Introduction

Avocado (Persea Americana Mill.) is the most important crop within the Laurel family (Lauraceae). This tropical tree produces an oily fruit used by indigenous people for at least 9,000 years and obtained in the wild in Meso-America, which was appreciated by Mayan and Aztec civilizations (Storey et al. 1986). Today, this fruit is quickly distributed in all regions of the world due to its favorable nutrient properties: it contains a large amount of monounsaturated fatty acids (FA) and valuable quantities of vitamins, minerals, and phytochemicals (Schaffer and Wolstenholme 2013). The fruits of avocado are mainly used for human consumption and to produce cosmetics, nutritional supplements, and livestock feed. The avocado pulp is generally used for culinary purposes, while the seeds and peels are usually disposed of by landfilling. In 2019, the world avocado production was 7,308,978 Tm (FAO, 2019), and from this production, the avocado by-products (AP) accounted for ~54.8% (wet basis) (Negesse et al. 2009). Historically, avocado seeds have been used in traditional Mesoamerican medicine for the treatment of rheumatism, asthma, various infectious diseases, and also for diarrhea and dysentery induced by intestinal parasites (Jiménez-Arellanes et al. 2013).

The criteria for designing feed formulas are based on improving body weight (BW), feed conversion efficiency, protein accretion, and milk and egg production. Regrettably, all criteria focus on productivity rather than on achieving healthy animals, and for this antibiotics are used (Guil-Guerrero et al. 2016a). This use is intended to avoid gastrointestinal infections and inflammatory bowel diseases. However, antibiotics are often misused in animal production farms, and thus antibiotic residues appear in the environment (Guil-Guerrero et al. 2016b). For avoiding this excessive use, plant-food by-products can be introduced into the diet of farm-animal, as these contain suitable concentrations of plant-derived compounds with antimicrobial and health-enhancing properties (Guil-Guerrero et al. 2016c).

A major problem with AP used as food or feed supplements is that although they contain protein and carbohydrates, the high concentration of polyphenols contained in it confers a bitter taste and can reach toxicity at high levels (Domínguez et al. 2014). However, their use in mixes with other plant food by-products or feed will reduce such flavor to acceptable limits, while providing interesting amounts of bioactive compounds for health promotion.

Previously, AP were partially reviewed about phenolic compounds content, and some biological activities, such as antioxidant ones (Araújo et al. 2018). However, despite the wide research carried out on the biochemical composition and biological activities of AP, as well as the studies on its use as animal feed, still, all this research remains un-reviewed as a whole. The present review discusses all relevant data about AP, focusing on their biochemical composition and related health benefits for farm animals, while potential directions for future studies are provided.

Biochemical composition and health benefits of avocado by-products

Approximately, 100 g of fresh fruits generate 12‒16 g of peel and 14‒24 g of seeds (Bressani et al. 2009); thus, finding new uses for this by-product is a desirable action. Considering the seeds, it highlights their protein content, which is highly dependent on processing methodology, ranging from 6.24 to 23.54 g·100 g^-1 (Talabi et al. 2016). The seeds also contain healthy fatty acids (FA), and the FA profiles of three avocado varieties (Fuerte, Bacon, and Hass) were studied in Japan by Takenaga et al. (2008). The main FA in seeds were linoleic acid (LA, 18:2n-6) (35.3‒38.2% of total FA), oleic acid (OA, 18:1n-9) (22.4‒22.1% of total FA), and palmitic acid (PA, 16:0) (17.7‒17.9% of total FA). These FA percentages are similar to those reported by Alkhalf et al. (2019).

Phytochemical studies on AP identified various classes of natural products: phenolic compounds, phytosterols, polyols, furan derivatives, acetogenins, carotenoids, abscisic acid, lignans, glucosides, as well as FA and hydroxylated ones. The levels of such compounds vary according to avocado variety, farming variables, and ripeness. Measured levels are also influenced by the method of extraction during experimentation.

Phenolics occur both in peels and seeds of avocado fruits and comprise phenolic acids (Fig. 1), phenolic alcohol derivatives (Fig. 2), flavonoids (Fig. 3), and procyanidins (oligomeric tannins, Fig. 4). Total phenolic amounts deeply differ between avocado peels and seeds (Table 1). Considering total phenolics reported as gallic acid equivalent (GAE), in avocado peels these ranged from 1.40–18.94 g·kg^-1 FW (Ramos-Aguilar et al. 2021) to 527.8 g GAE·kg^-1 DW (Rosero et al. 2019), while total phenolics in seeds ranged from 0.30 g GAE·kg^-1 g DW in cv. Criollo spp. (Cid-Pérez et al. 2021) to 292 g GAE·kg^-1 DW in avocado seeds (Pahua-Ramos et al. 2012). In AP, flavonoids showed low figures, but tannins occur in amounts similar to those of phenolic acids (Lee et al. 2008), and anthocyanins showed variable amounts in peels (Ashton et al. 2006).

Phenolic profiles are summarized in Table 2. The peels extracts contain a great variety of phenolic acids, such as hydroxycinnamic and hydroxybenzoic ones, and some flavonoids such as quercetin and catechin. The seed extracts contain mainly hydroxycinnamic acids, and flavonoids such as epicatechin catechin, and procyanidins. Summarized phenolic acids show interesting properties for human health and achieving a more sustainable and cleaner animal production. Simple phenolic acids have antibacterial actions, for instance against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Listeria monocytogenes. Saavedra et al. (2010) described synergy between streptomycin and phenolic acids against Gram-bacteria. Wen et al. (2003) indicated that phenolic acids mixtures have additive antilisterial effects, and reported a significant relationship between pH and antilisterial activity. Cueva et al. (2010) stated that phenolic acids act as growth inhibitors of several lactobacilli species and some pathogens (S. aureus and Candida albicans), but P. aeruginosa was not affected by these compounds. Flavonoids are secondary polyphenolic metabolites that have a ketone group and yellowish pigments, and the ones contained in AP have antibacterial activity. Such compounds have been successfully tested against oxacillin-resistant S. aureus, cariogenic Streptococcus mutans, and uropathogenic E. coli. The mechanisms related to the bacterial growth inhibition were diverse, e.g., destabilization of the cytoplasmic membranes and the deprivation of the substrates required for microbial growth, such as Fe and Zn (via chelation of molecules with these metals), and such depletion can severely limit bacterial growth (Dixon et al. 2005; Heinonen 2007). Other interesting phenolics are procyanidins, which are catechin- and epicatechin-oligomeric compounds, which exhibit chemoprotective properties against cancer (Jeong and Kong 2004), improve lipid metabolism (Puiggros et al. 2005), prevent infections in the urinary tract, and can modulate antioxidant enzymatic activities (Puiggros et al. 2005), among other bioactivities.

Tannins, i.e., water-soluble polyphenols, occur in avocado peel (Table 1, Negesse et al. 2009). These compounds, which have high molecular weight and many phenolic groups, are able to precipitate protein (Hagerman et al. 1998). Tannins affect rumen bacteria by inactivating several enzymes, e.g., glutamate dehydrogenase, proteases, and carboxymethyl cellulase. Furthermore, sulfur and iron bioavailability is limited to animals that consume tannin-rich tissues, thus, large consumption of tannins can induce toxicity (Kumar and Vaithiyanathan 1990). The phenolic compounds contained in AP are of great interest considering that these have high diversity, and therefore a synergistic antibacterial action due to such compounds can be expected through the consumption of AP, which could help to prevent many digestive pathologies in farm animals.

Fig. 1: Phenolic acids contained in avocado by-products

Fig. 2: Phenolic alcohol derivatives contained in avocado by-products

The occurrence of other compounds in AP is summarized in Table 3. Furan derivatives (Fig. 5) were found in seeds by Ding et al. (2007). These compounds have been subjected to structural modifications, and antibacterial, antifungal, and insecticidal activities were checked with positive results (Rodríguez-Saona and Trumble 2000). Aliphatic acetogenins, also known as “hydroxylated fatty alcohols”, were reported by Kashman et al. 1969a, b, which constitute a class of compounds almost exclusively isolated from avocado (Fig. 6). These have antimicrobial, antibacterial and spore germination inhibiting effects (Hernández-Brenes et al. 2013), and anti-inflammatory properties were cited for the acetogenins isolated from avocado seeds (Fig. 6, compounds 7‒11) (Rosenblat et al. 2011). Carotenoids were found in peels of Hass variety by Ashton et al. (2006). These comprise β-carotene, neoxanthin, violaxanthin, zeaxanthin, and α- and β-carotene (Fig. 7). These compounds have a great interest in animal health since carotenoids influence both cellular and humoral immunity, thus these can be used to prevent infectious and inflammatory processes. Furthermore, recent investigations on the role of carotenoids in angiogenesis, apoptosis, and gene regulation, revealed mechanisms of immune system regulation (Pechinskii and Kuregyan 2014). Polyols (ascorbic acid, mannoheptulose, and perseitol, among others), were reported by Tesfay et al. (2010) in avocado peels and seeds (Figs. 8, 9), and the latter was also found in Hass avocado peels by Figueroa et al. (2018). The consumption of such compounds has health effects on young monogastric mammals, which exhibited an increased survival rate, both over the intermediate portions of the pre-weaning period and over the entire pre-weaning period (Rodas et al. 2015). Avocado-derived compounds containing polyols, comprising D-mannoheptulose and/or perseitol, have been proposed for treating and preventing innate immunity modification diseases by increasing the production of antimicrobial peptides (e.g., Human β-defensin-2), without inducing inflammatory reactions, irritation, or intolerance (Piccirilli et al. 2015). Sterols (Fig. 10) occur mainly in the unsaponifiable obtained from the avocado peel, which contains 0.2% of saturated aliphatic hydrocarbons and more than 1% of sterols (Msika et al. 2013). This unsaponifiable includes stigmasterol, ß-sitosterol, campesterol, Δ⁵-avenasterol, Δ⁷-stigmasterol, and citrostadienol. Alkhalf et al. (2019) reported in avocado seeds cholesterol, stigmasterol, and β-sitosterol. Sterols-rich by-products could have interest in improving animal health since such compounds positively affect the wellness and health of farm animals through a variety of physiological

Fig. 3: Flavonoids contained in avocado by-products

Fig. 4: Procyanidins contained in avocado by-products

functions, e.g., antitumor effects, hormone-like actions, oxidation and inflammation resistance, immune modulation, and in vivo growth regulation (Guil-Guerrero et al. 2016a). Avocado seeds contain a lipid fraction in which occurs some FA as LA, OA, and PA, which store energy, while LA is an essential nutrient and OA is a bioactive compound (Takenaga et al. 2008). Besides these, other organic acids are present in avocado by-products, which are depicted in Fig. 11. Hass avocado peels contain quinic, citric, malic, and succinic acids (Figueroa et al. 2018), while two glycosylated abscisic acid derivates were isolated from seeds (Ramos et al. 2004). These molecules have been assayed in abscisic acid-fed mice and typified as healthy, since they decrease blood glucose concentrations in fasting, ameliorate glucose tolerance, adipocyte hypertrophy, tumor necrosis factor-α (TNF-α) expression, and macrophage infiltration, which were significantly improved (Guri et al. 2007). Therefore, such molecules could have positive health effects on farm animals, although the extent of this effect remains unknown. In addition to all the above-detailed compounds, Figueroa et al. (2018) found in avocado peels a lignan (nudiposide) and

Table 1: Total phenolics, anthocyanin, and flavonoids in avocado by-products

Variety	Seed	Peel	Reference
Total phenolics
Hass, Gwen, Fuerte	16.5-29.8 g GAE·kg^-¹ g FW	-	Torres et al. (1987)
-	88.2 g GAE·kg^-¹DW	-	Soong and Barlow (2004)
Several cultivars	19.2-51.6 g GAE.kg^-¹ FW	4.3-113.9 g GAE·kg^-¹ FW	Wang et al. (2010)
Hass	17.0–60.8 g GAE·kg^-1 g DW	32.9–90.0 g GAE·kg^-1 g DW	Rodríguez-Carpena et al. (2011)
Fuerte	20.3–69.1 g GAE·kg^-¹ DW	40.5–172.2 g GAE kg^--1 DW	Rodríguez-Carpena et al. (2011)
-	7.20 g GAE·kg^-1 g FW	8.39 g GAE kg^-1 g FW	Deng et al. (2012)
Hass	9.51 g CE·kg^-1 DW	25.32g CE·kg^-1 DW	Kosińska et al. (2012)
Shepard	13.04 g CE·kg^-1 DW	15.61 g·CE·kg^-1 DW	Kosińska et al. (2012)
-	292 g GAE·kg^-1 DW	-	Pahua-Ramos et al. (2012)
-	29.37 g GAE·kg^-¹ g DW	30.01 g GAE·kg^-¹ g DW	Oboh (2013)
Hass	57.3 g·GAE·kg^-¹ g DW	63.5 g·GAE kg^-¹ g DW	Daiuto et al. (2014)
-	1.553 g GAE·kg^-¹ g DW	12.523 g·GAE kg^-¹ g DW	Morais et al. (2015)
Hass	5.7 g GAE·kg^-¹ g DW	19.7 GAE·kg^-¹ g DW	Calderón-Oliver et al. (2016)
Hass	72.5 g·kg^-1	227 g·kg^-1	Melgar et al. (2018)
-	12.52-33.23 g GAE·kg^-1DW	12.42-31.10 g GAE·kg^-1 DW	Saavedra et al. (2017)
Hass	57.3 g GAE·kg^-¹ DW	63.5 g GAE·kg^-1 DW	Tremocoldi et al. (2018)
Fuerte	59.2 g GAE·kg^-¹DW	120.3 g GAE·kg^-¹DW	Tremocoldi et al. (2018)
Nariño	328.8 g GAE·kg^-1 DW	527.8 g GAE·kg^-1 DW	Rosero et al. (2019)
cv. Criollo sp.	0.30g GAE·kg^-1 g DW	-	Cid-Pérez et al. (2021)
Pinkerton	124 GAE·kg^-1 FW·min^-1	352 GAE·kg^-1 FW·min^-1	Skenderidis et al. (2021)
Hass and Hass type cv.	-	1.40–18.94 g GAE·kg^-1 FW	Ramos-Aguilar et al. (2021)
Hass	2 mg·kg^-1 DW	2-9 mg·kg^-¹ DW	Tesfay et al. (2010)
Total flavonoids
-	5.69 mg TE·kg^-¹	13.60 mg TE·kg^-¹	Lee et al. (2008)
-	2.32 g QE·kg^-¹ g DW	3.39 g QE·kg^-¹ g DW	Oboh (2013)
Hass	2.8 g QE·kg^-1 DW	10.9 g QE·kg^-1 DW	Calderón-Oliver et al. (2016)
Total tannins
-	137.2 mg TE·kg^-¹FW	223.45 mg TE·kg^-¹ FW	Lee et al. (2008)
-	-	49 g TE·kg^-1 DW Non tannin 10; tanin 39 Condensed tannin 22.1	Negesse et al. (2009)
Hass	0.09 g GAE·kg^-1g DW	0.04 g GAE·kg^-1g DW	Calderón-Oliver et al. (2016)
Total anthocyanins
Hass	-	0-230 g·kg^-1 FW, as cyanidin 3-O-glucoside	Ashton et al. (2006)

Abbreviations: CE: catechin equivalent; DW: dry weight; FW: fresh weight; GAE: gallic acid equivalent; QE: quercetin equivalent; TE: tannic acid equivalent

Fig. 5: Furans contained in avocado by-products

Table 2: Phenolic compounds occurrence in avocado by-products

Variety	Seeds	Peels	Reference
Several cultivars	Procyanidins: 23.7-55.6 kg^-¹ FW	Procyanidins 4.9-38.9 g GAE·kg^-1 FW	Wang et al. (2010)
Has	Procyanidins > hydroxycinnamic acids > catechins > hydroxybenzoic acids	procyanidins> hydroxycinnamic acids> catechins	Rodríguez-Carpena et al. (2011)
Fuerte	Procyanidins > hydroxycinnamic acids > catechins	Procyanidins > catechins > hydroxycinnamic acids > hydroxybenzoic acids	Rodríguez-Carpena et al. (2011)
-	Epicatechin 219.2, gallic acid 52.0 mg·kg^-1 DW	Catechin 520.8, chlorogenic acid 116.8, homogentisic acid 113.6, cyanidin 3-glucoside 31.6 mg·kg^-1 DW	Deng et al. (2012)
Has	Catechin/epicatechin gallate 152.8, procyanidin trimer A (II) 89.3, procyanidin trimer A (I) 81.7 mg·kg^-1 DW	Catechin 148.8, procyanidin dimer B (I) 135.4, chlorogenic acid 81.8, quercetin-3-O-arabinosyl-glucoside 80.4 mg·kg^-1 DW	Kosińska et al. (2012)
Shepard	Catechin/epicatechin gallate 105.4, procyanidin trimer A (I) 98.9, procyanidin trimer A (II) 73, 3-O-caffeoylquinic acid 53.5 mg CE·kg^-1 DW	Quercetin 3-O-galactoside 144.1, quercetin derivative (III) 81.9, quercetin-3-O-arabinoside 94.1, quercetin derivative (I) 63.7 mg CE·kg^-1 DW	Kosińska et al. (2012)
-	Protocatechuic 128.1, kaempferide 107.42, rutin 9.63, vainillic acid 28.67, syringic acid 2.51, kaempferol 2.19, chlorogenic acid 0.516 mg·kg^-1 DW	-	Pahua-Ramos et al. (2012)
-	-	Catechin hydrate 1.71 mg·kg ⁻¹DW Epicatechin 1.298 mg·kg ⁻¹ DW	Morais et al. (2015)
Hass	3-O-Caffeoylquinic acid 19 mg, B-type (epi)catechin 12 mg, epicatechin 46.5 mg·kg^-1 extract	4- and 5-O-Caffeoylquinic acid 40.9, epicatechin 46.5, catechin 20, B-type (epi)catechin 96, quercetin 7 mg·kg^-1 extract	Melgar et al. (2018)
	Catechin 242.6, chlorogenic acid 160.7, caffeic acid 136.9, ferulic acid 0.87 mg·kg^-1 DW	Chlorogenic acid 1376, p-Coumaric acid 17.4, ferulic acid 50.5 mg·kg^-1 DW	Saavedra et al. (2017)
Hass	-	Hydroxybenzoic acids: benzoic, p-hydroxybenzoic protocatechuic, gentisic acids. Hydroxycinnamic acids: caffeic acid, caffeoylquinic acids derivatives, p-coumaric acid. Flavanoids: naringenin, luteolin 7-O-(2″-O-pentosyl) hexoside, quercetin, quercetin glucosides, kaempferol glucosides, cinchonain I. Procyanidin dimers, trimers and tetramers. Lignans: nudiposide.	Figueroa et al. (2018)
Hass	Trans-5-O-caffeoyl-D-quinic acid 1.63 mg·kg^-1; Procyanidin B₁ 1.52 mg·kg^-1; catechin 3.64 μg·mg^-1; epicatechin 10.27 mg·kg^-1 DW	Procyanidin B2 48.38 μg·mg^-1, epicatechin 40.21 mg·kg^-1DW	Tremocoldi et al. (2018)
Fuerte	Trans-5-O-caffeoyl-D-quinicacid 5.74 mg·kg^-1; procyanidin B₁ 2.27 mg·kg^-1; catechin 8.13 mg·kg^-1; epicatechin 11.06 mg·kg^-1DW	Procyanidin B₂28.34 mg·kg^-1; epicatechin 30.40 mg·kg^-1 DW	Tremocoldi et al. (2018)
Nariño	Flavonols, catechins, hydroxycinnamic acids, quercetin glycosides and procyanidins, phloridzin	Flavonols, catechins, hydroxycinnamic acids, quercetin glycosides, procyanidins, phloridzin	Rosero et al. (2019)
Hass and “Hass type”		Chlorogenic acid (0.13-0.91), procyanidin B2 (0.29-5.86), Epicatechin (0.24-2.17), cyanidin 3-O-glucoside (0.09-0.83) g·kg^-1FW	Ramos-Aguilar et al. (2021)
Abbreviations: DW: dry weight; FW: fresh weight; GAE: gallic acid equivalent; RE: rutin equivalent; TE: tannic acid equivalent; QE: quercetin equivalent

two glucosides: the iridoid-type penstemide, and (1′S, 6′R)-8′-hydroxyabscisic acid β-d-glucoside, as well as hydroxylated FA. Phytates were described in avocado peels by Negesse et al. (2009). Phytic acid (Fig. 12), is the main storage form of phosphorus (P) in several plant tissues, especially bran and seeds, where it is found as phytate, including Mg, Ca, Na, and K. As phytate form, P is not available to humans and non-ruminant animals because they lack the digestive enzyme phytase, which releases phosphate from the inositol in the phytate molecule. This situation is different from that of ruminants, which can digest phytate with the aid of the phytase that several microorganisms produced in the rumen (Klopfenstein et al. 2002). Phytate can form complexes with protein, which are pH-dependent. Such complexes reduce the bioavailability of several mineral elements (Pallauf and Rimbach 1997). However, phytic acid can induce positive actions, for instance through the prevention of the formation of free radicals and by the reduction of the risk of high-fat diet-induced hyperglycemia via regulation of hepatic glucose enzyme activities (Kim et al. 2010). Phytic acid also decreases plasma triglycerides and cholesterol and induces a change in the carryover of heavy metals (Pallauf and Rimbach 1997).

All the reviewed compounds develop antibacterial, anti-inflammatory, and immunity-promoting actions, so the intake of PA would contribute to improving the health of the digestive tract of farm animals.

Biological activity of avocado by-products

Information on this topic is summarized in Table 4, the selected antimicrobial activity of avocado by-products extracts is detailed in Table 5, while information related to antioxidant properties is described in Table 6.

Antidiabetic, anti-inflammatory, antihepatotoxic, anti-toxic, and antihypertensive

These assays were accomplished through in vitro cell cultures and mice models. Uchenna et al. (2017), using a murine model demonstrated the effectiveness of raw avocado seeds against hyperglycemia and/or hypercholesteremia, while the seed extracts were characterized as antihypertensive and antihepatotoxic in Wistar rats by Imafidon and Amaechina (2010). Protection against UVB-induced damage and inflammation of the skin was reported by Rosenblat et al. (2011), who in vitro supplied polyhydroxylated fatty alcohols to keratinocytes from avocado seeds prior to UVB exposure. These compounds were able to reduce increasing cell viability and enhance DNA repair while decreasing the secretion of PGE2 and IL-6, thus PFA can act as photoprotective agents. Good anti-inflammatory activities of peel and seeds were reported for Hass and Fuerte varieties by Tremocoldi et al. (2018), and the main finding was that phenolic compounds-rich peel extracts were able to suppress the release of TNF-α and NO. Moreover, gastric ulcer can be prevented by seed extracts. Alkhalf et al. (2019) found that avocado peel extract was appropriate to reduce edema in mice at 10 g·kg^-1 BW, while the prevention of gastric ulcers due to oxidative stress was reported by Athaydes et al. (2019), who indicated a reduction of lipid peroxidation and an increasing of superoxide dismutase (SOD) enzyme activity. Anti-toxic and cardioprotective effects of the AE and EE of avocado seeds against doxorubicin (DOX)-induced toxicity were checked in a mice model by Shamlan (2020). Interestingly, avocado extracts mitigated the increased markers of cardiac dysfunction induced by DOX treatment.

Table 3: Other compounds occurring in avocado by-products

Variety	Seed	Peel	Reference
-	Abcisic acid	-	Ramos et al. (2004)
Hass	-	Carotenoids: total: 18-50; lutein: 10-21; β-carotene: 5-12; neoxanthin: 2-5 g·kg^-¹ FW. Others: anteraxanthin, violaxanthin, zeaxanthin, α-carotene. Total chlorophylls: 100-210 g·kg^-¹ FW	Ashton et al. (2006)
-	Alkanols, terpenoids, glycosides, furan ring derivatives, and a coumarin.	-	Ding et al. (2007)
-	Glycolipids, phospholipids	-	Takenaga et al. (2008)
Hass	Ascorbic acid: 12.6-22.7 Mannoheptulose: 5.34-7.95 Perseitol: 0.43-1.62 g·kg^-¹ DW	-	Tesfay et al. (2010)
-	-	Phytathe 9 mg TE·kg^-¹ DW	Negesse et al. (2009)
Avocado, several cultivars	Total carotenoids 0.7-6.3 mg kg^-¹ FW	Total carotenoids 9.3-17.7 mg kg^-¹FW	Wang et al. (2010)
Ettinger	Hydroxylated fatty alcohols, including persin	-	Rosenblat et al. (2011)
Hass	Hydroxylated fatty alcohols		Rodríguez-Sánchez et al. (2013)
-	1% sterols of unsaponifiable wt Sterols: ß-sitosterol, campesterol, stigmasterol, Δ5-avenasterol, Δ7-stigmasterol, citrostadienol	- -	Msika et al. (2013)
Hass	-	Organic acids: citric, malic, quinic, succinic. Polyols: perseitol. Glucosides: Iridoid-type: penstemide; and (1′S, 6′R)-8′-hydroxyabscisic acid β-d-glucoside. Hydroxylated fatty acids	Figueroa et al. (2018)
-	Fatty acids: mainly, linoleic, oleic and palmitic acids. Hydrocarbons: C₁₈-C₃₀, squalene. Sterols: cholesterol, stigmasterol and β-sitosterol.	-	Alkhalf et al. (2019)
Criollo sp.	Isoprenoids derivates, esters of fatty acids and their derivatives		Cid-Pérez et al. (2021)
Hass and Hass type cv.		Polyols, including perseitol and volemitol Organic acids: succinic, citric, malic, quinic, fumaric, oxalic. Sterols: β-sitosterol, stigmasterol, campesterol, cycloartenol; α- and δ-tocopherols Fatty acids: mainly oleic, linoleic, palmitic α- and δ-tocopherols; chlorophylls and pheophorbides; carotenoids (lutein and others)	Ramos-Aguilar et al. (2021)

Antitumor

These experiments were carried out using both peels and seeds. All trials were accomplished using cancer cell lines cultures, and cell growth inhibition by the MTT test was the more frequent antitumor assay. Alkhalf et al. (2019) found inhibition of the growth of hepatocellular and colon cancer cells by the lipid extracts of avocado peels. Dabas et al. (2019) checked methanol extracts (ME) for in vitro antitumor tests using several human cell lines, and a reduction in the viability of cells was found due to that ME downregulate the expression of cyclin D₁ and E_2, and the nuclear translocation of nuclear factor 𝜅B. Vo et al. (2019) found that the EE of seeds inhibited cancer cells growth and provided protection against H₂O₂-induced DNA damage and that AVM acts against NO production from cells. All these works concluded that avocado seeds constitute a raw source of healthy compounds and that these extracts can be used as ingredients for functional foods formulations.

Table 4: Biological activity of avocado by-products

Activity	Model	Variety	Extracted material	Test	Results	Concluding remarks	Reference
Antidiabetic	Sprague Dawley rats Spontaneous hypertensive rats	-	Raw seeds	Feeding avocado seeds	Avocado seeds lowered blood glucose and cholesterol and enhance liver glycogen storage	Possible uses of avocado seeds against hyperglycemia and/or hypercholesteremia.	Uchenna et al. (2017)
Antihepatotoxic	Wistar rats	Fuerte	Seeds AE	NaCl-treated rats	-AE reduce weight gain and blood pressure - AE reduces alkaline phosphatase activity	AE are antihypertensive and antihepatotoxic	Imafidon and Amaechina (2010)
Anti-inflammatory	In vitro cell cultures	-	Seeds PFA	In vitro keratinocytes cultures	Decreased IL-6 and cyclobutane pyrimidine dimers after UV-B radiation	PFA reduce UV-B-induced inflammation in skin	Rosenblat et al. (2011)
	Mice model	-	Peels and seeds lipid extracts	Carrageenan-induced edema in mice	Avocado peel extract reduce swelling at 10 g·kg^-1 of extract	Hydrocarbons, St and UFA have anti-inflammatory properties	Alkhalf et al. (2019)
	In vitro cell cultures	Hass and Fuerte	Peels and seeds AE and EE	-Lipopolysaccharides- stimulated macrophages - TNF-α production and MTT - NO by Griess reagent	Fuerte peel extract suppressed the release of TNF-α and NO	Phenolics from peels are anti-inflammatory	Tremocoldi et al. (2018)
Anti-inflammatory, hepatoprotective, antihypertensive	Mice model	-	Seeds EAE	Indomethacin-induced gastric ulcer in mice	Extract mitigates oxidative stress by reducing lipid peroxidation and increasing superoxide dismutase enzyme activity	Gastric ulcer can be prevented by seed extracts	Athaydes et al. (2019)
Anti-toxic and cardioprotective	Mice model: doxorubicin (DOX)-induced toxicity	Avocado	Seeds AE and EE	- DOX and DOX+ AE/EE- treated rats	DOX treatment increased markers of cardiac dysfunction, and avocado extracts mitigated it	-EE more effective than AE, as evidenced by biochemical markers.	Shamlan (2020)
Antitumor	In vitro cell cultures	-	Peels and seeds lipid extracts	HCT116 colon- and HePG2 liver-human cell line cultures. MTT test	Seed lipids inhibited hepatocellular and colon cancer cells growth	Seeds lipids have anti-cancer effects	Alkhalf et al. (2019)
	In vitro cell cultures	Hass	Seed colored ME	LNCaP cells - MTT test, cell cycle analysis, apoptosis	-Extracts reduced in vitro cancer cells viability, downregulated the expression of cyclin D₁ and E₂, associated with G₀/G₁ phase cycle arrest, and nuclear translocation of nuclear factor 𝜅B - Extracts induced apoptosis	Extracts can be used as a functional food ingredient	Dabas et al. (2019)
Antitumor and antioxidant	In vitro free radical scavenging and anti-proliferative activities	-	Seed EE	RAW 264.7 cells - NO production, MTT cancer cell growth inhibition, DNA oxidative assay	-High IC₅₀ for free radical scavenging fractions - Seed extract protect against H₂O₂-induced DNA damage - EE reduces NO production from lipopolysaccharide-stimulated cells - Extracts inhibited the proliferation of cancer cells	Avocado seeds are a source of healthy compounds	Vo et al. (2019).
Antimicrobial	Antifungal -Cladosporium cladoposioides	Avocado	Inmature peels, searching antifungal fractions	-Antifungal activity by TLC bioassay -Incubation of compounds with fungi	Trihydroxy fragments could be present in all active compounds	Preventive effects against avocado anthracnose	Adikaram et al. (1992)
	Antiprotozoal - Trypanosoma cruzi	Avocado	Seeds, EE, including six PFA	In vitro T. cruzi immobilization	PFA showed moderate activity against epimastigotes and trypornastigotes	PFA prevent T. cruzi disease, Chagas' disease	Abe et al. (2005)
	Amoebicidal, Giardicidal Trichomonicidal Antimycobacterial	Avocado	Seeds, CE and EE	In vitro cell cultures	- CE and EE activity against E. histolytica, G. lamblia and T. vaginalis (IC₅₀ <0.634 μg·ml^-1). - CE inhibited M. tuberculosis reference	Extracts active against all tested microorganisms	Jiménez-Arellanes et al. (2013)
	Antifungal -Candida spp., Cryptococcus neoformans, Malassezia pachydermatis	Avocado	Seeds HE and EE	Antifungal activity by microdilution in RPMI	Extracts active against all the yeast strains tested in vitro, with differing MIC	Candidates as antifungal agents	Leite et al. (2009)
	Antibacterial and antifungal	Avocado	Seeds ME and EAE	Disc diffusion method	ME and EAE had the lowest MIC against C. albicans	EA and EAE had higher activity than treptomycin	Idris et al. (2009)
	Antibacterial and antifungal	Hass’ and ‘Fuerte’	Peel, pulp, and seed extracts	Disk diffusion method	-Gram-positive bacteria more sensitive than Gram-negative. - Gram-positive Bacillus cereus and Listeria monocytogenes more sensitive - E. coli was the most sensitive Gram-negative bacteria	Good antimicrobial properties	Rodríguez-Carpena et al. (2011)
	Antibacterial	Avocado	Seeds: AE, ME and EE	Disk diffusion method	Seed extracts active against S. aureus and B. subtilis.	Seeds have highly antibacterial activity	Nagaraj et al. (20 10)
	Antibacterial	Fuerte, Hass, Shepard	Seeds and epicarp: AE and EE	Hole plate method	-Effect of ethanol extracts toward bacteria -AE activity only for Listeria monocytogenes (93.8–375.0 μg·mL^-1) and Staphylococcus epidermidis (354.2 μg·mL^-1)	Antimicrobial against S. enteritidis, Citrobacter freudii, P. areuginosa, and Enterobacter aerogenes	Chia and Dykes (2010)

Table 4: Continued

	Clostridium sporogenes vegetative cells and active endospores	Hass	Seeds AcE	Disk diffusion method	-All extracts inhibited vegetative cells and active endospores -MIC of molecules 7.8-15.6 μg·mL^-1 -Bactericidal for enriched fraction at 19.5 μg·mL^-1	Identified molecules inhibit Gram-positive spore-forming bacteria.	Rodríguez-Sánchez et al. (2013)
	Antimicrobial and antioxidant	Hass	- Seed and peel AE -Nisin, antimicrobial peptide	-Antimicrobial activity by turbidimetry -Listeria innocua, E. coli, Lactobacillus sakei, Weissella viridescens, and Leuconostoc mesenteroides.	- Polyphenols from peel extracts have antioxidant and radical scavenging properties - Peel and seed extracts and nisin have synergic antimicrobial properties	Avocado peel + nisin reduce the amount of nisin to achieve antioxidant and antimicrobial effects	Calderón-Oliver et al. (2016)
	Antioxidant, antimicrobial	Hass	Seed and peels AE and EE	- MIC, MBC, and MFC	- Bactericidal effects in Gram positive and negative strains. - Extracts from seeds displayed better MCB than peels - Fungicidal effect in 2 strains for kernel extracts.	-High activity against some bacteria and fungi strains	Melgar et al. (2018)
Larvicidal	Artemia salina and third stage Aedes aegypti larvae	Avocado	Seeds HE and ME	-Toxicity tests using A. salina -Larvicidal in A. agypti	Extracts active against larvae	Alternative dengue control agents	Leite et al. (2009)
Radioprotective	Sprague-Dawely rats	Avocado	Peel extracts	Exposition to 6 MV X-Ray	Avocado peel extract induced a greater recovery of lymphocytes, red blood cells, and platelets to irradiate rats when compared to rats without peel extract administration. SOD was further increased	Avocado peel extract acts as a radiation protective agent for blood cells and major organs	Kim et al. (2020)
Toxic	Acute and sub-acute toxicity in rats	Avocado	Seeds, EAE extract	- IP administration of seed extract - IP subacute toxicity for 14 d at 75 and 150 mg·kg^-1 BW	-Acute toxicity study showed low LD₅₀ - Liver and kidney had normal architecture after 14 d exposure -14 d-treatment decreased food consumption, BW, and blood parameters	Seed EAE had medium toxicity	Taha et al. (2008)
	Toxicity in rats	Avocado	Seeds, AE	- LD₅₀ and mortality - Sub-acute experiments, doses at a quarter of the maximum dose (10 mg·kg^-1 BW)	- Hematological parameters and ALT, AST, albumin and creatinine not significantly altered.	The seed extract was safe on acute and sub-acute basis	Ozolua et al. (2009)
	Oral acute toxicity in mice	Avocado	Seeds AE and ME	-Hypolipidemic test - LD₅₀ of seeds -Antioxidant -Oral Acute Toxicity	-100% mortality after 6 d in the group fed with 2500 mg seeds·kg^-1 BW -125 mg AS·kg^-1 BW reduced the elevated levels of total cholesterol	AE and ME can be used for treating hyperlipidemia	Pahua-Ramos et al. (2012)
	-Acute toxicity test in BALB/c mice -Genotoxicity	Avocado	Seed EE	-LD₅₀ -Genotoxicological study - Erythrocyte micronucleus test	-LD₅₀ EE: 1200.75 mg·kg⁻¹ BW -EE at 250 mg·kg⁻¹ BW inhibited micronucleus formation	- EE induces acute toxicity at 500 mg·kg⁻¹ - EE lacks mutagenic effects on blood cells	Padilla-Camberos et al. (2013)
	Wistar Albino Rats: -Hepatotoxicity -Liver enzymatic activity	P. americana seeds	Seed phenolics AE and EE	AST, ALT, and ALP activities	Hepatotoxic effect after oral administration of phenolic AE	- Liver damage - AE is hepatotoxic	Umar et al. (2016)

Abbreviations. AE: aqueous extract; AcE: acetone extract; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; BW: body weight; CE: chloroform extract; EAE: ethyl acetate extract; EE: ethanol extract; HE: hexane extract; IP: intraperitoneal; MBC minimum bactericidal concentrations, MFC: minimum fungicidal concentrations; ME: methanol extract; MIC: minimum inhibitory concentrations; PFA: polyhydroxylated fatty alcohols; RPMI: Roswell Park Memorial Institute; SOD: superoxide dismutase; St: sterols; UFA: unsaturated fatty acids

Antimicrobial

Information on this activity is summarized in Table 4, and selected data on MIC (mg·mL^-1) and inhibition zone (mm) at 100 mg·mL^-1 are displayed in Table 5. The most widely used method to check the antimicrobial activity of AP extracts was the agar disk technique, although the hole plate method and turbidimetry were also used. Jiménez-Arellanes et al. (2013) tested chloroform extract (CE) and EE of avocado seeds against Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, and amoebicidal, giardicidal, trichomonicidal, and antimycobacterial activities were found. The trypanocidal effects of ME of seeds against Trypanosoma cruzi were checked by Abe et al. (2005), and six hydroxylated fatty alcohols, i.e., acetogenins, were the bioactive compounds identified as responsible for such actions. These showed moderate activity against epimastigotes and trypomastigotes and thus can prevent T. cruzi disease, the etiological agent for Chagas' illness. Leite et al. (2009) checked the in vitro antifungal activity of avocado seeds against Cryptococcus neoformans, Candida spp., and Malassezia pachydermatis strains. The authors concluded that the extracts obtained from avocado seeds can be used as dengue control agents.

Several authors tested AP extracts against different pathogenic fungi: Adikaram et al. (1992) reported that dichloromethane extract of peels exercised antifungal activity against Cladosporium cladoposioides. Such activity Table 5: Selected antimicrobial activity of extracts of avocado by-products

Variety / extract	Bacillus cereus	Bacillus subtilis	Listeria monocytogenes	Staphylococcus aureus	Staphylococcus epidermidis	Streptococcus pyogenes	Escherichia coli	Klebsiella pneumoniae	Pseudomonas aeruginosa	Pseudomonas spp.	Salmonella typhimurium	Mycobacterium avium	Yarrowia lipolytica	Reference
MIC (mg·mL^-1)
Seed, ethyl acetate extract ^a	-	20	-	20	-	20	40	30	30	-	40	-	-	Idris et al. (2009)
Seeds, water extract	-	-	.09	-	0.35	-	-	-	-	-	-	-	-	Chia and Dykes (2010)
Seeds, ethanol extract	-	-	-	-	-	-	-	-	-	-	-	0.1	-	Jiménez-Arellanes et al. (2013)
Seeds, chloroform extract	-	-	-	-	-	-	-	-	-	-	-	0.02	-	Jiménez-Arellanes et al. (2013)
Chloramphenicol	-	-	240	70	-	80	60	60			60	-	-	Sreedevi and Pradeep (2016)
Peel, ethanol:water, (80:20 v/v)	0.015	-	0.030	0.030	-	-	0.30	-	0.030		0.10	-	-	Melgar et al. (2018)
Seeds, ethanol:water, (80:20 v/v)	0.020	-	0.030	0.030	-	-	0.15	-	0.030		0.030	-	-	Melgar et al. (2018)
Streptomycin	0.10	-	0.20	0.04	-	-	0.20	-	0.20		0.25	-	-	Melgar et al. (2018)
Inhibition zone (mm) at 100 mg·mL^-1
Seed, ethyl acetate extract^a	-	32	-	37	-	35	16	27	22	-	16	-	-	Idris et al. (2009)
Seed, methyl alcohol-water	-	16.7-32.7	-	20.8-30.5	-	-	-	-	-	-	-	-		Nagaraj et al. (2010)
Peel / Hass	-	-	5.07	5.80	-	-	5.73	-	-	5.73	-	-	6.00	Rodríguez-Carpena et al. (2011)
Seed / Hass	9.20	-	9.27	8.33	-	-	-	-	-	-	-	-	-	Rodríguez-Carpena et al. (2011)
Peel / Fuerte	6.33	-	5.80	5.33	-	-	-	-	-	5.73	-	-	5.53	Rodríguez-Carpena et al. (2011)
Seed / Fuerte	7.87	-	7.33	6.80	-	-	7.67	-	-	-	-	-	5.20	Rodríguez-Carpena et al. (2011)
Chloramphenicol	20.0	-	21.20	19.03	-	-	21.67	-	-	7.53	-	-	-	Rodríguez-Carpena et al. (2011)
Chloramphenicol	-	35.6	-	34.0	-	-	-	-	-	-	-	-	-	Nagaraj et al. (2010)

^a Extract displaying more potency among five different-polarity ones

was attributed to trihydroxy fragments, which later were identified as hydroxylated fatty alcohols. Idris et al. (2009) tested the activity of ethyl acetate extract (EAE), CE, and ME, which were compared favorably with the activity of the standard streptomycin, while ME and EAE had the lowest MIC value (10 mg·mL^-1) against C. albicans.

Nagaraj et al. (2010) tested several seed extracts against some bacterial pathogens, such as Bacillus subtilis, which causes foodborne illness, and S. aureus, the causative agent of impetigo, cellulitis, and scalded skin syndrome, being such potential diseases for humans easily transmitted by food animals. Authors assayed AE, EA, ME, and CE against the two above indicated bacteria, and significant activity was found. Chia and Dykes (2010) tested the antimicrobial activity of EA and AA of epicarps and seeds of several avocado varieties. The EA showed antimicrobial activity (0.104–0.417 mg·mL^-1) against Gram-positive and Gram-negative bacteria (except for E. coli), while AE was only active against Listeria monocytogenes and Staphylococcus epidermidis. No inhibition by either EA and AA were observed against Aspergillus flavus and Penicillium spp. Rodríguez-Carpena et al. (2011) found highly antibacterial activity for AP against several pathogens and spoilage microorganisms commonly found in meat products. The authors found Gram-negative bacteria less sensitive than Gram-positive, as demonstrated by the measured inhibition zone (mm) at 100 mg·mL^-1. Rodríguez-Sánchez et al. (2013) screened the AE of seeds, further fractionated, against active endospores and vegetative cells of Clostridium sporogenes. Authors performed bioassay-guided puriﬁcation of crude extracts from seeds, based on inhibitory properties, and linked antimicrobial activity to six acetogenin compounds (Fig. 6, compounds 1–6). Both vegetative cells and active endospores were inhibited by the extracts, and the MIC of isolated molecules was found in the 7.8 to 15.6

Fig. 7: Chemical structures of carotenoids contained in avocado by-products

Fig. 8: Chemical structure of phytic acid identified in avocado by-products

μg·mL^-1 range, while an enriched fraction at 19.5 μg·mL^-1showed bactericidal activity. The authors concluded that the isolated compounds should be used as natural alternatives to antibiotics and additives used by the food and pharmaceutical

Fig. 6: Chemical structures of acetogenins identified in avocado by-products. Active compounds 1−6 isolated from an avocado seed extract capable of inhibiting Clostridium sporogenes PA 3679 (ATCC 7955) vegetative cell growth and endospore germination. 1, (2S,4S)-1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene;2, persediene; 3, persenone-C; 4, persenone-A; 5, persenone-B; 6, persin (Rodríguez-Sánchez et al. 2013). Compounds 7-11 are anti-inflammatory, as described by Rosenblat et al. (2011)

industries to inhibit Gram-positive spore-forming bacteria.

Nisin (an antimicrobial peptide) was assayed in combination with AP extracts to improve the antimicrobial activity against some food-borne bacteria such as Listeria, as well as the antioxidant capacity (Calderón-Oliver et al. 2016). Both peel and seed extracts-containing mixtures showed antioxidant activity and radical scavenging capacity, which was attributed to their polyphenolic composition, and a synergic antimicrobial response was noted in the mixtures of both extracts in conjunction whit nisin. The highest antimicrobial and antioxidant activities were obtained for a mixture containing 61% of peel extract with 39% of nisin. Later, the encapsulation of a mixture of nisin and avocado peel extract by freeze and spray drying was optimized. Table 6: Selected antioxidant activities reported for avocado by-products

Variety	Organ	Antioxidant assays
Variety	Organ	ABTS radical µmol Trolox·g^-1	CUPRAC µmol Trolox·g^-1	DPPH IC₅₀ μg·mL⁻¹	DPPH µmol Trolox·g⁻¹	FRAP µmol FeSO₄g⁻¹	ORAC µmol Trolox·g⁻¹	TEAC μmol Trolox·g⁻¹	Reference
Avocado	Seed FW	236							Soong and Barlow (2004)
Avocado	Seed DW	725				1484			Soong and Barlow (2004)
Avocado	Seed DW	1571-1888				27287-3078			Soong and Barlow (2004)
Avocado	Peel DW			3.83					Lee et al. (2008)
Avocado	Seed DW			7.78					Lee et al. (2008)
Several cultivars	Seed FW				38-190		58.2-631		Wang et al. (2010)
Several cultivars	Seed FW				128-240		229-464		Wang et al. (2010)
Hass	Peel DW	1457				1457			Tesfay et al. (2010)
Hass	Seed DW	2593				1331			Tesfay et al. (2010)
Fuerte	Peel FW	35-242	104-456		35-175				Rodríguez-Carpena et al. (2011)
Fuerte	Seed FW	38-195	96-353		28-167				Rodríguez-Carpena et al. (2011)
Hass	Peel FW	16-104	56-218		18-89				Rodríguez-Carpena et al. (2011)
Hass	Seed FW	22-158	58-275		18-66				Rodríguez-Carpena et al. (2011)
Avocado	Peel DW	23.8						34.72	Deng et al. (2012)
Avocado	Seed DW	17.5						42.63	Deng et al. (2012)
Hass	Peel DW						470.0	161.0	Kosińska et al. (2012)
Hass	Seed DW						210.0	94.0	Kosińska et al. (2012)
Shepard	Peel DW						290.0	112.0	Kosińska et al. (2012)
Shepard	Seed DW						350.0	91.0	Kosińska et al. (2012)
Avocado	Seed DW	173.3							Pahua-Ramos et al. (2012)
Hass	Peel DW	792			310				Daiuto et al. (2014)
Hass	Seed DW	646			411				Daiuto et al. (2014)
Avocado	Peel DW			370.22		27.82			Morais et al. (2015)
Avocado	Seed DW			46.47		23.71			Morais et al. (2015)
Avocado	Peel FW				16.10	9.56 ^a			Rotta et al. (2015)
Avocado	Peel DW				763.02	422.8 ^a			Rotta et al. (2015)
Hass	Peel DW						216.8		Calderón-Oliver et al. (2016)
Hass	Seed DW						1.6		Calderón-Oliver et al. (2016)
Hass	Peel DW						12.41-31.10		Saavedra et al. (2017)
Hass	Seed DW						8.26-11.01		Saavedra et al. (2017)
Fuerte	Peel DW	1004.5			420.5	1881.4			Tremocoldi et al. (2018)
Fuerte	Seed DW	580.8			464.9	931.7			Tremocoldi et al. (2018)
Hass	Peel, DW	791.5			310	1175.1			Tremocoldi et al. (2018)
Hass	Seed DW	645.8			410.7	656.9			Tremocoldi et al. (2018)
Nariño	Peel DW			138.2				5,700	Rosero et al. (2019)
Nariño	Seed DW			320.1				3,200	Rosero et al. (2019)
Hass, Hass type	Peel FW							6.99-103.12	Ramos-Aguilar et al. (2021)
Ascorbic acid		8138.86	3009.68		54189.92				Tusevski et al. (2014)
α-tocopherol		2685.04	2488.81		2221.83				Tusevski et al. (2014)
BHA		2476.47	6422.31		4317.85				Tusevski et al. (2014)

^a expressed as Fe₂SO₄.7H₂O

Abbreviations: ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; CUPRAC: cupric reducing antioxidant capacity; DW: dry weight; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FW: fresh weight; FRAP: Ferric Reducing Antioxidant Power; ORAC: Oxygen Radical Absorbance Capacity; TEAC: trolox equivalent antioxidant capacity

The authors concluded that such microcapsules could be used as functional ingredients (Calderón-Oliver et al. 2017).

The antimicrobial activity measured as MIC and diameters of growth inhibition zones (mm) of AP extracts is compared with that of chloramphenicol in Table 5. Chia and Dykes (2010) and Jiménez-Arellanes et al. (2013) found extreme potency for all AP extracts. Idris et al. (2009), found higher activity developed by AP against some pathogenic bacteria than that showed by chloramphenicol, as demonstrated by MIC methodology. Concerning the activity evaluated by the diameter inhibition area methodology, Nagaraj et al. (2010) found growth inhibitory activity of avocado seed extracts against B. subtilis and S. aureus, which was similar to that exercised by chloramphenicol and approximately ~4‒6 times higher than that found by other authors for S. aureus. Melgar et al. (2018) found a high capacity of EA of AP against several bacterial and fungal strains. MIC, Minimum Fungicidal Concentration (MFC), and Minimum Bactericidal Concentration (MBC) were checked for 4 Gram-positive, 4 Gram-negative bacteria, and

Fig. 9: Chemical structures of polyols identified in avocado by-products

Fig. 10: Chemical structures of sterols identified in avocado by-products

7 microfungi. The activity was found in 7 bacterial strains, and the seeds extracts showed better MBC than peel extracts in 6 out of 8 strains, while the fungicidal effect was exercised only by seeds extracts against some strains.

Differences in antimicrobial activities of AP found by different authors could be related to diverse extraction procedures of AP, bacterial strains assayed or can be due to the various methodologies used for testing bacterial inhibition. In any case, all results indicate high antibacterial activity for avocado peel and seed extracts.

Text Box:

Fig. 11: Organic acids identified in avocado by-products

Fig. 12: Tocopherols identified in avocado by-products Larvicidal activity

Leite et al. (2009) tested HE and EE of avocado seeds against Artemia salina and evaluated larvicidal activity against Aedes aegypti. They concluded that the extracts obtained from avocado seeds merit further research to be used against dengue.

Toxicity of avocado by-products

Several experiments were performed to determine the potential toxicity of avocado seeds, which was mainly attributed to perseitol occurrence. Acute and sub-acute toxicity in rats was determined by intraperitoneal (i.p.) administration of EAE of avocado seeds (Taha et al. 2008). At the end of a 14 d-trial, it was found that the liver and kidney showed normal architecture and that the treatment decreased food consumption, BW, and blood parameters, concluding that the EAE induced relatively low toxicity. Another experiment was performed on rats to assess the possible toxicity of the AE of seeds (Ozolua et al. 2009). The authors calculated LD₅₀ and found that hematological parameters and the levels of albumin, creatinine, alanine aminotransferase (ALT), and aspartate Aminotransferase (AST), remained unchanged. Moreover, the seed extract was found to be safe on an acute and sub-acute basis. Pahua-Ramos et al. (2012) performed oral acute toxicity assays in mice-feed avocado seeds. They found hypolipidemic and antioxidant effects while feeding with 2.5 g of avocado seeds·kg^-1 BW for 6 d induced a 100% of mortality; however, it was noted that125 mg of avocado seeds·kg^-1 BW reduced the elevated levels of total cholesterol. Thus, it was concluded that both AS and ME or avocado seed flour can be used for treating hyperlipidemia. Padilla-Camberos et al. (2013) performed an acute toxicity test in male BALB/c mice, obtaining an LD₅₀ for the seed extract of 1200.75 mg·kg^-1. It was found that 250 mg·kg^-1 of extract and the negative control induced a low amount of micronucleated cells; however, in vivo mutagenicity on peripheral blood cells after seed extract supplementation was not observed. Umar et al. (2016) assayed hepatotoxic effects of AE and PE of avocado seed on liver enzymatic activity. They found that a daily oral administration of both AE and PE of seeds during 3 weeks at 500 mg·kg^-1 BW showed hepatotoxic effects. Such actions were also tested for liver enzymes by checking AST, ALT, and alkaline phosphatase (ALP) activities. It was found that a daily oral administration of AE and PE of seeds for 3 weeks at 0.5 g·kg^-1induced minor liver damage.

As seen, most experimentation showed weak toxicological effects, which occur only at high doses of seed extracts. Moreover, all experiments were conducted using mice and rats, which can be useful to discern the possible toxicity of AP for humans; however, other animal models designed to evaluate possible toxicity for farm animals remain poorly developed. This deprives knowing the precise use of such by-products to feed farm animals and given the appropriate nutrient composition and phytochemical profiles of AP, further experimentation on this subject is necessary.

Antioxidant activity

Oxidative stress has been cited as responsible for early events conducting to the development of important infectious diseases in farm animals, such as pneumonia and enteritis. Given that oxidative stress should be easily prevented with antioxidants, the use of antioxidant-rich feeds could be a positive action in farm animals (Lykkesfeldt and Svendsen 2007).

This capacity is usually determined through in vitro studies. But Oboh (2013) tested several phenolic extracts of different avocado organs in rat’s pancreas through a Fe²⁺ induced lipid peroxidation test. All the extracts caused a significant decrease in malondialdehyde contents in the pancreas in a dose-dependent manner, and the seed had the highest inhibitory effect on Fe²⁺ induced lipid peroxidation (IC₅₀ = 60.61 µg·mL^-1). The authors concluded that the phenolic extracts of several AP were able for protecting the pancreas from in vitro lipid peroxidation. This action was attributed to phenolics, due to their reducing power, Fe²⁺ chelating, and radical scavenging abilities.

In vitro performed colorimetric and fluorometric antioxidant capacity tests are based on Hydrogen Atom Transfer (HAT) mechanism. Usually, HAT-based methods monitor competitive reaction kinetics. These methods generally used a synthetic-free radical generator, e.g., the 2,2′-azinobis (3-ethylbenzothiazolline-6-sulfonic acid (ABTS). Generally, the antioxidant capacity usually checks the ability of the antioxidants in controlling the degree of oxidation, and the antioxidant capacity tests can be based on the peroxyl radical scavenging, such as those based on the total radical trapping antioxidant parameter (TRAP); oxygen radical absorbance capacity (ORAC); metal-reducing power such as ferric reducing/antioxidant power (FRAP); hydroxyl radical scavenging such as deoxyribose assay; organic radical scavenging such as ABTS, and 2,2-diphenyl-1-picrylhydrazyl (DPPH); and cupric reducing/antioxidant power (CUPRAC) (Karadag et al. 2009).

The results of the antioxidant activities of AP compared to ascorbic acid, α-tocopherol, and butylated hydroxyanisole (BHA) checked by various methodologies are displayed in Table 6. Notice that the results obtained for the antioxidant potential of the various extracts of seeds and peels for the same test are quite variable. This variability could be related to the use of by-products from different avocado varieties, and obtained from fruits in different maturation stages and cultivated following different agronomic protocols and under various climates. Good antioxidant activity was found in most of the extracts, albeit with many fluctuations, at about a tenth of that showed by checked pure molecules, i.e., ascorbic acid, α-tocopherol, and BHA. The antioxidant activity detected in both peels and seeds was found to be dependent on the type of test performed. For instance, Tesfay et al. (2010) found by the ABTS test higher antioxidant capacity for seeds than for peels; however, by the FRAP test the results were the opposite. On the other hand, an increase in the antioxidant capacity was detected in dried seeds and peels in comparison with fresh by-products. In this regard, Rotta et al. (2015) indicated an increase of up to 100 times this capacity for dried seeds, which is in good agreement with the findings of Soong and Barlow (2004), who reported that this phenomenon could be related to the production of Maillard-type antioxidants, although several other natural lipophilic antioxidants could have been degraded by drying. In addition, the surplus of the antioxidant activity might be related to the formation of polyphenols. As shown in Table 6, all tests indicate that both avocado peels and seeds have good antioxidant activities, which can be useful for increasing the health of farm animals.

Use of avocado by-products for feeding farm animals

Avocado has been cited as conditionally toxic for some animals, however ill effects of AP seem to be animal species-dependent, and healthy actions by feeding with AP have been found in some animals, such as pigs. The entire avocado fruits are commonly used as feed for pigs because of their high digestibility. However, it has been reported that the whole avocado fruit as pig feedstuff has lower nutritional value than the pulp and that the nutritive value of fruits varies according to cultivar. The seeds have higher nutritive value than peels, as evaluated in pigs by the mobile nylon bag technique (Carmenatti et al. 2015). The protein concentrations were found to be higher in pulps and peels than in the seeds of avocado varieties; however, the whole avocado fruit is easily digestible, and therefore fruit discards could be used for feeding pigs, although protein supplementation would be needed when AP constitute an important portion of pig diets (Ly et al. 2021). Overall, AP develop health actions in the muscles of pigs: it improves the FA composition, oxidation state and color stability of meat, and the composition of intramuscular fat. Also, the consumption of such by-products increases the degree of fat unsaturation, while the color of the muscles of pigs is better preserved from oxidation (Hernández-López et al. 2016). Positive results when feeding pigs with avocado peels were obtained also in rural Busia District, Kenya, by Mutua et al. (2012).

AP have been used successfully to feed ruminants. To this end, it has been proposed the avocado meal, which is an oil-extracted by-product from avocado fruits lacking commercial value, which contains high amounts of fiber. Given the high degradability of its dry matter, this AP has been proposed for ruminant diets (Skenjana et al. 2006). Generally, AP have been positively evaluated for feeding goats and sheep. For instance, it has been observed that a mix of avocado seeds and orange peels meal, in a 25:35 ratio, has the potential to positively replace guinea grass in the diet of West African Dwarf male growing sheep (Okoruwa et al. 2015). Moreover, a trial using avocado waste (a mixture of pulp and peels) included in multi-nutrient blocks for feeding goats was successfully conducted by Evan et al. (2020). These workers found an improvement in the quality of the FA profile of milk, while milk yield was unaffected. Authors reported that the intake of multi-nutrient blocks containing 14.8% AP was low, probably due to the oxidation and rancidity of avocado lipids. Interestingly, no changes were noted in milk production, however, feeding blocks with AP increased milk fat content with minor changes in the FA profile of fat milk.

In aquaculture, the use of AP is highly recommended. These can constitute an energy source in fish farms, which are highly dependent on local resources. This assumption was made mainly based on the contents of crude protein and fat, and also considering the effectiveness of removing anti-nutritional constituents (Kassahun et al. 2012). In this regard, the antioxidant properties of a tilapia (Oreochromis niloticus) diet with the inclusion of AP were evaluated. An increasing pattern was observed for both the antioxidant activity and the total phenolic compounds content as the level of AP inclusion increased. Therefore, AP was considered a good fed ingredient in aquaculture diets because of their antioxidant properties and the added value granted by its use (Jiménez-Ruiz et al. 2019).

In birds, the results appear to be contradictory. In this way, it was investigated the effectiveness of avocado seed powder-based supplements on meat quality and the liver and kidney physiology of culled female quails (Coturnix coturnix japonica). The results of the experimentation showed that avocado seed powder-containing supplements significantly improved the level of serum glutamic pyruvate transaminase (GPT), creatinine, urea, fat, protein, cholesterol, meat tenderness, and cooking loss. Thus, avocado seed powder-containing supplements improved meat quality and the liver and kidney physiology of the culled female quail (Tugiyanti et al. 2019). Similarly, Akinduro et al. (2021), proposed the use of avocado seed powder in broiler chicken diets. Authors demonstrated that avocado seed powder can be used in broiler chickens for up to 5.5% of the total diet, and such inclusion led to an improved feed conversion ratio and carcass growth. However, Van Ryssen et al. (2013) warmed against the inclusion of AP in broilers’ diets This was evidenced by some trials in that such waste induced poor performance of the birds, although no symptoms of toxicity were observed at the end of the experiments.

Despite the positive effects referenced, avocado fruits have been associated with congestive heart failure related to severe cardiomyopathy in goats, sheep, horses, and ostriches (Stadler et al. 1991). Low doses develop aseptic mastitis in horses and goats, while high intake of the fruits of some avocado cultivars has poisoned budgerigars, birds, cats, mice, rats, horses, rabbits, cattle and goats, and canaries, among others, and possibly dogs (Kellerman et al. 2005). Persin has been cited as responsible for the toxicity of avocado peels and seeds, being considered a fungicidal toxin found in the leaves and fruits of the avocado tree. The lethal dose is characteristic of each animal species. The mechanism by which avocado compounds act is by triggering fluid accumulation in the lungs, leading to difficulty breathing, and death takes place due to oxygen deprivation. Fluid accumulation can also occur in the pancreas, heart, and abdomen (Buoro et al. 1994).

To use AP as livestock feed, the excessive amount of bioactive compounds in AP could be decreased by hydrothermal treatment. This was successfully applied to avocado seeds to reduce bioactive compounds and fiber contents to safe levels, and different boiling times were investigated. Results showed a significant reduction in the fiber fractions and bioactive compounds as the boiling time increased when compared with the raw seeds, and iron was significantly higher in the treated samples than in the raw ones. Moreover, the energy values showed a slight increase from the raw at 30 min of hydrothermal treatment (Ibhaze 2017). Although persin was not analyzed in this work, this bioactive would probably follow the same trend as the other bioactive compounds.

The experimentation developed to feed PA to farm animals shows highly contradictory results. Probably, the toxicity or beneficial effects of PA in farm animals are due to the different levels of persin in the various PA, whose concentration is dependent on the type of avocado cultivar considered. Therefore, pending new research, it is advisable to use PA in mixtures with other by-products or, alternatively, to carry out hydrothermal treatments to reduce bioactive compounds included in PA to safe levels, according to the experimentation shown.

Conclusions

As exposed in this work, avocado waste, i.e., peels and seeds, contain suitable amounts of several health-enhancing compounds for farm animals. Avocado peels contain a great variety of phenolic acids and flavonol glycosides, while the seeds contain mainly flavanol monomers, procyanidins, and hydroxycinnamic acids. Other highly relevant phytochemicals found in avocado by-products are acetogenins, which have antimicrobial, antibacterial, and spore germination inhibiting effects, and whose occurrence is restricted to Annonaceae and Lauraceae. The utilization of avocado by-products to feed farm animals could become an important tool for adequate by-products management to ensure ecologically and sustainable production. Comprehensive experiments in farm animals are needed, including not only the use of pure phytochemicals but also feeding animals with raw by-products to ensure cheap exploitation and sustainability of the production processes. Also, rigorously toxicological experiments clarifying the possible toxic effects of avocado by-products cited in some farm animals are needed, while studies on nutritional aspects of avocado by-products such as the aminoacyl composition of the various seeds and peels varieties, will help to discern the nutritional potential of these by-products. Furthermore, the reduction of bioactive compounds contained in avocado by-products through different treatments, such as hydrothermal ones, should be enhanced. Not only in terms of the composition of the resulting product, but also in terms of the health of farm animals fed with such by-products.

Acknowledgements

The author acknowledge the financial support of Vicerrectorado de Investigación e Innovación of University of Almería (Project 2020/00001014), Junta de Andalucia (Proyect P20_00806), Campus de Excelencia Internacional Agroalimentario (ceiA3), and Centro de Investigación en Agrosistemas Intensivos Mediterráneos y biotecnología Agroalimentaria (CIAMBITAL).

Author Contributions

JLGG conducted the study, performed the literature search, the data extraction and wrote all parts of this paper.

Conflicts of Interest

The author declare no conflict of interest of any sort.

Data Availability

I hereby declare that the data relevant to this paper is available and will be provided on request.

Ethics Approval

This study does not involve human subjects. Thus, ethics approval is not required.

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