Part1: Effects Of Isorhamnetin On Diabetes And Its Associated Complications: A Review Of In Vitro And In Vivo Studies And A Post Hoc Transcriptome Analysis Of Involved Molecular Pathway

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Abstract: Diabetes mellitus, especially type2 (T2DM), is a major public health problem globally. DM is characterized by high levels of glycemia and insulinemia due to impaired insulin secretion and insulin sensitivity of the cells, known as insulin resistance. T2DM causes multiple and severe complications such as nephropathy, neuropathy, and retinopathy causing cell oxidative damage in different internal tissues, particularly the pancreas, heart, adipose tissue, liver, and kidneys. Plant extracts and their bioactive phytochemicals are gaining interest as new therapeutic and preventive alternatives for T2DM and its associated complications. In this regard, isorhamnetin, a plant flavonoid, has long been studied for its potential anti-diabetic effects. This review describes its impact on reducing diabetes-related disorders by decreasing glucose levels, ameliorating the oxidative status, alleviating inflammation, and modulating lipid metabolism and adipocyte differentiation by regulating involved signaling pathways reported in the in vitro and in vivo studies. Additionally, we include a post hoc whole-genome transcriptome analysis of the biological activities of isorhamnetin using a stem cell-based tool.

Keywords: isorhamnetin; quercetin; biological activities; diabetes; molecular pathways; microarray

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1. Introduction

Plants have been used as traditional medicines in almost all continents of the world since ancient times. Scientific research into the reasons for their medicinal uses has led to the exploration of bioactive molecules. Extremophile plants that grow in extreme environmental conditions are considered a good potential source of bioactive molecules of interest. In fact, these environmental constraints are at the origin of dysfunctional oxygen metabolism, which leads to oxidative stress by increasing reactive oxygen species (ROS)[1,2]. Some plants, such as halophytes, have a powerful antioxidant system to eliminate these harmful compounds. Among the bioactive molecules of interest, there are phenolic compounds. Several studies have valued plants rich in polyphenols with or without restrictive conditions in the laboratory [3-10]. Several biological effects are attributed to extracts rich in polyphenols, such as anti-inflammatory and anticancer activities [11-13]. Other studies showed the antioxidant, the antimicrobial [8,14-16] as well as anti-obesity, anti-diabetic and anti-hepatic steatotic [17-19] effects of isorhamnetin. Among these polyphenols, flavonoids are distinguished. This group includes several subgroups, such as flavonols. Isorhamnetin is one of the major compounds of flavonols. Isorhamnetin is a monomethoxy flavone or an O-methylated flavonol from the class of flavonoids. It is quercetin in which a methoxy group replaces the hydroxy group at position 3'. Some isorhamnetin derivatives are present in nature, such as isorhamnetin 3-O-β-d-glucopyranoside, isorhamnetin 3-O-neohesperidoside, and isorhamnetin 3-O-rutinoside from Calendula officinalis L. [20]. Isorhamnetin presents significant biological properties such as antioxidant [21], anticancer [22], antimicrobial [23], antiviral [24], anti-inflammatory and anti-diabetic effects [21,25-33].

In this review, the first focus is on the origin, chemical structure, isolation, and extraction methods, as well as the phytochemical aspect of isorhamnetin. Then, in the second part, we focus on describing the potential anti-diabetic effects of this flavonol through reducing diabetes-related disorders by decreasing glucose levels, ameliorating the oxidative status, alleviating inflammation, and modulating lipid metabolism and adipocyte differentiation. Finally, we performed a secondary analysis of our previously published whole-genome microarray data to explore diabetes-related bioactivities of isorhamnetin in a stem cell-based tool. We also aim to highlight the effect of this molecule on the regulation of the involved signaling pathways by reporting the in vitro and in vivo studies used in this field of research. In this review, we exposed the characteristics of the anti-diabetic activity of isorhamnetin in comparison with quercetin which is considered to be a metabolite of isorhamnetin and an important reference in the natural treatment of diabetes.

2. General Overview of Bioactive Molecules in Particular Polyphenols and Flavonoids 

2.1. Oxidative Stress as an Origin of Bioactive Molecules in Plants

Several plants can be subjected to various environmental conditions (salinity, drought, UV rays, heavy metals, extreme temperatures, nutrient deficiency, air pollution, and pathogen attacks). These constraints are at the origin of dysfunctions of oxygen metabolism, which generate oxidative stress by increasing reactive oxygen species(ROS). The oxygen molecule (O2)plays an important role in photosynthetic organisms. Originally, in higher plants and algae, gas exchange involving electrons occurs in chloroplasts by capturing carbon dioxide during the day and producing oxygen. This gas exchange involves electrons. Indeed, in the case of severe environmental constraints, a large part of oxygen is not reduced and can thus generate ROS in some organelles from plant cells [1,2] due to an imbalance in the proper functioning of chloroplasts and the transfer of electrons [1]. Moreover, under the environmental constraints mentioned above, many ROSs are produced, such as hydroxyl radical (OH), the superoxide anion radical(O,-), alkoxyl and peroxyl radicals(RO: and RO2, respectively), hydrogen peroxide(H2O2,), hypochlorite radical (-OCl), singlet oxygen (O2), nitric oxide radical (NO) and other lipid peroxides (such as malondialdehyde and 4-hydroxynonenal)[2,34,35]. Sometimes, ROS may have a role in cell signaling in the physiological behavior of plants, for example in the process of seed growth and development, development of tissues, and the transition from cellular proliferation to cell elongation during the earlier stages of differentiation [36]. At a high level, these molecules cause molecular damage such as lipid peroxidation of membranes, alteration of proteins and DNA, and cell death [1,35,37]. Some plants, such as halophytes, have the capacity to adapt well to these conditions through a powerful antioxidant system. Halophytes are known to be a source of secondary metabolites such as polyphenols [3-10]. These authors show that under severe conditions, polyphenols are synthesized to play an important role in protecting against stress-induced oxidative damage. The biosynthesis, the content, and the activities of these phenolic compounds are a function of several extrinsic(light, temperature, salinity, and dryness) and intrinsic(genotype, organ, and stage of development) parameters influencing their content and distribution in plants [8,38,39]. For example, in Pyracantha coccinea, some flavonoids such as flavanones, flavones, and flavonols are present in the shoots during the vegetative phase and in the roots exclusively during the reproductive one 40]. On the other hand, several authors have shown that these phenolic compounds have other biological properties such as anti-inflammatory and anticancer activities [11-13]. Other studies showed the antioxidant, antimicrobial [8,14-16] as well as anti-diabetic [18,41,42] effects of phenolic extracts.

2.2. Classification of Natural Antioxidants

The antioxidant system can be classified according to the nature of these components into enzymatic or non-enzymatic compounds. The first include superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase, and glutathione reductase [1,3542-44]. The genes relating to these enzymes revealed their importance during the postharvest physiological deterioration of storage root and in response to osmotic stress and abscisic acid as well as Xanthomonas axonopodis infection [44]. The second group contains mainly phenolic compounds, carotenoids, vitamins, and osmolytes [1]. Phenolic compounds are characterized by the presence of one or more benzene rings and differ in the complexity of the base molecule, the number and location of the hydroxyl, and the degree of polymerization. These compounds are secondary metabolites that fall into three broad groups: phenolic acids (derivatives of benzoic and cinnamic acids), flavonoids (flavonols, flavonols, flavanones, flavones, anthocyanins), and tannins (hydrolyzable tannins and proanthocyanidins). In addition to these molecules, stilbenes, lignans, and coumarins are also distinguished [45].

2.3. Origins and Biochemical Structure of Flavonoids, in Particular, Isorhamnetin

Phenolic compounds are secondary metabolites prevalent in plants. These compounds are with an aromatic ring with one or more hydroxyl groups(OH) and contain molecules ranging from simple phenolic acids to polymerized compounds such as tannins. The synthesis of phenolic compounds is a complex process that goes through several stages. Phenolic compounds are bioactive molecules with two origin pathways: on one side the shikimic acid and on the other side, the phenylpropanoid molecules. The biosynthesis of flavonoids such as isorhamnetin is based on these pathways. In fact, on the one hand, shikimate gives the basic skeleton of polyphenols which have one or more benzene rings (C6) carrying one or more hydroxyl functions. On the other hand, there is the synthesis of the C6-C3 base formed by the condensation of phenylalanine to cinnamic acid (Figure 1)[46].

More precisely, shikimic acid is at the base of several reactions, representing the skeleton of aromatic amino acids, which are the initiators of phenolic compounds. The first step consists of a combination of two molecules: phosphoenolpyruvate and erythrose 4-phosphate, which after four reactions, lead to forming the first skeleton of phenolics: the shikimate or shikimic acid (CHoOs). This later presents the first ring, which characterizes phenolics with two hydroxyl groups. The shikimate undergoes six reactions which end in the first amino acid: phenylalanine. Thanks to two key enzymes, phenylalanine ammonia-lyase (PAL) and cinnamate-4-hydroxylase(C4H), phenylalanine successively forms cinnamate and p-coumarate..In this step, the level of PAL activity could quantitatively regulate the accumulation of phenolic compounds. The p-coumarate molecule is at the origin of coumarin derivatives. The pathway for the synthesis of phenylpropanoid molecules is characterized by the presence of a key enzyme called 4-coumarate CoA ligase (4CL), which catalyzes in the presence of the thiol function of the coenzyme A(CoA) the p-coumaric acid into 4-coumaroyl CoA(C30Ha2N-O18P, S). The aromatic A-ring of flavonoids provides from the condensation of three molecules of malonyl-CoA (-C6).

Subsequently, the 4-coumaroyl CoA produced the naringenin chalcone, explaining the link between the aromatic B-ring and the 3C one of chalcone(C6-C3-). Chalcone is a key element in this topic because it is a precursor of all flavonoids based upon a fifteen-carbon skeleton consisting of two benzene rings. Afterward, chalcone is converted to naringenin (also called flavanone or trihydroxy flavone) by the action of chalcone isomerase (CHI).On the one hand, flavones such as apigenin, acacetin, chrysin, or luteolin are synthesized from naringenin in the presence of flavone synthase(FNS).On the other hand, different flavonols and succinic acid compounds are produced from naringenin in the presence of two enzymes; flavanone 3-dioxygenase (F3D) and flavonol synthase (FS). Apart from flavonol derivatives, other compounds, such as kaempferol, myricetin, and quercetin, are also produced. By a methyl group transfer from the S-adenosyl-L-methionine, the isorhamnetin is produced in the presence of the flavone 3'-O-methyltransferase (FMT)[47,48]. Then, isorhamnetin is a monomethoxy flavone or an O-methylated flavonol from the class of flavonoids. It is quercetin (precursor) in which the hydroxy group at position 3' is replaced by a methoxy group. Some isorhamnetin derivatives are present in nature, such as isorhamnetin 3-O-β-d-glucopyranoside, isorhamnetin 3-O-neohesperidoside, and isorhamnetin 3-O-rutinoside from Calendula officinalis L.[20].

In fact, flavonoids are considered one of the most important groups of the polyphenol family. They have a structure based on a diphenyl propane type with two benzene rings (ring A and B, see Figure 2) linked by a three-carbon chain that forms a closed pyran ring (C ring). Therefore, their structure is referred to as C6-C3-C6. O-glycosylation positions are C7 in flavones, isoflavones, flavanones, and flavonols, and C3 in flavonols and anthocyanidins. C-glycosylation positions are C6 and C8 in flavones [49]. In addition, the importance of the antioxidant role of phenolic compounds is related to the degree of hydroxylation of the molecule. Flavonoids include the isoflavones, flavones, flavanones, and their glycosides and flavonols as isorhamnetin, which is also named 3'-methoxy quercetin and 3-methyl quercetin [50]. In plants, an enzyme, the UDP-dependent glycosyltransferases, is responsible for the glycoside form of isorhamnetin (isorhamnetin 3-O-glucoside). This enzyme utilizes nucleotide diphosphate sugars, usually uridine diphosphate (UDP)-sugars, to transfer the methyl group to the cycle and associate the glycoside function with isorhamnetin [51].

2.4.Isolation and Analyses of Ilsorhamnetin Originated from Medicinal Plants

The distribution of isorhamnetin in medicinal plants is very wide and the methods of extraction and analysis are varied. Isorhamnetin derivatives are particularly wanted. The hydroxyl and methyl groups help in their characterization. Some methods are used to extract isorhamnetin, among them based on fractionation, using chemometric approaches, enzyme, and supercritical fluid extraction (SFE-CO2). Firstly, fractionation can be used to simplify the extraction by removing all oil and lipophilic pigments from lipid-containing samples. The defatted sample is also sonicated before maceration in a mixture of methanol and water. Chromatography can then be used for analyses of phenolic compounds, especially flavonoids [49]. For these last compounds, LC-MS is used and both electrospray ionization (ESI) and atmospheric-pressure chemical ionization are frequently applied, and the best sensitivity for flavonoids is generally achieved in the negative-ion mode [49]. For example, the phytochemistry of Calligonum Azel Maire plant fractions, collected from the Tunisian desert, have been assessed and an ultra-high performance liquid chromatography coupled to quadruple time of flight mass spectrometry UHPLC-ESI-QTOF was used to identify phenolics among them flavones and flavanols, which were the most abundant phenolic compounds identified [52]. More specifically, the presence of isorhamnetin glucoside and isorhamnetin glucosyl-rhamnoside was confirmed as the major compounds in the leaves of the edible halophyte Mesembryanthemum edule using LC/ESI-MS/MS technology [3]. This work was followed by others who have used LC-ESI-TOF-MS to characterize many polyphenols; among them, flavonoids were identified from aerial parts of the full flowering stage of halophytes such as Arthrocnemum indicum [5], Tamarix gallica [16], Glaucium flavum[13], and Salsola kali[8]. Similar to the last works, LC-ESI-TOF-MS and GC-MS profiling of Artemisia herbal were performed to identify phenolics, such as flavones, flavonols, and flavonoid alkaloids[7]. Another work was carried out on Pancratium maritimum and the analysis by HPLC-DAD-ESI/MS revealed the presence of flavonoids including flavonol as isorhamnetin with their pentoxide and hexoside conjugates such as isorhamnetin di-hexoside [53].

As a non-fatty compound, flavonols could be isolated by hexane. Then, a polar solvent such as ethanol can be used. A study on Limoniastrum guyonianum using HPLC showed the presence of many phenolics, among them isorhamnetin-3-O-rutinoside [54].

Far from phenolic extracts, phenolic compounds are also found in oils such as olive oil [55]. These compounds have the ability to protect oils against oxidation and improve the nutritional value of the oil. Isorhamnetin, as a phenolic compound, has also been detected in an oil extract from Tunisian black cumin (Nigella sativa L.)seeds obtained with a green solvent such as 2-methyl tetrahydrofuran (MeTHF) as an alternative to petroleum or hexane-based solvent to extract oil-enriched phenolic compounds [10]. The presence of isorhamnetin was approved by HPLC analysis. Analyses using Liquid Chromatography with Diode Array Detector(LC-DAD)revealed a high amount of isorhamnetin ranging between 6.3 and 6.6（μg/g oil） after oil extraction from black cumin by MeTHF and hexane， respectively. In addition, using H nuclear magnetic resonance(HNMR) and C nuclear magnetic resonance(CNMR), some flavonol glycosides such as kaempferol-3-O-rutinoside (nicotiflorin) and isorhamnetin-3-O-rutinoside (narcissism) were characterized from the aerial parts of Peucedanum aucheri Boiss collected in Marivan city, Kurdistan province, Iran [56]. The NMR method detects the property that certain atomic nuclei of the compound interact with a magnetic field. This property, which is to produce magnetic resonance at a certain frequency, provides information on the structure of the molecule. Secondly, mathematical models are chosen to evaluate the efficiency of flavonol extraction, especially to understand the best way to obtain isorhamnetin-3-O-rutinoside [20]. A multivariate factorial analysis was conducted using flowers of Calendula officinalis. Linear, quadratic, full cubic, and special cubic models were analyzed. The final full cubic was the most appropriate one, allowing greater efficiency in the extraction of isorhamnetin-3-O-rutinoside by 60%. For this study, the enzymes, Rapides Maxi Fruit and Viscozyme were used under some important factors affecting the enzyme activity under supercritical CO2 conditions such as pressure, temperature, pH, time, and aqueous ethanol solution. Finally, the supercritical fluid extraction method was used to extract phenolic compounds such as flavonols. Many years ago, the supercritical fluid extraction procedure was used on the Eucalyptus globulus bark for the first time, using pure and modified CO2 with water, ethyl acetate, and ethanol [57]. Authors showed that the supercritical CO2 combined with ethanol could extract significant amounts of phenolic compounds, including isorhamnetin. HPLC-MS quantification determined some flavonols, such as isorhamnetin-hexoside (0.26 g.g-I of ex-tract) and simple isorhamnetin (14.29 mg:g-I of extract). Ethanol was used as a co-solvent. The quantification was carried out with high-pressure liquid chromatography equipped with a photodiode array detector. Isorhamnetin 3-O-glucosyl-rhamnosyl-rhamnoside, isorhamnetin 3-O-glucosyl-rhamnosyl-pentoxide, isorhamnetin 3-O-glucosyl-rhamnoside, and isorhamnetin 3-O-glucosyl-pentoxide were the most abundant flavonols extracted from O. ficus-indica supercritical extracts. As mentioned before, high-pressure liquid chromatography or HPLC is a commonly used method for analyzing phenolic compounds. In apple juice and pear juice, isorhamnetin 3-O-glucoside was detected. In fact, the separation of the flavonol glycosides in a"Brettacher" apple extract by HPLC and mass spectrometry revealed the presence of two glycoside forms such as isorhamnetin 3-O-glucose and isorhamnetin 3-O-galactoside [58].