In plants, different types of ANFs have been found with diverse activities. In this review, we discussed some of the important ANFs and their medical importance. There are several reports on ANFs; however, the majority of them focus on their anti-nutritional characteristics, leaving wide knowledge gaps regarding their beneficial functions. Here, we systematically discuss the beneficial role of ANFs in treating human disorders and also highlight key points for their future research.

Protease inhibitors are ubiquitous in the kingdom Plantae, including in the seeds of legumes and cereals. Protease inhibitors are known to subdue the function of all four classes of proteolytic enzymes, namely, serine, cysteine, aspartyl, and metalloproteinases in the gastrointestinal tract of animals (12–15). Their antinutrient activity is linked to growth inhibition and hypertrophy of the pancreas. In animal studies, it was found that protease inhibitors are associated with pancreatic cancer and, hence, possess anti-carcinogenic effects and a wide range of therapeutic applications [Table 1; (29)]. Protease inhibitors act like competitive inhibitors, i.e., they bind to the active site of the enzyme and form a complex with a very low dissociation constant (10⁷–10¹⁴ M at neutral pH). The inhibitor imitates the substrate and therefore creates an inhibitor–enzyme complex which cannot be detached through the usual mechanism. This inhibits the active site of the enzyme and, in turn, the protease activity of the enzyme is silenced effectively (30). Inhibitors of serine proteases are the largest group of protease inhibitors. Two large families of protease inhibitors have been identified in legumes: the Bowman-Birk type (BBI) and the Kunitz-type inhibitors. BBI is the most widely categorized family of inhibitors in legume seeds and is composed of small peptide molecules and contains 71 amino acids. These inhibitors contain high levels of cysteine and seven disulfide bridges. The BBI peptide molecules are typically double-headed and so block two protease molecules simultaneously, which may be the same (such as trypsin) or different (such as one chymotrypsin and one elastase or trypsin). The BBI may be resistant to breakdown by heat (31). The Kunitz inhibitors are another class of inhibitors having a molecular weight of approximately 20 kDa. They are peptides of 181 amino acids, commonly found in soya beans and winged beans containing two disulfide bridges. These molecules are single-headed that inhibit the active site of one enzyme molecule (usually trypsin or chymotrypsin) simultaneously (32, 33). According to a recent study, protease inhibitors (PIs) operate as anticarcinogenic agents under in vivo and in vitro studies; however, the precise mechanism underlying PIs’ anticarcinogenic effect is not yet fully understood (34). It was found that PIs can affect both the early and later stages of carcinogenesis, but they have no impact on cells that have already undergone transformation (34). PIs have the ability to reverse the initial events, most likely by inhibiting a cellular activity that was initiated by carcinogen exposure. The ability of a PI to affect the expression of specific oncogenes and the amounts of specific types of proteolytic activity is central to its role in the repression of carcinogenesis (34). PIs with antineoplastic action have no discernible effect on normal cells, but they are capable of negating carcinogen-induced cellular changes in different studies (35, 36). The inhibitory profiles of PIs that affect transformation have been examined since not all PIs have the ability to prevent the transformation in vivo (37, 38). For instance, chymostatin - a very common and efficient chymotrypsin inhibitor is one of the most effective PI that suppresses the transformation. It has the ability to eliminate radiation–induced transformation in vitro with only picomolar concentrations in the medium (38). BBI family inhibitors are effective in suppressing transformation with nanomolar concentrations (39). It has been confirmed that BBIs from soybeans have the ability to inhibit prostate cancer cells’ formation of oxygen radicals and trigger DNA repair through a p53-dependent mechanism (40, 41). Moreover, experiments have shown that BBIs have impeded prostate tumor development by promoting the production of connexin 43 expressions in transgenic rats via its antiproliferative activity (42, 43).

PIs commonly found in raw legume seeds are trypsin and chymotrypsin inhibitors (44). Trypsin inhibitors (TIs) have been connected to increased pancreatic secretory activity, increased pancreatic hypertrophy, and decreased protein digestibility. Additionally, feeding studies including the administration of raw soybeans supplemented with partially purified trypsin inhibitors to rats and chickens resulted in considerable pancreatic hypertrophy and plethoric enzyme secretion (45, 46). Interestingly, a 22 kDa trypsin TI protein from the storage roots of sweet potato (Ipomoea batatas L.) has been shown in prior research to have an anti-proliferative activity and works by inhibiting the growth of NB4 promyelocytic leukemia cells (47). According to this study, TI causes NB4 cells to undergo apoptosis by impairing the cell cycle at the G1 phase and activating the caspase-3 and -8 cascades (Figure 3). Trypsin, chymotrypsin, and elastase were all inhibited by protease inhibitors (LC-pi I, II, III, and IV) that were isolated from the seeds of Lavatera cashmeriana and were recognized as Kunitz-type inhibitors based on their molecular size (48, 49). Moreover, all four classes of inhibitors exhibited anticarcinogenic activity under in vitro conditions. Among all, LC-pi I and II were treated as potential anticancer agents (50). Moreover, under in vitro conditions, a strong inhibitory effect of LC-pi I was observed at the initiation stage of cancer in the prostate (PC-3) and breast (MCF-7) cancer cell lines because of the presence of the protease inhibitor activity of trypsin, chymotrypsin, and elastase (49).

Tannins are polyphenolic substances with molecular weights ranging from 500 to 3,000 daltons. Chemically, they are classified as condensed tannins/pro-anthocyanidins and hydrolyzable tannins (119). Hydrolyzable tannins (HT) are compounds with a central polyol (D-glucose) core. The hydroxyl groups in these carbohydrate molecules are in part or completely esterified with phenol moiety-containing compounds, such as gallic acid (gallotannin) or ellagic acid (ellagitannin). HTs frequently occur in less quantity within plants and are easily hydrolyzed by mild acids and bases into carbohydrate and phenolic acids. Condensed tannins are naturally present in polyphenolic bioflavonoids, typically in the conformation of oligomers or polymers of polyhydroxy flavan-3-ol units, like (+)-catechin, (−)-epicatechin, flavan-3,4-diols, leucoanthocyanidins, or a combination of the two (120). Condensed tannins are commonly present in tree barks as barriers against microbes and are also found in other tissues of plants including buds, leaves, roots, seeds, and stems. The anti-nutritional effects of tannins are determined by their chemical conformation, dosage, and tolerable daily intake (29). Tannins have been documented to affect the digestibility of proteins and influence the bioavailability of non-heme iron, leading to less absorption of iron and calcium. They have an effect on carbohydrate digestibility, contributing to the reduced energy value of a diet (121).

Tannins are known to bind proteins through hydrogen bonds and hydrophobic interactions. They are known to interact with digestive enzymes, which exhibit anti-trypsin and anti-amylase activities, thus rendering them unavailable for digestion. It has been confirmed that condensed tannins triggered the testa-bound trypsin inhibitor activity (45). Tannins can also form a complex with vitamin B. They can also contribute to decreased palatability and lower growth rate (76). The implication of food tannins on human health is a common worry although they do have some beneficial effects as well. Some tannins possess antioxidant activity and are considered to be cardio-protective, anti-inflammatory, anti-carcinogenic, and anti-mutagenic (122, 123). Tannins are reported to inhibit lipid peroxidation and lipoxygenases under in vitro conditions. Recent studies indicate they have the ability to scavenge radicals, such as hydroxyl, superoxide, and peroxyl, which are considered to be important cellular prooxidants (124). According to an in vitro study using the human colon cancer cell line HT29, proanthocyanidin-rich apple polyphenol extract prevented the phosphorylation of epidermal growth factor (EGF), which in turn suppressed the development of these cells (125). Additionally, it has been shown that these substances inhibit the growth of cancer cells under in vivo conditions (126). Moreover, it is reported that tannins have the potential to enhance glucose absorption and prevent adipogenesis (127, 128). A class of tannins known as phlorotannins has been found to provide significant health benefits, including anti-oxidant, anti-inflammatory, anti-cancer, anti-proliferative, anti-bacterial, anti-mutagenic, anti-allergic, anti-diabetic, anti-obesity, and anti-hypertensive properties (83, 86–89). For instance, phlorotannins such as pyrogallol-phloroglucinol-6,6-bieckol, dieckol, 2,7-phloroglucinol-6,6-bieckol, and phlorofucofuroeckol A profoundly inhibit the dysfunction of suppressing monocyte migration, death of monocyte-associated endothelial cell, and inhibition of inflammation by monocyte-associated vesicles (129). All these studies strongly suggest that tannins are potential therapeutic agents against a wide range of diseases. Henceforth, it is critical to investigate the proper protocol for standardizing the dosage and troubleshooting its therapeutic effects through in vitro and in vivo studies.

Catechins (flavan-3-ol) are a group of secondary metabolites of flavonoids that have two benzene rings and a dihydropyran heterocycle. They are known to protect both involuntary and radiation-induced cancers as well as chemically generated mutations. Catechins promote phase I and II metabolic enzymes that enhance the formation and excretion of detoxified carcinogenic metabolites, inhibit cell division, and, thus, slow the emergence and spread of cancer. Catechins are strong antioxidants that prevent the oxidation of LDL cholesterol, lower cholesterol, and reduce body fat, all of which help to lower the risk of cardiovascular disease (122). Moreover, they help to reduce high blood pressure-induced strokes (122). Catechins and polyphenols present in tea leaves are efficient scavengers of reactive oxygen species (ROS) and act as indirect antioxidants by influencing transcription factors and enzyme activities. In humans, mild improvements in plasma antioxidant ability were observed following the consumption of catechins from green tea. Catechins present in tea and green tea have a very positive effect on the biomarkers of oxidative stress, particularly on the oxidative damage caused to the DNA in animal models, but human data on biomarkers of in vivo oxidative stress is limited. Greater human research studies examining the impact of tea and the catechins present in them on the biomarkers of oxidative damage to lipids, proteins, and DNA are required (130). Owing to its multiple therapeutic properties, it is vital to investigate the role of catechins in driving complex networks and unravel the underlying mechanisms to regulate the wide range of disorders.

Phytic acid is a myoinositol 1,2,3,4,5,6-hexakis dihydrogen phosphate found in plants, animals, and soil in the form of a salt of the mono and divalent cations K⁺, Mg²⁺, and Ca²⁺. It is the major cache of phosphorous encompassing 1–5% by weight in cereals, legumes, oil seeds, and nuts (131). Phytate promptly proliferates in the seeds during their ripening period. They are hoarded within leguminous seeds and oil seeds inside the globoid crystal in the protein bodies. Phytates also serve as reservoirs of cations, which are high-energy phosphoryl groups, and by chelating free iron, act as an efficient natural antioxidant (132). Monogastric animals like poultry and humans cannot break down phytic acid due to the absence of an adequate quantity of phytate-reducing enzymes in their alimentary tract (133–135). Phytates act as potent anions in a broad pH range and thus have an unfavorable impact on the bioavailability of divalent and trivalent mineral ions such as Zn²⁺, Fe^2+/3+, Ca²⁺, Mg²⁺, Mn²⁺, and Cu²⁺ in diet (29). The impact of an increased intake of a phytate-containing diet on mineral deficiency depends on what else is being consumed. The intake of phytate is a problem in regions of the world where cereal proteins are a significant and preponderant dietic component (25). Phytates substantially reduce the bioavailability of calcium, and the molar ratio of Ca²⁺: phytate has been recommended as a marker of Ca²⁺ bioavailability. The critical molar ratio of Ca²⁺: phytate is specified to be 6:1 (136). It has been reported that the interaction between phytate and carbohydrates (starch) reduces the bioavailability and breakdown of carbohydrates as the formation of the phytate-carbohydrate complex affects the rate of metabolism of starch (137). Nonetheless, recent data reveals positive health effects of phytic acid on hypocholesterolemia and hypolipidemia as an anticancer agent. The prophylaxis and suppression of tumor evolution and progression are primarily associated with the ability of IP6 to regulate the segregation, multiplication, and programmed cell death of tumor-forming cells (138, 139). This IP6-conferred anticarcinogenic protection is associated with its suppression of the generation of oxygen-free radicals by the preclusion of the Fenton reaction in iron chelation (140). Reports suggest that phytate inhibits the growth of human cell lines such as leukemic hematopoietic K-562 cell line (141), colon cancer HT-29 cell line (142), breast cancer cell lines (143), cervical cancer cell lines (144), prostate cancer cell lines, and HepG2 hepatoma cell line (145) in a dose- and time-dependent manner. Nonetheless, the phytate sensitivity of cells from different origins varies, indicating that phytate can affect different cell types by specific mechanisms of action. Phytates’ effectiveness as an anticancer agent was also shown in carcinogen DMBA-induced breast cancer rats. The animals treated with phytate showed a significant decline in proliferation. The study also compared the oncogenic indicators like serum total sialic acid (TSA), and documented grades of tissue nitric oxide to reflect carcinoma activity after administration of DMBA. Phytic acid administration in comparison to the control group considerably decreased the generation of TSA, increased programmed cell death, and suppressed oxidative stress linked with the generation of oxygen-free radicals from the carcinoma, therefore implying a probable medicinal value of phytic acid in breast oncogenesis. Phytic acid reduces the rate of cell multiplication in breast carcinoma, functions as an antioxidant, and also enhances programmed cell death (138). Dietary phytate may also aid diabetic patients as it suppresses the blood glucose levels by reducing the rate of metabolism of starch, hence slowing gastric emptiness. Similarly, phytate has also been proved to modulate the release of insulin (146). Across the western countries, cardiovascular disease is considered a leading cause of death. Elevated plasma cholesterol or elevated LDL-cholesterol concentrations have been shown to be one of the risk factors of heart diseases. Phytate, which is a component of fiber, is believed to affect the etiology of heart diseases. It has been shown that the reduction in serum cholesterol and triglyceride levels arises as a result of dietary phytate supplementation (147, 148). The decrease in serum Zn level and Zn-Cu ratio was correlated with this effect, and an imbalance in Zn-Cu metabolism is related with coronary heart disease. Phytic acid has been accepted as a superior preservative for juices (149) and meat products (150, 151). In addition, apple extract tempered with IP6 during distillation and packaging have exhibited a substantial diminution in the discoloration (brown color formation) due to polyphenol oxidase inhibition by IP6, whereas pigs fed with diet containing IP6 exhibited an enhanced shelf life of meat. The impact of IP6 on unsaturated fatty acids helped in maintaining the quality of meat and also enhanced the shelf life by suppressing lipid peroxidation (150–152). All these studies clearly approve the therapeutic potential of phytic acid and hence demands further investigation to unravel its proper use and applications.

Glucosinolates are an important class of phytochemicals present in Brassica vegetables within the range of 1.5–2.0 mg/g. They are primarily found in cabbage, broccoli, and Brussels sprouts. (153–155). Most glucosinolates are chemically and thermally stable but their hydrolytic derivatives are physiologically active (154). In raw vegetables, enzymatic hydrolysis occurs when cells are disrupted by masticating or refining the release of β-thioglucosidase (156). Glucosinolates are digested by microflora in the human GI tract and can therefore be utilized biologically in cooked vegetables even though cooking vegetables inactivates thioglucosidase. (153, 157). When plant tissue is damaged, the enzyme thioglucosidase breaks down the thioglucosidic bond, resulting in the creation of glucose and thiohydrosimate-O-sulphonate (an unstable aglycone) (155, 156). Depending on the reaction parameters such as pH and glucosinolate structure, different products can be obtained including isothiocyanates, nitriles, sulfides, thiocyanates, epithiitriles, oxazolidin-2-thiones, and indolyl compounds (154, 156). Hydrolytic decomposition products of glucosinolates, glucoraphanin, gluconasturtiin, and glucobrassicin exhibit antioncogenic properties. Furthermore, indol-3-carbinol, a component of glucobrasicin has the ability to inhibit human breast and ovarian cancers (158–160). Vegetables from the Brassica family comprise thioglycosides that are broken down into thiocyanates, which, in turn, impede the transport of iron and integration of iodine in thyroglobulin, ameliorating the augmentation of TSH secretion and thyroid cells. There is little epidemiological proof that the goitrogenic effects of glucosinolate degradation products lead to significant causes of human disease. There is significant doubt about the positive and negative repercussions of Brassica vegetables on health since there are major concerns regarding the breakdown products of glucosinolate and their antioncogenic effects. (161). Over 130 different glucosinolates have been identified as having antioxidant, anticancer, fungicide, and bactericidal properties (162, 163). For instance, isothiocyanates decrease redox imbalance levels in the system, alter chemokine function based on the reaction of the immune system, prompt programmed cell death, hinder the advancement of the cell cycle, prevent the formation and differentiation of blood vessels, and also show anti-bacterial, anti-viral, and anti-carcinogenic properties (160, 164–166). There are primarily two routes that have been proposed for isothiocyanates’ anticarcinogenic effects. First, phase II enzymes are activated concurrently with the inactivation of the phase I enzyme cytochrome P450s, which binds to isothiocyanate. Another mechanism involves the start of programmed cell death, which eliminates genetically compromised cells and halts the cell cycle (167–169). It has been proven that sulphoraphane from broccoli inhibits the growth of tumors by acting as the main inducer of cell-defensive phase II enzymes. As broccoli and Brussels sprouts are highly consumed, the extracts obtained from them are regarded as an appropriate tool for supplying sulphoraphane to humans (170). In another study, it was shown that 4-methylthiobutyl isothiocyanate specifically induced cytotoxicity in tumor-initiating cells via the p53-independent pathway; however, no apoptosis or necrosis was detected when used on normal liver cells. This compound was produced by the enzymatic breakdown of glucoerucin isolated from plants of the rocket species or by catabolism of sulphoraphane of isothiocyanate (165). In a clinical trial, the consumption of 250 g/day of broccoli and 250 g/day of Brussels sprouts significantly increased the clearance of the potentially cancer-causing chemical 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) found in properly cooked meat. It has been demonstrated that excess intake of vegetables from the Brassica family can minimize the risk of colorectal cancer by augmenting the excretion of PhIP and diet-containing heterocyclic amine carcinogens (171).

Saponins are non-volatile surface-active secondary metabolites that are typically found in plants. They are made up of a sugar moiety coupled with a steroid (or triterpene) group. These surface-active compounds are present in legumes in addition to some spices and herbs (172, 173). High concentrations of saponins impart astringency and a bitter flavor to food plants. They have historically been considered to be antinutrients due to their negative effects, for instance, growth degradation and their bitter taste, and their throat-irritating nature has led to their reduced consumption and is a key limiting factor in their application (174). Saponins have been shown to decrease the physiological availability of nutrients and enzymes and they also hinder the activity of certain metabolic catalysts such as trypsin and chymotrypsin and thus affect protein digestibility (175). Recent studies demonstrate saponins’ ability to lower cholesterol, boost the immune system, and prevent cancer (176). They also decrease the peril of coronary heart disease in humans. Foods rich in saponin are essential for regulating the level of cholesterol in blood plasma, preventing peptic ulcers and osteoporosis, and decreasing the risk of heart diseases (177). Saponins are used as adjuvants in viral (e.g., Quillaja saponaria-21) and bacterial vaccine (e.g., Quillaja saponins) applications (174). A diet high in saponins can be used to treat acute lead poisoning, prevent dental cavities, inhibit platelet aggregation, and cure hypercalciuria in people (178).

Alkaloids are secondary metabolites found in a wide range of plant species that possess therapeutic properties. Alkaloids, which primarily represent themselves as specialized molecules participating in metabolism, are nitrogen-containing chemicals having the capacity to react with acids to produce salts (179). Alkaloids appear to be present in distillate from roots, seeds, leaves, or barks of affiliates of at least 40% of plant families. Families including Amaryllidaceae, Compositae, Leguminosae, Liliaceae, Papaveraceae, and Solanaceae are especially rich in alkaloids. Solanine and tomatine are common examples. Solanine is found in little concentrations in potatoes, whereas tomatine is present in tomatoes. Alkaloids are premeditated to be anti-nutrients due to their effect on the nervous system, hindering or erroneously enhancing electrochemical transmission. The glycoalkaloids, solanine, and chaconine found in potato and Solanum spp. (180) are hemolytically active and harmful to fungi and humans. There have been studies on the infertility effects of certain plant alkaloids (181) although less intake of alkaloids arbitrate pivotal therapeutic functions, such as pain reduction, blood pressure control, tumor cell destruction, and stimulation of circulation and respiration (175).