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Pharmacological Activities of Soursop (Annona muricata Lin.)

Soursop (Annona muricata Lin.) is a plant belonging to the Annonaceae family that has been widely used globally as a traditional medicine for many diseases. In this review, we discuss the traditional use, chemical content, and pharmacological activities of A.muricata. 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%). Several biological activities and the general mechanisms underlying the effects of A.muricata have been tested both in vitro and in vivo. 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.


Introduction
Presently, the use of natural ingredients as treatments for various diseases is increasing. Plants are a source of natural ingredients that are widely used as medicines. The compounds present in plants are responsible for their activities against various diseases, and studies can be performed to identify the active compounds in plants and determine their pharmacological activities against diseases [1].
Many studies on plants, their contents, and the pharmacological activities of their constituents have been conducted. Annona muricata Lin., commonly called soursop, is part of the Annonaceae family, which comprises more than 130 genera and 2300 species. A. muricata L. contains various compounds with pharmacological activity. This plant is widely grown in tropical and subtropical areas, such as Southeast Asia, South America, and the rainforests of Africa. 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 [1,2]. Different parts of A. muricata have different activities. 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 [3].
The main active components of A. muricata are acetogenin, alkaloids, and flavonoids [4]. Analysis of the compounds in A. muricata leaf extract revealed secondary metabolites such as flavonoids, terpenoids, saponins, coumarins, lactones, anthraquinones, glycosides, tannins, and phytosterols [5]. A. muricata has been widely used to treat many disorders, such as parasitic infections, inflammation, diabetes, and cancer [8]. All parts of A. muricata are used in traditional medicine by people who live in tropical areas, with the leaves, stem bark, roots, and seeds primarily used as medicinal ingredients [9]. A. muricata leaves are used to treat headaches, insomnia, cystitis, and cancer, the seeds are used to treat parasitic infections [1], and the fruit is used to treat diarrhea and neuralgia, eliminate worms and parasites, increase milk production in lactating women, and reduce fever [10].

Traditional Uses
Ethnobotanical studies have reported that A. muricata is used to treat bacterial and fungal infections, as it possesses anthelmintic, antihypertensive, anti-inflammatory, and anticancer activities. It has also been used as an analgesic and to treat fever, respiratory and skin illnesses, diabetes, and internal and external parasites. In several tropical sub-Saharan countries such as Uganda, all parts of the plant are used to treat malaria, stomach ache, parasitic infections, diabetes, and cancer [7,11].
Additionally, the seeds are used as anthelmintic and antiparasitic treatments, and the leaves, bark, and roots of A. muricata have been used for their anti-inflammatory, antihypertensive, sedative, antidiabetic, smooth muscle relaxant, and antispasmodic effects [1,12,13]. The leaves are used to treat cystitis, diabetes, headaches, hypertension, insomnia, and liver problems and as an antidysenteric, anti-inflammatory, and antispasmodic agent. The cooked leaves are applied topically to treat abscesses [14]. In tropical African countries, including Nigeria, the leaves are traditionally used to treat skin diseases [12].
In South America, A. muricata fruit juice is used to treat many diseases, such as heart and liver disease, and has antidiarrhea and antiparasitic effects [15]. The fruit flesh is used to increase breast milk production after childbirth and treat rheumatism, arthritic pain, fever, neuralgia, dysentery, heart and liver diseases, and skin rashes, and it has antidiarrhea, antimalarial, antiparasitic, and anthelmintic properties [1,16]. Table 1 summarizes the results of previous studies on the pharmacological activities of A. muricata and the underlying molecular mechanisms.
[21] Disrupts MMP function, causes leakage of cytochrome C from mitochondria, and activates caspase-3, caspase-7, and caspase-9 expression in HT-29 colon cancer cells. [22] Apoptosis mechanism mediated by a decrease in Bcl-2 expression and an increase in caspase-3 and caspase-9 expression in MCF7 breast cancer cells. [23] Seeds Increases caspase-3 cleavage and DNA fragmentation in endometrial cancer cells. [24] Antiulcer Leaf Activates prostaglandin synthesis and supresses aggressive factors of gastric mucosa.
Among the major compounds, acetogenin is the most abundant in A. muricata. Acetogenin is a long-chain fatty acid derivative that is widely present in the Annonaceae family and is produced via the polyketide pathway. Acetogenins have a long aliphatic chain of 35-38 carbons bonded to a g-lactone a-ring, terminally substituted by b-unsaturated methyl, with tetrahydrofurans (THF) located along the hydrocarbon chain ( Figure 2) [38,39]. The most abundant alkaloid compounds in A. muricata are reticuline and coreximine ( Figure 3).  The most common alkaloids present in this plant are the isoquinoline, aporphine, and protoberberine types [4,40]. The most common flavonoid is quercetin ( Figure 4) [41][42][43], although the most abundant flavonoid in the leaf extract is rutin, followed by quercetin and kaempferol [21].

Anticancer
The anticancer activity of A. muricata is related to its cytotoxic activity against cancer cells. Table 3 shows the effects of A. muricata against cancer cells.
Extracts from several parts of A. muricata act as anticancer agents via several mechanisms. Reportedly, the extracts of the fruit, stems, seeds, and twigs of A. muricata administered to fibrosarcoma cells (HT1080) inhibited matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9, which play important roles in cancer progression [17]. Extracts from the leaves, twigs, and roots inhibited the proliferation of the human leukemia cell line HL-60 by disrupting MMPs, reactive oxygen species (ROS) generation, and the G0/G1 cell cycle arrest that led to the inhibition of cancer cell growth [10] (Figure 5). Treatment of the A549 lung cancer cell line with the ethyl acetate extract of A. muricata leaves induced apoptosis via the upregulation of Bax and downregulation of Bcl-2 expressions. It has also been reported that apoptosis induced by the ethyl acetate extract of A. muricata leaf is related to cell cycle arrest at the G0/G1 phase [18]. The leaf extract induced apoptosis by enhancing the expression of caspase-3 in the colorectal cancer cell line COLO-205 [19] and the breast cancer cell line MDA-MB-231 [20]. Another study showed that the ethanol extract and ethyl acetate fractions of A. muricata leaves were active against MCF7 cells via an apoptosis mechanism mediated by decreased Bcl-2 expression and increased caspase-3 and caspase-9 expression [23]. Additionally, the presence of annonaceous acetogenins along with flavonoids in A. muricata leaves inhibited the proliferation of the human prostate cancer cell line PC-3 [21]. Several compounds isolated from A. muricata also show antiproliferative effects. Annomuricin E inhibited HT-29 cell growth by disrupting MMPs, causing leakage of cytochrome c from mitochondria, and activating the pro-apoptotic factors caspase-3, caspase-7, and caspase-9 [22]. Annonacin inhibited the proliferation of endometrial cancer cell lines ECC-1 and HEC-1A via annonacin-mediated apoptotic cell death, which was associated with an increase in caspase-3 cleavage and DNA fragmentation [24]. Another mechanism related to the anticancer activity of A. muricata is the modulation of antioxidant enzyme activities. A study reported that 50% ethanol extract of A. muricata leaves led to the upregulation of the expression of the antioxidant enzyme superoxide dismutase-1 (SOD1), which catalyzes the breakdown of superoxide into oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ), preventing cellular damage [73]. A study that administered 300 mg of A. muricata leaf water extract to patients with colorectal cancer in capsule form after breakfast reported the inhibition of colorectal cancer cell growth (DLD-1 and COLO 205). The A. muricata leaf water extract has selective inhibitory activity against colorectal cancer cells and does not inhibit normal cell growth. The inhibition of cancer cell growth is modulated by acetogenin activity in the complex I mitochondrial electron transport chain, hampering the process of ATP formation needed for cancer cell growth [22] (Figure 5). Other studies also showed the inhibitory effects of acetogenin against colon cancer cells. Consumption of 5 g of leaf extract powder and seeds of A. muricata three times per day accompanied by lifestyle modifications was shown to help the healing process in patients with colon cancer. Another study showed that one of the acetogenins in A. muricata, annocherimolin, has cytotoxic potential against HT-29 colon cancer cells [74].

Antiulcer
A. muricata contains a high concentration of flavonoids, tannins, and phenolic acids, which possess therapeutic effects due to their antioxidant, anti-inflammatory, and gastroprotective properties [25,75,76]. A survey revealed that the leaves and bark of A. muricata are popularly used to make tea to treat gastrointestinal problems such as gastritis and poor digestion [25].
In several studies, A. muricata has been reported to improve gastric lesions. A study that used a hydroalcoholic extract of A. muricata leaves to treat ulcers in absolute or acidified methanol-or indomethacin-induced gastric lesions in rats showed that the extract reduced the ulceration process by activating prostaglandin synthesis as a gastro protector and suppressed aggressive factors of the gastric mucosa [25]. In another study, A. muricata ethyl acetate extract showed antiulcer activity via ROS-scavenging and gastric wall damage protection in rats with ethanol-induced gastric injury. Additional mechanisms of A. muricata antiulcer activity include the upregulation of Hsp70 and the downregulation of Bax, which are involved in gastric injury suppression [26]. The minimal inhibitory concentration of A. muricata leaf extract against H. pylori is 20 mg/mL [77]. A. muricata also showed antiulcer activity by downregulating the expressions of Bax and MDA and upregulating the expressions of catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), nitric oxide (NO), PGE2, glycogen, and Hsp70 [27]. A. muricata also inhibited inflammatory mediators such as IL-1β, TNF-a, and IL-6 [78] (Figure 6).

Antidiarrhea
Diarrhea is a common gastrointestinal disorder caused by bacterial infections. It is characterized by abdominal pains, watery stools, and increased bowel movement frequency [79]. Antibiotics have been used as antidiarrhea drugs; however, disadvantages such as bacterial resistance and adverse side effects limit their usefulness [80]. Traditional plants are also widely used to treat diarrhea. The bark and fruit of A. muricata are widely used by West Africans to treat diarrhea, and their antidiarrhea effects have been reported [81,82]. The fruit of A. muricata showed antidiarrhea activity at a dose of 400 mg/kg body weight in mice with castor oil-induced diarrhea. Flavonoids, triterpenoids, and saponins of A. muricata play a role in its antidiarrhea activity by inhibiting intestinal motility and secretions that cause diarrhea [28].

Antidiabetic
A. muricata also exhibits antidiabetic activity. It contains flavonoids that inhibit αglucosidase activity through hydroxylation bonding and substitution at the b-ring. This inhibition suppresses carbohydrate hydrolysis and glucose absorption and inhibits carbohydrate metabolism into glucose [83].
A. muricata fruit extracts were reported to exert antioxidant and antidiabetic effects by inhibiting key enzymes relevant to type 2 diabetes mellitus, such as α-amylase and α-glucosidase, in vitro. A study showed that the pericarp of A. muricata has the highest inhibitory enzyme and antioxidant properties [33]. The fruit pulp and leaf extract also showed high abilities to inhibit α-amylase and α-glucosidase and minimize the rate of glucose assimilation into the blood after feeding compared with the standard drug [84]. The aqueous extract of A. muricata shows antidiabetic effects via antioxidant mechanisms. A. muricata leaf extract given to streptozotocin-induced diabetic mice induced a decrease in lipid peroxidation processes, which are a sign of oxidative stress, and indirectly affected insulin production and endogenous antioxidants [12].
A. muricata seed oil also showed potential antidiabetic activity against type 1 diabetes induced by streptozotocin. The study showed that an experimental model treated with A. muricata seed oil had significantly reduced blood glucose levels compared with the control group. The preserved area of the pancreatic islets was also improved compared with that in the control group [85]. Another study indicated that diabetic rats treated with A. muricata had significantly reduced blood glucose levels. That study also reported that daily intraperitoneal administration of 100 mg/kg A. muricata extracts to diabetic rats for 15 consecutive days resulted in a statistically significant increase in body weight despite a decrease in food and fluid intake, which is an indicator of improved glycemic control [86].

Antiprotozoal
Diseases caused by parasitic protozoa such as toxoplasmosis, trypanosomiasis, leishmaniasis, and malaria are the most common protozoal diseases worldwide [30]. The lack of treatment options for parasitic protozoal infections implies the importance of developing therapies for protozoal diseases by utilizing the potential of medicinal plants.
Several studies have been conducted to determine the antiprotozoal activity of A. muricata. A study reported that A. muricata ethanol leaf extract showed antiprotozoal activity against Toxoplasma gondii with an IC 50 of 113.3 µg/mL [29]. Another study reported that A. muricata ethyl acetate leaf extract showed antiprotozoal activity against Leishmania spp. and Trypanosoma cruzii with IC 50 s of < 2 5 µg/mL [30]. Moreover, some studies detected antiprotozoal activities in several compounds isolated from A. muricata. Two acetogenins, annonacinone and corossolone, that were isolated from the seeds of A. muricata showed antileishmanial activity, with an IC 50 ranging from 13.5 to 37.6 µg/mL [87]. A. muricata ethanol leaf extract also has antiprotozoal activity against Plasmodium falciparum with an IC 50 46.1 µg/mL [29]. Additionally, the dichloromethane fractions and subfractions of A. muricata bark and roots showed antiprotozoal activity against P. falciparum, with IC 50 values ranging from 0.07 to 3.46 µg/mL. Furthermore, gallic acid compounds isolated from the bark and roots of A. muricata showed activity against P. falciparum with an IC 50 3.32 µg/mL [32].

Antibacterial
A. muricata extracts showed antibacterial activity against Gram-positive and Gramnegative bacteria compared with the standard antibiotic streptomycin. However, the solvent used for extraction can affect the bioactive efficacy of the extracts. The combination of A. muricata ethanolic extract and antibiotic treatment decreased the potential of antibiotic multidrug-resistant Escherichia coli and Staphylococcus aureus strains [88][89][90]. Another study reported that the bioactive compounds in A. muricata, such as alkaloids (annonaine, asimilobine, liriodenirine, nornuciferine, etc.), attack the bacterial membrane (plasma and outer membrane), resulting in broad-spectrum antibacterial activity [34].

Antiviral
A. muricata extract has been reported to possess antiviral activity, for example by interfering with the replication process of HIV-I. In another study, ethanolic extracts from the stem and bark of A. muricata showed in vitro antiviral activity against herpes simplex virus [91,92]. Another study showed that acidified ethanolic extract of A. muricata decreased viral replication after 1 h of contact time. This activity may be due to the presence of phenolic compounds such as rutin [93]. Acetogenins such as annomuricin a, annomuricin b, annomuricin c, muricatocin c, muricatacin, cis-annonacin, annonacin-10-one, cis-goniothalamicin, arianacin, and javoricin were shown to possess good inhibitory activity against SARS-CoV-2 spike proteins (in silico). Cis-annonacin had a low binding energy and greater hydrogen bond formation ability, which indicated that it was the most potent among the acetogenins tested in the study. This result shows that annonaceous acetogenins can be viewed as potential anti-SARS-CoV-2 agents and should be studied in vitro and in vivo [94].

Antihypertensive
Research has revealed that A. muricata fruit extracts exhibit antioxidant and antihypertensive properties through angiotensin-I-converting enzymes in vitro [33]. A. muricata leaf extract was also shown to show antihypertensive activity in normotensive rats. The suggested hypotensive mechanism of action of A. muricata extract is via the blockage of calcium ion channels, which lowers blood pressure [35]. Another study showed that A. muricata aqueous extract had antihypertensive properties; combinations of A. muricata and other plants, such as Persea americana, also exhibited antihypertensive activity, providing a safe and effective solution for hypertension prevention and treatment [95].

Wound Healing
A. muricata is also known for its wound healing activity. Two doses of A. muricata ethyl acetate extract showed significant wound healing activity in both macroscopic and microscopic analyses of wounds. Wound treatment with an ointment containing A. muricata ethyl acetate extract caused a significant increase in antioxidant levels and a decrease in the MDA level in wound tissues compared with those in the vehicle control [96]. A. muricata bark and leaf extracts also showed wound healing effects compared with that in untreated wounds [97].

Toxicology
Several studies have been conducted to determine the toxicity of A. muricata. The level of toxicity of A. muricata depends on the part of the plant as well as the solvent. A study showed that A. muricata aqueous extract had an LD 50 > 5 g/kg, whereas that of the ethanolic extract was >2 g/kg [14]. Another study reported an LD 50 of >211 mg/kg for A. muricata leaf aqueous extract, which is higher than the recommended daily consumption limit for humans. The aqueous extract of A. muricata with doses of >1 g/kg can cause hypoglycemic conditions and hyperlipidemia, and doses of >5 g/kg can cause damage to the kidneys [98].
A study reported that acetogenin in A. muricata is a neurotoxin that has the potential to cause neurodegenerative disorders [99]. Acetogenin causes an increase in tau phosphorylation, which is associated with neurodegenerative tauopathy [100]. In addition, some alkaloids in A. muricata are believed to have an influence on nerve cells [1]. Annonacin, the most abundant acetogenin in A. muricata, as well as some types of alkaloids, such as reticuline, solamin, and coreximine, disrupt the energy formation process in dopaminergic cells [4]. In murine model tests, annonacin was demonstrated to penetrate the brain barrier, decrease ATP levels in brain cells, and damage the basal ganglia [101]. In mice, annonacin caused a decrease in ATP levels in the striatum and disrupted energy production by mitochondria, resulting in the disruption of tau cells, which led to symptoms of neurodegenerative disease [99].
Although some compounds in A. muricata have been reported to play a role in neurodegenerative disorders, the doses that produced negative effects were equivalent to consuming one fruit every day for 1 year [102]. Research on the neurotoxicity of annonacin showed that neurodegenerative conditions caused by these compounds arise due to continuous exposure or consumption. Thus, to avoid the occurrence of neurodegenerative conditions that may occur due to compounds present in A. muricata, continuous excessive consumption is not recommended [103].

Conclusions
A. muricata is widely used as a traditional medicine. Parts of the A. muricata plant, such as the leaves, fruit, seeds, bark, and roots, have pharmacological properties. From the 49 research articles that we obtained, it was reported that its pharmacological properties included anticancer (25%), antiulcer (17%), antidiabetic (14%), antiprotozoal (10%), antidiarrhea (8%), antibacterial (8%), antiviral (8%), antihypertensive (6%), and wound healing properties (4%) (Figure 7), because of the various compounds contained in A. muricata. Meanwhile, from 35 reference articles, 101 single compounds of A. muricata were reported. The main active compounds in A. muricata are acetogenins (49%), alkaloids (26%), flavonoids (19%), and others (6%), which are reported to be responsible for the pharmacological activities listed above ( Figure 8). However, not all secondary metabolites of A. muricata have been identified. Many studies on A. muricata were conducted in the past three decades; however, no preparations produced from A. muricata have been tested and approved by the FDA or EMA. Acetogenins, which are the main active compounds, are difficult to obtain because they are thermolabile, creating challenges for the scale-up and production of stable raw materials. Thus, developing drug preparations is difficult, despite the empirical evidence regarding the bioactivity of acetogenin compounds. Moreover, high doses of acetogenins can be neurotoxic and may cause neurodegenerative disorders. Some alkaloids present in A. muricata are also believed to affect nerve cells. However, research on the neurotoxicity of annonacin states that neurodegenerative conditions caused by these compounds arise due to continuous exposure or consumption. Further research on the toxicity of A. muricata and clinical trials testing the pure compounds are needed to fully elucidate its pharmacological activities and ensure the safety of A. muricata as a potential drug for various diseases.