants which are widespread in distribution across various continents such as Asia, South and Central America, Australia and Africa. The genus of Annona is one of 120 genera of the Annonaceae family and contains more than 119 species of trees and shrubs. Most species are found in tropical America, where over 105 species have been identified. Due to its edible fruits and medicinal properties, Annona is the most studied genus of Annonaceae family. To date, only a limited number of these species have economic value, including A. squamosa L. (sugar apple), A. cherimola Mill. (Cherimoya), A. muricata L. (guanabana or soursop), A. atemoya Mabb. (atemoya), a hybrid between A. cherimola and A. squamosa, A. reticulata L. (custard apple), A. glabra L. (pond-apple) and A. macroprophyllata Donn. Sm. (ilama). Phytochemically, several classes of secondary metabolites, including acetogenins, essential oils, alkaloids, terpenoids and flavonoids. The pharmacological activities of Annona species leaves and seeds include antibacterial, anticancer, antidiabetic and anti-inflammatory properties.


Introduction
In 1789, the Annonaceae family was cataloged by de Jussieu [1,2]. It encompasses tropical and subtropical plants, which are widespread in distribution across various continents such as Asia, South and Central America, Australia and Africa [3]. It is one of the largest Mangnoliidae families and the number of its genera and species is still debated [4][5][6]. Bailey and Popenoe believe that it has between 40 and 50 genera and from 500 to 600 species [6]; however, many studies have indicated that the Annonaceae family is comprised of more than 2400 species distributed in approximately 120 genera [4,5]. The family of Annonaceae involves trees, lianas and bushes arranged in four large subfamilies: Malmeoideae, Annonoideae, Ambavioideae and Anaxagoreoideae [7,8]. Economically, species of Annonaceae are important as a source of edible fruits, for instance, the pawpaw (Asimina), custard apple, sweetsop, soursop and cherimoya [1]. It has also been reported that some oils from the seeds might be used for the production of edible oils and as an ingredient in soaps, and the woods of some species have been reported for alcohol production [3]. Chemical studies of Annonaceae species have reported the isolation of a wide diversity of phytochemical components, including acetogenins, alkaloids and flavonoids from the bark, fruits, leaves, seeds and pulp of Annonaceae [9]. This review aims to provide a comprehensive summary of the botanical features, phytochemistry, pharmacological properties, and the traditional and ethnomedicinal uses of the Annonaceae family and, specifically, Annona species.

Traditional Uses
Annonaceae species are famous in tropical regions and used traditionally across tropical regions due to their widespread distribution. Various parts of the species are used traditionally, including leaves, seeds, bark, fruit, stem, roots and twigs. A range of different methods for preparation is reported, such as infusions, pastes and decoctions [11]. For instance, the fresh fruit of Annona dioica is used for wound healing in Brazil [11]. The dried leaves of Annona muricata are used orally for analgesic effects in some parts of Indonesia [11]. In Burkina-Faso, the bark and roots of Annona muricata are used for dysentery and as an anthelmintic medicine, whereas the leaves are utilized for both fever and dysentery [12]. In the northwestern part of Brazil, both leaves and twigs of Duguetia chrysocarpa are ground and the extract of this mixture are utilized for treating gastrointestinal ulcers as well as a remedy for bowel disease [11]. A decoction of the stem bark of Annickia chlorantha is used orally as a remedy for the treatment of wounds and fever in Cameroon [13]. Further data on the traditional uses of the most widely used Annonaceae species are presented in Table 1. Insertion of root pieces into rectum and leaves burnt and smoke inhaled [22]

Phytochemistry of Annonaceae Family
A wide array of chemical compounds from various parts of Annonaceae plants have been discovered, isolated and characterised. The results of both phytochemical investigations and biological studies on various plants from this family have led to the identification of a wide diversity of compounds such as annonaceous acetogenins, flavonoids, alkaloids and essential oils, as summarized in (Table 2). These phytochemical constituents have been found to exhibit a broad range of biological activities such as immunosuppressive, antineoplastic, cytotoxic, antimicrobial, anti-inflammatory effects (Table 3). However, it is the Annona genera that are the most widely used as a food source and in traditional medicines.

Annona Genera
The genus of Annona is one of the 120 genera of the Annonaceae family and contains more than 119 species of trees and shrubs, most of them distributed in tropical areas of the Americas and Africa [6]. The majority of these species are found in tropical America, with more than 105 species (26 of them are endemic) and 10 species distributed in tropical Africa [10,34]. It has been reported that this genus is the second or the third largest genus in the Annonaceae family [35]. Its generic name derives from the Latin Hispaniolan Taino "annual harvest" [6,35]. Due to its edible fruits and medicinal properties, Annona is the most important genus of Annonaceae family [2]. Numerous Annona species furnish edible fruits like Annona muricata ("graviola"), Annona crassiflora ("araticum") and Annona sqaumosa ("fruta do conde") [2]. Most of the fruits are consumed either in fresh form or used in desserts, juices and ice cream preparations [34]. Despite Annona having many species, only limited species of this family are economically important such as A. squamosa L. (sugar apple), A. cherimola Mill. (Cherimoya), A. muricata L. (guanabana or soursop), A. atemoya Mabb. (atemoya), a hybrid between A. cherimola and A. squamosa, A. reticulata L. (custard apple), A. glabra L. (pond-apple) and A. macroprophyllata Donn. Sm. (ilama) [6]. Phytochemically, several classes of secondary metabolites such as acetogenins, essential oils, alkaloids, terpenoids and flavonoids have been described in this genus [34,36]. A variety of pharmacological activities have been reported from various parts of Annona species specially leaves and seeds including applications against antibacterial [37], antinociceptive [38], anticancer [39], anticonvulsant [40], antidiarrhea [41], antidiabetic [42], antimalarial [39], anti-inflammatory [43], antioxidant [44], antileishmanial [45], antiulcer [46] and antidepressant [47].

Botanical Features
Generally, Annona species are small trees or shrubs with a height from 5 to 11 m depending on various factors including soil, climate, species, and crop management [2]. In relation to the botanical characteristics of Annona species, the majority of them are moderately erect with brown bark that is frequently furrowed (Table 4) [10]. The stems are rust-coloured (ferruginous) and covered with densely matted hairs (tomentose) when young, becoming smooth and hairless (glabrous) as they mature [6,10]. It has thin lateral roots and a taproot that is not generally pronounced [2]. With regard to the flowers, they are hermaphrodites, solitary or fascicle containing from two to four flowers. The flowers are usually fragrant, with six petals and three green sepals, in a circular arrangement of two verticils [6]. Flowering of the plant usually starts at 3 to 4 years and flower opening usually occurs by separation of the apex of external petals [6,10]. Finally, the leaves may be shiny or hairy and have an impressed vein on the upper side, and the fruits are syncarpous and comprised of seeds and many carpels [6,10].

Annona Cherimola
Annona cherimola Mill (Cherimoya) belongs to the genus Annona in the Annonaceae family in magnolias order, which means "cold seeds", and is a small tree that produces heart-shaped and conical edible fruit [55]. It is a steep, semi-momentary and a low bunched tree that is widespread in Ecuador and Peru and distributed throughout Asia, South Europe, America and Africa [56]. In Mexican traditional medicine, this plant has been used to treat various diseases such as diabetes, cough, fever, headache, worms and inflammation either alone or in combination with other plant species [48,[57][58][59][60]. Recently, various parts of A. cherimola have been phytochemically profiled and contain various polyphenols and alkaloids. The leaves were found to be a source of bioactive compounds with potential for use as treatments for skin and eye diseases and gastric, cardiovascular and intestinal disorders [55].

Annona Squamosa
Annon squamosa L., commonly known as custard apple, is a tropical, endemic species of the West Indies, Ecuador, Peru, Brazil, South and Central America, Mexico, Bahamas, Bermuda, and Egypt [61]. This plant is extensively cultivated in various states of India, including Maharashtra, Gujarat, Madhya Pradesh, Chhattisgarh, Assam, Uttar Pradesh, Bihar, Rajasthan, Andhra Pradesh, and Tamil Nadu. The total area of cultivation has been reported by the Indian Council of Agricultural Research (ICAR) as 40,000 ha [62]. Its tree grows as a small sapling from 3 m to 8 m, with large branches having brownish or light brownish bark and it has thin leaves and is known for its edible fruit [61]. In the Aligarh district village in Uttar Pradesh, A. squamosa is well-known for its antidiabetic properties [63]. Its seeds, bark and leaves possess various pharmacological properties, mainly anti-tumour properties [64].

Annona Muricata
Annona muricata is a commonly known as soursop and graviola and is native to Central and South America. It is a small tree 5-10 m tall and 15-83 cm in diameter; it has low branches and edible fruit that are used commercially for the production of candy, juice and sherbets [65]. Traditionally, the aerial parts of this plant have been used to treat various diseases like diabetes and malaria and nowadays, it is widely used by people diagnosed with cancer [66]. Moreover, this species possesses several pharmacological properties, including vasodilator, cardio-depressive, antispasmodic, antimutagen, anticonvulsant, antiviral, antidiabetic and antihypertensive effects [67]. Both the leaves and seeds of A. muricata have been evaluated for their constituents resulting in the identification and isolation of more than 50 mono-THF acetogenins, alkaloids, terpenoids, saponins, flavonoids, coumarins, cardiac glycosides, phenols, tannins and anthraquinones [67].

Annona Reticulata
Annona reticulata Linn is a traditionally important plant utilized in traditional medicines [68]. It is indigenous to the West Indies and widely distributed in tropical and subtropical regions of the world [54]. It is a small tree with a height between 6 and 7.5 m and contains numerous lateral branches [54]. It has a cylindrical stem that contains lenticels and very short coffee-colored hairs [54]. The leaves of A. reticulata are lanceolate, membranous, oblong, and rounded or curate at the base. Fruits are edible, rough, somewhat heartshaped and yellow in color that shifts to yellowish-red on ripening, and the seed is smooth and blackish in color [69]. Traditionally, A. reticulata has been utilized for the treatment of epilepsy, dysentery, cardiac problem, constipation, haemorrhage, bacterial infection, parasite and worm infestations, fever, ulcers and as an insecticide [68,69]. Its leaves are used for helminthiasis treatment while bark is a powerful astringent and used as a tonic [68,69].

Annona Coriacea
Annona coriacea Mart. is a species belonging to the Annona genera, commonly known as "marolo", "araticum" and "araticum-liso" [70]. This plant is distributed across Paraguay and Brazil, with little available information about its ethnomedicinal uses [71]. It is a small tree (3-6 m) and its edible fruit consist of an ovoid-obtuse syncarp and weighing up to 1.5 kg [72]. The leaves are glabrous on the ventral surface, obovate, and the base is frequently cordate and margin undulate [73]. The flowers are terminal, thick, solitary and having fleshy petals with colors shifting between orange and pink [73]. The leaves are traditionally used as carminatives, anthelmintics, antirheumatics and in the treatment of stomatitis, headaches, abscesses, neuralgia, rheumatism, ulcers and dermatitis [74,75]. Both seeds and fruits are toxic when crushed and exhibited effects against ectoparasites like lice [75].

Annona Senegalensis
Annona senegalensis is a small tree 2-6 m tall that is commonly known as wild custard apple and wild soursop [76]. This plant is native to tropical east and northeast, west and west-central, and southern Africa and islands in the western Indian Ocean [76]. Its leaves are simple, alternate, oblong, green to bluish-green, ovate or elliptic, and mainly lack hairs on the upper surface and brownish hairs on the lower surface [77]. This plant has been used in traditional medicine as a pain reliever, antioxidant, antidiarrheal, antitrypanosomal, antimalarial, anti-inflammatory, antimicrobial, antiparasitic, anticonvulsant and as an antisnake venom [78]. It has been reported that the leaves of A. senegalensis are used for the treatment of tuberculosis, yellow fever and smallpox, whereas stem bark is reported for the treatment of injury from venomous animals [79]. The root was also reported for treating erectile dysfunction, tuberculosis, gastritis, reproductive deficiency and in the management of malaria and diabetes [80].

Annona Vepretorum Mart
Annona vepretorum is commonly recognized as 'bruteira', is a small tree of 2.5-10 m high native to the Brazilian biome Caatinga [81]. The fruits of A. vepretorum can be consumed either raw or as juice for nutritional purposes [82]. Traditionally, a decoction of the leaves have been used to bathe in for the treatment of allergies, yeast, skin diseases and microbial infections, whereas the root is traditionally used to treat snake and bee bites, inflammatory conditions and heart pain [81].

Annona Salzmannii
Annona salzmannii is a tree of 6-20 m high that known as "araticum-da-mata" and "araticum"apé" [83]. It is commonly cultivated in Brazil especially in the States of Bahia, Pernambuco, and Paraíba [83]. Its root, seeds and leaves are used in folk medicine for treating several illnesses like ulcers, dysentery and inflammatory conditions [83]. The leaves and bark of A. salzmannii are utilized for the treatment of tumors, diabetes and inflammatory conditions [84].

Annona Crassiflor
Annona crassiflor is known as araticum of cerrado or cerradão [85]. It is a small tree that bears a typical fruit known as araticum of cerrado or cerradão [86]. The fruits are highly consumed "in natura" by native people and can be used to make juice, jelly and ice-cream [85,86]. In folk medicine, the seeds are used to treat scalp infections, and infusions of the leaves and seeds are utilized for their antidiarrheal and antitumor properties [85]. For more details about the botanical characteristics and traditional uses of Annona species, see Tables 4 and 5.  [11,33]

Traditional and Ethnomedicinal Uses of Annona Genus
Traditionally, the Annona species have been used widely. For instance, antidiarrheal effects have been reported for A. reticulata, A. muricata and A. salzmannii, whereas A. cherimola, A. squamosa and A. reticulata have been reported for their antiparasitic effects (Table 5) [10]. Moreover, both A. vepretorum and A. salzmannii have been also reported for anti-inflammatory effects [10]. A. purpurea and A. reticulata have been used to treat fever, while anticancer effects have been reported for A. senegalensis and A. muricata [105,106]. Furthermore, A. foetida, A. muricata and A. glabra have been traditionally used to treat rheumatism [107], while A. reticulata, A. salzmannii, A. foetida and A. squamosa have been described for treating ulcers [4]. In Indonesia, the fruit juice of A. muricata has used as a diuretic and to treat liver ailments and leprosy [108], whereas leaves was used to treat spasms, boils and as an aphrodisiac [36]. The leaves of A. diversifolia have been used as anti-inflammatory, anticonvulsant and analgesic agents [52]. Ethnobotanically, despite reports of the toxicity of A. muricata seeds, the powder of toasted seeds has been reported to be used as an emetic and cathartic in the traditional Mexican pharmacopeia [36]. To Southeast Asian people, the immature fruit of A. reticulata was used to treat both dysentery and diarrhea, and a decoction of roots was used to cure toothache and as an antipyretic [109]. Additionally, a decoction of leaves has been used internally against worms and topically to treat abscesses and boils [109]. Finally, the leaves of A. squamosa have been used as tonic and cold remedy in tropical America and systemically to cure dysentery in India [108].

Phytochemistry of Annona Species
A wide range of secondary metabolites, including acetogenins, flavonoids, alkaloids and essential oils (Figure 1), from nearly every part of Annona plants, have been discovered, isolated and characterised ( Table 6). The plants of the Annona genera are also found to be rich in minerals and vitamins, for instance, calcium, potassium, magnesium, sodium, copper, zinc, selenium, phosphorus, iron, vitamin C, pantothenic acid B 5 , thiamine and riboflavin [6].

Pharmacological Properties of Annona Species
The species of the Annona genera have a been reported to elicit a diversity of biological activities such as antitumor, anti-inflammatory, antioxidant, antinociceptive, antiprotozoal, antipyretic, antiulcer, antihyperglycemic, anthelmintic, antileishmanial, antimalarial, antidiarrheal, antifungal and antimicrobial promoted by whole extracts, fractions, or pure compounds (Table 7).

Antibacterial Activity
The antibacterial activities of Annona species have been reported in many studies, for example, both methanolic and ethanolic leaf extracts of A. muricata exhibited antimicrobial activity against Staphylococcus aureus, and this activity was attributed due to the presence of flavonoids, alkaloids and steroids in the extract [165,166]. In contrast, an aqueous extract of the peel of A. muricata did not show any activity [165,166]. The root of A. reticulata has also been investigated for its antibacterial activity against Gram-positive Staphylococcus aureus, Bacillus cereus and Bacillus subtilis, and Gram-negative Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi [54]. The root extract was found to possess pronounced activity against Bacillus cereus as well as notable inhibition against all tested strains [54]. Moreover, the leaves of A. cherimola have been reported for antibacterial activity against Staphylococcus aureus and Bacillus subtilis with growth inhibition zone diameters of 11 mm and 14 mm, respectively [37]. The aqueous and methanolic seed extracts of A. squamosa have reported activity against Staphylococcus aureus with Minimum Inhibitory Concentrations (MIC) of 50 mg/mL and Minimum Bactericidal Concentrations (MBC) of 100 mg/mL [167]. The activity of the isolated compounds from Annona species has been reported in various studies; for instance, the fatty alcohol 11-hydroxy-16-hentriacontanone isolated from leaves of A. squamosa has a reported activity against Gram-positive and Gram-negative bacterial strains, with MIC values of 25-50 µg/mL [168]. Additionally, the alkaloids liriodenine, annonaine, asimilobine, reticuline and cleistopholine isolated from A. salzmannii demonstrated activity against a range of Gram-positive bacteria, including Kocuria rhizophila, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis with MIC values from 25 to 500 µg/mL [36]. Notably, annonaine and asimilobine had activity equal to or better than the control chloramphenicol (MIC 50 µg/mL) against many of the species tested [36].

Anticancer and Antiproliferative Activity
Various studies have reported the anticancer activity of either crude extracts or isolated compounds from Annona species. For example, the leaves extract of both A. squamosa and A. reticulata exhibited potent antiproliferative effects against two human T-lymphotropic virus type = 1 infected cell lines (MT-1 and MT-2) with EC50 values from 0.1 to 1 µg/mL [169]. In in vitro studies, the ethanolic extract of A. muricata leaves was reported for its cytotoxicity against promonocytic leukemic cells (U-937) with an LC 50 = 7.8 µg/mL [170].  [112].
The antiproliferative activity of methanolic extracts from the leaves and seeds of A. coriacea was tested in vitro against a range of human tumor cell lines; including melanoma (UACC-62), non-small cell lung cancer cells (NCI-H460), colon cancer cell line (HT29), breast cancer (MCF-7) and leukemia (K-562). The seed extracts displayed potent antitumor activity with GI50 values between 0.02 and 3.83 µg/mL, and the leaf extracts exhibited anticancer activity at concentrations ranging from 0.02 to 0.08 µg/mL [35,171]. The cytotoxicity of annonacin, found in many Annona species, has been reported against various cell lines derived from cervical cancer (HeLa and HeLa S3] with IC 50 0.219 and 0.426 µg/mL, and ovarian cancer (PA-1 and SKOV3) with IC 50 s of 0.452 and 0.411 µg/mL [172]. The cytotoxicity of annonacin has also been demonstrated against bladder cancer (T24), breast cancer (MCH7) and skin cancer (BCC-1) with IC 50 s 0.324, 0.433 and 0.427 µg/mL, respectively [172]. The cytotoxicity of five other acetogenins (squamocin M, annofolin, isolongimicin B, glaucanisin, and annotacin) isolated from A. cornifolia against human breast cancer (MCF-7) was reported, with IC 50 s of approximately 0.3 µM [173]. In an in vitro study, the annoaceous acetogenins laherradurin and cherimolin-2 isolated from A. diversifolia were shown to have ED 50 s of 0.015 and 0.05 µg/mL, respectively, against the cervical cancer cell line (HeLa) [174].
In clinical studies, the anticancer activity of A. muricata has been reported in a small number of studies. A patient diagnosed with breast cancer has maintained stable disease activity with no reported side effects after using an aqueous extract of A. muricata leaves for more than five years [175]. Another patient with metastatic ovarian cancer experienced disease stability after starting to take a complementary medication containing A. muricata as a tablet [176]. Finally, the effect of A. muricata leaves extract revealed higher cytotoxicity in the supplemented group with colorectal cancer compared with the placebo group in a randomized controlled trial [177].

Antidabetic and Antilipidemic Activity
Multiple studies have investigated the antidiabetic activity of various extracts from Annona plants such as A. cherimola, A. squamosa, A. muricata and A. reticulata. The ethanolic leaf extract of A. cherimola (300 mg/kg) was administered to alloxan-induced type 2 diabetic rats, and four hours later, blood glucose level had decreased from 331.5 mg/dL to 149.2 mg/dL [58]. The young leaves of A. squamosa, often in combination with black pepper (Piper nigrum), have been used in northern Indian traditional medicine as an anti-diabetic, and are still in use today. Administration of aqueous A. squamosa leaf extract to streptozotocinnicotinamide type-2 diabetic rats resulted in decreased blood glucose and increased levels of serum insulin [178]. Another traditional Indian medicine used as an antidiabetic and anti-lipidemic is a polyherbal formulation of A. squamosa fruits and Nigella sativa seeds. The polyherbal formulation administered over a one-month period, dose-dependently decreased blood glucose and increased insulin in streptozotocin-induced diabetic rats, with a dose of 200 mg/kg showing similar to the effects of a dose of 250 mg/kg tolbutamide [179]. A single dose of 100 or 200 mg/kg of aqueous leaf extract of A. muricata did not inhibit blood glucose levels in normal rats; however, the same doses administered to the diabetic rats effectively lowered blood glucose levels by 31.77% and 45.77%, respectively [180]. Finally, a dose of 100 mg/kg of both methanolic extract and the residual fractions of A. reticulata leaves decreased blood glucose levels from 432.33 to 371.67 mg/dL and 417.83 to 402.50 mg/dL, respectively, in streptozotocin-induced diabetic rats [181]. A. cherimola leaf extract was also found to decrease HbA1c by 7% and lead to a significant decrease in urine glucose over a 28-day subchronic study in streptozotocin-induced diabetic mice [182]. These studies support the traditional use of Annona species as antidiabetics, suggesting that further identification of the active constituent(s) with antidiabetic properties and clinical studies of a longer duration are warranted.
Limited studies have also investigated the antilipidemic activity of some Annona species. The polyherbal formulation of A. squamosa fruits and Nigella sativa seeds (200 mg/kg) administered to streptozotocin-induced diabetic rats for one month also resulted in significant inhibition of the formation of both lipid peroxide and tissue lipids [179]. Administration of an extract of A. muricata extracts resulted in reductions in the serum total cholesterol, lowdensity lipoprotein cholesterol and triglycerides in diabetic rats [183]. The tea infusion of leaves from A. cherimola (1.5 g) also elicited a reduction in the total cholesterol, triglycerides, and low-density lipoprotein by 15.4, 21.9 and 63.2%, respectively, in streptozotocin-induced diabetic rats [182].

Anti-Inflammatory Activity
The anti-inflammatory effects of Annona plants have been reported in many studies; for instance, after one day of orally administered doses of 200 and 400 mg/kg of A. squamosa root extract in an acute carrageenan-induced rat paw edema model, significant inhibition was produced with 24% and 47% inhibition respectively compared to diclofenac sodium inhibiting inflammation by 72% [184]. In an in vitro study, the chloroform extract of A. muricata leaves significantly inhibited activity of phospholipase A 2 [185]. With doses of 0.2-0.6 mg/mL, the enzyme activity was inhibited by 23.91%-43.48% [185]. In the same study, the chloroform extract of A. muricata leaves at 0.5 and 1.0 mg/mL also inhibited prostaglandin synthase activity by 87.46% and 82.92%, respectively, compared to the positive control indomethacin at 1 mg/mL which reduced enzyme activity by 87.46% [185].
Extracts of A. senegalensis roots were assessed for anti-inflammatory activity through in vitro inhibition of protein denaturation, hyaluronidase and xanthine oxidase. The ethyl acetate fraction was found to have the greatest activity inhibiting protein denaturation (70.6%), hyaluronidase (72.2%) and xanthine oxidase (78.7%) at a concentration of 100 µg/mL [186]. The ethanolic extract of A. muricata leaves exhibited anti-inflammatory effects in the carrageenan-induced rat paw acute edema model. Paw edema was reduced after orally administered doses of 200 and 400 mg/kg with (23.16% and 29.33%) and (29.50% and 37.33%), respectively, after 60 and 90 min of treatment [187]. Additionally, A. muricata fruit has been shown to exert an anti-inflammatory effect in a xylene-induced ear edema test [188]. Additional information regards pharmacological activities of Annona species, see (Table 7).
The lyophilized fruit extract at 50 mg/kg and 100 mg/kg inhibited the xylene-induced ear edema by 82.35% and 76.47%, respectively, compared to prednisolone, which reduced ear edema by 47.06% [188]. At intraperitoneal doses of 25, 50 and 100 mg/kg, the ethanolic extract of A. vepretorum leaves, inhibited carrageenan-induced leukocyte migration to the peritoneal cavity by 62%, 76% and 98%, compared to dexamethasone (2 mg/kg, i.p.). which reduced leukocyte migration by 89% [104]. The flavonoids, quercetin and kaempferol isolated from leaves of A. dioica exhibited potent dose-and time-dependent anti-inflammatory activity in a carrageenan-induced paw oedema model with IC 50 s of 8.53 and 10.57 µg/mL, respectively. The crude methanolic extract of A. dioica also reduced myeloperoxidase activity 6 h after the induction of paw oedema with a maximal inhibition of 51% at a dose of 300 mg/kg. [189]. Finally, hinesol, z-caryophyllene and beta-maaliene isolated from leaves of A. sylvatica also inhibited leukocyte migration at concentrations from 36.04 to 45.37 µg/mL in both carrageenan-and complete Freund's adjuvant-induced mouse paw edema [33].

Antioxidant Activity
An extract of A. coriacea seeds was investigated for its antioxidant activity using free radical 2,2-diphenyl-1-picrilhidrazil (DPPH) and bleaching of β-carotene, and a moderate antioxidant effect was reported of 31.53% in the DPPH test and 51.59% for the β-carotene bleaching test [190]. The pulp of A. coriacea fruit displayed a weaker antioxidant activity compared to the seeds, with 13.49% for the DPPH test and 32.32% for the β-carotene assay [190]. Additionally, various parts of A. muricata, including bark, leaves and stem, exhibited antioxidant activity using DPPH assay and the EC 50 value was recorded as 90 mg/g for bark, 290 mg/g for leaves, and 116 mg/g for the stem, compared to ascorbic acid with 157.5 mg/g [44]. Finally, an ethanolic extract of A. squamosa leaves was also reported as having antioxidant activity when evaluating DPPH, nitric oxide and superoxide radical assays. The activity was reported as 75.12%, 34.69% and 10.29%, respectively, at a concentration of 100 µg/mL [191].

Antileishmanial Activity
Many extracts and pure compounds from Annona plants have been tested against Leishmania, such as methanolic seed and leaf extracts of A. squamosa, for activity against L. amazonensis, with resulting showing IC 50 s of 46.54 µg/mL and 28.32 µg/mL, respectively [192]. Alkaloids and acetogenins isolated from both leaves and seeds of A. squamosa were also reported for their activity against promastigote forms of L. chagasi, with the EC 50 value reported as 23.3 µg/mL for alkaloids and from 25.9-37.6 µg/mL for acetogenins [192]. Alkaloids like liriodenin isolated from the leaves of A. mucosa exhibited antileishmanial activity against promastigote forms of L. braziliensis, L. guyanensis and L. amazonensis with IC 50 s of 55.92 µg/mL, 0.84 µg/mL 1.43 µg/mL respectively [193]. Finally, O-methylarmepavine isolated from leaves A. squamosa displayed antileishmanial activity against both promastigote and amastigote forms of L. chagasi with EC 50 s of 23.3 µg/mL and 25.3 µg/mL, respectively [45].

Antiviral Activity
The antiviral activity of various Annona species was reported in several studies using either whole extract or pure compounds. For instance, 16ß,17-dihydroxy-ent-kauran-19-oic acid was isolated from the fruits of A. squamosa and showed significant activity against human immunodeficiency virus (HIV) replication using H9 lymphocyte cell assay with EC 50 value of 0.8 µg/mL [194]. The ethanolic extract of A. squamosa seeds at 0.15 µg/mL, 0.25 µg/mL and 0.35 µg/mL also exhibited dose-dependent antiviral activity against the Avian influenza virus with the percentage of antiviral activity at 33.33%, 43.06% and 59.72%, respectively [195]. The leaves of A. squamosa extract were also tested against dengue virus type-2 (DENV-2) in Vero cells using Viral ToxGLo TM assay. At a concentration of 6.25 µg/mL, DENV-2 replication was reduced with IC 50 73.78 µg/mL in Vero cells [196]. The methanolic extracts from the peels of A. squamosa and A. reticulata demonstrated antiviral activity against human immunodeficiency virus 1 (HIV-1) using a non-radioactive immune/colorimetric assay. Both A. squamosa and A. reticulata revealed high antiviral activity by inhibition of HIV-1 reverse transcriptase with values of 96.45% and 78.63% [197].

Pharmacological Activity of Isolated Compounds from Annona Species and their Mechanism of Action
The in vitro and in vivo biological activity of compounds that have been isolated from various parts of Annona plants will be discussed. Squamins C-F were isolated from the seeds of A. globifora and tested in vitro against trophozoites of Acanthamoeba spp. strains such as A. castellanii Neff, A. polyphaga, A. griffin and A. quina (Table 8) [200]. All tested compounds exhibited antiamoeboid activity against the strains by inducing programmed cell death [200]. The same compounds were also tested for their cytotoxicity against murine macrophage cell line J774A.1 (ATCCTIB-67) and showed no cytotoxicity effect with CC 50 values greater than 200 µM [200]. Rollinicin and rolliniastatin-1 isolated from the seed of A. mucosa were also reported for their larvicidal effect against Aedes aegypti and Aedes albopictus larvae [201]. Rolliniastatin-1 exhibited the best larvicidal effect against both Aedes aegypti and Aedes albopictus with LC 50 s of 0.43 and 0.20 µg/mL −1 , respectively. Rollinicin displayed similar activity against Aedes aegypti and Aedes albopictus with LC 50 s of 0.78 µg/mL and 1.128 µg/mL, respectively [201]. However, the larvicidal mechanism action of these compounds was not reported. Annonacin isolated from the seed of A. muricata was evaluated for its larvicidal activity on Aedes aegypti and Aedes albopictus larvae [202]. The greater larvicidal activity was reported against Aedes aegypti with a LC 50 of 2.65 µg/mL compared to Aedes albopictus with LC 50 of 8.34 µg/mL. The mechanism of action was reported as being inhibition of their metabolic enzymes, particularly proteases and amylases that are important for the development of Aedes spp. larvae [202]. Twelve acetogenins isolated from the seed of A. cornifolia were tested for their antioxidant activity against DPPH [90]. These acetogenins were identified as 9-hydroxyfolianain, 4-desoxylongimicin, squamocin M, squamocin L, folianin A, folianin B, annofolin, isolongimicina B, bullatacin, asimicin, cornifolin and anotacin, and showed a strong DPPH radical scavenging with IC 50 s ranging from 0.99 ± 0.18 to 1.95 ± 0.34 µg/mL compared to ascorbic acid with an IC 50 1.62 ± 0.35 µg/mL [90]. It has been suggested that the antiradical activity of acetogenins may be related to the α,β-unsaturated lactone ring moiety, which is also present in ascorbic acid [90]. Furthermore, the antioxidant activity of pure compounds from the bark of A. salzmanni after isolation of five alkaloids identified as liriodenine, anonaine, asimilobine, reticuline and cleistopholine [203]. The antioxidant activity was assessed through the Oxygen Radical Absorbance Capacity (ORAC) assay and asimilobine was found to be the most active alkaloid with ORAC value of 2.09 [203]. The rest of the compounds exhibited antioxidant activity with ORAC values ranging from 0.25 to 0.85 [203]. These same compounds were also examined for their antimicrobial activity against Kocuria rhizophila, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis with MIC values from 25 to 100 µg/mL [203]. In an in vitro study, the antimicrobial activity of isolated alkaloids from aerial parts of A. senegalensis was assessed in a microdilution assay [154].
Many compounds isolated from various Annona species have demonstrated cytotoxicity against different cancer cell lines (Table 9). Three alkaloids were isolated from leaves of A. crassiflora identified as crassiflorine, xylopine and stephalagine and tested for their activity against colon carcinoma cells (HCT-116) using MTT assay [204]. The cytotoxicity activity for the tested compounds was reported with IC 50 values of 143.4 µM, 30.2 µM and 48.5 µM, respectively [204]. Muricin J-K isolated from the fruit of A. muricata exhibited anticancer activity against human prostate cancer cell lines (PC-3) through inhibition of the mitochondrial complex I in an in vitro study [205]. The anticancer activity was also reported for the alkaloid coclaurin isolated from aerial parts of A. squamosa. Cytotoxicity studies against human breast cancer cells (MCF-7), human colon cancer cells (HCT116) and human liver cancer cells (HEPG-2) reported IC 50 values of 15.345 µg/mL, 8.233 µg/mL and 1.674 µg/mL, respectively [206]. Bullatacin isolated from A. cherimola demonstrated inhibition of tumor growth at a dose of 15 µg/kg in mice bearing HepS and S180 xenografts and tumor growth was reduced by 63.4% and 65.8%, respectively [207]. In the same study, annonacin administrated orally (10 mg/kg) to hybrid mice (BDF-1) models significantly reduced lung cancer by 57.9% [207]. However, the mechanism of action of these acetogenins was not described in this study. Finally, stephalagin, an alkaloid isolated from the peel of A. crassiflora fruit, was reported as pancreatic lipase inhibitor with an IC 50 of 8.35 µg/mL −1 in vitro study [208].

Toxicity and Interactions
Generally, the safety of natural medicines can be assessed according to their effects and drug-drug interactions. An epidemiological study has reported that consumption of fruits of Annonaceae led to the prevalence of atypical parkinsonism in Guadeloupe due to the presence of acetogenins in the plant fruits [238]. According to Champy et al. 2005, the amount of annonacin per a single fruit is approximately 15 mg, and an adult who consumes a daily intake of one fruit for one year is equivalent to the amount of annonacin injected into rats, which induced the brain lesions [239]. It has been suggested that the toxicity might be related to the capacity of the tetrahydrofuran ring to chelate calcium ions [35]. Moreover, the fruit of A. squamosa has been analysed for its quantity of squamocin using HPLC-MS and reported 13.5-36.4 mg of squamocin for each fruit, and that long-term consumption of A. squamosa fruit may be a risk factor in the development of neurodegenerative disorders [240]. Additionally, the use of a dietary supplement sold in the USA containing an extract of A. muricata has been found to exhibit neurotoxic effects in human neuron cultures [241].
The interactions of Annona species with other drugs have been reported in other studies; for instance, administration of capsules of A. muricata leaves in combination with glibenclamide resulted in improved glycaemic control compared to patients who received only glibenclamide [242]. An additional study reported that a combination of aqueous custard apple leaf extract and glipizide enabled a decrease in the dose of glipizide by up to half in rats with type-2 diabetes and reduced the risk of requiring insulin therapy [243]. These outcomes suggest the potential use of certain Annona species in conjunction with antidiabetic medications to maximize the efficacy of a lower therapeutic dose.

Conclusions
This review provides a comprehensive summary of the botanical features, ethnomedical uses, pharmacology and phytochemistry of the main species of Annonacae family and, in particular, the Annona genera used in traditional medicine practices. Of the many members of the Annonacae family, the Annona species is heavily used in traditional medicines across the world. Among the 30 reviewed Annona species, six species A. squamosa, A. muricata, A. cherimola, A. senegalensis, A. reticulata and A. coriacea are the most widely studied for their pharmacological activities and phytochemical profiles of their bark, leaves, fruits and seeds. Various pharmacological properties have been reported, including antidiabetic, hepatoprotective, anti-inflammatory, antiprotozoal, antitumor, antioxidant, antimicrobial and anticonvulsant activity.
With regard to the phytochemistry of Annona species, the main classes of constituents identified to date are acetogenins, alkaloids, phenols and essential oils. The alkaloids are mainly present in the leaves, whereas acetogenins are present in the seeds and found in smaller quantities in the pulp and leaves of Annona species. The chemical profiles of the acetogenins present in different species have been extensively studied and their anticancer activity investigated, with low concentrations exhibiting chemotoxicity against several cancer cell lines. These preclinical results, along with the reported case studies, suggest that further clinical studies evaluating the role of acetogenins in the treatment of various types of cancers are warranted. Importantly, formulations, including the parts of the Annona species used, agricultural practices and the extraction methods vary considerably, leading to likely variations in the phytochemical and pharmacological profiles. In this respect, further characterization of standardized formulations of Annona species is required to predict likely clinical effects. Additional interesting results on the antidiabetic effects of fruits from Annona species also warrant further investigation as nutraceuticals to assist in the therapy of diabetes. Funding: This research received no external funding.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.