Structures, Sources, Identification/Quantification Methods, Health Benefits, Bioaccessibility, and Products of Isorhamnetin Glycosides as Phytonutrients

In recent years, people have tended to consume phytonutrients and nutrients in their daily diets. Isorhamnetin glycosides (IGs) are an essential class of flavonoids derived from dietary and medicinal plants such as Opuntia ficus-indica, Hippophae rhamnoides, and Ginkgo biloba. This review summarizes the structures, sources, quantitative and qualitative analysis technologies, health benefits, bioaccessibility, and marketed products of IGs. Routine and innovative assay methods, such as IR, TLC, NMR, UV, MS, HPLC, UPLC, and HSCCC, have been widely used for the characterization and quantification of IGs. All of the therapeutic effects of IGs discovered to date are collected and discussed in this study, with an emphasis on the relevant mechanisms of their health-promoting effects. IGs exhibit diverse biological activities against cancer, diabetes, hepatic diseases, obesity, and thrombosis. They exert therapeutic effects through multiple networks of underlying molecular signaling pathways. Owing to these benefits, IGs could be utilized to make foods and functional foods. IGs exhibit higher bioaccessibility and plasma concentrations and longer average residence time in blood than aglycones. Overall, IGs as phytonutrients are very promising and have excellent application potential.


Introduction
Phytonutrients are chemical compounds that are only present in natural plants and are beneficial to the human body [1]. They are widely used in food and nutraceuticals due to their health-promoting benefits [2]. Flavonoids are a class of polyphenolic compound distributed in many fruits, vegetables, and plants [3]. The six major subclasses of flavonoids, which include flavones (e.g., luteolin), flavonols (quercetin), flavanones (hesperidin), catechins or flavanols (epicatechin), anthocyanidins (cyanidin), and isoflavones (daidzein), have been reported to represent various families of phytonutrients [4]. Accumulating evidence based on observational and clinical studies shows that a plant-based dietary pattern rich in fruits, vegetables, and whole grains has a clear effect on the prevention of various chronic diseases [5], and people also tend to consume dietary flavonoids from fruits and vegetables. Flavonoids are widely found in food, and most of them exist in their glycosidic forms [6,7].
Isorhamnetin glycosides (IGs), as natural flavonol compounds, are primarily extracted from various plant-based foods or medicinal plants such as Opuntia ficus-indica, Hippophae rhamnoides, and Ginkgo biloba [8][9][10]. IGs are biologically important flavonols with proven beneficial properties that give them medicinal value [11,12]. They possess diverse biological and pharmacological properties, such as antioxidant, anti-inflammatory, anti-cancer, antidiabetic, anti-obesity, and hepatoprotective properties [13][14][15][16][17]. Due to their beneficial biological activities, IGs have been considered a significant potential class of phytonutrients, and an increasing number of products containing IGs are circulating on the market in many diverse biological and pharmacological properties, such as antioxidant, anti-inflammatory, anti-cancer, antidiabetic, anti-obesity, and hepatoprotective properties [13][14][15][16][17]. Due to their beneficial biological activities, IGs have been considered a significant potential class of phytonutrients, and an increasing number of products containing IGs are circulating on the market in many countries, including the United States, Canada, Mexico, China, India, and some European countries [18,19].
Here, for the first time, a review of all studies that describe the biological activity of IGs is presented, with particular emphasis on molecular signaling pathways and mechanistic explanations for their health-promoting potential. This review also introduces the structure of IGs and the primary sources of IGs. Moreover, current methods for the analysis and quantification of IGs are summarized. Furthermore, this paper also focuses on the main bioaccessibility of IGs. Overall, this article strongly supports the use of IGs as phytonutrients.

Sources of IGs
IGs as nutritional supplements can be obtained from some foods and medicinal plants. Commonly consumed foods containing IGs include Hippophae rhamnoides, Opuntia ficusindica, Vaccinium corymbosum, Vaccinium myrtillus, Brassica juncea, rice, and onions. The main medicinal sources of Igs are Ginkgo biloba, pollen Typhae, Microctis folium, Sambucus nigra, and Calendula officinalis ( Figure 3).

Opuntia ficus-indica
Opuntia ficus-indica, otherwise known as the prickly pear or nopal cactus, is a multipurpose crop that grows wild in the arid and semi-arid regions of the world [70]. It is used not only in the diet to provide food and feed, but also for healthcare due to its antioxidant, anti-inflammatory, and anxiolytic properties [71,72].

Hippophae rhamnoides
Hippophae rhamnoides (also named sea buckthorn) [20] constitutes a rich source of IGs [10]. Its berries have been categorized as a "medicine food homology" fruit by China's National Health Commission for both nutritional and medicinal purposes [19]. Hippophae rhamnoides has a wide range of positive biological, physiological, and medicinal effects, such as antioxidative, anti-inflammatory, antidiabetic, anticarcinogenic, hepatoprotective, and dermatological effects [75].
IGs have been found in all parts of the sea buckthorn plant, including the berries, leaves, and seeds [76]. An investigation of six cultivated Hippophae rhamnoides varieties revealed that the berries contained an average of 917 mg/100 g DW of flavonol glycosides [77], whereas the content of flavonol glycosides in leaves was higher than that in berries, with an average of 1118 mg/100 g DW. Isorhamnetin

Opuntia ficus-indica
Opuntia ficus-indica, otherwise known as the prickly pear or nopal cactus, is a multipurpose crop that grows wild in the arid and semi-arid regions of the world [70]. It is used not only in the diet to provide food and feed, but also for healthcare due to its antioxidant, anti-inflammatory, and anxiolytic properties [71,72].

Hippophae rhamnoides
Hippophae rhamnoides (also named sea buckthorn) [20] constitutes a rich source of IGs [10]. Its berries have been categorized as a "medicine food homology" fruit by China's National Health Commission for both nutritional and medicinal purposes [19]. Hippophae rhamnoides has a wide range of positive biological, physiological, and medicinal effects, such as antioxidative, anti-inflammatory, antidiabetic, anticarcinogenic, hepatoprotective, and dermatological effects [75].

Ginkgo biloba
Ginkgo biloba is one of the most commonly used herbal supplements in the world [79], and is also a crucial source of IGs [80]. It has been demonstrated that Ginkgo biloba has various remarkable biological properties, including neuroprotective, anticancer, cardioprotective, and stress-alleviating properties, and could affect tinnitus, geriatric conditions, and psychiatric disorders [81]. The major compounds of Ginkgo biloba are terpene lactones and flavone glycosides [82]. Flavonol glycosides are most prevalent in Ginkgo biloba leaves, and have been identified as derivatives of the aglycones quercetin, kaempferol, and isorhamnetin, which are, by themselves, present in only small amounts in the leaves. The dominant flavonol glycosides of Ginkgo biloba leaves were found to be kaempferol-3-Orutinoside and isorhamnetin-3-O-rutinoside (24), and content of the latter ranged from 30 to 80 mg/100 g [9].

IG Identification and Quantification Methods
Different techniques have been used for the characterization, identification, and quantification of IGs, including spectral techniques and chromatographic techniques. The following review addresses the applicability of the ultraviolet-visible spectrum (UV), infrared spectroscopy (IR), nuclear magnetic resonance (NMR), mass spectrometry (MS), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), ultraperformance liquid chromatography (UPLC), and high-speed counter-current chromatography (HSCCC) methods developed for the determination of IGs.

Spectral Techniques and Mass Spectrometry
Various spectral methods have been employed for the identification and quantification of IGs. UV, IR, MS, and NMR have been used to determine the structure of IGs.

UV
The UV absorption spectra of flavonoids mainly have two absorption bands in MeOH, i.e., band I, which is caused by the electron transition of the cinnamoyl group, and band II, which is caused by the electron transition of the benzoyl group. Regarding UV in flavonols, band II absorption usually occurs in the region of 240-280 nm, and is relatively affected by increased hydroxylation of the A-ring; meanwhile, band I absorption occurs in the region of 328-385 nm and is relatively affected by increased hydroxylation of the B-ring and C-ring. The addition of diagnostic reagents (NaOMe, NaOAc, NaOAc/H 3 BO 3 , AlCl 3 , and AlCl 3 /HCl) has a certain impact on the UV spectrum [104]. For example, the UV spectrum (38) showed two absorption maxima: 359 nm for band I, and 258 nm for band II. A large bathochromic shift (up to 56 nm) in band I with NaOMe was observed, and was attributed to the presence of free 4 -OH. A free 7-OH group occurred with small bathochromic shift (16 nm) in band II upon the addition of a NaOAc reagent. Additionally, a 5, 7-dihydroxy A-ring was expected to result from the AlCl 3

IR
IR can be used to determine the characteristic functional groups of IGs. For example, the characteristic functional groups of isorhamnetin-3-O-α-L-arabinoside-7-O-β-Dglucoside (26) isolated from the Callianthemum genus were determined using IR. Its spectrum showed the characteristic absorption bands of a hydroxyl (3444.87 and 3429.43 cm −1 ), a carbonyl (1653.00 cm −1 ), and a phenyl group (1600.92 and 1490.97 cm −1 ) [57]. If the IR spectrum contained a band of 1725 cm −1 for ester carbonyl, it indicated that a hydroxyl was acylated [92]. For example, the IR spectrum of isorhamnetin-3-O-(6-acetyl-glucoside) (7) showed a band at 1725 cm −1 , which indicated the presence of an ester carbonyl [106].

NMR
NMR is a widely used spectroscopic technique for structure identification. The 1 H NMR and 13 C NMR spectra were used to determine chemical shifts in the functional groups and carbon skeleton of IGs.
Strong regularity in the 1 H NMR spectrum of IGs can be found. The chemistry shifts of H-6 and H-8 of the A-ring are in the ranges 6.00~6.20 and 6.30~6.50 ppm, respectively, and appear as doublets, with a coupling constant of 2.5 Hz, because of two aromatic protons in the meta position. In the B-ring, H-2 , in the range of 7.20~7.90 ppm, appears as a doublet with a coupling constant of 2.5 Hz; H-5 , in the range of 6.70~7.10 ppm, appears as a doublet with a coupling constant of 8.5 Hz; H-6 , in the range of 7.20~7.90 ppm, appears as a doublet of doublets, with coupling constants of 2.5 and 8.5 Hz; and a singlet at 3.80 ppm belongs to 3 -OMe [23,57,107]. Some information on sugar linkage can also be obtained from the 1 H NMR spectrum. The chemical shift in the H-1 (anomeric) proton varies according to the glycosylation pattern, e.g., 7-O-glucosides occurred at 4.8~5.2 ppm, while 7-O-rhamnosides occurred at 5.1~5.3 ppm; moreover, 3-O-glucosides occurred at 5.7~6.0 ppm, while 3-Orhamnosidesoccurred at 5.0~5.1 ppm [105].
The A 13 C NMR spectra of IGs can determine the number and environment of each carbon [57]. Moreover, the 1 H and 13 C-NMR signals and the linkages of each saccharide can easily be assigned using 2D-NMR, including COSY, HSQC, and HMBC technology. For example, an analysis of the HMQC spectrum of isorhamnetin-3-O-α-L-arabinopyranose-7-β-D-glucopyranoside (26) can enable all the protons and corresponding carbons in the structure to be assigned. In the HMBC spectrum, correlations between H-1" of arabinose and C-3, and between H-1 of glucose and C-7, indicated that arabinose was attached to the C-3 of the aglycone, and glucose was attached to the C-7 of the aglycone, respectively. Thus, they were combined to form isorhamnetin-3-O-α-L-arabinopyranose-7-β-Dglucopyranoside (26) [107].

MS
MS analysis is based on the mass-to-nucleus ratio and is used to determine molecular structure and weight. The loss of some ion fragments from a molecular or pseudomolecular ion is very characteristic of the mass spectra of IGs.
Electrospray ionization (ESI), an ionization technique, is often used for the MS analysis of IGs. The collision-induced dissociation of a pseudomolecular ion caused a characteristic fragment ion of isorhamnetin glycoside at m/z 315, which was assigned to isorhamnetin [108]. MS is also used in the determination of the attachment of sugars in IGs. In the mass spectrometry of isorhamnetin-glucoside-di-rhamnoside, a precursor ion at m/z 769 originated from the product ion at m/z 315, which is the characteristic ion of isorhamnetin aglycone, and the loss of 454 Da corresponded exactly to two rhamnose units (2 × 146 Da) and one hexose unit (162 Da) [109].
Atmospheric pressure chemical ionization (APCI) is another choice of method for detecting the molecular structure and weight of IGs. The regularities of the characteristic ions of isorhamnetin 3-O-glucoside (4) obtained in APCI-MS were analyzed; a pseudo molecular ion of m/z 477 and a second fragment of m/z 315 were provided, a characteristic fragment ion of m/z 315 was assigned to isorhamnetin, and the loss of 162 Da corresponded to one glucose unit [108].
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful new technique that can rapidly identify and quantify IGs [110].

Chromatographic Techniques
IGs can be distinguished from each other on the basis of chromatographic techniques. Therefore, the analysis, characterization, and quantification of IGs are usually performed using the following chromatographic techniques: TLC, HPLC, UPLC, and HSCCC.

TLC
TLC is a method that can be used to detect IGs, and has the advantages of rapidity, simplicity, and economy. TLC is usually carried out in ascending mode on standard silica gel plates or microcrystalline cellulose. IGs can be eluted on thin-layer chromatography plates along with the standard compounds and distinguished by their retardation factor (R f ). TLC on silica gel layers for flavonol glycosides is often eluted with an EtOAc-Pyr-H 2 O-MeOH system, an n-BuOH-HOAc-H 2 O system, an EtOAc-methyl ethyl ketone-HOAc-H 2 O system, anEtOAc-HOAc-H 2 O system [111], a buthanol-EtOH-H 2 O system [23], or another developing solvent system [107]. Generally, the spots with IGs on a TLC plate can be observed directly under UV light, and the spots are dark. They will appear yellow or green under UV light after the addition of NH 3 (gas) or a 1:1 mixture of 2% diphenyl-boric acid-ethanolamine complex in EtOH and 10% polethylenglycol 4000 in MeOH stain [112].
Moreover, a 1% ethanolic solution of ferric chloride or aluminum chloride is often used as a TLC dipping solution.

HPLC and UPLC
HPLC is suitable for analyzing active components in natural extracts due to its simplicity, sensitivity, precision, and selectivity. In order to identify and quantify IGs, the chromatographic conditions of HPLC mainly include the use of a reverse-phase C 18 column, acidic water, and MeOH or MeCN as a mobile phase [23,92,113].

HSCCC
High-speed counter-current chromatography (HSCCC), a new, continuous, and efficient liquid-liquid partition chromatography, eliminates the irreversible adsorptive loss of samples onto solid support matrix columns, and has excellent sample recovery compared with certain conventional methods [117,118]. IGs can be separated and purified efficiently through multiple distribution processes using HSCCC. Isorhamnetin-3-O-glucoside (4) (13 mg) was obtained via one-step HSCCC separation from a 240 mg sample of the medicinal herb lotus plumule [119]. HSCCC was also successfully applied to the preparative isolation of IGs [120].

The Health-Promoting Effects of IGs
IGs possess a variety of biological properties, including antioxidant, anti-inflammatory, and anti-cancer properties. Research has recently been undertaken to investigate their pharmacological benefits for the treatment of various diseases, such as diabetes, obesity, hepatic diseases, and thrombosis. Their health-promoting effects are summarized below.
Evaluation of the antioxidant properties of IGs were also carried out using various cell type experiments and animal models. The oral administration of isorhamnetin-3,7diglucoside (18) to streptozotocin-induced diabetic rats significantly reduced their levels of 5-(hydroxymethyl) furfural (5-HMF), which is an indicator of the glycosylation of hemoglobin, and of stress [95]. Similarly, isorhamnetin 3-O-robinobioside (22) exhibited significant antioxidant effects on the human chronic myelogenous leukemia cell line K562 [131]. IGs had the ability to inhibit the formation of H 2 O 2 -induced radicals in the surrounding environment of intestinal epithelial cells [132]. Moreover, the transcriptional genes of the antioxidant system and the DNA repair pathway were upregulated after incubation with isorhamnetin 3-O-neohesperidoside (15) in pKS plasmid DNA [133]. Narcissin (24) and isorhamnetin 3-O-glucoside (4) demonstrated strong inhibition of reactive oxygen species (ROS) production in the oxidative burst activity of whole blood, neutrophils, and mononuclear cells [134]. Plant extracts rich in IGs also exhibited antioxidant activity. IG-rich concentrate from Opuntia ficus-indica juice had the ability to inhibit the formation of H 2 O 2induced radicals in the surrounding environment of intestinal epithelial cells [135]. The total antioxidant activity of Hippophae rhamnoides berry extracts, evaluated via ORAC and PSC, was significantly associated with total phenolics, including isorhamnetin-3-rutinoside (24) and isorhamnetin-3-glucoside (4) [136].

Anti-Inflammatory Activity
IGs have anti-inflammatory properties due to different mechanisms. As an important inflammatory mediator, high-mobility-group protein 1 (HMGB1) contributes to organ damage and inflammation [138]. Isorhamnetin 3-O-galactoside (8) (5 µM) has been demonstrated to significantly inhibit the release of HMGB1 and reduce HMGB1-dependent inflammatory responses in human endothelial cells. It was found that 8 (4.8 mg/mouse) could also inhibit HMGB1 receptor expression, the HMGB1-mediated activation of NF-kB, and the production of tumor necrosis factor (TNF-α) in mice [139].

Hepatoprotective Ability
The liver is the most essential and functional organ in the body, and it is where primary detox and metabolic events occur [167]. Liver injury can be caused by various factors, including alcohol, microbial infection, drugs, biological toxins, and chemical agents [168]. Flavonoids in many different foods and medicinal plants have therapeutic potential in liver disease [169].
Zhang G et al. observed that isorhamnetin-3-O-β-D-glucopyranoside-7-O-α-L-rhamnoside (20) (40 µM) exhibited a profound inhibitory effect on the activation of hepatic stellate cells (HSCs) induced by transforming growth factor-β (TGF-β), and decreased the levels of inflammatory factors. It over-regulated the proteins of the DNA damage signaling pathway, including the ataxia telangiectasia mutated gene (ATM), Rad3-related gene (ATR), checkpoint kinase1 (Chk1), checkpoint kinase2 (Chk2), p53, and alpha-smooth muscle actin (α-SMA) ( Figure 5B) [176]. In addition, the active components of sea buckthorn berry (20 and 40 mg/kg) had inhibitory effects on the development of fibrosis in rats after bile duct ligation, and they attenuated liver injury and inflammation by downregulating the expression of αSMA, while over-regulating the DNA damage signaling pathways and their related genes.

Antidiabetic Activity
The antidiabetic properties of IGs may appear through different functions. IGs inhibit various pathways associated with the progression of diabetes, including the regulation of glucose metabolism and enhancing insulin secretion [179].

Anti-Obesity Activity
Flavonoids could protect against obesity-related pathology by inhibiting adipogenesis and exerting anti-inflammatory activity [192]. Sea buckthorn leaf extract contains a high content of flavonoid glycosides, especially isorhamnine-3-glucoside (4) and quercetin-3glucoside [78]. Flavonoid glycosides extracted from sea buckthorn leaves (SLGs) could suppress diet-induced obesity in C57BL/6J mice [98]. In this study, the authors mentioned that 12 weeks of oral administration with a high-fat diet (HFD, 60 kcal% fat) + 0.04% (w/w) SLGs significantly prevented adiposity and dyslipidemia by suppressing lipogenesis and the absorption of dietary fat. This anti-obesity effect was explained by the improvement of inflammation and a decrease in gluconeogenesis. Narcissin (24) and 4 (30 µM) showed moderate inhibitory effects on triglyceride and glycerol-3-phosphate dehydrogenase activity in a 3T3-L1 preadipocyte [193]. Furthermore, it was demonstrated by Chang-Suk Kong et al. that 4 (20 µM) potently suppressed adipogenic differentiation by downregulating peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding proteins, sterol regulatory element-binding protein 1, and the adipocyte-specific proteins in 3T3-L1 preadipocytes. Furthermore, the specific mechanism mediating its action occurred through the activation of AMPK [194].
IG-rich plant extracts also have obvious anti-obesity effects. César Rodríguez-Rodríguez et al. have demonstrated that oral treatments of HFD, with a low (0.3%) or high (0.6%) dose of OFI-E rich in isorhamnetin glycosides, to C57BL/6 mice for 12 w ameliorated the development of HFD-induced obesity-related metabolic abnormalities by reducing weight gain, increasing insulin secretion, and enhancing energy expenditure in mice [70]. Further mechanistic studies verified that OFI-E and IGs could reduce fatty acid synthesis and increase fatty acid oxidation, leading to reduced fat accumulation in adipose tissue, thereby preventing adipocyte hypertrophy. OFI-cladode infusions (1%, administered daily in the drinking water) reduced proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the colon, adipose tissue, and spleen in Swiss male mice fed an HFD, as well as IL-6 and TNF-α in the plasma. These results suggested that OFI-cladode ameliorated HFD-induced obesity-related inflammation [195]. The results showed that intragastric administration of the extract from Hippophae rhamnoides seeds with concentrations of 100 and 300 mg/kg led to anti-obesity, triglyceride-lowering, and hypoglycemic effects in obese mice. It markedly inhibited macrophage infiltration into adipose tissue by regulating PPARγ and PPARα gene expression and inhibiting adipose tissue inflammation [196]. Oral sea buckthorn flavonoid administration (0.06% and 0.31% w/w, mixed in the diet) was able to alleviate body weight gain and insulin resistance in high-fat-and high-fructose-diet-induced C57BL/6J mice [197]. An extract of black soybean leaves (EBL), which mainly contains quercetin glycosides and isorhamnetin glycosides, inhibited HFD-induced obesity. Dietary supplements with 1% (wt/wt diet) EBL significantly reduced weight gain, improved glucose homeostasis, and decreased the glucose, insulin, HbA1c, and HOMA-IR index levels in HFD-fed mice. Mechanistic studies revealed that EBL inhibited hyperglycemia and hepatic steatosis through the adiponectin and AMPK signaling pathways, while isorhamnetin 3-O-α-L-rhamnopyranosyl (1→2)]-β-D-galactopyranosid (33) (50 µM) directly reduced lipid accumulation in HepG2 cells by enhancing AMPK activity [62].

Antithrombotic Activity
Thrombosis is a critical event in diseases correlated with atherosclerosis, myocardial infarction, and stroke [198]. The aggregation of platelets at the site of injury, as well as thrombin generation and fibrin formation triggered by the activation of tissue factors, are involved in thrombosis formation [199]. Therefore, the therapeutic mechanism includes the inhibition of platelet activation, adhesion, and aggregation, the improvement of fibrinolytic system function, and the regulation of coagulation system function [200].
Sae-Kwang Ku et al. assessed the antithrombotic activity of isorhamnetin 3-O-galactoside (8) from Oenanthe javanica. Studies have confirmed that it (10 µM) could significantly prolong the activated partial thromboplastin time and prothrombin time, inhibit the activity of thrombin and factor X, and inhibit the thrombin in human umbilical vein endothelial cells activated by TNF-α and the generation of factor X. In addition, isorhamnetin 3-O-galactoside (2.5 mg/kg) also elicited consistent anticoagulant effects in mice [201]. IGs isolated from sea buckthorn fruits showed marked anticoagulant and antiplatelet activity [202]. A thrombus-formation analysis system indicated that inhibited the TNF-α-induced production of plasminogen activator inhibitor type 1 (PAI-1) and reduced the ratio of PAI-1 to tissue-type plasminogen activator (tPA) [201].

Toxic Effects
Flavonoids are natural components of fruits, vegetables, tea, wine, traditional medicines (such as ginkgo biloba), and a considerable number of herbal dietary supplements. With growing interest in alternative medicine, the general population is consuming more flavonoids [203]. Since flavonoids are common edible ingredients in our daily diets, research on their potential cytotoxicity is warranted.

Bioaccessibility of IGs
The bioaccessibility of bioactive compounds refers to the maximum fraction of the compound released from the food matrix into the lumen of the gastrointestinal tract to be absorbed [208]. Most flavonoids exist in nature as glycosides, in which sugar residues modify the absorption mechanism and their ability to enter cells or interact with transporters and cellular lipoproteins [209,210]. Flavonoid glycosides exhibit better bioavailability both in vitro and in vivo, which is probably due to their higher aqueous solubility and stability during digestion [8]. At the same time, the gut microbiota plays an important role in improving the bioavailability and enhancing the absorption of flavonoids [211]. The deglycosylation of flavonoid glycosides by the gut microbiota enhances the bioavailability of flavonoids [212].
It was also reported that the antidiabetic, anti-inflammatory, and antiallergic activities of flavonoid glycosides were similar or even higher than those of aglycones when provided orally [216][217][218][219]. The effect of flavonoid glycosides is beneficial, probably due to the fact that flavonoid glycosides maintain higher plasma concentrations and have a longer mean residence time in the blood than aglycones [220]. Typhaneoside (45) and isorhamnetin-3-O-neohesperidoside (15) were detected immediately after the oral administrations of pollen typhae extract in rats, indicating that they were rapidly absorbed after oral administration [86,221]. IGs in sea buckthorn berries were monoglucuronidated in humans and were readily bioavailable [222]. Following the ingestion of lightly fried onions, flavonols were absorbed into the plasma of humans as glycosides, with a higher accumulation of isorhamnetin-4 -glucoside (9) in the plasma and urine than quercetin conjugates, which indicated that 9 may be preferentially absorbed [223]. Similarly, the results of a randomized crossover supplementation trial in female volunteers showed that 9 underwent significant elevation in the plasma after the ingestion of onion powder [224]. Antunes-Ricardo et al. reported that IGs found naturally in O. ficus-indica have a longer elimination half-life than isorhamnetin, suggesting that they can maintain constant plasma concentrations, and thus, prolong their biological effects [8].
Planar lipophilic polyphenols, such as curcumin, epigallocatechin gallate, quercetin, and genistein, are known as Pan-Assay Interference Compounds (PAINS) or Invalid Metabolic Panaceas (IMPS) because of their ability to interfere with membrane dipole potential [225]. Ana Marta de Matos et al. demonstrated that compounds produced via C-glycosylation are no longer able to alter the membrane dipole potential [226]. However, O-glycosylated compounds are easily hydrolyzed in the gut, so they are not suitable for this strategy. There are no more studies on the interference of isorhamnetin glycosides on membrane dipole potential, so further research in this field is warranted.

Marketed Products Related to IGs
In recent years, there has been increased interest in natural phytonutrients. Phytonutrients, such as beta-carotene (representative food, e.g., carrots), lutein (collard), isoflavones (soybeans), resveratrol (red wine), and anthocyanins (grapeseed), are known to provide a variety of significant benefits to humans and improve human well-being [227]. IGs as phytonutrients have been used in food and as a remedy against different health disorders, and processed into various products.

Food and Functional Food Products Using Opuntia ficus-indica
The cultivation for Opuntia ficus-indica is scattered across various parts of the world, such as Central and South America, Southern Spain, the Mediterranean Sea, Angola, Australia, India, and South Africa [228][229][230]. Opuntia ficus-indica has long been marketed in different forms, such as fresh, frozen, or pre-cooked, and used as fresh greens and in salads in Mexico, Latin America, South Africa, and the Mediterranean area [231]. As a popular dietary supplement in the United States, Opuntia ficus-indica products could be potentially utilized for body weight control and liver function support.
Opuntia ficus-indica can be processed into many food products ( Figure 6). Its cladodes have been used as a vegetable, usually eaten freshly peeled, in salads, cooked (boiled, fried, or deep-fried), or made into a juice or sauce [232,233]. Its fruit can be squeezed and used to produce juices, jams, candies, beverages, ice creams, and teas [234][235][236], and has also been added to rice field bean flour to produce an innovative gluten-free pasta [237]. Its peel has been utilized as a substitute for vitamin E, as an antioxidant in margarine preservation [238]. Its seed can be used to make oil [239]. Freeze-dried pulp can be added to rice or corn flour, resulting in a puffed flavanol-rich snack [240]. Its cladodes, pulp, or seeds, or whole plant, can be made into flour, which can partly substitute wheat or corn flour in doughs, bread, cookies, snacks, or desserts [18,241,242]. Opuntia ficus-indica-related products on the market have been listed in Table 3.
During the processing of Opuntia ficus-indica products, the processing technology used preserves the fruit's nutritional and sensory characteristics, and increases the content of IGs. It was reported that the extrusion or the preparation of concentrated juice pretreated with a pulsed electric field of Opuntia ficus-indica allowed for an increase in isorhamnetin glycoside content, especially isorhamnetin-3-O-rutinoside (24) [243,244].

Food and Functional Food Products of Hippophae rhamnoides
Hippophae rhamnoides possesses abundant bioactive compounds that can be utilized in the preparation of functional food products [19]. The berries, seeds, leaves, and even bark can be processed into supplemental products that gave the body all-natural assistance for many different functions. Hippophae rhamnoides leaves have gradually begun to be used in the food industry for tea processing [245]. A wide variety of products-jams, jellies, juices, powder, and seed oils-can be formulated from Hippophae rhamnoides berries [76]. Over the years, Hippophae rhamnoides products have increased in popularity (Table 4) [246]. Hippophae rhamnoides product consumption as part of the regular diet is common in Asia, the United States, and some European countries [247]. It was found that isorhamnetin derivatives were the most important flavonoids in Hippophae rhamnoides fruit juice [248]. The treatment of by-products in juice production via solvent-free microwave hydrogenation diffusion and gravity technology obtained more flavonoids, such as isorhamnetin, isorhamnetin 3-O-glucoside (4), isorhamnetin 3-O-rutinoside (24), than conventional solvent extraction [249].

Conclusions and Prospects
IGs are bioactive flavonoids found in various plants, such as Opuntia ficus-indica, Hippophae rhamnoides, and Ginkgo biloba. Routine and innovative assay methods, such as IR, TLC, NMR, UV, MS, HPLC, UPLC, and HSCCC, have been widely used for the characterization and quantification of IGs. Numerous lines of findings have elucidated the pharmacological activities of IGs. These studies have focused on multiple properties of IGs, such as their antioxidant, anti-inflammatory, or anticancer capacities. In recent years, IGs have attracted more attention due to their health-promoting effects on diabetes, obesity, liver injury, and thrombosis. Furthermore, the sugar residues of IGs make them more bioaccessible than aglycones. Meanwhile, IGs maintain higher plasma concentrations and longer average residence time in the blood than aglycons. This indicates that IGs are potent phytonutrients with potential health-promoting effects.
Growing evidence based on observational and clinical studies suggests that a plantbased diet based on fruits, vegetables, and whole grains has a significant effect on preventing various chronic diseases, including cancer, diabetes, and obesity [5]. IG traces have been identified in Hippophae rhamnoides, Opuntia ficus-indica, Vaccinium corymbosum, Vaccinium myrtillus, Brassica juncea, rice, onions, Ginkgo biloba, pollen Typhae, Microctis folium, Sambucus nigra, and Calendula officinalis, among their dietary and medicinal components [8][9][10]. People are more comfortable consuming phytochemicals and nutrients in their daily diets, such as fruit, vegetable juice, and tea [250]. They make vegetables and fruits into salads, blend them in juices, and process them into by-products. Hippophae rhamnoides could be served in pure juices, wine, and health supplements [251]. Meanwhile, Opuntia ficus-indica is used in many forms, including in food, feed, health, and nutrition, and is also used in formulated products, including teas, jams, and juices [252]. Additionally, IGs could be ingested from these plants. The extensive studies herein provide a sufficiently solid basis to discuss the health claims and health-promoting biological activities of IGs in humans. However, the clinical pharmacological effects of Igs still require further study so that their protective effects can be fully exploited in medical or pharmaceutical settings. The pharmacological mechanism of IGs also needs to be further elucidated to provide a material basis for their clinical investigation and application.

Conflicts of Interest:
There are no conflicts to declare.