Seed Waste from Custard Apple ( Annona squamosa L.): A Comprehensive Insight on Bioactive Compounds, Health Promoting Activity and Safety Proﬁle

: Annona squamosa L. (custard apple or sugar apple), belonging to the Annonaceae family, is a small tree or shrub that grows natively in subtropical and tropical regions. Seeds of the custard apple have been employed in folk medicines because of the presence of bioactive chemicals/compounds such as alkaloids, ﬂavonoids and phenolic compounds and acetogenins and cyclopeptides that are responsible for various biological activities. The seeds also show the presence of tannins, vitamin C, vitamin E and a higher content of amino acids. From investigations, it has been shown that the seeds of A. squamosa have considerable potential to be used as an antibacterial, hepatoprotective, antioxidant and antitumor/anticancer agent. Cyclosquamosin B, extracted from the custard apple seed, possesses vasorelaxant properties. Tocopherols and fatty acids, notably oleic acid and linoleic acid, are also found in the seed oil. A. squamosa seeds contain a high amount of annonaceous acetogenins compounds, which are potent mitochondrial complex I inhibitors and have high cytotoxicity. A survey primarily based on the nutritional, phytochemical and biological properties showed that A. squamosa seeds can be used for the discovery of novel products, including pharmaceutical drugs. Although there are sufﬁcient in vitro and in vivo experimental investigations supporting the beneﬁts of seeds, clinical investigations/trials are still needed to determine the health contributing beneﬁts of A. squamosa seeds.

search was performed from April to August 2022. Thus, it was concluded that the A. squamosa seed lacks a compilation of important information on its nutritional and phytochemical profile and biological activities. Hence, the present review highlights the crucial information regarding the nutritional, pharmacological and biological properties of A. squamosa seeds and also specifies the research areas that are less focused or that have not been previously studied.

Figure 1.
Custard apple fruit and its seed by-products.

Proximate Composition of Custard Apple Seeds
The fresh fruits of A. squamosa are commonly eaten in various regions of India, but the seed oil has not been reported yet for edible purposes. The seed of the custard apple is mainly composed of a seed coat (32.4%) and a seed kernel (67.7%). On a dry weight basis, the investigation shows a 22.2% content of crude fatty oil in seed kernels. The method of gas chromatography-mass spectroscopy (GC/MS) was used for studying the methyl esters of custard apple seed's fatty oil to determine its chemical composition, and results showed a total of 11 fatty acids, among which linoleic acid (22.9%), oleic acid (47.4%), palmitic acid (12.1%) and stearic acid (13.6%) were present in higher amounts. 11eicosanoic acid (0.2%), dihydro sterculic acid (0.1%), eicosanoic acid (0.9%), heneicosanoic acid (2.3%) and margaric acid (0.2%) were all found in lesser amounts in the oil. These 11 fatty acids together constitute nearly 99.8% of the oil. 17-methyloctadecanoic acid (0.1%) and palmitoleic acid (0.01%) were both identified in traces or in a minimum amount. The high quantity of unsaturated fatty acids (UFAs) was also determined in further investigations of the oil. About 70.3% of the oil was contributed by linoleic acid (22.9%) and oleic acid (47.4%); similarly, 25.7% of the oil consisted of palmitic acid (12.1%) and stearic acid (13.6%) [21]. Mariod et al., [22] have demonstrated the presence of leucine, isoleucine, glutamic acid, phenylalanine-tyrosine, aspartic acid, serine, alanine, methionine-cystine, histidine, arginine, glycine, valine, threonine and lysine in the amounts of 0.845, 0.464, 0.995, 0.671, 0.684, 0.299, 0.594, 0.106, 0.139, 0.704, 0.392, 0.642, 0.324 and 0.407 g/100g protein, respectively [22]. A proximate composition analysis of A. squamosa seeds demonstrated the presence of carbohydrate, fat, fiber, ash, protein and moisture at concentrations of 66.64, 29.21, 32.64, 1.90, 2.25 and 3.92 g/100g dry weight (DW) (%), respectively [23]. On the other hand, minerals (in mg/kg) such as K (56.47%) and Ca (46.90%) are present in Figure 1. Custard apple fruit and its seed by-products.

Phytochemical Profile of Custard Apple Seeds
Phytochemical investigations reported cyclopeptides and annonaceous acetogenins as the chief constituents in the seeds of A. squamosa [9,23]. Different parts of A. squamosa contain several phytochemicals involving alkaloids, such as aporphine, norcorydine, roemerine, corydine, glaucine, anonaine and norisocorydine, in different parts of the plant [30,31]. The seeds of A. squamosa were found to contain acetogenins (polyketide), namely annotemoyin−1 and 2, cholesteryl, coumarinoligans, glucopyranoside, squamocin, and squamocins B-N [32]. It was demonstrated that the custard apple seeds are toxic, but they are used as a biopesticide or an insecticide (its preparation may cause eye irritation that results in damage to the cornea). Custard apple seeds contain a high oil content and can be used for the production of soap and/or, if treated, can be used as an alternative to cooking oil [33]. Seeds contain volatile substances such as 12,15-cis-squamostatin-A, bullatacin, β-caryophyllene, α-pinene, β-pinene, anonaine, camphene, spathulenol, germacrene, squamocin, duvariamicin-III, myrcene, liriodenine, annonacin and molvizarin [34]. In a study, the identification of phytochemicals present in the seed extract of A. squamosa was carried out via Fourier-transform infrared (FTIR) analysis [35]. The result of the investigation shows the presence of alkenes, imine, oxime, quinone or conjugated ketone, nitro compounds, amides, nitroso compounds, sulfone, aromatics, sulphate ester, alkyl halides, phosphine, ethers, phosphonate, trimethylsilyl, amine oxide, phosphor amide, carboxylic acids, thiocarbonyl esters, phosphine oxide, phosphate, organosilicon, phosphite esters and amines in the methanolic seed extract of A. squamosa. The atmospheric pressure chemical ionization mass spectrometry (APCI-LC-MS) analysis revealed the presence of numerous acetogenins such as squamocin, annonacin and annonacin VI and cyclopeptides such as cyclosquamosin A and B and cyclosquamosin H in the seed extract of A. squamosa [35]. The earlier studies reported the medicinal properties of squamocin and annonacin present in the seeds of A. squamosa, suggesting that these compounds play a major role in the insecticidal, anti-inflammatory and anticancer properties [34,[36][37][38]. In a study, the seed extract of A. squamosa in different solvent systems (petroleum ether, water and methanol) was tested for phytochemical screening. The presence of alkaloids, carbohydrates, flavonoids, glycosides, phenols, proteins, saponins, sterols, tannins and terpenoids was observed in the methanolic and aqueous seed extract, whereas the presence of only alkaloids, flavonoids and tannins was observed in the petroleum ether extract [39]. The effects of the extraction conditions and the solvent nature on the total flavonoid content (TFC) and the total polyphenol content (TPC) have been demonstrated in different studies. Nguyen et al., [40] determined the TPC and TFC from A. squamosa seed extract under different reaction conditions and, from the results, the values of TFC and TPC were observed to be 189.15 mg quercetin equivalent (QE)/100 g DW and 234.17 mg gallic acid equivalent (GAE)/100 g DW, respectively, in the ethanolic seed extract, which are found to be higher in comparison with the methanolic seed extract (183.90 mg QE/100 g DW and 232.01 mg GAE/100 g DW) and the water seed extract (84.90 mg QE/100 g DW and 113.89 mg GAE/100 g DW).
A study by Leite et al., [29], has also determined the TPC and TFC in the methanolic seed extract of A. squamosa and, interestingly, the results showed that the seeds have a significantly higher flavonoid content (893.30 µg QE/g extract), and phenolic content (32.53 µg GAE/mg extract). Flavonoids such as isoquercetin, rutin, quercetin and gallic acid have been identified in the seeds [41]. The presence of leucoanthocyanins, polyphenols, tannins, triterpenes and unsaturated sterols have also been demonstrated in chemical studies of the A. squamosa seeds [42]. The seeds of A. squamosa also show the presence of annoglaxin, annosquacin A-D, annosquamin A-C, bullatencin, dotistenin, murisolin, cyclosquamosin B-I, squamin A and B, uvariamicin I-III and many other annonaceous acetogenins and cyclopeptides as the main constituents, each responsible for different functions such as vasorelaxant, cytotoxic against various cancer cell lines, antibacterial and nematicidal, etc. [9]. In a recent study of A. squamosa, phenolic compounds were determined in the seed extract and the result revealed the presence of cinnamic acid and its derivatives involving ferulic acid (5.08 mg/100 g), o-coumaric acid (49.02 mg/100 g) and p-coumaric acid (1.96 mg/100 g), gallic acid, p-hydroxybenzoic acid, salicylic acid and syringic acid in the seeds of A. squamosa. The seeds of the custard apple also show the presence of flavonoids, phenolic compounds, sulfated polysaccharides, tannins and triterpenoids [23]. In a study conducted by Janicke et al., [43], it was observed that dietary fiber abundant in ferulic acid, hydroxycinnamic acid and p-coumaric acid are protective against cancer. Furthermore, p-coumaric acid and o-coumaric are reported to be responsible for antimutagenic, anti-inflammatory, antioxidant, antitumor and anticancer activity [43,44]. Recently, fruits of A. squamosa have attracted a lot of attention due to their numerous health benefits. However, this current study provides information on the seeds of A. squamosa with its numerous health-related biological properties. The major nutritional and phytochemical components of A. squamosa seeds are shown in Figures 2 and 3. against cancer. Furthermore, p-coumaric acid and o-coumaric are reported to be responsible for antimutagenic, anti-inflammatory, antioxidant, antitumor and anticancer activity [43,44]. Recently, fruits of A. squamosa have attracted a lot of attention due to their numerous health benefits. However, this current study provides information on the seeds of A. squamosa with its numerous health-related biological properties. The major nutritional and phytochemical components of A. squamosa seeds are shown in Figures 2 and 3.

Pharmacological Properties
Recently, custard apple seeds have emerged as a potential ingredient for the development of supplementary foods because of its significant nutraceutical and phytochemical composition. However, the development of by-products by integrating the bioactive compounds from the custard apple seeds are well-endowed with novel pharmacological properties. These properties of A. squamosa or the custard apple seed have been exten-

Pharmacological Properties
Recently, custard apple seeds have emerged as a potential ingredient for the development of supplementary foods because of its significant nutraceutical and phytochemical composition. However, the development of by-products by integrating the bioactive compounds from the custard apple seeds are well-endowed with novel pharmacological properties. These properties of A. squamosa or the custard apple seed have been extensively studied for the antimicrobial, antidiabetic, anti-inflammatory, anticancer, antitumor, antioxidant, hepatoprotective, antiproliferative, antiheadlice, antihelminthic and antilarval activities ( Figure 4). The important pharmacological properties/activities are well discussed in the following Sections 4.1-4.7.

Antimicrobial Activity
Antimicrobial activity refers to all the active agents that inhibit the growth of bacteria, prevent the development of microbial colonies and sometimes kill the microorganisms (microstatic); however, microorganisms have become resistant to many antibiotics, which results in immense clinical challenges/issues in treating infectious diseases. This issue has forced researchers to find new antibacterial substances, especially plant-based ones. In a study, the chloroform extract of A. squamosa seeds was tested against different bacterial strains, involving Escherichia coli, Bacillus subtilis, Staphyllococcus aureus, Klebsiella pneumoniae, Salmonella typhi and Proteus mirabilis, to check the effectiveness of the seed extract against human pathogenic bacteria. The chloroform seed extract (10-60 µg/mL) showed significant antibacterial activity with a minimum inhibitory concentration of 13.6 µg/mL, 16 µg/mL, 33.1 µg/mL, 37.7 µg/mL, 50 µg/mL and 51 µg/mL and an inhibitory effect of 37-56%, 40-60.75%, 36-64%, 48.5-63%, 35-53.5% and 34-47% for K. pneumoniae, B. subtilis, E. coli, P. mirabilis, S. typhi and S. aureus, respectively. The results of the investigations showed that custard apple seeds (chloroform extract) might be used to treat bacterial infections [45]. Likewise, in another study, three different solvent extracts of the seeds of A. squamosa, viz. petroleum ether, methanol and chloroform, were investigated for their antibacterial property. The findings of the study showed that the petroleum ether extract exhibits the highest antimicrobial effect for S. aureus with a zone of inhibition (ZOI) of 12 mm and the lowest for Pseudomonas aeruginosa with ZOI of 7.8 mm, while the methanolic extract exhibits significant inhibition against K. pneumoniae with ZOI 12.8 mm and against Bacillus subtilis with ZOI 9.2 mm and the chloroform extract against E. coli with ZOI 14.8 mm and B. subtilis with ZOI equal to 1.7 mm [46]. In a different study, conducted by Aamir et al., [47], the methanolic seed or cotyledon extract of A. squamosa was investigated for its antimicrobial effect against microbial strains S. aureus, K. pneumoniae, S. typhi, E. coli, Enterococcus faecalis, Salmonella paratyphi and Pseudomonas aeruginosa. The findings of the study reported that the methanolic seed extract (50 mg/mL) inhibits the growth of the tested bacterial strains with ZOI equal to 27-30 mm for E. coli, 31 mm for S. typhi, 27-32 mm for S. aureus, 23 mm for E. faecalis, 22-24 mm for P. aeruginosa, 22-30 mm for S. paratyphi and 11-20 mm for K. pneumoniae. Similarly, when the authors of the abovementioned study used the A. squamosa methanolic seed extract in combination with the Phoenix dactylifera seed extract (2:1), it resulted in strong synergistic effects against all the tested microorganisms (ZOI: 18-30 mm), except for P. aerugenosa and S. paratyphi [47]. The results of these investigations indicate that the seed extracts of this plant could be used to treat enteric diseases. Similarly, combinational antimicrobial activity (for S. aureus, K. pneumoniae, E. coli, S. typhi, E. faecalis, P. aeruginosa and S. paratyphi) was also observed for the methanolic seed extracts of Annona squamosa and Prunus persia (1:2), using the agar disc diffusion method [48]. From the results, it was observed that the seed extract of both plant species when used in combination exhibited strong inhibitory effects against all the tested pathogens, with ZOI ranging between 18-34 mm [48]. In the latest study, it was revealed that the seed extract of A. squamosa exhibited the best antimicrobial activity against various pathogenic bacteria, including E. coli, B. subtilis, Candida albicans, K. pneumoniae, S. aureus and Salmonella senftenberg, owing to the fact that these pathogens act as foodborne pathogens, with an inhibition zone of 9.50, 12.50, 9.53, 10.33, 12.30 and 6.50 mm, respectively [23]. However, due to the advances in the field of nanomedicine and nanotechnology, some reports suggest that plant-mediated copper oxide nanoparticles (pm-CuO NPs) from the aqueous extract of the custard apple showed significant antibacterial effects against the plant pathogenic bacterial strain Xanthomonas oryzae, which is responsible for bacterial blight in rice crops [49]. Further, the well-diffusion assay against X. oryzae exhibited sensitivity towards 500 mg and 1000 mg pm-CuO NPs, demonstrating 9 mm and 15 mm ZOIs, respectively [49]. The results proved that the seed extract of A. squamosa has promising properties for combating microbial infections, even agricultural pathogens. However, further studies are still required related to the utilization of nanoparticles prepared from the A. squamosa seed extract before its on-field utilization as an antibacterial agent.

Antidiabetic Activity
Diabetes is one of the common endocrine disorders, characterized by altered carbohydrate, insulin and protein metabolism as a consequence of pancreatic insulin deficiency or insulin dysfunction [50]. According to the reports of the World Health Organization (WHO), 80-90% of people above 40 years old are more prone to non-insulin-dependent diabetes mellitus [51]. In a study, the ethanolic and methanolic seed extract of A. squamosa was administered to alloxan-induced diabetic rats (150 mg/kg body weight (BW)) to check its effect on blood glucose levels in diabetic rats [52]. The ethanolic (dose: 200 mg/kg) and the methanolic (dose: 200 mg/kg) seed extract of A. squamosa exhibited significant dosedependent 43.96% and 45.99% antihyperglycemic activity, respectively. From the results, it was observed that the ethanolic extract was less effective than the standard used (glibenclamide) for hyperglycemic activity [52]. Compounds such as saponins, flavonoids, acetogenins, phenolic compounds and alkaloids are known to be active antidiabetic agents [1,52]. The antidiabetic property of the methanolic and ethanolic seed extract of the apple may be due to the presence of more than one antihyperglycemic agent mentioned above [52]. Further, the action mechanism will be explained by pharmacological and biological studies that help in presenting the seeds of A. squamosa as therapeutic agents in antidiabetic research. However, there are very limited studies investigating the antihyperglycemic effect of A. squamosa seeds. It has been concluded that there is a need to conduct investigations in the same field.

Anti-Inflammatory
Inflammation is the human body's defensive system, regulated by pro-and antiinflammatory mediators (chemokines, cytokines, etc.) [53]. Many factors may cause inflammation in the human body, including exposure to allergens, physical trauma, thermal or chemical stimuli and microbial infection [54]. However, inflammatory disorders such as chronic asthma, rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis may be caused by internal or external factors that disrupt anti-inflammatory mediators [55]. For instance, the seed extract of A. squamosa causes a decrease in IL-6 and TNF-α levels in the lipopolysaccharide (LPS)-stimulated macrophage J774A.1 cell line [56]. It was shown that two parallel synthesized cyclic cyclopeptides extracted from the seeds of the custard apple, i.e., cyclosquamosin D and cyclopeptide B, were demonstrated to have anti-inflammatory actions by inhibiting the generation/production of IL-6 and TNF-α. From the results, it was concluded that cyclopeptides have strong anti-inflammatory effects, reducing the levels of IL-6 and TNF-α in the bloodstream (~25%), with an IC 50 value of 1.22 and 9.2 µM, which is significantly higher than observed with natural products or natural cyclic peptides [56]. Similarly, in another study, cyclosquamosin D inhibited the generation of pro-inflammatory cytokines in Pam 3 Cys-stimulated and LPS-stimulated J774A.1 macrophages in a dose-dependent manner. For TNF-α a 60-20%, whereas for IL-6 a 50-10% reduction was observed at a dosage of 5-50 µg/mL, respectively [57]. From the findings of different studies, it was deduced that cyclic peptides extracted from custard apple seeds might be utilized as an anti-inflammatory agent, though further investigation is needed for their anti-inflammatory effect.

Anticancer/Antitumor Activity
Cancer is a genetic disorder, caused by the mutations that happen to take control of genes in our body and control how cell functions, grow, multiply and die [58][59][60]. The reports of Cancer Research UK estimate that there are more than eight million cancer-related deaths worldwide per year and this may increase in the future [61]. In a study conducted by Chen et al., [18], the in vivo and in vitro antitumor activity of acetogenins isolated from the custard apple seed oil was investigated against human tumor cell lines. Two major acetogenins isolated from the seed oil of A. squamosa, i.e., 12, 15-cis-squamostatin-A (47.98 mg/g) and bullatacin (256.18 mg/g), were detected and quantified by high-performance liquid chromatography (HPLC). The result of the study indicates that seed oil shows considerable antitumor properties against A-549, Hela, MCF-7 and HepG2, especially for Hep G2 (IC 50 : 0.36 mg/mL) and MCF-7 (IC 50 : 0.25 mg/mL) cells in vitro [18]. Furthermore, the oral treatment of custard apple seed oil also prevents growth of H 22 tumor cell lines in mice, with a reduction rate of 69.55% with no post-treatment side effects, suggesting that the seeds of the custard apple may be used as a potent ingredient for the production of anticancer drugs [18]. Similarly, the seed oil (dose: 0.5-1.0 mL/Kg) of the custard apple shows considerable antitumor activity in H 22 xenograft-bearing mice, with an inhibitory rate of 53.54% against the development of H 22 cell lines [62]. It was found that seed oil shows antitumor effects by inhibiting the interleukin-6/Jak/Stat3 signaling pathway by reducing the production of interleukin-6, Janus kinase and activators of transcription (p-Stat3) and phosphorylated signal transducer expressions [62]. Acetogenins such as squafosacin B, squadiolin A and squadiolin B are well-known cytotoxic acetogenins found in the seeds of the custard apple [63]. Hence, in a study, it was proved that squadiolin A and squadiolin B have a significant cytotoxic effect on MDA-MB-231, with an IC 50 value of 0.63 and 0.28 µM, respectively. Furthermore, squafosacin B also exhibits cytotoxicity against HepG2 (IC 50 : 0.71 µM), Hep 3B (IC 50 : 0.72 µM) and the MCF-7 cell line (IC 50 : 0.96 µM) [63]. In another study, A. squamosa seed-oil-synthesized nanoparticles (ASSO-NPs) exhibited strong antitumor activity against the 4T1 mouse breast cancer cell line and showed more enhanced properties than the free seed oil (in vivo) [64]. The findings of the investigation revealed that the ASSO-NPs group (15 mg/kg) has the highest tumor growth inhibitory rate of 69.8%, significantly greater than the free seed oil group (135 mg/kg, 52.7%, p < 0.05) in a 4T1 tumor-bearing mice model [64]. In addition to antitumor activity, it was noticed that there was no significant change in the weight of the mice, indicating that ASSO-NPs have good safety [64].

Antioxidant Activity
Oxygen free radicals or reactive oxygen species (ROS) have a dual role in the biological system. They can be both beneficial and/or harmful [65,66]. ROS play a role in a wide range of diseases as well as in food spoilage through autoxidation of lipids and enzymatic oxidation during storage and processing of fats, oils and other fat-based products [67,68]. In a study, Kothari and Seshadri [69] evaluated the free radical scavenging activity of the A. squamosa seed extract (solvent used: acetone, hexane, water, chloroform: methanol (2:1) and ethanol (50%), using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay). From the results, it was concluded that the highest and the lowest antioxidant activity was observed for the water (777.64 g GAE/g) and the hexane (268.75 g GAE/g) seed extract of A. squamosa, respectively. Apart from the water and the hexane seed extract, other solvent extracts, i.e., acetone (229.29 g GAE/g), chloroform methanol (203.81g GAE/g) and ethanol (427.14 g GAE/g), also exhibited antioxidant potential [69]. Similarly, in a related study, the seed extract exhibited antioxidant activity with an IC 50 value of 7.88 µg/mL, using a DPPH assay [23]. In a different study, ethanolic seed extract was assessed for antioxidant activity in alcohol-induced liver impairment in Sprague Dawley rats (150-210 g) by oral treatment of the seed extract (200 and 400 mg/kg po) once a day continuing for 8 days [32]. It was demonstrated that treatment with the ethanolic seed extract significantly increased the level of antioxidant markers such as superoxide dismutase (SOD) (p < 0.01-p < 0.001), glutathione (GSH) (p < 0.05-p < 0.001) and catalase (CAT) (p < 0.05-p < 0.001), while decreased the level of thiobarbituric acid reactive substances (malondialdehyde) (TBARS (MDA)) (p < 0.05-p < 0.001) [32]. Thus, treatment with ethanolic A. squamosa seed extract causes the restoration of altered antioxidant enzymes towards normal. However, it was observed that there are not many studies related to the antioxidant potential of A. squamosa seeds, although several studies have derived a correlation between the phenolic content and the antioxidant property of plants [23,69,70]. Further studies for the identification of bioactive compounds responsible for antioxidant activity are required. Such research investigations may be helpful in product development, cosmetics, nutraceuticals and biopharmaceuticals in the race against illnesses such as cardiovascular disease, cancer and neurological disorders and may also contribute to the database of significantly important medicinal plants.

Hepatoprotective Activity
The liver is a critical organ in vertebrates and is prone to various disorders globally, including liver damage due to alcohol, pharmaceutical drugs (paracetamol, chemotherapeutic treatments) and toxic materials [71,72]. Alcohol is the third most frequent cause of mortality following smoking and hypertension [73]. The long-term use of alcohol causes alcoholic liver disease (ALD), a global concern without an effective solution, and chronic hepatotoxicity, which may develop to liver fibrosis and cirrhosis [74]. ALD has the highest impact in Europe (12%) and accounts for 4% of mortality worldwide and 5% of disabled life [75,76]. In a study, the ethanolic extract of custard apple seeds was used to treat liver damage caused due to alcohol in a rat animal model [32]. The liver toxicity in the tested animals was induced by the administration of 50% alcohol at a dosage of 12 mL/kg for a time interval of 8 days. Oral treatment followed, with seed ethanolic extracts at a dose level of 200 and 400 mg/kg, once for the following 8 days. The antihepatotoxicity effect of the seed extract was assessed in the rat animal model via measuring alanine aminotransferase (ALT), lactate dehydrogenase (LDH), aspartate transferase (AST), alkaline phosphatase (ALP), serum bilirubin levels (SBL), total cholesterol, albumin and total protein levels. From the results, it was proposed that treatment with the ethanolic seed extract prior to ethanol administration in the tested animal model significantly reversed the effects of alcohol toxicity by decreasing the ALT (p < 0.05-p < 0.001), ALP (p < 0.01-p < 0.001), AST (p < 0.05-p < 0.001), LDH (p < 0.05-p < 0.001), SBL (p < 0.01-p < 0.001) and cholesterol (p < 0.05-p < 0.001), while increasing the level of albumin (p < 0.01-p < 0.001) and total protein (p < 0.05-p < 0.001) levels [32]. Thus, the seed extract of A. squamosa reversed the effects of liver toxicity induced due to alcohol treatment in rat in a dose-dependent manner. Similarly, in another pre-clinical study, the hydroalcoholic seed extract of A. squamosa was evaluated for its hepatoprotective effect in CCl 4 -induced hepatotoxicity in rats, by quantifying serum enzymes such as serum glutamic pyruvic transaminase (SGPT), ALP, serum glutamic-oxaloacetic transaminase (SGOT) and total bilirubin for liver function tests. From the results, it was demonstrated that treatment with the hydroalcoholic seed extract (100-400 mg/kg body weight (B.W), for 7 days) resulted in a reduction in the level of SGOT (51.22-87.37 U/L), SGPT (38.21-96.22 U/L), ALP (98.28-159.25 U/L) and total bilirubin (0.71-1.47 mg/dL), indicating the hepatoprotective effect of A. squamosa seed extract in a concentration-dependent manner [20]. From the results of the above-mentioned studies, it was concluded that the seeds of A. squamosa can be used against liver damage.

Other Activities
As mentioned, the seeds of A. squamosa also exhibit various other pharmacological activities. For example, cyclosquamosin B extracted from custard apple seeds possesses vasorelaxant properties on the aorta of the rat animal model as a result of the reduction of Ca 2+ released from the extracellular medium via voltage-gated Ca 2+ -channels, at a concentration of 10 −5 M [77]. In an investigation, custard apple seed powder was stated to have anti-implantation activity in rabbits, but the results of the investigation are not promising to be used pharmacologically. Researchers have also demonstrated the antiheadlice activity of A. squamosa seeds that, when used along with coconut oil in ratio of 1:2, can kill head lice (98%). It was also demonstrated that the petroleum ether seed extract of A. squamosa in coconut oil eliminates lice (90%) in vitro in 26 min [78]. The seed extract of A. squamosa also exhibits antihelminthic properties when tested on Pheritima posthuma (i.e., earthworm). It also shows antihelminthic properties for Haemonchus contortus, the nematode worm of goats and sheep [79]. In addition, the ethanolic seed extract of A. squamosa inhibits larval growth (20-fold) in Spodptera litura compared with A. muricata [80]. All the biological activities of A. squamosa seeds are mentioned in Table 2 and Figure 4. The results of all the above-mentioned studies prove the pharmacological importance of A. squamosa seeds-that are recognized as waste-thus evidencing their pharmacological value. Annona squamosa seeds Antimicrobial Aqueous seed extract (500 and 1000 mg pm-CuO NPs)

Toxicity of Annona squamosa Seeds
A. squamosa seeds have been employed in the traditional medical system since time immemorial for skin exfoliation and elimination of headlice [87,88]. The seeds of the custard apple include biologically active substances such as polyphenols, alkaloids, acetogenins and cyclohexapeptides [89,90]. The pharmacological properties of certain acetogenins, including wound healing capabilities, anti-lice, mosquitocidal characteristics, anticancer, antifungal and antioxidant properties have been investigated in the past few years [91]. Custard apple seeds are toxic mainly due to the presence of a high amount of annonaceous acetogenins (neurotoxins) that are stated to cause irritation in mucosa and the eye and vomiting (taken orally). Safety concerns are stated concerning the use of the plant in dietary supplements and is cited in the poisonous plants database of the Agence Francaise de Securite Sanitaire des Aliments (AFSSA) and the American Food and Drug Administration (FDA) [92]. Others have claimed that when crushed seeds come into contact with the eyes, they may cause conjunctival irritation that ultimately leads to eye ulcers. When a toxic extract of the custard apple seed was tested on rat eyes, it resulted in a conjunctival infection and delayed damage to the corneal epithelium [93]. Some examples show that this plant's seeds induce severe symptoms of toxic keratitis after inadvertent contact with the custard apple seed. For instance, a patient utilized the oil combined with A. squamosa seed powder for the control of headlice. Results demonstrated that the patient exhibited considerable conjunctival congestion, blepharospasm, coarse punctate epithelial diffuse erosions and dense stroma and no anterior chamber reaction was seen in either of the patient's eyes. Individuals in a different study experienced a similar pattern after being exposed to custard apple seeds for 8 to 12 h. Slit-lamp examination revealed widespread erosions with coarse punctate epithelium in both eyes [87]. Furthermore, it is concluded that the seeds or the seed extract/powder of the custard apple are highly toxic and an irritant, causing conjunctivitis and corneal epithelium damage with a high risk of secondary infection. However, people need to learn about the toxicity of the seeds of the custard apple, which requires further investigations to acquire the knowledge of its safety aspects and dosage.

Conclusions and Future Perspectives
A. squamosa has gained popularity due to a recent increase in investigations/studies conducted on the health benefit and the bioactivities of different parts of the plant such as the seed parts, the bark, the leaves, the fruits, etc. A. squamosa has been employed in indigenous folk medicine worldwide and likely used in the food industry, as 50-80% of the fruit is edible. The pulp is used as a flavoring agent in ice cream and contains vitamin B1 (thiamine), dietary fiber, potassium and sodium in considerable amounts. The seeds of A. squamosa are a rich source of phytochemicals such as polyketides, annonaceous acetogenins (neurotoxins), cyclopeptides, carbohydrates, proteins, lipids, oleic acid and linoleic acid. Based on in vivo and in vitro experiments A. squamosa seed extracts were found to be effective in various bioactivities such as antitumor, antimicrobial, antifungal, antidiabetic, hepatoprotective, anticancer and antifertility. A few studies were available on the phytochemical profile and the molecular mechanism of various bioactivities of A. squamosa seeds. However, more pharmacological studies need to be performed to determine the nutraceutical and food supplement potential of the seeds. Based on the studies available, A. squamosa seeds may likely be used as an ingredient in the nutraceutical and food/nutrition industry, especially as anticancer drugs and antitumoral dietary supplements, benefiting human health.