hussain2020 compressed

(1)

Contents lists available atScienceDirect

Pharmacological Research

journal homepage:www.elsevier.com/locate/yphrs

Review

Flavonoids and type 2 diabetes: Evidence of e ﬃ cacy in clinical and animal studies and delivery strategies to enhance their therapeutic e ﬃ cacy

Tarique Hussain

^a,b,c

, Bie Tan

^a,b,

*, Ghulam Murtaza

^d

, Gang Liu

^a,b

, Najma Rahu

^e

, Muhammad Saleem Kalhoro

^f

, Dildar Hussain Kalhoro

^e

, Tolulope O Adebowale

^b

,

Muhammad Usman Mazhar

^c

, Zia ur Rehman

^g

, Yordan Martínez

^h

, Shahzad Akber Khan

ⁱ

, Yulong Yin

^a,b

aCollege of Animal Science and Technology, Hunan Agricultural University, Changsha, 410128, Hunan, China

bLaboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, Hunan, China

cAnimal Sciences Division, Nuclear Institute for Agriculture and Biology (NIAB), P. O. Box: 128, Jhang Road, Faisalabad, 38000, Pakistan

dShaheed Benazir Bhutto University of Veterinary & Animal Sciences, Sakrand, 67210, Sindh, Pakistan

eDepartment of Veterinary Microbiology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, Sindh, 70050, Pakistan

fFood Engineering and Bioprocess Technology, Asian Institute of Technology, Bangkok, 12120, Thailand

gKey Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal 19 Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

hPan-American School of Agricultural Sciences and Production Department, P. O. Box: 93, Francisco Morazan, Honduras

iFaculty of Animal Husbandry and Veterinary Sciences, University of Poonch, Rawalakot, 12350, Pakistan

A R T I C L E I N F O

Keywords:

T2DM Oxidative stress

Inﬂammation andﬂavonoids

A B S T R A C T

Diabetes mellitus type 2 (T2DM) is a metabolic disorder develops due to the overproduction of free radicals where oxidative stress could contribute it. Possible factors are defective insulin signals, glucose oxidation, and degradation of glycated proteins as well as alteration in glutathione metabolism which induced hyperglycemia.

Previous studies revealed a link between T2DM with oxidative stress, inflammation and insulin resistance which are assumed to be regulated by numerous cellular networks such as NF-κB, PI3K/Akt, MAPK, GSK3 and PPARγ. Flavonoids are ubiquitously present in the nature and classified according to their chemical structures for example,flavonols,flavones,flavan-3-ols, anthocyanidins,flavanones, and isoflavones. Flavonoids indicate poor bioavailability which could be improved by employing various nano-delivery systems against the occurrences of T2DM. These bioactive compounds exert versatile anti-diabetic activities via modulating targeted cellular signaling networks, thereby, improving glucose metabolism,α-glycosidase, and glucose transport or aldose re- ductase by carbohydrate metabolic pathway in pancreaticβ-cells, hepatocytes, adipocytes and skeletal myo- fibres. Moreover, anti-diabetic properties offlavonoids also encounter diabetic related complications. This review article has designed to shed light on the anti-diabetic potential offlavonoids, contribution of oxidative stress, evidence of efficacy in clinical, cellular and animal studies and nano-delivery approaches to enhance their therapeutic efficacy. This article might give some new insights for therapeutic intervention against T2DM in near future.

https://doi.org/10.1016/j.phrs.2020.104629

Received 10 October 2019; Received in revised form 23 December 2019; Accepted 2 January 2020

Abbreviation:AGEs, advanced glycation end products; AGTL, adipose triglycerides lipase; ARE, antioxidant response element; EGCG, epigallocatechin gallate; ERK, extracellular signal regulating kinase; ETC, electron transport chain; FoxO1, forkhead box proteinO1; GSIS, glucose-stimulated insulin secretion; GSK3, glycogen synthase kinase-3; HDAC7, histone deacetylase 7; IGF-1, insulin-like growth factor; KEAP1, kelch like-ECH-associated protein 1; MGL, monoacylglycerol lipase; NF- κB, nuclear factor kappa B cells; NSCLC, non-small cell lung carcinoma; PDX1, pancreas-duodenum homebox-1; PKC activation, protein kinase C; ROS, reactive oxygen species; RNS, reactive nitrogen reactive species; TLR4, toll like receptors 4; TNF-5, tumor necrosis factor-5; UCP2, mitochondrial uncoupling protein-2

⁎Corresponding author at: College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, Hunan, China.

E-mail address:[email protected](B. Tan).

Pharmacological Research 152 (2020) 104629

Available online 07 January 2020

T

(2)

1. Overview of diabete

Diabetes is a common chronic disorder arises when body fails to control glucose homeostasis within the cells or impaired glucose uptake to various tissues and instability in maintaining glucose levels by liver [1]. T2DM is a widely prevalent form, classified by metabolic disorders with hyperglycemia and considered as a main health concerned issue comprising higher morbidity and mortality [2]. The global evidences of diabetes occurrences ranging from 1%–8% and expected to be 333 million in 2025, and 430 million in 2030 [3]. Literature revealed that > 90% T2D cases are reported as type 2 diabetes mellitus (T2DM) [4]. However, it is difficult to differentiate both types of diabetes mellitus on population levels, but genetic composition may help in some extent to illustrate individual response against environmental fluctuations [5]. Possible factors of fast growth rate of diabetes are aging, overpopulation, increase growth rate, progressive urbanization, obesity, high caloric diet and fast food [6]. The incidences of occur- rence are higher in Asian countries such as India, Japan and China due to socio-economic growth and industrialization [7]. Epidemic scenario in China and India develops due to the onset of loss of body weight at young age, than western countries [8]. Survey based study in China documented that in 2010, more than 113.9 million adults were affected by diabetes mellitus and 493.4 million adults had predicted pre-diabetes mellitus condition [9]. Another report in India highlighted that 62 million individuals showed diabetes mellitus and 77 million indicated pre-diabetes mellitus in 2011 [10]. Apart from that, Thailand and Malaysia, spent 11%–75% per capita income in 2007 without T2 diabetes mellitus complications, while those who exerted complications spending up > 3 times than those without complications [11]. A growing literature exhibited that USA was ranked third having highest number of diabetes mellitus patients in 2015 [12]. It is further indicate that in North America and Caribbean region spending 85 fold on diabetes mellitus and that in Southeast Asia spent more than the rest re- gions of the world [12]. Moreover, mounting evidences shown that T2DM cases could be avoided through consuming healthy diet, healthy body weight, daily exercise, skipping smoking and consuming moderate alcohols [13]. As the disease attempting to grow worldwide rapidly, the vascular complications have now become a serious health disorder.

Diabetic complication arises when patient’s leaves untreated for example, neuropathy, nephropathy, vision disorder, erectile dysfunction, heart disease, stroke and peripheral vascular disease, which later converted into ulcers, gangrene and amputation [14].

Several classical anti-diabetic drugs are commercially available in the market but their side effects like stress, allergic reaction and in- fection subsequently takes long time resulting in lower efficacy of the drug to the target tissues [15]. Being the available of wide spectrum drugs, none of the therapy possess favorable effects and their responses varies among patients might be due to the individual immune status, secondary complications and micro and macro-vascular damages [16].

Considering these drug obstacles, World Health Organization (WHO) suggests 1200 herbal plant species around the globe, which exerts strong anti-diabetic properties and it could be e used as an alternative therapeutic intervention against prevention of T2DM [17]. Natural plants particularlyflavonoids are abundantly exist in nature, exhibiting strong anti-diabetic activities in-vitro and in-vivo. Therefore, proper approaches and advanced tools must be employed to develop drug exerts less toxic effects [18]. This review paper extends the role of flavonoids in oxidative stress triggered T2DM, molecular mechanism involved, evidence of efficacy in clinical, cellular and animal studies and nano-delivery approaches to enhance their therapeutic efficacy.

Thesefindings might have clues regardingflavonoid based anti-diabetic drug, which could have more favorable effects than existing drugs.

2. Diabetes and pathological mechanism of T2DM

Type 1 diabetes mellitus (T1DM) occurs approximately in 10% of

the diabetes cases around the globe, but it seems to be appeared in earlier life. It prevails in the response of self-immune destruction ofβ cells of the endocrine pancreas. Whereas; few patients having this metabolic disorder represent < 10% and is characterized as type1 diabetes, not showing any evidence of autoimmune disease and pathogenesis and known as idiopathic [19]. Increased chances of occurrences have been reported in relative’s having higher degree of genetic risk and genetic identity correlated with the risk [20]. Gene variants in one main locus, human leuckocyte antigen (HLA), indicating 50%–60%

genetic susceptibility through interacting with HLA protein binding to antigenic proteins and then antigen produced by T cells [21]. More than 50 more genes individually attribute smaller effects [22]. These gene variants modify immune function and tolerance [23] variants which modulate viral responses, and impairs environmental signals and endocrine functions [24] and shows the expressions in pancreaticβ-cells [25]. Genetic impact induced autoimmunity and progression of diseases have been reported in relatives [26]. Moreover, these gene variants indicate 80% type 1 diabetes heritability. Epigenetic, expression of genes and RNA profiles mayfluctuate according to the time and impact on disease progression thus, showing a dynamic risk [27]. However, other factors like environmental signaling, dysfunction of pancreaticβ -cells, diet and gut microbiota and autoimmunity etc are well ascribed by [28].

Gestational diabetes mellitus (GDM) a pregnancy complication where hyperglycemic condition occurs during pregnancy [29]. A recent article published by International Diabetes Federation [30] explores that GDM affects about 14% pregnancies around the world and af- fecting 18 million births annually [31]. Prominent factors of inducing GDM include obesity due to overweight, western food, and lack of micronutrients, maternal overage, family anamnesis of insulin resistance or diabetes. It can be resolved followed by delivery despite of having health complications consisting of T2DM, cardiovascular diseases (CVD) in mother as well as in child at later stages. Published literature revealed that GDM shown their existences in three forms of diabetes and vast majority (80%) cases of GDM appears in context toβ- cell dysfunction due to the insulin resistance [32]. Hence, affected women having increased level of insulin resistance in accordance with normal pregnant women and further declining glucose utilization and promote production of glucose and free fatty acids (FFA) concentrations [33]. It is believed that detrimental effects on β-cells cause overproduction of insulin due to overuse of energy intake and insulin resistance, leading to cellular exhaustion. Due to the fact that pathology is closely resembles with T2DM, needs more discussion between the etiologically indistinct diseases [34]. Moreover, the detailed informa- tion regarding GDM is well illustrated by [35]. Type-1 Diabetes and Gestational diabetes do not come under the scope of this review; we have given little touch to these two types.

Diabetes mellitus is characterized in type 1 and type 2 diabetes, where T2DM accounts approximately in 95% individuals [36]. T2DM is caused by insulin resistance (IR) and deﬁcient level of insulin secretion [37] and may turn into organs injury and other several obstacles [38].

Acute complications of the disease are linked with higher mortality and the chronic complications of the diabetes associated with demoralizing consequences perused via longer duration of increased blood glucose level. Microvascular damages results in diabetic retinopathy, diabetic nephropathy and diabetic neuropathy [39] while, the macrovascular consequences such as cardiovascular and cerebrovascular diseases have also been observed [40]. It is furthermore, evident that T2DM leads to cause morbidity, depression [41] sexual dysfunction [42] and dementia [43]. Chronic feature of T2DM has made it expensive ailment for patients, their families and health system [44]. Insulin resistance andβ cell dysfunction results could be due to the deﬁcit insulin amount that is known to be the cause of occurrences of T2DM. Sensitivity of insulin can be inﬂuenced by high rise of glucose and lipids, oxidative stress, adipokines, autophagy and improper insulin synthesis [45]. Obesity is also considered as a major predisposing factor, display key function in

(3)

T2DM condition [46] and it leads to oxidative stress along with IR, production of cytokines, dysfunction in lipid regulation and inﬂuencing protein kinase phosphatase signaling [47]. Oxidative scenario in T2DM, develops due to overproduction of free radicals that may results in DNA oxidation, injury in organs which interfere cellular function [48].

Overconsumption of nutrient intake produces more calories which stored in adipose tissues. However, excessive storage of calories in fat cells for longer period, leading to cause hypoxia in adipose tissues and it results stimulation of hypoxia inducible factor-1 (HIF-1) [49] and it enhances the expressions of c-Jun N-terminal kinase (JNK) and in- hibitor nuclear factor kappa-B kinase (IKK) to establish inflammation in adipose tissues [50]. Due to the persistent inflammatory response, cytokines are produced which further enhanced insulin resistance (IR) [51] and lipolysis [52]. Moreover, inflammatory cytokines may decline peroxisome proliferator-activated receptor γ (PPARγ), activity and promote adipose cellular death and inflammation [53]. Due to the development of IR, the action of insulin acts as lipolytic. The hyperinsulinemia may stimulate lipoprotein lipase by the lipoprotein triglycerides hydrolysis. Whereas; endoplasmic reticulum (ER) stress is attributed and JNK pathway is activated (Fig.1) [54]. As a result, IR and enhancedflux of FFA in adipose tissues may form a vicious cycle. In- creased amounts of FFA produced from fat cells pour into circulation, transfer and gathered in other organs in order to persuade lipotoxicity and accelerate the systematic IR [55].

Liver is the key organ which display pivotal role in balancing stable blood glucose level, via adjusting the equilibrium among glycogenesis and glycolysis of stored glycogen [56]. Fatty deposition in the liver, responsible for establishing IR [57]. Kupffer cells (macrophages in liver), stimulates and establish inflammation via cytokines through activation of nuclear factor κB (NF-κB) [58]. In contrast, enhanced Level of FFA inducesthe accumulation of diacyglycerol (DAG) and ceramide. DAG suppress insulin activity by stimulation of protein kinase C isoforms (PKC), and influencing signal transduction through serine phosphorylation of insulin receptor (IRS) [59]. In addition, ceramide a strong stimulator of inflammation may activate JNK and NF- κB/IKK signals that are closely affiliated with IR [60]. IR condition also establishes in skeletal muscle, a profound indicator of glucose uptake [61,62]. The increased intensity of FFA levels of lipotoxicity may

trigger theβcells dysfunction. Moreover,βcells are more susceptible to damages mediated by inflammatory cytokines [63]. Due to fact, thatβ cells may not be able to make insulin level stable in normal way, which ultimately cause hyperinsulinemia and hyperglycemia, that increases the development of T2DM. Apart from above mechanism, numerous factors are also involved in developing T2DM, comprising autophagy [64] endoplasmic reticulum stress [65] protein folding [66] post- translational modifications [67], DNA methylation and histone modifications and microRNAs [68]. Despite, this contributory function is not independent and have interaction with each other and ultimately cause T2DM.

3. Relationship between oxidative stress and diabetes

Reactive oxygen and reactive nitrogen metabolites consists of hydroxyl radical, superoxide anion, peroxyl radicals, lipid peroxyl, nitric oxide, peroxynitrite and nitrogen dioxide which are unstable and highly reactive species due to the presence of unpaired electron [69]. While, reactive nitrogen species (RNS) originated from nitric oxide pathway [70]. Other sources of free radical productions are mitochondrial d- amino acid oxidase [71] dihydrolipoamide dehydrogenase [72]α-keto acid dehydrogenase complex [73] and xanthine oxidase [74]. The non- radical species of ROS are hydrogen peroxide, singlet oxygen, lipid peroxide (LOOH) and others. Among this, the nature of hydrogen peroxide is highly reactive and may cause to profound cellular damage along with transition metals yielding the production of hydroxyl radical through the Fenton reaction [69]. The ROS and RNS are produced in normal metabolism, their physiological activity become evident once, they produced at lower concentration to maintain redox homeostasis [75]. As aforementioned that ROS are unstable and highly reactive which can be detrimental to biomolecules including lipids, proteins and DNA. Body is well equipped with antioxidant system which not only controls ROS in limit but also exhibit encountering eﬀects through displaying various mechanisms. Such antioxidant system consists of superoxide dismutase, catalase, peroxiredoxins, thioredoxins, and glutathione peroxidases [76]. Mitochondria are the key source of energetic metabolism by which ATP is produced via oxidative phosphorylation.

The oxidation of nutrients generates two molecules such as NADH and Fig. 1.Ameliorative effects of flavonoids in oxidative stress and T2DM Oxidative stress induces insulin resistance and metabolic disorders. Pancreatic β cells produce insulin which exert various functions such as reduction in glucose production and output in liver, enabling glucose transport in skeletal muscles and reduced lipolytic effect in adipose tissues.

Overproduction of inflammatory cytokines for example, interleukin-1 (IL-1) interleukin-6 (IL- 6), C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α) interrupt β cell function, inducing inflammatory response via crosstalkwith monocytes andmacrophages resulting in insulinresistance and ultimately decrease insulin dependent signaling. Hence, the production of insulin contributes to target tissues for example higher concentration of glucose, fatty acids and in skeletal muscles, liver and adipose tissues which triggers numerous metabolic disorders. Flavonoids such as genistein, kaempferol, pectolinarin and quercetin target through interaction with pro-inflammatory cytokines mediated β cells dysfunction, apoptosis, and reduction in pro-inflammatory cytokines, enhancing insulin dependent signaling and promoting glucose uptake in various cell types [217].

(4)

FADH2 which further oxidized in electron transport chain and thereby producing ATP, ROS and oxygen. Due to this, mitochondria are the vital source of intracellular ROS, but also exert antioxidant enzymes which balance the cellular redox status. The prevalence of SOD in mitochondria, manganese superoxide dismutase (MnSOD), is responsible for deactivation of superoxide radical and thus keeps ROS in limit to maintain homeostasis [76]. Glucose is the pivotal source of nutrient for energy which is generated in ETC from NADH and FADH2. This is not unforeseen that ROS are actively participated in pathophysiology of diabetes. Indeed, imbalance of redox homeostasis in T2DM patients [77] and other studies have reported a prominent evidences of oxidative stress in patients with diabetes mellitus [78]. Moreover, this condition leads to dysfunction in mitochondrial ETC associated with diabetes related mitochondria diseases [79]. Additionally, mitochondrial genome translates proteins which regulate ETC function for production of ATP and it is well highlighted that occurrences of mitochondrial mutation make patients susceptible to diabetes [80]. Current studies indicate that individual having mitochondrial disease have recorded more chances of endocrine disorders particularly diabetes mellitus [79]. Therefore, it exerts strong association between mitochondrial diseases with higher evidences of oxidative stress has been observed [81]. This data revealing a relationship between T2D and oxidative stress contributing mitochondria, hence, it is clearly understood that mitochondria depict dynamic role in etiology of diabetes and overriding of ROS production play key role in disease development and its complications [82].

Expanding the further role of mitochondria, it serves for generating metabolic signals which allow exocytosis through the activation ofβ cells. Perturbation in the function of oxidative phosphorylation may cause impairment inβ-cell activity closely related with T2DM. Thus, it shows that T2DM patients exhibit defective mitochondrial features [83]. Moreover, PPAR-γis a protein tightly regulates metabolic function in various cell lines such asβcells and PGC-1α, and decreases insulin secretion in T2DM patients [84]. Positive eﬀects of enhanced Forkhead box protein O1 (FoxO1) activity inβ- cells have been reported by suppression of mitochondrial lipid metabolism [85]. The histone deacetylase 7 (HDAC7), is an enzyme mediates mitochondrial gene expression, contributes in T2DM and create hindrances in energy production through modulating oxidative phosphorylation [86]. Pre- valence of excessive glucose in mitochondrial energy production causes diabetes which promotes ROS production and provokes to secondary micro-vascular complications [87]. Overwhelming activity of ROS inβ cells triggers uncoupling protein-2 (UCP-2), which is responsible for protein leakage through uncoupling oxidative phosphorylation during ATP production [88]. Approximately, more than 98% of oxygen consumption takes place in mitochondria [89]. Out of that 0.2%–2.0%

converted into generation of ROS production [90]. Disruption in mitochondrial function induces oxidative stress in skeletal muscles liver, fat, and pancreas as shown inFig.1 [90,91]. It is further noted that, adenosine triphosphate (ATP) is a key fuel for cell survival and its production can be inﬂuenced by insulin signals in various cell lines.

Thus, it is truly conﬁrmed that oxidative stress regulates diabetes.

However, excessive superoxide radicals production through the enhanced complex-1 activity in electron transport chain of mitochondria, could be a causative factor for inducing diabetes [92]. Therefore, it is considered as the promising target for reducing NADH concentration and amelioration of oxidative stress [93]. Commercially available drug metformin, targets complex-1 and antagonizes its activity, thus it is known as potential anti-diabetic drug [94].

Endoplasmic reticulum (ER) is a cell organelle play vital role in biosynthesis of cellular proteins [95] and regulates various functions of protein synthesis, modiﬁcations, folding and maturation activities [96].

Mitochondria being the key source of ROS mediated oxidative stress in diabetes but other sources endoplasmic reticulum stress are also involved in diabetes [97]. Mitochondrial ROS formation through ETC has already discussed above while, ER generates ROS production via

disulﬁde bond formation during the process of protein folding. These two processes contribute in diabetic pathogenesis. Evidences have shown that prominent consequence of T2D is the higher amount of circulating glucose [76]. The susceptibility of Mitochondrial DNA damage due to ROS which leads to mutation and altered formation of ETC proteins. This ultimately results in disturbances of ETC function and production of more ROS, hence it highlighting oxidative stress in mitochondria. In line with oxidative stress in ER, gathering of unfolded proteins results in overwhelming oxidative stress [98]. Same way, within the cell, ROS produced more oxidative stress, and thereby redox homeostasis is disturbed. However, the relationship between mitochondria and ER are tightly interlinked and thus play important role for maintaining cellular homeostasis. During the exchange of metabolites and ions within mitochondria and ER, each molecule is aﬀected by the oxidative stress of other [99]. Moreover, ER lumen serve as the place of calcium storage, and act as pivotal source for regulation of ROS signals [100]. The ER stressed cells, calcium is liberated through ER and uptake by mitochondria, thereby enhances ROS via indirect mechanism. The stimulation of Krebs cycle enzymes and oxidative phosphorylation and suppression of complex III of ETC are another mechanism in which calcium promotes mitochondrial ROS production. To maintain redox status in balance within mitochondria and ER, calcium display key role as it witnessed in the presence of higher level of IP3Rs (inositol 1,4,5 trisphosphate receptors), calcium-handling protein in MAMs [101]. Enhanced ROS level promote mitochondrial mediated superoxide in endothelial cells [102] and endoplasmic reticulum [103]

thereafter, decline antioxidant system and make vulnerable to cellular and enzymatic damage, and lipid peroxidation which ultimately results in progression of insulin resistance and hyperglycemia [104]. Thus, aforementioned evidences clearly shown that development of T2D arises due to the ROS mediated oxidative stress from variety of sources including mitochondria and endoplasmic reticulum.

4. Classiﬁcation, biochemical properties and biological functions ofﬂavonoids

Flavonoids are the parts of polyphenol compounds which can be sub-classified into six distinct types consisting offlavonols,flavones, flavan-3-ols, anthocyanidins, flavanones, and isoflavones. Theflavo- noid skeleton is comprising of (C6– C3 – C6) and may involve on several constitutes [105]. Available literature revealed that more than 9000flavonoids varieties have been investigated so far [106] and their daily consumption may varies from 20 mg to 500 mg in the form of tea, red wine, apples, onions and tomatoes [107]. These are the low molecular weight compounds display pivotal role and existing in the form of glycosylated or esterified forms, comprising of C6 C3 C6 rings, known as A and B linked by three-carbon-ring C (Fig. 2) [108]. Flavonoids originate from various sources such as medicinal plants, fruits, vegetables, nuts, seeds, stem,flowers tea [109]. Flavonoids, being the low molecular weight constituents consist of 2-phenyl-carbon nucleus and are synthesized by shikimic acid pathway. Conventionally,flavonoids are categorized by degree of oxidation, detection of ring C and its linkage with ring B. Of note, thatflavones andflavonols contains huge number of compounds exerting the narrow senseflavonoids, such as 2- benzo-γ- pyrone category. Flavanones andflavanonols consists of sa- turated C2-C3 bonds, and rarely comprises onflavones andflanonols in plants. Flavonoids biosynthesis takes place due to precursor, chalcones which display key role in coloration of plants. Flavonols are pathway the reductive products of dihydroflavonols for example flavan-3-ols broadly present in plant kingdom, also recognized as catechins. Inter- estingly, few otherflavonoids also existing the lack of C6-C3-C6 skeleton, such as biflavones, furan chromones and xanthones. Glycosides represent distinct chemical features and shown their predominating forms offlavonoids [110]. Flavonoids possess health promoting properties either directly or indirectly. Previous evidences indicate that flavonoids (proanthocyanid) exerted antioxidative properties [111].

(5)

These bioactive compounds are cell signaling modulators and induce gene expressions in various animal models as well as in human epidemiological studies [112]. Notably, some classes offlavonoids distribu- tion are mainly found in food whereas, others classes are restricted to certain foods. For example,flavonols can be observed in fruits, vegetables and teas while, isoflavones are found in food whereas, others classes are limited to certain foods. For instances,flavonols are mainly reported in fruits, vegetables and teas while, isoflavones are found in leguminous plants [113]. Consumption of dietaryflavonoids may vary within population according to the availability of dietary sources, dietary practices and food habits in various cultures [114].

Innumerable epidemiological studies have submitted undesirable correlation among intake of medicinalflavonoids and progression of variety of diseases [115]. Flavonoids having typical pattern of diverse structures may influence enzymatic system which contribute in crucial pathways, exerting active polypharmacological behaviors [116]. Sev- eral in-vivoandin-vitro animal evidences documented the promising health promoting effects of dietaryflavonoids on glucose homeostasis for the eradication of diabetes and obesity. Flavonoids perform multiple functions such as carbohydrate digestion, adipose deposition, insulin release, and glucose uptake in insulin-responsive tissues via different cell-signaling pathways [117]. Hence, it is not shocking that the asso- ciations among chemical features and activities have been widely de- liberated.

5. Antidiabetic evidences ofﬂavonoids

Flavonoids are the plant secondary metabolites exerting strong anti- diabetic properties for example, quercetin, naringin, hesperidin, epigallocachetin gallate, baptigenin, myricetin, and anthocyanins. These possess massive antioxidant and anti-inflammatory properties. Current evidences have revealed thatflavonoids compounds have shown gene regulatory effects [118]. Cultured cells treated with cocoaflavonoids indicating the poorly understood mechanism in-vivo studies. By employing different cell lines, results are depicted inTable 1. It has been observed that cocoa flavanols may promote glucose homeostasis through mediating carbohydrate function in the gut [119]. Several studies suggested the ameliorative effects of cocoa treated cells against apoptotic factors promote glucose synthesis, activate insulin secretion and persuade cellular replication. Therefore, catechin enriched cocoa flavanol increased glucose triggered insulin secretion, on the other hand, cultured cells treated with total cocoa extract or polymeric procyanidin-rich fractions did not show any positive effects at the level of (0.75 −25μg/mL) [120]. Ingestion of diet supplemented with 10%

cocoa to Zucker diabetic fatty (ZDF) rats for 9 weeks declined hyperglycemia promotes insulin sensitivity and enhanced cell mass function [121]. Cocoa and cocoaﬂavanols and its diabetic eﬀect in human are shown inTable 2.

Moreover, increased glucose level induced insulin secretion treated with catechin enriched fraction associated with improved mitochondrial respiration. It is therefore, indicating the improvement in redox Fig. 2.Chemical structures offlavonoids having anti-diabetic properties Flavonoids are the secondary metabolites exist in the nature in huge amounts. These can be sub-classified into six distinct groups such asflavones,flavonols,flavanones,flavanols, isoflavone and anthocyanidins and exert strong anti-diabetic properties.

Individual compound can be identiﬁed on the basis of hydroxyl group and their alkylation and glycosylation.

(6)

state, enhanced or declined glutathione and nuclear factor erythroid 2- related factor 2 (Nrf2) in nucleus for transcription of targeted genes which are responsible for promoting mitochondrial function and GABPA protein factors [120]. Epicatechin (EC) at the dose of 0.5–10μM down regulates the expression of peroxisome proliferator-activated receptors (PPARs) and reduced DNA targeted binding in 3T3-L1 adipocytes [122]. Moreover, EC also suppressed tumor necrosis factor (TNF) signaling which contributes in insulin resistance [122]. In-vitroevi- dence of inclusion kaempferol at (10μM) improved cell viability, decreased cell apoptosis, and declined caspase-3 activities inβcells and human islets exposed to hyperglycemic conditions. Such functions are belonging to enhanced expression of anti-apoptotic serine/threonine- speciﬁc protein kinase (AKT) and B-cell lymphoma-2 (Bcl2) proteins, increased cyclic adenosine 3,5-monophosphate (cAMP) signaling, and promoted secretion and synthesis of insulin inβcells [123]. Myricetin treatment in diabetes triggered GLUT4 expression [124] and enhanced the phosphorylation of AKT and insulin receptor substrate 1 (IRS1)

[123]. Myricetin at 0.12% supplementation to mice fed on high-fat high-sugar diet lead to reduce body weight and promoted hypercho- lesterolemia and hypertriglyceridemia [125] showing that myricetin may improve insulin secretion and reduce diabetes and obesity. In 3T3- L1 adipocytes (a cell line derived from mouse), addition of naringenin suppressed glucose uptake [126] and decreased phosphoinositide 3- kinases (PI3K) and Akt phosphorylation naturally potentiated by insulin, hence, it regulating insulin-induced glucose transporter type-4 (GLUT-4) translocation [127]. Apart from that, naringenin also suppressed dyslipidemia and promoted glucose metabolism via reduced level of blood glucose and lipids through independent channels viaﬁ- broblast growth factor-21 (FGR-21) [128]. Moreover, supplementation of pure synthetic daidzein to hamsters at (16 mg/kg body weight/day) signiﬁcantly reduced blood glucose and plasma total cholesterol levels in response to casein fed rats [129]. A recently investigation on mice revealed that daidzein from soy supplementation diet at 198 ppm and 286 ppm respectively from conception to adulthood increased lipid Table 1

Anti-diabetic function ofﬂavonoidsin-vitroandin-vivomodels.

Cocoa Flavanol Treatment Anti-diabetic eﬀects Cells/Animal models References

Glucose uptake

Cocoa liquor 0.05–10μg/mL, 15 min ↑glucose uptake,↑GLUT-4 translocation; L6 (skeletal muscle) [279]

procyandin extract Insulin signaling Cocoa extract or

0.75−25μg/mL, 24 h ↑insulin secretion,↑mitochondrial complex INS-1E (pancreas) [120]

Polymeric enriched III-V,↑ATP,↑GSH,

fraction

EC 5–20μM, 20 h ↓ROS,↓carbonyls,↓cell death,

↑IRS-1,↑IRS-2,↑p-AKT,↑p-GSK-3,↑p-

Epicatechin 1–10μM, 24 h AMPK HepG2 (insulin-resistant cells) [233]

Cocoaﬂavonoids 100–200μg/mL, 4 h ↓p-ERK,↓p-AKT 3T3-L1 (adipocyte) [280]

Procyanidin fortiﬁed cocoa 10 and 25μM, 2 h ↑glycogen synthesis,↑glucose uptake Human primary skeletal muscle cells [281]

extract Animal models

Cocoa powder 10% cocoa powder for 9 weeks ↓Glucose,↓insulin,↓HOMA-IR,↓TG Zucker diabetic fatty (ZDF) rats [222]

cocoa powder 8% cocoa powder for 10 weeks = Glucose,↓insulin↓HOMA-IR,↓IL-6 High-fat-fed obese C57BL/6 J mice [282]

600 mg cocoa polyphenols/Kg

cocoaﬂavonoids =Glucose, =insulin, =HOMA-IR Obese-diabetic (ob-db) rats [283]

body weight/day for 4 weeks 25 mg oligomeric procyanidins/Kg

oligomericprocyanidins ↓Glucose,↓insulin High-fat-fed obese C57BL/6 J mice [284]

body weight/day for 12 weeks cacao liquor 0.5% and 1% cacao liquor

↓Glucose,↓fructosamine High-fat-fed obese C57BL/6 J mice (Adipose [285]

proanthocyanidins Proanthocyanidins for 3 weeks tissue and skeletal muscle)

The arrow indicates an increase (↑) or decrease (↓) in the levels or activity of the diﬀerent parameters analyzed.“=”symbol designates unchanged parameters.

Abbreviations: GLUT4 Glucose transporter type-4; ATP Adenosine tri-phosphate; GSH Glutathione; INS-1E Insulin-secreting rat insulinoma; IRS-1 2 Insulin receptor substrate-1-2; p-Akt Phosphorylation of serine/threonine-protein kinase; p-GSK-3 Phosphorylation of glycogen synthase kinase-3; p-AMPK Phosphorylation of 5′ AMP-activated protein kinase; p-ERK Phosphorylation of extracellular Signal-Regulated Kinase; HOMA-IR Homeostatic model assessment of insulin resistance; TG Total glycerides; IL-6 Interleukin-6.

Table 2

Cocoa and cocoaﬂavanols and its diabetic eﬀects in humans.

Cocoa and chocolate

Population Anti-diabetic eﬀects Design References

Treatment

50 mg epicatechin Diabetic = HOMA-IR, =BP, =LDL-Cho Randomized crossover [286]

450 mgﬂavonoids Diabetic ↓HbA1c,↓Glucose Randomized, placebo controlled [287]

↑GSH,↑SOD,↑Catalase

100 mg epicatechin Diabetic Open label protocol [288]

10 g cocoa powder Diabetic ↓LDL-Cho,↓inﬂammatory markers Randomized [289]

Flavanol-rich cocoa

Diabetic =glycaemic parameters Randomized crossover design [290]

(963 mgﬂavanols/day)

The arrow indicates an increase (↑) or decrease (↓) in the levels or activity of the diﬀerent parameters analyzed.“=”symbol designates unchanged parameters.

Abbreviation: HOMA-IR, Homeostatic model assessment of insulin resistance; BP, Blood pressure; LDL-Cho, Low density lipoprotein cholesterol; HbA1c, Hemoglobin A1c; GSH, Glutathione, SOD, Superoxide dismutase.

(7)

peroxidation and glucose metabolism [130]. In addition, daidzein or genistein supplementation at 0.02% may decrease diabetes occurrences and augment glucose homeostasis via intactness of pancreatic β-cell function in non-obese diabetic (NOD) mice [131]. Another evidence of oral apigenin induction (0.78 mg/kg body weight) for approximately 10 days was investigated to reverse the suppression of hepatic antioxidants in alloxan triggered insulin dependent diabetic mice, exerting the scavenging activity of free radicals. Molecular mechanism of anti-diabetic properties ofﬂavonoids is well illustrated inFig. 3.

6. Bioavailability ofﬂavonoids and implication of nano-tools

In human, bioavailability of flavonoids affected during phase-2 metabolism [132]. Normally, many of theflavones undergo to sulfa- tion, methylation and glucuronidation via small intestine and liver [133]. While, the conjugated metabolites can be present in plasma followed byflavonoid ingestion [134]. Generally,flavonoids metabolites exert less bioactivity than its parent compound [135]. Despite, bioactivity of various in-vitro systems, bioavailability of flavonoids would be a main indicator of bioactivity in-vivo. It is therefore, important to improve bioavailability in order to increase health beneficial effects in-vivo[136]. As the molecular diversity found in flavonoid compounds, therefore, proper tools should be implicated to improve stability, bioavailability and bio-efficacy of the compounds. The complex nature of theflavonoid structures and its molecular weight may be responsible for lower bioavailability [137]. To enhance the efficacy of flavonoid metabolism by increasing intestinal absorption through absorption enhancers [136] improving intestinal absorption from colon to small intestine [138] enhancing metabolic stability [139] and implying novel delivery systems [140].

The nanotechnology is a newﬁeld to improve compound structural modiﬁcations to enhance solubility by nano-systems [141] including chitosan, polymers, cyclodextrins and dendrimers. Therefore, these advanced technologies could be employed (e.g. microencapsulation, nano-delivery systems, microemulsions, enzymatic methylation). As

nanotechnology is an emergingﬁeld used to dispense drug to the particular site to improve compound eﬃcacy and its utilization [142].

Moreover, this technology avoids by passing natural barriers and metabolic alternation, which are assumed to be responsible for poor absorption. Notably, efficacy of the drug and its bioavailability could be influenced by material composition and physico-chemical properties of nano-particles [143]. In addition, competition and inhibition in intestinal cell transports can also increase the bioavailability of the components [144].In-vitroexperiments reveal thatflavonoid bioavailability of hesperidin can be improved through inhibiting ABC trans- porters via competition with otherflavonoids such as quercetin leading to reduce efflux of hesperidin [145]. Multiple nano-delivery systems have been employed to enhance bioavailability of phytocompounds to ensure strong anti-diabetic properties [146]. Some examples offlavonoids embedded nano-delivery system are given below.

Multiple nano-delivery tools systems have been implied to enhance bioavailability of phyto-compounds to ensure strong anti-diabetic properties [146]. For example, baicalin is successfully implicated with nanostructured lipid carrier (NLC) delivery systems with particle size 92 nm thereby; it exerts potent anti-diabetic properties through oral route [147]. Another, compound stevioside elicit increased anti-diabetic activities by polyethylene glycol-polylactic acid nanoparticle delivery systems with particle size 150 nm [148]. Moreover, researchers are trying to ﬁnd out compounds for eﬀective delivery materials to systematic circulation, which must be non-toxic and non-mutagenic having good water solubility. In addition, variety of nano-delivery systems play important role in delivery system to many diseases [149].

A very few nano-delivery systems for example, solid nanoparticles, nano-phytosome and nano-emulsion have exerted efficient delivery of phytoactive compounds. Currently, a new emerged drug delivery system was studied by employing nano-wire systems [150] but their drug efficacy is still lower. Efficient nanodelivery system to improve bioavailability of polyphenol compounds are indicated in Fig. 4. An increased bioavailability of quercetin was observed in quercetin-loaded NLC developed by using phase inversion method having particle size Fig. 3.Molecular mechanism of anti-diabetic properties offlavonoids. This schematic shows that insulin is recognized by insulin receptors and hence it stimulates intrinsic kinase activity that leads to autophosphorylation and recruit substances, for example, IRS1-4 proteins, Cbl and SHC. Phosphorylation of IRS proteins as- sists proteins with Src homology-2 SH2 do- mains. Many proteins are considered as adaptor molecules for instance, performing regulatory function of PI3K or adaptor molecule growth factor receptor bound protein-2 (Grb2) that ultimately stimulate Ras-MAPK pathway. Whereas; PI3k enzyme is based on the regulatory (p85) and catalytic (p110) sub- units that catalyzes the formation of lipids second messenger PIP3 within cellular milieu, and activates phosphoinositide-dependent kinase (PDKs). PDK targets consist of PKB/Akt and the atypical PKC isoforms. Along with PI3K, stimulation of PKB/Akt and PKCs are contributed in the insulin activated GLUT translocation, glucose uptake and glycogen synthesis.

(8)

∼32 nm [151]. Further evidences, reported that quercetin-loaded NLC systems was established having particle size 47 nm, leading to higher bioavailability upto 60% [152]. Currently, cationic-modiﬁed NLC delivery system was designed with particle size ∼126 nm, illustrating higher bioavailability in lung, kidney and liver tissues [153]. In addition, numerous phyto-based anti-diabetic compounds persistently re- leased via oral route, but their bioavailability in diabetic model is still limited. Hence, NLC system could be referred as novel delivery system which continuously releases phytocompounds.

Nano-emulsion system highly increases delivery of numerous li- phophilic bioactive compounds which elicit anti-dibetic activities with enhanced efficacy and bioavailability [154]. The mechanism of lipid based nano-carriers drug absorption is displayed inFig. 4. Currently, bitter gourd seed oil nano-emulsion consisting of 50% α-eleostearic acid was studied in diabetic rat model. The findings suggested that bitter gourd seed oil nano-emulsion with particle size < 100 nm was extremely stable and could deliver increased anti-diabetic activity by oral induction [155]. Same researchers further illustrated effect of gourd seed oil nano-emulsion having increased cellular uptake and enhanced anti-oxidative properties [156]. It is further indicate that curcumin and quercetin have showed elevated bioavailability via nano- emulsion delivery systems. Moreover, a curcumin-encapsulated with nano-emulsion was developed with a particle size∼130 nm, and exhibited efficient bioavailability along with liver protection [157].

Available literature highlight that naturally exist bioactive compounds may be proﬁciently delivered through oral route via nano-emulsion.

Although, more evidences are needed to collect particular individual compound response in diabetic model together with delivery systems implications.

Nanoliposomes are known to effectively deliver bioactive compounds such as hydrophilic and hydrophobic having increased stability, efficacy and bioavailability of lower particle size [158]. Currently, an anti-diabetic compound derived from herbal extract named Orthosi- phon stamineu, successfully prepared with nanoliposome system with particle size 152 nm to increase its antioxidant efficiency [159]. Studies on resveratrol encapsulated with nanoliposome indicated two times higher bioavailability than control group [160]. Moreover, catechin was fruitfully loaded with nanoliposome system, may effectively bypass numerous factors and it might be used as possible efficient food to deliver catechin [161]. Several phytobioactive compounds were studied

successfully to enhance bioavailability in anti-diabetic animal models but limited studies have been reported.

Nanosuspensions are defined as delivery techniques through oral route of bioactive ingredients via liquid form having particle size < 1μm, and it can be employed by different techniques for example wet or dry milling [162]. A berberine, anti-diabetic compound has shown to increased anti-diabetic activity against T2DM animal models upon induction of low dosage level. Same way, phyto-compounds such as quercetin, has elicited prominent anti-diabetic effect through obtaining higher efficacy and bioavailability by using nanosuspension system. However, quercetin nanosuspensions having uniform size, showed higher bioavailability and improved oral delivery with particle size 70× as compared to control group [151]. Another proof, with curcumin loaded nanosuspension were developed with particle size 210 nm, and it indicates higher absorption rate such as increased confirmation and fluidity of intestinal mucosal membrane [163]. Such studies gives clues to understand underlying mechanism to prepare nano-suspension based phytoconstituents for anti-diabetic studies in different animal models.

Polylactic-co-glycolic acid nanoparticles (PLGA NPs) can be utilized efficiently by oral delivery system for different phytobioactive compounds in order to improve bioavailability and stability of the compounds [164]. The anti-diabetic function of quercetin and curcumin are profoundly captured and absorbed in PLGA nanoparticles having size > 100 nm by implying different methods like solvent evaporation or nano-precipitation, such delivery system has increased anti-diabetic effects [165]. The quercetin embedded with PLGA NPs having uniform particle size ∼179 nm was prepared. Animal based studies reported that PLGA NPs effect with apart of 5 days is similar to daily intake of quercetin. Hence, the delivery systems of quercetin encapsulated with PLGA NPs are very efficient and reduced the frequency of the drug [166]. Moreover, fenugreek seed extract embedded with PLGA NPs exerted stronger anti-diabetic function in alloxan-induced diabetic models [167] with evidence of increased antioxidant and anti-lipid peroxidation activity. Blach seed derived thymoquinone showed pow- erful anti-diabetic property and loaded with PLGA NPs documented increased antioxidant effect with constant production in simulated gastro-intestinal system [168]. Further evidences illustrated that costus speciosus extract embedded with PLGA NPs were reported to enhance efficacy in anti-diabetic model, and such study may effectively Fig. 4.Underlying mechanism of increasing drug absorption of lipid nanocarriers. The carrier materials are based on the natural polymers or synthetic biodegradable having high molecular polymers. Synthetic biodegradable polymers include polylactic-glycolic acid.

While the natural poylmers are distributed into categories such as polysaccharides and proteins. The polysaccharide compounds are derived from plants like pectin,cellulose etc, starch, guna Arabic, carrageenan,alginate) and polysaccharide originate from microbial or animals (xanthan gum, chitosan). Meanwhile, proteins consist of albumin, gelatine, soy proteins, casein. The nanoparticle are made up of polysaccharides, it possess unique properties and are best carriers to deliver compounds without aﬀecting physiological barriers and can successfully deliver drug into the system.

Natural biomaterials, polysaccharides are stable, safe, non-toxic, hydrophilic and biodegradable. However, polyphenols are the cost eﬀective, abundantly exist in the nature (Figure courtesy by [278].

(9)

controlled glucose level [169]. Quercetin is aflavonoid showing potent bioactivity in different oxidative stress related diseases like T2DM [170]. The anti-diabetic potential of quercetin has been well observed in different cell and animal studies, however, its efficacy via oral route is low [171]. Therefore, nano-delivery approach of quercetin is quite promising to enhance its antidiabetic activities. Currently, PLGA encapsulated quercetin NPs were prepared having particle size∼179 nm, indicating increased bioavailability in streptozotocin-induced diabetic rat model and proved beneficial effects by PLGA delivery systems [166]. Other study revealed that curcumin embedded with PGLA NP was developed for oral delivery having particle size∼158 nm, effectively reported increased solubility and bioavailability [172].

7. Clinical trials ofﬂavonoids against T2DM in humans

Several studies revealed that higher consumption of totalflavonoids have been linked to reduced risk of diabetes in numerous human trials [173]. Although, individual intake offlavonoids seems to show no any effect on diabetes; though, limited data on human clinical trials have been reported [147]. The meta-analysis of randomized controlled trial indicated that tea catechins could considerably decline fasting blood glucose [174] whereas; tea or tea extracts seems not exert hypogly- cemia in T2D patients in a double-blind, placebo-controlled, randomized trial [175]. Different clinical trials exhibited that extract of green tea and polyphenol may decrease bone resorption markers [176] and albuminuria in diabetes patients [177]. Furthermore, results of anti- diabetic function in human studies were reported other compounds of soy instead of isoflavones [178]. However, isoflavones were associated with reduced risk of T2 diabetes [179] whereas; intake of isoflavones was not indicated any effect on fasting insulin, glucose and HbA1c [180]. Individual isoflavones rarely have shown effect on glycemic control and insulin sensitivity in a clinical trial [181]. Research on assessing anti-diabetic activities of anthocyanins, consisting of de- creasing blood glucose [182] glucosuria and HbA1c [183] and hence enhancing insulin secretion [184] and resistance [185] with and without anthocyanins and their glycosides may promote glucose homeostasis via interactingβ-cell mass and function, insulin sensitivity, and glucose uptake. Although, a very few studies on non-diabetic healthy human subjects reported that anthocyanins did not exert changes in blood glucose or plasma insulin concentration [186].

Further studies on clinical trials indicate different outcomes due to the different flavonoid compounds like silymarin [187] and silybin- beta-cyclodextrin [188] promoted glycemic and lipidemic status in T2D patients. Same way, intake of cranberry juice for 3 months enhanced glycemic control in T2D subjects [186]. Moreover, rats were offered 2%

whole cranberry powder with normal rodent chow from 6 to 22 months old. Results revealed the enhanced level of insulin and thus by exerting insulinotropic effect [189]. However, samefindings of cranberry juice have also been observed with chokeberry juice for 3 months in T2D patients [190]. In addition, extract of grape seed give clues of inflammation markers in glycemic control in obese T2D subjects [191]

whereas; grapes consumption for 3 weeks reduced plasma LDL-cho- lestrol in obese subjects [192]. More, results report that consumption of catechins at 582.8 mg for 3 months reduced weight loss in obese subjects with T2D, with minor improvement in glucose control [193].

Likewise, evidence demonstrated that intake of catechin-rich green tea at 615 mg for 1 month enhanced postprandial glucose in T2D subjects [194]. Although, it is unpredictable that catechins are helpful against T2D. Excessive dose of tea extract, catechins alone, compared with mixture, could be eﬀective strategy for preventive and curative approach against T2D in humans. However, metabolic status predicts treatment duration in human subject studies.

A double-blind, placebo-controlled trial of 48 T2D patients provided 12 week supplementation of Pycnogenol at 125 mg, rich source of procyanidins and bioﬂavonoids, could increase diabetes control, suppress CVD risk factors, and decease hypertensive medicine use

compared with controls [195]. Another, double blind study, based on 80 T2D patients, Brazilian green propolis (226.8 mg/day) with high contents of polyphenols andflavonoids for 8 weeks, was reported to prevent T2D patients from development of severe blood uric acid and assessed glomerularfiltration rate [196]. A randomized, double-blind, placebo-controlled trial offered acacia polyphenol (flavonoid) (250 mg/

day) to 34 subjects might enhance glucose homeostasis in non-diabetic subjects where glucose tolerance was found [197]. The Eugenia puni- cifolia (Kunth) DC. (Myrtaceae), dried powder of leaves enrichedfla- vonol glycosides, is potent as an adjuvant against T2D patients. In a pilot non-controlled study, intake of this powder for 3 months was observed to significantly reduced hemoglobin and basal insulin levels [198]. In summary,flavonoids, the part of polyphenols provide beneficial effects on T2D patients, by controlling diabetes, suppressing severe blood uric acid, and maintain insulin levels. Further studies of clinical trials on human subjects are warranted on differentflavonoid compounds alone or with mixture. Still the evidences are insufficient;

therefore, more studies are needed on clinical aspect ofﬂavonoids in T2DM.

8. Flavonoids targeted approaches in modulating T2DM

Flavonoids are structurally different molecules enormously present in the nature. The anti-oxidative effects offlavonoids come into central zone due to vast properties to overcome oxidative stress in diabetic patients over the past decade. We have provided evidences that diabetes develops from oxidative stress. Numerous efforts have been taken to minimize oxidative stress induced cellular insult in diabetes by antioxidant supplementation [199]. Flavonoids display strong anti-oxidant and anti-inflammation activitiesin-vitroandin-vivo[200] and also modulate transcription factors and pro-inflammatory mediators by interaction with receptors [201]. Currently, pancreatic islet isolation and its transplantation model might have therapeutic implications in T2DM [202]. The best way is to imply both strategies such asflavonoids with transplantation process based on pancreas which might give some new therapeutic insights. It is further indicated that uptake multipleflavo- noid compounds irrespective of single in excessive amounts are most important. Of note, that numerous literature on human and animals studies revealed appropriate doses of pure singleflavonoid increase glycaemia because most flavonoids exerts activity by influencing digestion of complex sugars and absorption of glucose [203]. Anti-diabetic mechanism offlavonoids is shown inTable 3.

As mentioned above, signaling pathways display vital roles in pathogenesis of oxidative stress induced diabetes, while some of them are considered as potential target for therapeutic intervention. The Flavonoids such as epigallocachetin gallate (EGCG) have shown to exert anti-inﬂammatory activities in pancreatic β -cells [140] by encountering mitochondrial mediated oxidative stress thus, inhibiting cytochrome-c release from mitochondria into cytosol and thereby suppress caspase activity [140]. The 5′AMP-activated protein kinase (AMPK) is basically known as a promising target for therapeutic intervention of diabetes [204]. At cellular level, EGCG activates the AMPK pathway that prevents hepatic gluconeogenesis [205] and enhances fatty acid oxidation [206] as well as regulates mitochondrial biogenesis [207]. In addition, AMPK activation elevates GLUT4 expression in skeletal muscles [208] thus, it promotes glucose uptake.

Flavonoids such as naringin and hesperidin have shown to reduce GLUT2 protein expressions by liver and increase expressions GLUT4 in WAT [209]. Furthermore, apigenin is 200 times stronger than metformin, an activator of AMPK. At cellular level, exposure of HepG2 cells to high glucose and apigenin reduces the ACC phosphorylation and hamper lipid accumulation [210]. Thus, it reveals the beneﬁcial eﬀects of apigenin on diabetes through mediating AMPK, energy metabolism.

Molecular mechanism of ﬂavonoids anti-diabetic properties are illustrated inFig. 3.

The PPAR-γ is the proteins belonging to superfamily of nuclear

(10)

receptors. These receptors are found in different cell lines, such as adipose tissues, muscles, brain and immune cells, and are responsible for fatty acid storage, glucose metabolism, lipid uptake and adipogen- esis. PPAR-γcan response to various factors, such as uptake glucose, fatty acids, transcript factors such as (aP2), GLUT4 [211] as well as fatty acid transport proteins acyl-CoA synthetase. It has been pro- claimed that PPAR-γregulate glucose metabolism in tissues, and its activation leads to development of T2DM. Supplementation of hesperidin and naringin is known to stimulate PPAR-γof diabeticdb/dbmice [209]. Thus, it shows that stimulation of PPAR-γleads to fat cell dif- ferentiation, increase in glucose metabolism via stimulation of GLUT4 production and inhibition phosphoenol pyruvate carboxykinase (GTP) and glucose-6-phosphatase (G6Pase). Moreover, hesperidin and naringin may provide protection against hyperglycemia in T2DM through the modulatory effects of PPAR-γ, a main target for development of T2DM drugs. Of note, naringin may activate PPARγ and GLUT4 to regulate hepatic enzyme expression contributing in glycolysis and gluconeogenesis, thus promoting hyperglycemia in T2DM affected mice [209]. Kaempferol, aflavonoid compound found in Ginkgo biloba L., cruciferous vegetables, grapefruit, tea and some edible berries [212].

Orally induction of kaempferol was clearly reduced fasting blood glucose, serum HbA1c levels and increased insulin resistance. Liver gene expression analysis revealed that kaempferol reduced PPAR-γ and SREBP-1c expression. It is admitted that kaempferol possess anti-obese and anti-diabetic effects which are mediated by SREBP-1c and PPAR-γ regulation via stimulation of AMPK pathway [213]. Thus, it suggests that reduced genetic expression of PPAR-γ mediated by regulating AMPK activation [214]. Moreover, eriodictyol, a flavonoid exists in lemon fruits; depict positive effect on obesity and diabetes [215]. Evi- dences shown that dietary approach of eriodictyol in diabetic rats can effectively reduce oxidative stress [216] while, eriodictyol treatment has upregulated the expression of PPARγ2 and the adipocyte-specific fatty acid binding protein [217].

Nrf2 signals promotes antioxidant response element which subsequently stimulates transcription genes responsible for endogenic antioxidant enzymatic system. Physiologically, these proteins reside in cytoplasm under the assembly of Keap1 proteins. Upon occurrences of

oxidative insult, Nrf2 protein kinase stimulates from Keap1 (cytoplasm) and translocate into nucleus where it transcript genes for antioxidant response [218]. After exploring the key role of Nrf2 in ameliorating oxidative stress, thus, it is considered as the therapeutic target for preventing insulin related disorders as well as micro and macro vascular complications. The promising effect of Nrf2 is to reduce the intensity of oxidative stress mediated damages which ultimately prevents the complications [219]. Dietaryflavonoids possess capabilities to activate Nrf2 signaling pathways and modulate critical enzymatic systems [220] where its preliminary function depends upon the cellular dis- tribution/phosphorylation [220]. Flavonoids such as green tea catechins, reduced lipid peroxidation and enhanced plasma total antioxidant capacity, and also attenuated signaling pathways, prooxidant enzymes and increasing antioxidant enzymes such as SOD, glutathione peroxidase and catalase [221]. Moreover, epicatechin (EC) and cocoa phenolic extract (CPE) suppressed high glucose induced antioxidant defenses and p-MAPKs, and sustained Nrf2 stimulation [222]. Studies on rats exhibited that quercetin mitigated the expressions of oxidative stress and inflammation, like Nrf2, heme oxygenase-1, and NF-κB, proposing that quercetin, a flavonoid compound possess anti-inflammation effects on adipose tissue might be linked with decreased body weight [223]. Similarly, Kobori et al. [224] documented that quercetin included in the diet tend to restore cell proliferation in diabetic mice. Thus, these compounds are considered as potential targets of Nrf2 stimulation in context of T2DM occurrences.

Glycogen synthase kinase-3 (GSK-3) is a conserved enzyme derived from muscles to phosphorylate glycogen synthase [225]. The GSK3 pathway contributes in variety of processes such as energy metabolism, transcription factor activation, cell growth, proliferation, inflammation, and microtubule stability [226]. Based on different substrates, the in- terruption in GSK3 function causes numerous pathogenic processes including diabetes [227]. At cellular milieu insulin attach and stimulate insulin receptor (IR), via phosphorylation of key tyrosine residues. After tyrosine phosphorylation of insulin receptor substrates (IRS) andfinally causes stimulation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) pathway [228]. Whereas; stimulation of AKT causes suppression of GSK-3 by phosphorylation that results in Table 3

Flavonoid compounds and their anti-diabetic mechanism.

Compounds Sources Mechanistic view Model References

↑NF-κB pathway HFD-induced in

Luteolin Treatment of 10 mg/kg orally for 4 weeks [153]

Obesity male C57BL/6 J mice Feed supplementation with 200 mg/kg for 8 weeks Activation of AMPK pathway HFD-induced

Tangeretin [291]

and its anti-inﬂammation eﬀects obese C57BL/6 J mice 18 h treatment with quercetin at the dose of ↑glycogen synthase Murine H4IIE and human HepG2 50μM

Quercetin [292]

cells.

↑cAMP signalling↑PKA

Genistein 1 g of genistein/kg for 8 weeks HG-induced diabetic db/db mice [293]

activation

↓apoptosis,↓caspase-3

Kaempferol kaempferol at diﬀerent doses 0.01, 0.1, 1 and INS-1Eβ-cells [123]

10μM for 4 days

in beta-cells

Orally at 10 mg/kg for 2 Anti-adipogenic eﬀect, C57BLKS/JLeprdb/ Leprdb

Wogonin weeks [294]

↑PPARαand MAPK pathways mice and 3 T3-L1 cells

Administration of chrysin at the dose of ↓TNF-α HFD-STZ-induced

Chrysin [295]

40 mg/kgbw for 16 weeks by gastric gavage

↓proinﬂammatory cytokines Diabetic wistar albino rats

Abbreviation: NF-κB, Nuclear factor kappa B proteins; AMPK, 5′AMP-activated protein kinase; cAMP, Cyclic adenosine monophosphate; PKA,Protein kinase-A;

MAPK, peroxisome proliferator-activated receptors; TNF-α, Tumor necrosis factor alpha; INS-1E, Insulin-secreting rat insulinoma; HFD-STZ, High fed diet streptozotocin-treated rat model, 3T3-L1, cell line derived from mouse; C57BLKS/JLeprdb/ Leprdb, A mouse model for non-insulin dependent diabetes mellitus.