Modulatory effect of caffeic acid in alleviating diabetes and associated complications

Abstract

Diabetes mellitus (DM) is one of the most common metabolic disorders characterized by elevated blood glucose levels. Prolonged uncontrolled hyperglycemia often leads to multi-organ damage including diabetic neuropathy, nephropathy, retinopathy, cardiovascular disorders, and diabetic foot ulcers. Excess production of free radicals causing oxidative stress in tissues is often considered to be the primary cause of onset and progression of DM and associated complications. Natural polyphenols can be used to induce or inhibit the expression of antioxidant enzymes such as glutathione peroxidase, heme oxygenase-1, superoxide dismutase, and catalase that are essential in maintaining redox balance, and ameliorate oxidative stress. Caffeic acid (CA) is a polyphenolderived from hydroxycinnamic acid and possesses numerous physiological properties includ-ing antioxidant, anti-inflammatory, anti-atherosclerotic, immune-stimulatory, cardioprotective, antiproliferative, and hepatoprotective activities. CA acts as a regulatory compound affecting numerous biochemical pathways and multiple targets. These include various transcription factors such as nuclear factor-B, tumor necrosis factor-α, interleukin-6, cyclooxygenase-2, and nuclear factor erythroid 2-related factor 2. Therefore, this review summarizes the pharmacological properties, molecular mechanisms, and pharmacokinetic profile of CA in mitigating the adverse effects of DM and associated complications. The bioavailability, drug delivery, and clinical trials of CA have also been discussed.

Key Words: Diabetes mellitus; Caffeic acid; Diabetic foot ulcer; Retinopathy; Nephropathy

Core Tip: Diabetes mellitus has emerged as one of the most common metabolic disorders worldwide which can lead to other complications such as retinopathy, nephropathy, neuropathy, and foot ulcers. Free radical-induced oxidative stress is one of the primary causes of diabetes. Caffeic acid (CA) is a natural polyphenol obtained from various fruits and vegetables. CA and its derivatives act as an antioxidant and regulate the signaling pathways involved in lipid and carbohydrate metabolism. CA also exerts anti-diabetic effects by modulation of inflammatory cytokines and transcription factors.

INTRODUCTION

Diabetes mellitus (DM) is a metabolic disorder marked by elevated blood sugar levels that stems from the complete loss or dysfunction of insulin producing pancreatic β-cells and subsequently results in other complications in several organs of the body.DM is one of the most frequently occurring metabolic diseases worldwide and is the leading cause of death due to comorbidities[1,2]. The main subtypes of DM are type 1 diabetes (T1DM) and type 2 diabetes (T2DM). T1DM, also referred to as insulin dependent DM, is an autoimmune condition that is mediated by the dysfunction of pancreatic β-cells with complete loss of insulin production[3]. T2DM is the insulin resistance type that occurs when pancreatic β-cells are incapable of producing enough insulin. T2DM affects 90%–95% of diabetic individuals globally[4]. Several reports suggest that around 400 million people worldwide would be affected by DM by the year 2025[5]. Both types of DM are frequently linked to long-term consequences such as higher risk of cardiovascular diseases (CVD), retinopathy, neuropathy, nephropathy, foot ulcers, and other vascular anomalies. These complications consequently lead to blindness in diabetic patients, end-stage renal disease, atherosclerosis, and even mortality[6]. Compared to non-diabetic individuals, T2DM patients are at much higher risk of foot injuries and cardiovascular morbidity like atherosclerosis[7]. Studies have demonstrated that metabolic variables, oxidation/glucoxidation, and changes in vascular reactivity are some of the major factors that contribute to diabetic atherosclerosis[8]. Although the pathophysiological mechanism linking DM to its complications is yet to be extensively explored, oxidative stress appears to be a key factor[9-11]. Several reports have suggested that increased oxidative/nitrosative stress and cellular redox disturbances facilitate the etiology and development of both T1DM and T2DM. Uncontrolled hyperglycemia causes oxidative stress and further damages the cells primarily by targeting various metabolic pathways such as enhancement of polyol pathway, increased synthesis of advanced glycation end products (AGEs), activation of protein kinase C, and upregulated hexosamine pathway[9,10]. Therefore, hyperglycemia results in elevated levels of reactive oxygen species and reactive nitrogen species (RNS) in the majority of organs. Moreover, a decrease in cellular antioxidant defences is linked to an increase in oxidative stress in diabetic individuals[9,12,13]. The primary factor contributing to endothelial cell failure in diabetic complications may be due to increased lipid peroxidation caused by oxidative stress. Endothelial dysfunction in DM has been attributed to excessive generation and/or insufficient clearance of free radicals by the antioxidant defencesystem[14] (Figure 1). Since oxidative stress is involved in the development of T1DM, T2DM, and diabetes-associated complications, use of antioxidants as a counter measure could be beneficial. When cells are exposed to chemicals/oxidants, natural polyphenols can be used to induce or inhibit the expression of enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1) that are essential in maintaining cellular homeostasis[15]. Natural polyphenols are secondary metabolites having lower risk of adverse effects when employed in conventional and alternative medicine[16].

Figure 1 Multiple factors responsible for the onset and progression of diabetes mellitus and associated complications including diabetic nephropathy, neuropathy, retinopathy, and cardiovascular disorders. Hyperglycemia leads to the formation of advanced glycation end products and activation of protein kinase C. This further results in oxidative stress-mediated dyslipidemia, hypertension, activation of polyol pathway, and inflammatory stress. AGE: Advanced glycation end products; TNF-α: Tumor necrosis factor-α; IL: Interleukin; VEGF: Vascular endothelial growth factor; IGF: Insulin-like growth factors; NF-κB: nuclear factor-κB.

Caffeic acid (CA) is a polyphenolic derivative of hydroxycinnamic acid, formed as a product of secondary metabolism in fruits and vegetables[17-19]. CA can be present in simple monomeric form as amides, glycosides, sugar, and organic acid esters, or in complex oligomeric forms as derivatives of flavonoids. CA can also be found attached to some cell wall proteins and polymers[19-20]. CA inhibits the growth of bacteria, fungi, and insects, protects plants from ultraviolet-Bradiations, and contributes to plants’ defensive mechanism against predators, pests, and illnesses[21]. Numerous biological effects of CA and its derivatives have been demonstrated through experimental studies, including antibacterial, antiviral, antioxidant, anti-inflammatory, anti-atherosclerotic, immune-stimulatory, cardioprotective, antiproliferative, and hepatoprotective activities[21-25]. Propolis, derived from honeybee, is rich in CA phenethyl ester (CAPE), a common naturally occurring derivative of CA having widespread applications in research and industry[26]. CAPE acts as a regulatory compound affecting numerous biochemical pathways and multiple intracellular targets including several transcription factors, namely, nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase (iNOS), activated T-cell nuclear factor, and hypoxia-inducible factor-1[26-30]. Most of these pathways are usually involved in the regulation of inflammatory and oxidative stress markers. Numerous studies have reported the efficacy of CAPE in the treatment of stress-induced pathologies. Recent studies have shown the protective ability of CAPE against nephrotoxicity induced by a number of xenobiotics (methotrexate, doxorubicin, cisplatin, toluene, carbon tetrachloride, etc.) or by diverse toxic conditions[31]. Several reports suggest the application of CAPE in experimental and clinical studies for the treatment of several diseases such as cancer, thyroid, liver diseases, hepatic insulin resistance, non-alcoholic fatty liver disease, or hepatocellular carcinoma[32,33]. In vivo studies have reported that oral ingestion of CAPE stalled the progression of atherosclerosis in mice deficient in apolipoprotein E[32]. In addition, involvement of CAPE in molecular signaling pathways suggests that CAPE has therapeutic efficacy in diverse inflammatory diseases and cancer[31-32]. Similarly, CA treatment has also exhibited protective efficacy in various organs such as the brain, kidneys, lungs, ovaries, and heart from diabetes-induced damage[34-36]. Therefore, this review reports the structural and pharmacological properties of CA and its derivatives with special emphasis on the key mechanisms of action and pharmacokinetic properties of CA, especially in DM and associated complications.

SOURCES AND CHEMISTRY OF CA

CA occurs naturally in several vegetables and fruits including kiwis, blueberries, plums, cherries, apples, cereals, carrots, and cabbage. CA can also be found in propolis, which is a resinous substance made by honeybees[37]. Different plant species have variable amounts of CA[38]. It is a very prevalent phenolic acid that accounts for 75 to 100 percent of the total hydroxycinnamic acid in fruits[39]. Structurally, CA is a phenylalanine-derived hydroxycinnamic acid with a 3,4-dihydroxyaromatic ring connected to carboxyl group through a trans-ethylene bond[37]. CA is synthesized naturally in plants via the endogenous shikimate pathway[23,37]. The biosynthesis of CA begins with precursor shikimic acid and involves three enzymatic reactions: (1) Phosphorylation by shikimate kinase; (2) The conjugation of phosphoenolpyruvate by 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase; and (3) Formation of intermediary metabolite chorismic acid by the enzyme chorismatesynthase[23,37]. Cinnamic acid is produced from the deamination of L-phenylalanine by enzyme phenylalanine ammonia lyase; it is converted into p-coumaric acid by the action of cinnamate-4-hydroxylase, which is subsequently converted into CA by enzyme 4-coumarate 3-hydroxylase[23] (Figure 2). CA is generally extracted from plant materials and by microbial synthesis using organisms like Escherichia coli. Two enzymes can be produced by genetic modifications in Escherichia coli strains: Tyrosine ammonia lyaseand 3-hydroxylase hydroxyphenylacetate which act on L-tyrosine to produce L-dopa and coumaric acid, respectively, leading to the synthesis of CA[36].

Figure 2 Biosynthesis of caffeic acid via the shikimic acid pathway. ATP: Adenosine triphosphate; EPSP: 5-enolpyruvylshikimate-3-phosphate; PLP: Pyridoxal phosphate; NAD: Nicotine adenine dinucleotide; PAL: Phenylalanine ammonia lyase; C4H: Cinnamate-4-hydroxylase; C3H: Coumarate 3-hydroxylase.

PHARMACOKINETICS OF CA

CA has a molecular weight of 180.16 g/mol and is typically found as a white, amorphous powder. The partition coefficient (logP) for CA ranges from 1 to 1.3[40,41]. In addition, propolis contains large amounts of naturally occurring derivative of CA, the CAPE that appears as a white crystalline solid and has a molecular weight of 284.31 g/mol. The intriguing aspect of CAPE is its ability to traverse the blood-brain barrier, which can be attributed to logP values of CAPE ranging between 3.2-13.8[41-43]. CA is essentially found in food in esterified form with chlorogenic acid, thus limiting its absorption in the body[44]. Human tissues such as the intestinal mucosa, stomach, and liver, and biological fluids such as plasma, duodenal fluid, and gastric juice lack the esterase enzymes that hydrolyze chlorogenic acid to release CA. Thus, it is hydrolyzed by intestinal microflora before its absorption[42,44]. As a result, the pharmacokinetic process starts when CA is consumed and enters the stomach in its esterified state, where a small amount of CA is absorbed[41-44]. Thereafter, the intestinal mucosa absorbs up to 95% of CA in its free form after the bacterial esterases in the colon break the ester part of CA[42-44]. Monocarboxylic acid transporters are involved in the active transport of CA across membranes into intestinal cells[36,42,44]. The peak plasma concentration of CA occurs after 1 h of meal digestion, and it takes repeated dosage every 2 h to sustain high levels of CA in plasma[36,42]. Under anaerobic conditions, gut bacteria having tyrosine decarboxylase can cause decarboxylation of CA, producing a compound known as 3-(3-hydroxyphenyl)-propionic acid that has stronger antioxidant activity than CA[45]. Sulfotransferases, uridine diphosphate-glucuronosyltransferases, and catechol-o-methyltransferases catalyze three main enzymatic conjugation processes of sulphation, glucuronidation, and methylation of CA, respectively, that occur immediately after absorption. This increases the hydrophilic properties of CA, thus reducing its toxicity and speeding up elimination. The liver and kidney are the major sites of CA metabolism. The primary elimination route of CA (5.9% to 27%) is via urine[43-45].

ANTI-DIABETIC EFFECTS OF CA

DM is characterized by hyperglycemia, altered lipid and carbohydrate metabolism, and oxidative stress[1,2,46]. The successful control of high blood sugar levels with natural polyphenols may be significant in minimizing diabetic complications, particularly micro- and macro-vascular disorders. Plant products used in traditional medicine constitute a potential alternative for effective control of diabetes, owing to their affordability, high efficacy, and minimal negative effects[47-49]. CA is a natural compound that is known to promote insulin secretion, inhibit α-amylase and β-glucosidase, prevent sodium-dependent glucose transporter-1 from absorbing glucose in the gut, and lower hepatic glucose output. Besides its anti-diabetic efficacy, CA also modifies the microbiome, facilitates insulin-dependent glucose uptake, activates adenosine monophosphate-activated protein kinase, and has immunomodulatory, antimicrobial, hypocholesteremic, and antioxidant properties[36,49]. Experimentally, in streptozotocin (STZ)-induced diabetic rats, and Balb/c and C57BL/KsJ-db/db mice, CA exhibited potential antihyperglycemic effects along with antioxidant and anti-inflammatory properties[50,51]. CA may exert its protective effects by activating and safeguarding intracellular antioxidant enzymes, and by transferring hydrogen atoms and single electrons, as well as by chelating metal ions[52]. In addition, CA helps to upregulate the transcription factor nuclear factor erythroid2-related factor 2 (NrF2) which controls the expression of over 200 genes involved in the cellular antioxidant and immune regulatory mechanism by binding with antioxidant response elements, which is also linked with the detoxification of xenobiotics. CA also regulates β-cell and adipocyte GLUT4 functions, increases activity of glucokinase in hepatocytes, inhibits glucose-6 phosphatase and phosphoenolpyruvate carboxykinase, and reduces glycosylated haemoglobin, thus resulting in controlled DM. CA aids in enhancing the utilization of glucose and glycogen synthases. This leads to reduced cholesterol biosynthesis and prevention of lipogenesis. CA suppresses iron-induced elevation of cholesterol and improves the levels of plasma insulin, C-peptide, and leptin[53]. In a study with STZ-induced diabetic rats, a significant decrease in malondialdehyde (MDA) level and SOD and CAT activities was observed in the liver, retina, and heart, post CA treatment. Insulin-like growth factors (IGFs) are known to be associated with the progression of DM where reduced serum IGF-I levels have been linked with poor glycemic control in DM, while elevated plasma IGF-II levels have been linked to the progression of DM[54,55]. In STZ-induced diabetic rats, the effects of CA administration led to amelioration of changes in gene expression as well as changes in the levels of IGF-I and IGF-II in the blood, liver, heart, and kidney[35,56].

ROLE OF CA IN DIABETES-ASSOCIATED COMPLICATIONS

Diabetic foot and wound healing

Chronic wounds below the ankle or foot lesions in diabetic patients that penetrate the dermis layer are known as diabetic foot ulcers (DFU)[57]. People with diabetes have a lifetime risk of developing foot ulcers in 25% cases, which may lead to 50%–70% of total non-traumatic amputations[58-61]. In recent years, amputation rates have increased significantly, which in turn has raised the rate of morbidity and death[62-65]. The wound healing cascade in diabetic patients is often hindered and delayed due to high blood sugar levels[66]. Hyperglycemia leads to a series of events such as formation of AGEs, non-enzymatic glycosylation, activation of the polyol pathway and the diacylglycerolprotein kinase C pathway, and hyperactivity of the hexosamine pathway[67,68]. These alterations are linked to a prolonged inflammatory phase causing stiffening of endothelial walls, which makes it challenging for blood to pass via tiny arteries near the surface of the incision[69]. As a result, there is also a lack of oxygen release and nutrition at the wound site, causing further elevation of blood sugar levels in the wound area. Therefore, the wound healing cascade is prolonged, leukocyte migration is reduced, and macrophage introduction is delayed[70]. Additionally, hyperglycemia also activates an inflammatory reaction by triggering NF-κB light-chain-enhancer of activated B cells[71,72]. Moreover, oxidative stress, dyslipidemia, and insulin resistance play a significant role in the development of DFU[73,74]. Thus, management of all these factors is crucial for the treatment of DFU.

Studies have shown that low glycemic index of CA and its derivatives is mainly responsible for their antidiabetic, antioxidant, and anti-inflammatory properties which aids in managing foot ulcers[75-78]. An early study on STZ-induced diabetic mice revealed that topical administration of propolis is well tolerated and aids in healing of human DFU[79]. CAPE increases wound contraction and re-epithelialization by reducing oxidative stress and accelerates cutaneous wound healing, which is mediated by its antioxidant action[80,81]. In another study on diabetic mice, topical application of propolis was found to stimulate the release of vascular endothelial growth factor (VEGF) in smooth muscle cells and facilitate the relaxation of arteries via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway which accelerated the healing of cutaneous diabetic wounds in mice[82,83] (Figure 3 and Table 1).

Figure 3 Mechanism of diabetic wound healing mediated by caffeic acid. Hyperglycemia leads to formation of advanced glycation end products (AGEs), hypoxia, and inflammation at the site of injury. Caffeic acid stimulates the inflammatory cascade which inhibits the formation of AGEs and elevates the levels of vascular endothelial growth factorand insulin-like growth factors. This results in vascular angiogenesis and re-epithelialization at the site of injury. VEGF: Vascular endothelial growth factor; IGFs: Insulin-like growth factors; TNF-α: Tumor necrosis factor-α; IL: Interleukin.

Table 1 Protective effects of caffeic acid and derivatives in diabetes and associated complications.

Type of study/condition	Dose	Mode of action	Ref.
STZ-induced diabetic mice	Topical administration of propolis at 20 μL	Healing of human DFU	[79]
	CAPE at 5 μmol/kg and 10 μmol/kg	Increased wound contraction and re-epithelialization by reducing oxidative stress	[80,81]
Diabetic mice with DFU	Topical application of propolis	Stimulated VEGF and activated NO/cGMP pathway	[82,83]
Diabetic mice with renal damage	CA at 5%	Decreased AGEs, IL-1b, and IL-6, and reduced activity of renal AR and SDH.	[92]
STZ-induced diabetic mice, nephropathy	CA at 10-50 mg/kg	Modulation of autophagy pathway	[93]
	CA at 40 mg/kg	Improved renal parameters, and downregulated the expression of miR-636	[94]
	CAPE and CAPE-pNO2 at 20 μmol/kg/d	Inhibited inflammation through the Akt/NF-κB pathway and prevented renal fibrosis through the TGF-β/Smad pathway	[95]
Diabetes induced in HUVECs	CAPE treatment at 3-10 μM	Reduced VEGF-induced angiogenesis	[96]
STZ-induced diabetic rats, retinopathy	CAF6 and CAF12 at 250 mM	Modulation of ERK1/2 and protein kinase-B/Akt signaling pathways	[98]
STZ-induced diabetic rats, neuropathy	CAPE at 10 μM/kg/d	Inhibition of iNOS enzyme	[102]
Alloxan-induced diabetic mice, CVD	CA at 50 mg/kg	Reduced atherogenic indices such as TG, LDL-c, VLDL-c, and TC	[53]
	CA at 2%	Improved glycemic control and lipid metabolism, increased plasma antithrombin-III and protein C activities, and decreased MDA, IL-β, IL-6, and TNF-α levels	[34]
STZ-induced T1D rat model, CVD	CAPA at 3 and 15 mg/kg	Reduced myocardial infarction and amelioration of cardiac dysfunction	[103]

STZ: Streptozotocin; DFU: Diabetic foot ulcer; CAPE: Caffeic acid phenethyl ester; VEGF: Vascular endothelial growth factor; NO: Nitric oxide; cGMP: Cyclic guanosine monophosphate; CA: Caffeic acid; AGEs: Advanced glycation end products; IL: Interleukin; AR: Aldol reductase; SDH: Sorbitol dehydrogenase;CAPE-pNO2: Caffeic acid para-nitro phenethyl ester; NF-κB: Nuclear factor-κB; TGF-β: Transforming growth factor-β; HUVECs: Human umbilical vein endothelial cells; ERK: Extracellular signal regulated kinase; iNOS: Inducible nitric oxide synthase; TG: Triacylglycerol; LDL-c: Low density lipoprotein cholesterol; VLDL-c: Very low-density lipoprotein cholesterol; TC: Total cholesterol; T1D: Type1 diabetes; MDA: Malondialdehyde; TNF-α: Tumor necrosis factor-α; CVD: Cardiovascular disorder;CAPA: Caffeic acid phenethyl amide.

Diabetic nephropathy

Diabetic nephropathy is a consequence of prolonged uncontrolled DM causing damage to the renal blood vessel clusters. The pathogenesis of diabetic nephropathy and other complications of diabetes have been linked to non-enzymatic glycation, with the formation of AGEs, also recognized as Maillard reaction products. These AGEs include glycated haemoglobin, glycated albumin, pentosidine, and carboxymethyllysine (CML)[84-86]. In addition, disruption in Th1-Th2 cytokine balance and over-production of pro-inflammatory cytokines result in increased inflammatory stress in diabetic patients, which further accelerates diabetic nephropathy[87,88]. Early investigations have suggested that CA lowers blood glucose by modulating the polyol pathway. Aldose reductase (AR) is the first and rate-limiting enzyme in the polyol pathway that reduces glucose to sorbitol, which could be further metabolised to fructose by the enzyme sorbitol dehydrogenase (SDH)[89,90]. The generation of AGEs was increased by the flux through SDH and an elevated fructose level, which enhanced diabetes-induced microvascular abnormalities[91] (Figure 4). In diabetic mice, CA significantly decreased the production of AGEs, inflammatory cytokines like IL-1b and IL-6, levels of plasma HbA1c, urinary glycated albumin, renal CML, pentosidine, sorbitol, and fructose, and considerably reduced the activity of renal AR and SDH along with suppression of renal AR mRNA expression[92]. In an in vivo study with STZ-induced diabetic rats, CA in a dose range of 10-50 mg/kg attenuated diabetic nephropathy via modulation of autophagy pathway by inhibiting autophagy-regulating miRNAs[93]. In another study on STZ-induced diabetic rats, oral treatment of CA at 40 mg/kg mitigated renal damage and significantly reduced fasting blood glucose, cholesterol, and triglyceride in diabetic rats. CA treatment also improved histological parameters in the diabetic kidney and downregulated the expression of miR-636[94]. In another study in STZ-induced diabetic mice, intraperitoneal treatment with CA derivatives CAPE and CA para-nitro phenethyl ester (CAPE-pNO₂) at 20 μmol/kg/d resulted in improved renal biochemical parameters such as decreased serum creatinine, MDA, 24-h albumin excretion, blood urea nitrogen, myeloperoxidase levels, and SOD activity in diabetic mice. CAPE and CAPE-pNO₂ also inhibited inflammation via the Akt/NF-κB pathway and prevented nephropathy through the transforming growth factor-β/Smad pathway[95] (Table 1).

Figure 4 echanism of protective action of caffeic acid in diabetic nephropathy and retinopathy. Hyperglycemia induces formation of advanced glycation end products and reactive oxygen species in renal and retinal tissues, which in turn causes mitochondrial dysfunction by inhibiting antioxidant enzymes such as manganese superoxide dismutase, glutathione peroxidase, catalase, and activates the production of inflammatory cytokines like tumor necrosis factor-α, interleukin-6, nuclear factor-κB, polyol, and hexosamine signaling pathways. Caffeic acid increases the levels of antioxidant enzymes and suppresses inflammatory response, thus protecting the tissues from diabetic nephropathy and retinopathy. AGEs: Advanced glycation end products; ROS: Reactive oxygen species; Mn-SOD: Manganese superoxide dismutase; CAT: Catalase; SOD:Superoxide dismutase; GSH: Glutathione; NF-κB: Nuclear factor-κB; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α.

Diabetic retinopathy

Long-term DM results in diabetic retinopathy characterized by aberrant retinal blood vessel proliferation and microvascular retinal alterations, resulting in partial vision loss or even complete blindness. One of the major factors causing diabetic retinopathy is VEGF-driven angiogenesis. In a study using human umbilical vein endothelial cells (HUVECs), CAPE treatment in the dose range of 3-10 μM decreased VEGF-induced angiogenesis, indicating possible positive effects in the treatment of diabetic retinopathy[96] (Figure 4). In another study, HUVECs treated with CAPE at 5-20 μg/mL exhibited a reduction of VEGF-induced neovascularization and proliferation, tube formation, and migration. The protective efficacy of CAPE can be attributed to the inhibition of VEGF-induced VEGF receptor-2 activation and associated downstream pathways[97]. An in vivo study in a STZ-induced diabetic rat model demonstrated the protective efficacy of CA hexyl (CAF6) and dodecyl (CAF12) amide derivatives in diabetic retinopathy. Treatment with CAF6 and CAF12 at a dose of 250 mmol/L led to increased retinal SOD levels, and improved thickness of the whole retinal layer, outer nuclear layer, and ganglion cell count.The CA derivatives ameliorated diabetic retinopathy via modulation of the extracellular signal regulated kinase (ERK)1/2 and protein kinase-B/Akt signaling pathways[98] (Table 1).

Diabetic neuropathy and cardiovascular complications

The brain is another organ which is adversely affected by prolonged uncontrolled hyperglycemia, and cerebral dysfunction in diabetic patients is known to be a multifactorial process[99]. Free radical-mediated oxidative stress induced by hyperglycemia plays an important role in the pathogenesis of diabetic neuropathy[100]. It stimulates the production of the inflammatory cytokine TNF-α and promotes the expression of NF-κB[101]. The NO radical in the central nervous system acts as an important regulator leading to the generation of RNS via the enzyme iNOS and results in elevated oxidative stress in brain. In an in vivo study, STZ-induced diabetic rats post intraperitoneal treatment with CAPE at a dose of 10 μM/kg/day showed reduced NO radical and lipid peroxidation, and increased activities of antioxidant enzymes such as SOD, CAT, and GPx in the rat brain. In addition, CAPE was shown to inhibit the activity of iNOS enzyme, thus preventing excess production of RNS[102].

Hyperglycemia combined with dyslipidemia, oxidative stress, and inflammation cause CVD such as hypertension, cardiac myopathy, and atherosclerosis. DM-mediated CVD is characterized by elevated levels of triacylglycerol (TG), low density lipoprotein (LDL), very low-density lipoprotein (VLDL), and total cholesterol (TC). Atherogenic dyslipidemia in diabetic patients leads to increased risk of cardiac failure. Studies on alloxan-induced diabetic mice have revealed that CA at a dose of 50 mg/kg acts as a potent agent in controlling hyperglycemia and reducing atherogenic indices such as TG, LDL-c, VLDL-c, and TC. Thus, successful restoration of lipid and glucose metabolism parameters in mice by intraperitoneal CA administration led to improved cardiac function[53]. In another study, diabetic mice when orally fed2% CA, exhibited improved glycemic control and lipid metabolism. CA treatment led to a significant increase in plasma antithrombin-III and protein C activities, and decrease in MDA, IL-β, IL-6, and TNF-α levels[34]. Studies on a STZ-induced T1DM rat model demonstrated that intraperitoneal pre-treatment with CA phenethyl amide at doses 3 and 15 mg/kg led to reduced myocardial infarction and amelioration of cardiac dysfunction[103] (Table 1).

BIOAVAILABILITY AND DRUG DELIVERY OF CA

Plant-derived natural products including CA have several applications in the treatment of a wide range of diseases. However, there are many limitations that come in the way of using phytochemicals as alternative medicine. To ascertain the optimum utilization of plant-derived compounds in clinical investigations, it is important to design novel carriers for the delivery of natural products[104,105]. The use of CA as a pharmaceutical is constrained by a number of physicochemical and pharmacokinetic factors including poor water solubility and lack of specific tissue targeting[106]. Studies have also shown that CA has low oral bioavailability (14.7%) and low intestinal absorption (12.4%) in a rat model[107]. Therefore, numerous nanoparticles (NPs) have been created for the delivery of CA and related compounds in disease therapy with positive outcomes, including polymeric NPs, metal NPs, carbon nanomaterials, and lipid nanostructures[108,109]. The use of NPs for targeted delivery of CA is well reported. The combinations of gold and iron NPs (Au-Fe₃O₄) with CA, quercetin, and 5-fluorocytidine have been formulated for use in breast cancer treatment. Studies related with formulation and development of CA-NPs are mostly targeted for cancer therapy. The release of quercetin and CA from these nanostructures inhibits lactate secretion and prevents glycolytic reprogramming[106,107]. Additionally, NPs have also been designed for CAPE delivery for the treatment of cancer. In a recent study, methoxy poly (ethylene glycol)-b-poly(-caprolactone) was used to create polymeric nanostructures, which were subsequently loaded with CAPE[45,110,111]. There is still much work to be done in terms of NP formulation and design. Hence, further studies are required to examine the potential functions of NPs of CA for better delivery, in treatment of other diseases including DM.

CLINICAL TRIALS AND FUTURE PROSPECTS

Phytometabolites are pharmacologically active compounds and their clinical applications are constantly increasing[45,112,113]. Several reports have shown that approximately one fourth of all clinical compounds used as drugs are derived from natural products[114,115]. The pharmacological action of CA and its derivatives, particularly CAPE, as hepatoprotective, reno-protective, antioxidant, anti-diabetic, anti-inflammatory, and anticancer agents have been well documented. The activity of antioxidant enzymes such as SOD, CAT, and HO-1 is positively modulated by CA. CAPE treatment leads to protection against oxidative stress-mediated diabetic complications by regulating the transcription factors NF-κB, Nrf2, and COX-2 and associated molecular pathways. Moreover, CAPE shows notable efficacy in both in vitro and in vivo diabetic models with no substantial negative effects. CA exerts anti-diabetic efficacy in vitro and in vivo via reduced VEGF angiogenesis and decreased MDA, TNF-α, IL-β, IL-6, and other inflammatory and oxidative stress markers.

In order to examine the clinical trials’ data with respect to CA and related compounds in diabetic patients, we searched the largest clinical trial database at ‘https://clinicaltrials.gov’. No search results were obtained with the keywords ‘CA/CAPE and diabetes’. Since propolis found in beehive is a major source of CAPE, therefore we searched with keywords ‘propolis and diabetes’ on the database. Three studies were found in which propolis was administered orally or applied topically to diabetic patients. The results are summarized in Table 2.

Table 2 List of clinical trials conducted on propolis against diabetes mellitus.

Number	Treatment	Condition	Outcome	ClinicalTrials.gov Identifier, phase, and status of trial
1	Propolis 300 mg twice a day for 12 wk	Type 2 DM	Propolis administration modified the glycemic control in patients with type 2 DM	ClinicalTrials.gov
				Identifier: NCT03416127
				Phase: 2
				Status: Completed
2	Propolis 400 mg for 6 mo, after performing scaling and root planing	Type 2 DM, periodontitis	Improvement in HbA1c, FPG, serum CML, and changes in periodontal parameters	ClinicalTrials.gov
				Identifier: NCT02794506
				Phase: 4
				Status: Completed
3	Propolis spray at the site of injury	Diabetic foot ulcer	Propolis possesses anti-inflammatory and antioxidant effects and its topical application is well tolerated, improving the healing of human diabetic foot ulcer	ClinicalTrials.gov
				Identifier: NCT03649243
				Phase: Not applicable
				Status: Completed

DM: Diabetes mellitus, HbA1c: Hemoglobin A1c, FPG: Fasting plasma glucose, CML: Carboxymethyllysine.

CA has potential application in the treatment of several diseases including diabetes and associated complications. However, more in vivo research needs to be done for a better understanding of the mode of action of CA in DM and associated problems, particularly the role of cytoprotective enzymes like HO-1. Additionally, pharmacokinetic studies are required to entirely understand the metabolic pathway of CA post oral administration. Thus, further clinical investigations in humans are needed to determine the pharmacological potential of CA in major illnesses like diabetes.

CONCLUSION

DM has emerged as one of the most common metabolic disorders worldwide which can lead to other complications such as retinopathy, nephropathy, neuropathy, and foot ulcers. Free radical-induced oxidative stress is one of the primary factors causing DM. CA is a natural polyphenol obtained from various fruits and vegetables. CA and its derivatives act as an antioxidant to regulate the signaling pathways involved in lipid and carbohydrate metabolism. CA also exerts anti-diabetic effects by modulation of inflammatory cytokines and transcription factors. Furthermore, novel delivery strategies are being used for transport of CA to enhance its bioavailability, which has enabled the widespread use of CA in various disease therapies.

ACKNOWLEDGEMENTS

Risha Ganguly and Ramesh Kumar acknowledge financial support from University Grants Commission/Council of Scientific and Industrial Research, New Delhi, India in the form of UGC/CSIR-Senior Research Fellowships. Shiv Vardan Singh acknowledges UGC for Dr DS Kothari Fellowship. Kritika Jaiswal acknowledges financial support from University Grants Commission, New Delhi, India in the form of UGC-CRET Fellowship. All the authors also acknowledge DST-FIST and UGC-SAP facilities of the Department of Biochemistry, University of Allahabad, Prayagraj, India.