Enzymatically Modified Isoquercitrin: Production, Metabolism, Bioavailability, Toxicity, Pharmacology, and Related Molecular Mechanisms

Quercetin and its glycosides, such as isoquercitrin or rutin, are among the most ubiquitous flavonoids present in plants. They possess numerous health-promoting properties, whose applicability is, however, limited by poor water solubility and absorption issues. Enzymatically modified isoquercitrin (EMIQ) is an isoquercitrin derivative obtained from rutin via enzymatic transformations that greatly enhance its bioavailability. Due to advantageous reports on its safety and bioactivity, EMIQ is currently gaining importance as a food additive and a constituent of dietary supplements. This review summarizes the thus-far-conducted investigations into the metabolism, toxicity, biological properties, and molecular mechanisms of EMIQ and presents a comprehensive characterization of this valuable substance, which might represent the future of flavonoid supplementation.


Introduction
Flavonoids represent a large group of plant polyphenols, with over 10,000 compounds identified until now. Based on the differences in the basic structure, they might be subdivided into several subclasses, such as flavonols, flavan-3-ols, flavanones, flavones, isoflavones, chalcones, and biflavonoids. In recent years, there has been growing interest in flavonoids, particularly flavonols, due to the increasing evidence for their prominent role in the prevention and treatment of various human diseases. Quercetin (3,3 ,4 ,5,7-pentahydroxyflavone; QU) is the best-known, prototypical representative of the flavonol subclass, which is among the most-widespread and most-studied types of flavonoids. Plants contain QU not only as an aglycone, but also in various conjugated forms such as glycosides, among which isoquercitrin (quercetin 3-O-β-D-glucopyranoside; IQ) and rutin (quercetin 3-O-β-D-rutinoside; RT) are the most ubiquitous [1,2]. These compounds are the main health-promoting components of black elderberry, capers, cloves, black chokeberry, grapes, buckwheat, onions, and olives, among others [3,4]. Accumulating in vitro and in vivo data suggest that QU and its glycosides have numerous beneficial properties, including antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective, anti-inflammatory, and antidiabetic effects [3,[5][6][7][8]. However, their low solubility in water and other hydrophilic media, resulting in poor bioavailability [9], severely limits the potency of their positive health effects and applicability in medicine, pharmacy, and the food industry. Therefore, improving the water solubility of flavonols without compromising their original physiological activities has become one of the relevant topics in flavonoid research [10][11][12].
Numerous studies have shown that the oligoglycoside moiety added to QU via enzymatic modification might increase the hydrophilic properties and enhance the bioavailability of the obtained substance. Such a product dissolves easily in the gastrointestinal [10][11][12].
Numerous studies have shown that the oligoglycoside moiety added to QU via enzymatic modification might increase the hydrophilic properties and enhance the bioavailability of the obtained substance. Such a product dissolves easily in the gastrointestinal fluids and is readily digested at the intestinal mucosa membranes to yield QU ready for absorption. Enzymatically modified isoquercitrin (EMIQ) is one such innovative substance. It is a water-soluble quercetin oligoglucoside produced from RT by a series of enzymatic transformations. The bioavailability of EMIQ is approximately 17-times that of QU and 44-times that of RT; this molecule is also more easily absorbed in humans [10,13].
Despite the advance that has been made in research on the different pharmacological properties of EMIQ, no comprehensive reviews are available. Thus, this paper summarizes the findings on the production methods, physicochemical properties, biological effects, and therapeutic potential of this molecule.

Figure 1.
A simplified scheme for EMIQ production. RT obtained from natural sources is transformed using bacterial enzymes first into IQ and then into isoquercitrin oligoglucosides with 1 to 10 α-glucosyl moieties. For more details, see the manuscript text.
In the first stage of the production process, α-L-rhamnosidase (from Penicillium decumbens; EC. 3.2.1.40) leads to the derhamnosylation of RT to produce IQ. Next, an aqueous solution of IQ is combined with dextrin and treated with cyclomaltodextrin Figure 1. A simplified scheme for EMIQ production. RT obtained from natural sources is transformed using bacterial enzymes first into IQ and then into isoquercitrin oligoglucosides with 1 to 10 αglucosyl moieties. For more details, see the manuscript text.
In the first stage of the production process, α-L-rhamnosidase (from Penicillium decumbens; EC. 3.2.1.40) leads to the derhamnosylation of RT to produce IQ. Next, an aqueous solution of IQ is combined with dextrin and treated with cyclomaltodextrin glucanotransferase (from Paenibacillus pseudalcaliphilus or Paenibacillus macerans; EC.2.4.1.19), which catalyzes transglycosidation and incorporates α-glucose moieties to form EMIQ. The solution is heated (98 • C for 5 min at pH 4.2) to inactivate both enzymes, then filtered and evaporated under vacuum. The residue is dissolved in ethanol and subjected to crystallization, which removes impurities, including QU. The final ethanolic solution is evaporated in vacuo yielding EMIQ [15]. All enzymes used in the production of EMIQ are on the List of Existing Food Additives in Japan's Food Sanitation Law and are produced under food additive good manufacturing practices (GMPs) in Japan [15,35].

Physicochemical Properties
EMIQ is a yellow/orange/yellow-brown powder with a distinctive odor [14,15]. The octanol-water partition ratio (logP = −0.77) expressing the lipophilicity of a compound shows that the EMIQ has substantial hydrophilic properties [13]. The solubility of EMIQ in distilled deionized water at 25 • C is 130 g/L, which is~81,500-and~8400-times higher than observed for QU (0.0016 g/L) and RT (0.0155 g/L), respectively [9]. Moreover, EMIQ has a very high resistance to oxidative degradation and higher thermal stability than QU. It completely resisted the oxidation process in the presence of Cu 2+ ions (0.1 mM, pH 3.5) for up to 14 h at 25 • C, whereas QU was degraded entirely under the same conditions. The change in reaction conditions, i.e., an increase in the concentration of Cu 2+ ions, alkalinity, and temperature, caused EMIQ to decompose faster, but its retention was still higher than that of QU. During thermal exposition at 100 • C for 3 h, the amount of EMIQ was reduced by 55%, while QU was undetectable by HPLC after 3 h at 80 • C. On the other hand, oligoglucosylation does not protect flavonoid aglycones from degradation by irradiation with ultraviolet C light (UV-C) at 254 nm [9].

Application in the Food Industry
EMIQ was first developed in 1987 and approved by the Japanese Ministry of Health and Welfare for use as a food additive in 1996 [36]. Its monograph with quality requirements is contained in the latest edition of Japan's Specifications and Standards for Food Additives [14]. The EMIQ producer distributes it under the commercial names SANMELIN ® AO-3000 and SANMELIN ® POWDER R-20 and recommends EMIQ as an antioxidant for preserving flavor, color, and freshness [34].
In the USA, the Expert Panel of the Flavor and Extract Manufacturers Association concluded that EMIQ might be generally recognized as safe (GRAS) in 2005 [36]. The U.S. FDA also granted GRAS status for EMIQ. Currently, in the United States, EMIQ is approved as an antioxidant (for protection of flavors and colors) in multiple food categories (i.e., fruit and vegetable juices, beverages, milk products, puddings, jams, jellies, bakery products) at 75-150 milligrams per kilogram food (mg/kg) and in chewing gum at 1500 mg/kg [15].
The usability of EMIQ as a color protector in beverages was demonstrated, e.g., by Yan et al. [37], who proved that EMIQ remarkably improves the thermal and light stability of anthocyanins at different pH levels. It was also shown to prevent, to some extent, the lipid peroxidation of a walnut beverage emulsion induced by thermal treatment and UV light. However, in that case, its activity was worse than that of a standard synthetic antioxidant, BHT. Moreover, the emulsion system with EMIQ was prone to the loss of its stability [38]. Thus, as a strongly hydrophilic molecule, EMIQ might not be particularly suited for use in lipid emulsions.

Upper Gastrointestinal Tract
The metabolic pathway of EMIQ after oral ingestion ( Figure 2) starts already in the mouth. In vitro assays showed that salivary α-amylase is able to cleavage parts of the α-(1→4) linkages and deoligomerize the EMIQ components into simpler ones. The conversion is the faster the longer the α-glucosyl side chain is. Even after 1 min of contact, substantial degradation was observed, especially for IQG 6 and IQG 7 , of which only about 20% or less was left intact. On the other hand, IQ, IQG 1 , and IQG 2 were resistant to the α-amylase digestion even after a longer exposure time [39]. enzyme reactivity were noticed that resulted in different product distributions. Nevertheless, the enzymatic cleavage was still hardly effective towards IQ, IQG 1 , and IQG 2 , which constitute the final digestion products at this stage. It was also established that IQG 3 and IQG 4 degrade mainly into IQ and IQG 1 , while IQG 2 is the dominant product of IQG 5 and IQG 6 transformation. The overall results of the study suggest that, upon arriving in the small intestine, most of the EMIQ might be already converted into IQ, IQG 1 , and IQG 2 [39].

Figure 2.
Metabolic pathway of EMIQ after oral ingestion. (A) Oral ingestion of EMIQ in the form of isoquercitrin oligoglucosides with 1 to 10 α-glucosyl moieties; (B) after passing the upper gastrointestinal system, the α-oligoglucosyl chain of EMIQ components is shortened to at most two moieties due to the activity of salivary and pancreatic amylases; (C) the enzymes of the intestinal brush border free QU from the glycosides with IQ as an intermediate product; QU is then absorbed into the enterocytes via passive diffusion; (D) the metabolites of EMIQ detectable in the plasma are formed in the enterocytes and in the liver by coupling QU with glucuronic acid (Gla), sulphates (Sph), or by methylation of QU to form isorhamnetin (IR) and tamarixetin (TX).

Lower Gastrointestinal Tract
In the small intestine, the remaining α-glucosyl derivatives might be degraded into QU in a two-stage process, with IQ as an intermediate product. The in vitro study with the homogenate of rat intestinal epithelium showed that the process goes rapidly, and the inhibition of α-glucosidase activity hindered the first phase of the transformation. Thus, mucosal maltose-glucoamylase (MGAM) was suggested to be involved [10,18]. The experiments with artificial gastric juice showed that all EMIQ components are resistant to the low pH of the stomach for up to 3 h at 37 • C, which suggests that no substantial digestion processes take place in this part of the gastrointestinal system [39]. Further degradation of EMIQ occurs in the duodenum and is due to the action of pancreatic α-amylase. In comparison to its salivary counterpart, some variations of the enzyme reactivity were noticed that resulted in different product distributions. Nevertheless, the enzymatic cleavage was still hardly effective towards IQ, IQG 1 , and IQG 2 , which constitute the final digestion products at this stage. It was also established that IQG 3 and IQG 4 degrade mainly into IQ and IQG 1 , while IQG 2 is the dominant product of IQG 5 and IQG 6 transformation. The overall results of the study suggest that, upon arriving in the small intestine, most of the EMIQ might be already converted into IQ, IQG 1 , and IQG 2 [39].

Lower Gastrointestinal Tract
In the small intestine, the remaining α-glucosyl derivatives might be degraded into QU in a two-stage process, with IQ as an intermediate product. The in vitro study with the homogenate of rat intestinal epithelium showed that the process goes rapidly, and the inhibition of α-glucosidase activity hindered the first phase of the transformation. Thus, mucosal maltose-glucoamylase (MGAM) was suggested to be involved [10,18]. MGAM is one of the α-glucosidases of the brush border epithelial cells that takes part in the final stage of starch decomposition. It cleaves glucose units from the non-reducing end of the sugar chain by hydrolysis of α-(1→4)-glucosidic linkages [40]. Thus, it might be at least partially responsible for the observed conversion. However, more detailed experiments with EMIQ components are still required to explain the role of MGAM (or other α-glucosidases) at this stage of their digestion.
The transformation of IQ into QU is a better-recognized process. In the experiments of Makino et al. [10,18], lactase-phlorizin hydrolase (LPH) was found to be the primary enzyme responsible for the conversion. LPH is a membrane-bound member of the βglycosidase family present in the mammalian intestine. It is the only β-glycosidase that acts extracellularly; thus, it can operate on substrates prior to the absorption. It is known to be responsible for the hydrolysis of various dietary flavonoids, including IQ (K m = 46 µM) [41]. As phlorizin did not influence IQ hydrolysis, it was suggested that the lactase domain of LPH participates in the process [41,42]. The glucose released during the reaction is actively transported into the enterocytes by a sodium-dependent glucose transporter (SGLT 1), while the aglycone is now lipophilic enough to be absorbed via passive diffusion [18,41].
As the metabolites of EMIQ are present in the blood as early as 15 min after oral administration, most researchers indicated the small intestine as the leading absorption site [10,18]. It is, however, known that some amounts of the polyphenols may reach the large intestine, where they undergo transformation by gut microbiota. Among the bacterial catabolites of QU are, e.g., 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenyl acetic acid, and 3-(3-hydroxyphenyl)-propionic acid [43,44]. However, none of the studies on EMIQ's bioavailability have looked at its fate beyond the small intestine, and the plasma concentrations of the products of bacterial QU degradation have yet to be measured. Thus, this topic is undoubtedly worthy of further investigation.

Plasma Metabolites and Bioavailability
The primary metabolites of EMIQ thus far detected in plasma after oral administration to animals and humans are QU conjugates. Some methylated derivatives have also been detected, in particular the conjugates of isorhamnetin and tamarixetin ( Figure 3, Table 1) [10,13,45,46]. The transformation of QU to glucuronides and sulfates mostly takes place in the enterocytes due to the action of UDP-glucuronosyltransferase and phenol sulfotransferase, respectively [42,44]. On the other hand, catechol O-methyltransferase, responsible for the methylation process, is present in larger quantities in the liver than in the intestine [10]; thus, at least to some extent, this part of the metabolic transformation may occur after the absorption of QU conjugates to the blood. Following this hypothesis, the plasma levels of methylated metabolites remain at a plateau for a long time (in contrast to the levels of QU conjugates, which gradually decrease after the initial rise) [10,13].
the final stage of starch decomposition. It cleaves glucose units from the non-reducing end of the sugar chain by hydrolysis of α-(14)-glucosidic linkages [40]. Thus, it might be at least partially responsible for the observed conversion. However, more detailed experiments with EMIQ components are still required to explain the role of MGAM (or other α-glucosidases) at this stage of their digestion.
The transformation of IQ into QU is a better-recognized process. In the experiments of Makino et al. [10,18], lactase-phlorizin hydrolase (LPH) was found to be the primary enzyme responsible for the conversion. LPH is a membrane-bound member of the β-glycosidase family present in the mammalian intestine. It is the only β-glycosidase that acts extracellularly; thus, it can operate on substrates prior to the absorption. It is known to be responsible for the hydrolysis of various dietary flavonoids, including IQ (K m = 46 µM) [41]. As phlorizin did not influence IQ hydrolysis, it was suggested that the lactase domain of LPH participates in the process [41,42]. The glucose released during the reaction is actively transported into the enterocytes by a sodium-dependent glucose transporter (SGLT 1), while the aglycone is now lipophilic enough to be absorbed via passive diffusion [18,41].
As the metabolites of EMIQ are present in the blood as early as 15 min after oral administration, most researchers indicated the small intestine as the leading absorption site [10,18]. It is, however, known that some amounts of the polyphenols may reach the large intestine, where they undergo transformation by gut microbiota. Among the bacterial catabolites of QU are, e.g., 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenyl acetic acid, and 3-(3-hydroxyphenyl)-propionic acid [43,44]. However, none of the studies on EMIQ's bioavailability have looked at its fate beyond the small intestine, and the plasma concentrations of the products of bacterial QU degradation have yet to be measured. Thus, this topic is undoubtedly worthy of further investigation.

Plasma Metabolites and Bioavailability
The primary metabolites of EMIQ thus far detected in plasma after oral administration to animals and humans are QU conjugates. Some methylated derivatives have also been detected, in particular the conjugates of isorhamnetin and tamarixetin ( Figure 3, Table 1) [10,13,45,46]. The transformation of QU to glucuronides and sulfates mostly takes place in the enterocytes due to the action of UDP-glucuronosyltransferase and phenol sulfotransferase, respectively [42,44]. On the other hand, catechol O-methyltransferase, responsible for the methylation process, is present in larger quantities in the liver than in the intestine [10]; thus, at least to some extent, this part of the metabolic transformation may occur after the absorption of QU conjugates to the blood. Following this hypothesis, the plasma levels of methylated metabolites remain at a plateau for a long time (in contrast to the levels of QU conjugates, which gradually decrease after the initial rise) [10,13].  Animal research showed that the true bioavailability (AUC oral /AUC intravenous (%)) of EMIQ after oral administration, assessed as total plasma QU levels, was 35%. Thus, it was about 17-, 3-, and 44-times higher than that of QU (2%), IQ (12%), and RT (0.8%), respectively [10].
In the only study with human participants, quercetin 3-glucuronide, quercetin 3sulphate, and isorhamnetin 3-glucuronide have been reported as the main metabolites. A substantial variation was observed between individual patients in terms of the quantitative ratios between different metabolites. However, in general, the metabolite profile of EMIQ was similar to that after IQ administration. The maximal plasma concentration of the metabolites was achieved after 1.5-2 h and, similar to the case of animal studies, was about three-times higher than that obtained for IQ [13].

Elimination and Accumulation
EMIQ's metabolites are at least partially eliminated through urine, and the elevated levels of QU conjugates in the urine samples of laboratory animals were detected even 28 days after exposure. This fact suggests that the metabolites may to some extent accumulate in the body. Indeed, the quantifiable amounts of quercetin 3-glucuronide, isorhamnetin, QU, and kaempferol were detected in the bones, cerebrum, and fat of Sprague-Dawley rats after 14 days of oral administration of EMIQ (1.5, 3, and 5% in the diet). The accumulation was dose-dependent and caused the characteristic yellow discoloration of the femur. The data suggest that the effect is reversible and not connected with any histopathological changes. On the other hand, this is evidence of the systemic availability of EMIQ metabolites, which might be advantageous for its health benefits [36].

Safety
The so-far collected data on EMIQ toxicity, performed on animal models, indicate that EMIQ is safe, does not induce severe adverse effects, and does not exhibit cytotoxic, genotoxic, embryotoxic, or allergenic effects ( Table 2).

General Toxicity
The acute toxicity assessment of EMIQ was commissioned by San-Ei Gen F.F.I. for the GRAS notification [15]. The rats were treated with a single dose of EMIQ up to 25 mg/kg and then observed for 14 days for adverse reactions. No deaths were recorded during the follow-up period; thus, the LD 50 for rats was estimated to be greater than 25 mg/kg. Moreover, no abnormal behavior or other toxic effects were documented, and the necropsy found no significant abnormalities.
The toxicity tests with prolonged EMIQ exposure in rat/mouse models were carried out within 4-104 weeks and used EMIQ concentration in the diet up to 5%. No treatmentrelated mortality or severe clinical symptoms was reported in any of the studies [15,45,[47][48][49]. In animals consuming higher concentrations of EMIQ (≥2.5%), a tendency for lower body weight was noticed that mostly was unrelated to lower food consumption [45,47,49]. Dosedependent yellow coloration of bones (e.g., femur, calvarium, cranium) was reported in every study; however, it was not connected with any histopathological changes in the bone structure [15,45,47,49]. Increased urine ketone level (only in males on 2.5% EMIQ dose) and unnaturally yellow urine coloration was observed by Tamano et al. [47], but generally, the urinalysis did not present any treatment-related changes of toxicological importance [45,48]. Statistically significant, but mild changes in the weights of different organs were reported in almost every study. They were not connected with any pathological findings and occurred only in one of the genders, and no clear trend seemed to be observable across the studies [45,[47][48][49]. In a study by Nyska et al., no changes in automated motor activity were found, which suggest no neurotoxic effects [45]. Sporadic non-dose-dependent changes in hematological and biochemical parameters were found by Tamano et al. [47], Nyska et al. [45], Salim et al. [48], and Mahapatra et al. [49], which were considered of no toxicological meaning.
The subacute toxicity was also evaluated in a model of juvenile Göttingen minipigs. No toxic effects were found during a 10-day treatment with EMIQ in doses up to 1000 mg/kg/day. Compared to untreated control animals, there were no effects on weight gains and food consumption and no significant differences in hematological parameters and organ weights. The histological examination did not reveal any pathologies that could be related to the EMIQ-administration [46]. No severe adverse effects were found during a 4-week treatment with the same doses of EMIQ. All pigs fed with 1000 mg and two males treated with 300 mg of EMIQ had yellow discoloration of the femur and coccyx. However, microscopic observations presented no change in bone growth or histological changes. The consumption of EMIQ also affected the color of urine, without affecting any of the parameters of the urinalysis [46].
In the studies with human subjects, EMIQ (42 mg/day/6-times a week for 4 months) was well tolerated and no adverse effects were reported. The hematological and biochemical parameters of serum stayed within the reference ranges during the treatment time, and no significant changes connected with EMIQ supplementation were observed [50].

Genotoxicity
A comprehensive assessment of the genotoxic potential of EMIQ and IQ was conducted following the current EFSA (European Food Safety Authority), OECD (Organisation for Economic Co-operation and Development), and FDA guidance on genotoxicity and toxicity testing. Both substances (in doses up to 5000 µg/plate) tested positive in Salmonella typhimurium strains (TA98, TA100, TA1537) reverse mutation assays, and the exposure to IQ induced moderate chromosomal aberrations in Chinese hamster ovary cells. However, all other in vitro micronuclei and chromosomal aberration assays in mammalian cells gave negative results. Moreover, no in vivo genotoxicity was demonstrated in a micronuclei/comet assay in rats and a Muta TM mouse mutation assay, thus supporting the safety of EMIQ usage [51].

Embryotoxicity
EMIQ was evaluated for embryo/fetal survival, developmental toxicity, and maternal side effects in female rabbits. No maternal toxicity was observed after oral administration of EMIQ in doses up to 1000 mg/kg/day. The reproductive parameters, such as gravid uterine weight, number of implantations, implantation loss, live young, the ratio of malesto-females, placental, litter and fetal weights, and litter size, were not affected. In fetal development, sporadic cases of kidney and ureter absence were recorded; however, they were considered unrelated to the treatment due to the lack of reproducibility. Based on the study results, the NOAEL for maternal toxicity and embryo/fetal development for EMIQ was specified as 1000 mg/kg/day [52].

Allergenic Effects
EMIQ exhibited no skin irritant properties when applied topically to murine ear in concentrations up to 50%. The compound did not induce lymphadenopathy, ear swelling, erythema, irritation, and other skin lesions. The skin sensitization potential of EMIQ was assessed by the Local Lymph Node Assay ex vivo, and according to the assay protocol, EMIQ has been classified as a non-sensitizer at all tested concentrations [53].

Bioactivity
The animal studies conducted so far showed that EMIQ administered orally can exhibit significant systemic effects (Table 3). Within the research interest were, e.g., anti-allergic, cardioprotective, and chemopreventive effects, which to a large extent are connected with the antioxidant and anti-inflammatory potential of EMIQ [19,20,24]. As the metabolite profile of EMIQ is similar to that of natural quercetin glycosides, it is often regarded as the more advantageous source of this multi-functional flavonol [20,54].

Cardioprotective and Metabolic Effects
In a study carried out on rats with spontaneous hypertension, it was found that EMIQ (3 and 26 mg/kg/day) has a significant hypotensive effect. EMIQ reduced systolic blood pressure in the tested rats by about 16-21 mmHg, but did not impact the diastolic blood pressure and heart rate. The EMIQ effects were higher than those of QU, but weaker than those of diltiazem (a positive control) [20].
The atheroprotective and plaque-stabilizing effects of EMIQ were examined using apolipoprotein E-deficient mice, which spontaneously develop hypercholesterolemia and atherosclerosis when fed a high-fat diet. After 14 days of therapy, EMIQ (0.026% in the diet) significantly reduced the atherosclerotic aortic lesions and the plaque area in the aortic sinus compared to the untreated group. In addition, EMIQ decreased the accumulation of macrophages (by about 47%), increased collagen content in the plaque (by about 41%), and almost doubled the number of smooth muscle cells, suggesting a plaque-stabilizing effect. However, the reduction of atherosclerosis was not connected with normalizing the lipid metabolism, as no impact on total cholesterol, high-density cholesterol, and triacylglycerol levels was revealed. The authors connected the beneficial effects of EMIQ with its antioxidant capacity and hindering lipid peroxidation. Moreover, they suggested that EMIQ acts directly in the arterial wall, which was evidenced by the decrease (about 47%) in the levels of 4-hydroxy-2-nonenal (a major aldehyde arising due to lipid peroxidation) in atherosclerotic lesions. On the other hand, the levels of TBARS in the serum were not affected [21].
Contrary effects as to the impact of EMIQ on lipid metabolism were found by Jiang et al. In their study, the authors compared the effect of QU and its glycosides (IQ, RT, and EMIQ) on the prevention of hyperglycemia and obesity in mice on a high-fat diet. All tested compounds reduced the amount of white adipose tissue in the mesentery. Moreover, supplementation with QU and its glycosides normalized the glycemia parameters, reduced the weight gain, lowered plasma lipid levels, and decreased markers of adipocyte differentiation in adipose tissue. According to the authors, the observed effects result from the increased phosphorylation of AMP-activated protein kinase (AMPK) and subsequent inhibition of cholesterol and triglyceride synthesis, suppression of adipocyte differentiation and lipogenesis, and activation of lipolysis. Furthermore, the investigated analytes induced the translocation of the muscle glucose transporter 4 (GLUT4), which plays a vital role in regulating whole-body glucose homeostasis [54].
In a recent study by Im et al. [55] in mice also, a synergistic effect of EMIQ in combination with a heat-transformed green tea extract (100 + 100 mg/kg bw) was observed in the reduction of body weight, fat mass, and adipocyte size. The therapeutic effects were comparable to that of mirabegron (10 mg/kg bw), an agonist of the β3-AR signaling pathway, the primary regulatory mechanism involved in lipolysis. Furthermore, in in vitro models of C3H10T1/2 adipocytes and immortalized brown pre-adipocytes, it was proven that co-treatment (50 + 50 mg/kg bw) contributes to an increase in mitochondrial metabolism and a decrease in lipid content in adipocytes.
On the other hand, the consumption of EMIQ (7 g/kg bw) in combination with soybean fiber (50 g/kg bw) ameliorated glucose intolerance induced by a high-fat high-sucrose diet in rats. The effect was ascribed to EMIQ metabolites, which were suggested to increase the secretion of GLP-1 [56]-an incretin that regulates the postprandial increase in glucose by several mechanisms, including promoting insulin gene transcription, stimulating pancreatic β cell proliferation and neogenesis, inhibiting β cell apoptosis, and blocking glucagon release [57]. However, most observed effects were not significant in the groups administered single treatments. Such results were explained by the increased bioavailability of QU from EMIQ during co-ingestion of soybean fiber, which was confirmed by the increased levels of its conjugates in blood. As the soybean fiber mainly impacts the fermentation in the large intestine, the elevated absorption of QU may result from changes in the growth and behavior of intestinal microflora [56].

Anti-Allergic and Anti-Inflammatory Effects
The studies of Makino et al. [18] showed that EMIQ might be effective in hindering allergic reactions. Its activity (single oral dose of 4 mmol/kg) in inhibiting passive cutaneous anaphylaxis in mice sensitized by intraperitoneal ovalbumin injection was comparable to that of azelastine (3.3 µmol/kg). EMIQ treatment (50 and 100 mg/kg) also effectively reduced mice paw edema induced by histamine and compound 48/80. The higher dose of EMIQ was as efficient as sulfasalazine (100 mg/kg) and, similar to the control drug, completely prevented fatal anaphylactic reactions. In addition, the histopathological data revealed that EMIQ prevented mast cell degranulation and reduced histamine release [19].
In two small (20-24 participants) double-blind, placebo-controlled clinical trials, the potential of EMIQ to alleviate pollinosis symptoms was evaluated. In both cases, during 8-week treatments with EMIQ (100 mg/day), a clear trend was noticed toward reducing ocular symptoms such as itching, lacrimation, and congestion. While the decreases in the scores for individual symptoms were mainly not statistically significant, a significant reduction in the total scores for ocular symptoms was recorded. On the other hand, no differences were found in the nasal symptoms. In one of the studies, after a 4-week treatment, an improvement was noticed regarding the impact of the symptoms on daily activities; however, the overall quality of life was not affected in both trials. Moreover, there were no changes in the plasma IgE, IL-4, IL-5, IL-12, IL-13, IFN-γ, and eotaxin levels. Nevertheless, the potential of EMIQ to alleviate allergic reactions in human subjects is undoubtedly worthy of further investigation [16,17].
The anti-inflammatory properties of EMIQ have also been studied in the context of digestive system disorders. For example, EMIQ (50 and 100 mg/kg, orally) demonstrated significant protective effects in mice with gastric ulcers induced by water-restraint stress. At the higher dose, no major gastric mucosa lesions were found, and only a slight hemorrhage and a few exposed superficial mucus cells were observed. The authors related the results with the decrease in histamine release and prevention of its ulcerative impact on the stomach. The gastroprotective effect of EMIQ may also have antioxidant mechanisms. In particular, a dose-dependent increase of gastric reduced glutathione levels and a reduction in gastric lipid peroxidation markers (malondialdehyde) and inflammation mediator (nitric oxide) were noticed [19].
EMIQ treatment (1.5% in diet) also helped to counter the adverse inflammatory responses in dextran-sodium-sulfate-induced colorectal mucosal injury in mice by reducing the expression of pro-inflammatory mediators, especially TNF-α, IL-6, IL-17, and a keratinocyte-derived cytokine. Histopathological findings showed that the animals treated with EMIQ had milder submucosal edema and colonic mucosa damage than those not taking the drug [31,58]. Microarray gene expression analysis showed that the antiinflammatory effects could result from the regulation of the various signaling pathways, i.e., mitogen-activated protein kinase (MAPK), transforming growth factor β (TGF β), IFN-γ, or focal adhesion [58].

Chemopreventive Effects
Yokohira et al. [32] were the first to investigate the chemopreventive effects of EMIQ. The study was motivated by the high antioxidant properties of the substance and its ability to increase the antioxidant capacity of rat plasma when administered with the diet. In the rat model of liver carcinogenesis induced by N-diethylnitrosamine (DEN), a correlation was found between EMIQ dose (0.01, 0.1, and 1% in the diet) and the decrease in the number of preneoplastic lesions (glutathione-S-transferase-placental-form (GST-P)-positive foci). However, the individual inter-group differences were not statistically significant. As one of the possible reasons for the lack of significance, a generally poor response to the carcinogenic effect of DEN was given.
In the following years, a series of experiments was performed using a two-stage hepatocarcinogenesis model, in which the administration of other tumor promoters followed DEN treatment. In all of the studies, EMIQ treatment (0.2% in drinking water or 0.5% in the diet) significantly decreased the number and area of GST-P-positive foci in the liver [23,24,26,27,29]. The effects of EMIQ were also expressed by diminishing the number of proliferating cells and increasing the number of apoptotic cells inside the lesions [26,27,29]. The studies were, however, inconclusive as to the potential mechanisms behind such results.
In the study of Nishimura et al. [23], in which the carcinogenesis was induced by co-treatment with DEN and oxfendazole, EMIQ administration reduced the transcription of the Cyp2b2 gene, which increased due to the carcinogens exposure. Cytochrome P450 2B2 (CYP2B2), coded by that gene, is one of the enzymes of phase I drug metabolism and might be involved in the generation of oxidative stress, which, in turn, might be responsible for the formation of preneoplastic lesions. Thus, the antioxidant properties of EMIQ were considered responsible for its chemopreventive activity. An in vitro study in which EMIQ hindered the NADPH-dependent ROS production in hepatic microsomes further supports this hypothesis. Moreover, in the study by Hara et al. [24], EMIQ suppressed the lipid peroxidation induced by DEN/piperonyl butoxide treatment, which was evidenced by the decrease in the TBARS level in rat liver [29]. The antioxidant mechanism was also implicated in EMIQ's prevention of cancer development induced by DEN/β-naphthoflavone. In this case, the impacted genes were Gstm1 and Yc2, involved in phase II drug metabolism and protection against oxidative stress.
On the other hand, Morita et al. [26] did not confirm the antioxidant effects of EMIQ in the DEN/phenobarbital-induced model of liver cancer. In the study, there were no differences between the levels of ROS and oxidative stress markers (TBARS and 8-OHdG) in the livers of rats treated with EMIQ and untreated control. No significant changes were recorded for the mRNA expression levels of the genes coding enzymes of the cytochrome P450 family, including CYP2B2. As the primary mechanism of the chemopreventive effects in that model, the inhibition of nuclear translocation of constitutive androstane receptor (CAR) was suggested. CAR is one of the xenobiotic receptors regulating the expression of numerous drug-metabolizing enzymes and is known to be responsible for the carcinogenic effects of phenobarbital and other non-genotoxic chemicals in rodents [59,60]. However, the exact way EMIQ interferes with the process still needs to be explained. 6-week-old male apoE-deficient mice Diet-induced atherosclerosis EMIQ (0.026% in the diet) 7-10/group No effect of food intake, body weight, and lipid profile ↓ (~50%) area of aortic atherosclerotic lesions ↓ (~24%) plaque area in the aortic sinus ↓ lipid peroxidation product (4-HNE) in the plaque area ↓ macrophage accumulation in the plaque ↑ plaque stabilization (↑ collagen levels and smooth muscle cell accumulation) [21] 4-week-old male ICR mice Diet-induced obesity and hyperglycemia EMIQ, QU, IQ, RT (0.1% in the diet) 5/group ↓ weight gain ↓ glucose, insulin, ↓ HOMA-IR ↓ total cholesterol, triglycerides, NEFA ↑ AMPK phosphorylation, no effect on AMPK expression ↑ GLUT4 translocation to the plasma membrane of skeletal muscle No effect on JAK/STAT-pathways ↑ ACC phosphorylation in white adipose tissue and the liver ↓ adipocyte differentiation ↓ UCP1, PGC-1α, PRDM 16 expression in white adipose tissue ↓ FAS, SREBP1, ↑ CPT1, PPARα expression in the liver [54] 5-week-old male Wistar-ST rats Diet-induced obesity and hyperglycemia EMIQ (0.7% in the diet) Soybean fiber (5% in the diet) EMIQ + soybean fiber (0.7% + 5% in the diet) 7/group ↓ visceral fat (soybean fiber) ↓ glucose, insulin, HOMA-IR (EMIQ + soybean fiber) [56]      The arrows ↑ and ↓ designate the increase or decrease in a particular parameter, respectively.
The role of the anti-inflammatory effects of EMIQ was indicated in the studies of Shimada et al. [24] and Fujii et al. [27]. The evidence was, among others, the decrease in the expression of genes connected with the inflammatory response, including Cox2, Nfkbia, and Serpine1, and the decrease in the number of COX-2-positive cells. Furthermore, according to Fujii et al. [27], the anti-cancer influence of EMIQ in DEN/thioacetamide-induced carcinogenesis also relies specifically on the suppression of the activation of ED2-positive hepatic macrophages.
Some evidence also suggested the impact of EMIQ on the mitogen-activated protein kinase (MAPK) cascade involved in cell growth. However, the collected data are ambiguous and indicated a rather indirect influence [26].
The latest study in a similar model of DEN/malachite-green-induced liver carcinogenesis showed no hindering impact of EMIQ on the development of neoplastic lesions [30]. However, in that case, the rats were additionally fed a high-fat diet to imitate steatosisrelated liver promotion, which, combined with a different carcinogen used, might be a reason for the lack of EMIQ's effectiveness. The concurrently tested apocynin, an inhibitor of NADPH oxidase (NOX), exhibited a chemopreventive effect, which was suggested to be connected with the reduced expression of one of the NOX membrane regulatory subunits (p22phox) in the lesions. A tendency to suppress p22phox expression was also observed for EMIQ, but the effect was not statistically significant. Nevertheless, EMIQ was able to diminish total cholesterol levels and the levels of alkaline phosphatase, one of the signs of liver dysfunction [30].
On the contrary, no chemopreventive effects of EMIQ were observed in the renal carcinogenesis model with karyomegaly induced in rats by administration of ochratoxin A. The authors suggest that the genotoxic effects of ochratoxin A is unrelated to oxidative stress, as the treatment did not impact the TBARS and reduced the glutathione levels [61]. This result might be indirect evidence for the role of antioxidant mechanisms in the chemopreventive properties of EMIQ.
Further confirmation of the role of anti-inflammatory mechanisms came in turn from the study of Kangawa et al. [58] in which EMIQ inhibited the colon carcinogenesis induced in mice by azoxymethane and dextran sodium sulfate treatment. The EMIQ administration reduced the number of proliferative lesions in vivo, hindered the macrophage infiltration in the intestinal wall, and decreased the histological inflammatory score. The impact on Wnt/β-catenin signaling was also implicated, but further studies are required to clarify the details.

Neurological Effects
Oxidative stress is also known to impact the neurological connections with the central nervous system and impair cognitive functions. Thus, some researchers decided to investigate the potential of EMIQ to mitigate those disorders.
Okada et al. [62] studied the impact of EMIQ on the synaptic plasticity in the hippocampus during the developmental stage in rats. The rats were treated with EMIQ (0.5% in the diet) starting from the pregnancy period up to adulthood. No impact of the treatment on short-term spatial memory was found, as judged by the results of the Y-maze test. However, the fear extinction tests revealed a decrease in the freezing time in the third trial, thus suggesting that EMIQ might support fear extinction learning. Moreover, the increase in synaptic plasticity was indicated by induced transcription of some proteins associated with long-term memory and memory rewriting (e.g., Fos, Kif21b, Grin2d) in the hippocampal dentate gyrus, amygdala, and prefrontal cortex. In the subsequent investigation, it was determined that EMIQ exposure starting already in the fetal stage is required to obtain the advantageous effects. Thus, some EMIQ-induced changes occur very early in brain development. Additionally, more detailed immunohistochemical assays revealed a much more complex impact on the proteins involved in synaptic plasticity [63].
In the study by Okano et al. [64] in a rat model, it was also demonstrated that EMIQ treatment (0.25 and 0.5% in the diet), initiated in late pregnancy and continued into adulthood, suppressed the LPS-induced neurobehavioral changes in the offspring, similar to the symptoms of autism spectrum disorder. The detailed histopathological and immunochemical assays revealed that EMIQ ameliorates the pro-inflammatory and oxidative responses caused by LPS that lead to brain damage.

Musculotropic Effects
Oxidative stress is one of the causes of aging and, among others, may result in muscle atrophy. Thus, the effect of EMIQ supplementation (36 mg/day/kg) on the physiological changes of skeletal muscles related to age was investigated in old mice. Six months of EMIQ consumption were ineffective for age-related muscle loss; however, this significantly improved locomotor activity. As muscle is a critical organ in regulating the metabolism of the whole body, a metabolic profile under sitting and exercise conditions was also determined. Chronic administration of EMIQ prevented the age-related decrease in lipid oxidation and slightly improved the antioxidant capacity and oxidative stress in skeletal muscle, increasing the expression of genes for the antioxidant enzyme glutathione peroxidase and reducing the level of carbonyl proteins [66].
On the other hand, in the study by Kohara et al. [65], EMIQ (4 mg/kg/day) impacted muscle growth in functionally overloaded mice during a 3-week treatment. The overload initiated by the ablation of gastrocnemius and soleus muscles induced hypertrophy of the plantaris muscle. The effect was enhanced by EMIQ treatment alone and in combination with whey proteins. Moreover, muscle hypertrophy was also observable in sham-operated mice.
Evidence for muscle hypertrophy induced by EMIQ was also found in humans. In the study by Omi et al. [50], the effect of EMIQ consumption on the body composition and antioxidant status of men (18.5-30 years old), who regularly undergo intense exercise, was assessed. In the randomized, double-blind, placebo-controlled study, 40 participants took part. One group ate 20 g of supplemental whey protein powder with 42 mg EMIQ, and the other consumed whey protein only (six-times a week, immediately after practice for 4 months). The obtained results suggested that EMIQ supplementation may optimize the effect of exercise and increase muscle hypertrophy. The effects were evidenced by a significant increase in lower limb fat-free mass and muscle mass, exceeding the impact of the training alone. The authors suggested that the results may be related to the antioxidant properties of EMIQ as improvement in antioxidant status was observed. Other mechanism are, however, possible, and require further investigation.

Methods
The present literature review was conducted by searching the PubMed, Web of Science, Google Scholar, and Science Direct databases using the term "Enzymatically modified isoquercitrin/EMIQ/AGIQ/α-glycosyl isoquercitrin/α-oligoglucosyl quercetin 3-Oglucoside" as descriptors. The inclusion criteria to collect the literature for this review were: (a) articles written in English and published in peer-reviewed journals; (b) articles that reported the manufacturing method, structural aspects, and physicochemical properties of EMIQ; (c) articles that assessed or reported the pharmacological/bioactive effects of EMIQ; (d) articles that reported the safety data of EMIQ; (e) articles that assessed the metabolism and bioavailability of EMIQ. A total of 57 research articles were extracted; the selection was based on their title, abstracts, and keywords. The oldest publications were from 1999, and the most recent one was from August 2022.

Conclusions
EMIQ is an enzymatically transformed mixture of isoquercitrin α-oligoglucosides with improved bioavailability compared to natural dietary flavonoids such as quercitrin, isoquercitrin, or rutin. It is also characterized by enhanced water solubility, stability, and thermal resistance, which make it particularly suited for food and nutraceutical purposes. EMIQ has already been approved as an antioxidant food additive in the USA and Japan. As the bioactivity data show, it has also considerable potential as a constituent of dietary supplements and nutraceuticals with health-benefiting properties. The conducted animal research demonstrates that EMIQ is a safe product with cardioprotective, anti-allergic, chemopreventive, neuroprotective, and musculotropic properties. In our estimation, EMIQ is an innovative substance with high applicability potential; however, there is still research to be performed before its broader use in nutrition and pharmacy. More clinical trials with human subjects are required to explain in detail the possibilities and limitations of EMIQ's health effects. It would be undoubtedly interesting to also perform further investigation into the metabolic fate of EMIQ in human intestines, which would broaden the general knowledge on flavonol's absorption mechanisms.
The case of EMIQ suggests also that α-glucosylation is indeed a very effective method for the bioavailability increase of quercetin. Thus, in our opinion, the development of similar transformation procedures for other valuable phenolics might be worthwhile and constitute an important future direction in plant metabolite supplementation that is beneficial for human health.