Oleanolic, Ursolic, and Betulinic Acids as Food Supplements or

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Oleanolic, Ursolic and Betulinic Acids as Food Supplements or Pharmaceutical agents for Type 2 Diabetes – Promise or Illusion? Filomena S.G. Silva, Paulo J. Oliveira, and Maria Fatima Duarte J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06021 • Publication Date (Web): 25 Mar 2016 Downloaded from http://pubs.acs.org on March 29, 2016

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Journal of Agricultural and Food Chemistry

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Oleanolic, Ursolic and Betulinic Acids as Food Supplements or Pharmaceutical agents for

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Type 2 Diabetes – Promise or Illusion?

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Filomena S.G. Silva†, Paulo J. Oliveira‡, Maria F. Duarte†*

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Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja),

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ABSTRACT

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Oleanolic (OA), ursolic (UA) and betulinic (BA) acids are three triterpenic acids (TAs) with

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potential effects for treatment of type 2 diabetes. Mechanistic studies showed that these TAs act

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as hypoglycemic and anti-obesity agents mainly through (i) reducing the absorption of glucose;

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(ii) decreasing endogenous glucose production; (iii) increasing insulin sensitivity; (iv) improving

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lipid homeostasis; and (v) promoting body weight regulation. Besides these promising beneficial

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effects, it is believed that OA, UA and BA protect against diabetes-related comorbidities due to

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their anti-atherogenic, anti-inflammatory and anti-oxidant properties. We also highlight the

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protective effect of OA, UA and BA against oxidative damage which may be very relevant for

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the treatment and/or prevention of T2DM. In the present review, we make an integrative

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description of the anti-diabetic properties of OA, UA and BA, evaluating the potential use of

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these TAs as food supplements or pharmaceutical agents, in order to prevent and / or treat

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T2DM.

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KEYWORDS: Oleanolic acid, ursolic acid, betulinic acid, anti-diabetic, hepatic disease,

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cardiovascular disease, anti-obesity.

Apartado 6158, 7801-908 Beja, Portugal. *Corresponding Author Phone: + 351 284314399, email: [email protected] CNC, Center for Neuroscience and Cellular Biology, UC-Biotech Building, Biocant Park, University of Coimbra, 3060-107 Cantanhede, Portugal.

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INTRODUCTION

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Diabetes is a chronic metabolic disorder that is currently assuming epidemic proportions.

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According to statistics, it was estimated that about 9% of the adult worldwide population had

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diabetes in 2014, with 90% among those having type 2 diabetes mellitus (T2DM).1 Type 2

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diabetes is characterized by hyperglycemia resulting from insulin resistance, as well as

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insufficient insulin secretion by pancreatic β-cells. Accumulating evidence demonstrates that

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T2DM is associated with obesity,2 as well as to development of several comorbidities, including

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hepatic, cardiac and renal disorders.3 Management of T2DM requires an integrated approach,

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which includes hyperglycemia and obesity treatments, as well as prevention of diabetic

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comorbidities. However, the currently available anti-diabetic drugs have limited efficacy and/or

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safety concerns, and only temporarily improve blood glucose levels, failing in the treatment of

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obesity, as well as in the prevention of diabetes complications. Therefore, the identification of

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new therapeutic agents, which may have the potential to prevent, or treat diabetes and its related

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comorbidities, are an unmet need.

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Consumption of different natural compounds is known to result in anti-diabetic effects, offering

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exciting possibilities for the future development of successful therapies. Terpenoids are a diverse

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group of phytochemical agents derived from squalene or related acyclic and cyclic 30-carbon

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precursors, which are currently receiving attention because of their wide range of biological

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activity.4 An increasingly number of publications has focused on pentacyclic triterpenoids,

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lupane, oleanane and ursane-type structures, particularly oleanolic (3β-hydroxyolean-12-en-28-

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oic, OA), ursolic (3β-hydroxyurs-12-en-28-oic, UA) and betulinic (3β-hydroxy-lup-20-en-28-

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oic, BA) acids (Figure 1) due to their anti-diabetic properties (reviewed in5-7).


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Oleanolic, ursolic and betulinic acids have several important biological activities, including anti-

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tumoral, anti-inflammatory and anti-oxidant effects,4 as well as hepato-,8-11 cardio-12-14 and

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nephroprotective8,

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potential application for T2DM management, and its associated comorbidities. The biological

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effects mainly result from their anti-hyperglycemic,16-19 anti-hyperlipidemic,20-23 anti-

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atherogenic,24-26 anti-oxidant27-29 and anti-inflammatory27,

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that OA, UA and BA, or plants extracts rich in those TAs reduce the absorption and uptake of

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glucose, lower endogenous glucose production, increase insulin biosynthesis, secretion and

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sensitivity, and protect against diabetic complications.6, 7 These protective effects occur without

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noticeable hepatotoxicity, at the dose used in the experimental studies, as indicated by the

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decreased values of aspartate aminotransferase and alanine aminotransferase measured.21,

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This relevant fact is contrary to most currently used anti-diabetic drugs, including sulfonylureas,

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α-glucosidase inhibitors, biguanides and thiazolidinediones, which can cause liver toxicity.33, 34

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The low toxicity profile of these compounds has also been evidenced in several in vivo studies,

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where OA (10-1700 mg/kg)35-39, UA (25-2500 mg/kg)10, 40, 41 and BA (25-50 mg/kg)42 protected

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the liver from oxidative damage, at concentrations equivalent or higher to those used in diabetic

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studies, when administered to rodents pre-treated with different hepatotoxicants. The protective

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effect of OA, UA and BA against oxidative damage may be critical for their use in the treatment

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and prevention of diabetes and their comorbidities.

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In this context, in the present unique review, we make an integrative description of the

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hypoglycemic, anti-obesity properties of OA, UA and BA, as well as their effects on diabetic

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comorbidities in the management of T2DM. We here underline the main targets of action and

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limitations of these compounds, exploring the current knowledge and open questions. The

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roles. The three triterpenic acids (TAs) described above also have a

30, 31

action. Published data indicate

22, 32



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present work focuses on the main issues that research in this field should take into account,

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emphasizing what should be clarified in the future, and evaluating the potential use of these TAs

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as food supplements or pharmaceutical agents, in order to prevent and / or treat T2DM.

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MECHANISMS BEHIND HYPOGLYCEMIC EFFECTS

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Numerous research described hypoglycemic effects for OA, UA and BA by using in vitro and in

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vivo models. The description of hypoglycemic effects of these three TAs is summarized in Table

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1.

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Absorption and uptake of glucose:

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Inhibition of carbohydrate hydrolysis, and down-regulation of glucose transporters in the

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gastrointestinal tract decreases post-prandial hyperglycemia,43, 44 suppressing, at least in part, the

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development of T2DM.45

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Oleanolic, ursolic and betulinic acids inhibit pancreatic α-amylase activity in vitro ([TAs]: 5–50

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µg/mL ≈ 10–100 µM),46,

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(HFD)-induced obese mice.32, 48, 49 Additionally, other authors also showed that these three TAs

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have also the capacity to decrease α-glucosidase (IC50: 0.1–231 µM)47, 50-54 activity in vitro. Also,

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OA inhibits human pancreatic α-amylase activity (IC50 0.1 mg/mL ≈ 200 µM),55 and decreases

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post-prandial hyperglycemia in diabetic rats by inhibiting the activities of α-amylase (IC50 3.60

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µM), and α-glucosidase (IC50 12.40 µM), and simultaneously down-regulating the expression of

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sodium-dependent glucose co-transporter (SGLT-1) and glucose transporter -2 (GLUT-2) in the

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small intestine16 (Table 1). Taken together, these results suggest that inhibition of α-amylase and

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α-glucosidase, as well as down-regulation of glucose transporters by the above mentioned TAs

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can delay glucose absorption in the small intestine in diabetes, consequently preventing post-

47

and in vivo (5-20 mg/kg/day during 15 weeks) on high-fat diet


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prandial plasma glucose increase. However, the reduced number of animal testing and the lack of

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human studies raise questions about the true effectiveness of these compounds in vivo,

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highlighting the need for more studies in living systems.

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Endogenous glucose production and glycogen synthesis:

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T2DM increases endogenous glucose production and reduces the synthesis of glycogen by

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increasing gluconeogenesis, glycogenolysis, and by reducing glycogenesis.56

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Oleanolic, ursolic and betulinic acids (IC50: 14.9, 9 and 43 µM, respectively) inhibit glycogen

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phosphorylase (GP),57 the rate-limiting step of glycogenolysis, which decreases glycogenolysis

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and also stimulates glycogen synthesis.58 Moreover, UA treatment (5 mg/kg/day or 0.01-0.05%,

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w/w) increases glycogen content by increasing hepatic glucokinase activity in vivo mice

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models,17,

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glucose levels by inhibiting hepatic glucose-6-phosphatase (G6Pase) activity,17, 20, 59 the ultimate

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step in glucose production.61 In line with increased hepatic glycogen, UA (2 mg/kg/day) also

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increases glycogen deposition in healthy rats,62 most likely by promoting the phosphorylation

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and inactivation of glycogen synthase kinase-3 (GSK-3), an enzyme which inhibits glycogen

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synthesis pathway.63 Additionally, OA, UA and BA (10 µM) stimulate glucose uptake and

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glycogen synthesis in human HepG2 hepatoma cells via 5′ AMP-activated protein kinase

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(AMPK)-GSK-3β pathway.64 Once activated, AMPK, an energy sensor that regulates cellular

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metabolism, leads to the inhibition of glucose production by increasing GSK-3β

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phosphorylation. Activation of AMPK decreased gluconeogenesis by regulating gluconeogenic

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targets, such as phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase.65 In vivo treatment

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with OA (20 mg/kg/day) decreased G6Pase and PEPCK protein contents,18 which may result in

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lower gluconeogenesis rates. A similar in vitro study, but using BA instead (10 and 20 µM) 5

20, 59

a key regulatory step in liver glycogen formation,60 and also decreases blood

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demonstrated an effect on hepatic gluconeogenesis by decreasing PEPCK and G6Pase mRNA,

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resulting from AMPK activation19. Another study also evidenced that once activated by BA, the

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AMPK pathway promoted glucose utilization and glucose transport across the cell membrane, by

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induction of glycolysis and by up-regulation of GLUT-1 and -2 protein expression in mouse

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embryonic fibroblasts. This study also demonstrated that glycolysis stimulation by BA was

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accompanied by a reduction of glucose oxidation66 (Table 1). According to the results, OA, UA

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and BA reduced glucose availability in the body by limit endogenous glucose production, also

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promoting glycogen synthesis in T2DM, contribute to the hypoglycemic effect of these

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compounds. However, future studies are warranted to confirm these results using more in vivo

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assays, including in humans.

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Insulin resistance:

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T2DM mainly results from insulin resistance and β-cell dysfunction. The improvement of insulin

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sensitivity represents a good strategy to prevent T2DM development.67

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Numerous studies have supported the notion that OA, UA and BA improve insulin responses by

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acting as insulin secretagogue and insulinomimetic agents in different contexts.68-73 According to

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different reports, the three TAs interfere with insulin biosynthesis, secretion and sensitivity

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involving

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phosphatases,74-79 by stimulating phosphatidylinositol 3-kinase-dependent (PI3KB)/protein

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kinase Akt pathway,72, 73, 80-82 by acting as TGR-5 receptors 83-86, by improving the survival of β-

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cells68, 69, 87 and/or by acting as anti-inflammatory and anti-oxidant agents 27, 28, 31.

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Protein tyrosine phosphatases and PI3KB/Akt pathway: Several in vitro studies provided

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evidence that OA, UA and BA directly inhibited protein tyrosine phosphatase 1B (PTP-1B),74-79

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thus improving insulin resistance in T2DM and obesity.88-91 PTP-1B is a molecule that 6

multi-target

mechanisms,

mainly

by inhibiting

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different

protein

tyrosine

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negatively regulates insulin signaling. Insulin regulates glucose homeostasis through: i) binding

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to its receptor to initiate a signaling cascade; ii) activating and promoting phosphorylation of

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insulin receptor substrate proteins; and iii) mediating the PI3K/Akt pathway.92, 93 Akt pathway

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activation facilitated glucose uptake into adipose tissue, cardiac muscle and skeletal muscle, by

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mediating the translocation of glucose transporter -4 (GLUT-4) to the plasma membrane, and by

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inhibiting gluconeogenesis.5 However, PTP-1B inhibited the PI3K/Akt signaling pathway

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resulting in insulin resistance. The mechanism is thought to result from inhibition of GLUT-4

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translocation to the plasma membrane, decreasing glucose re-uptake and gluconeogenesis.

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Therefore, the direct inhibition of PTP-1B by OA, UA and BA may improve insulin sensitivity.

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Nevertheless, it was described that UA acted directly in the insulin receptor (IR).80 In vitro

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studies performed by Jung et al. in cultured adipocytes and Chinese-hamster ovary cells

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expressing human IR80 showed that UA is an insulin sensitizer, leading to an increase in IRβ

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auto-phosphorylation and to a subsequent activation of downstream PI3K signaling pathway.

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The activation of this pathway would result in phosphorylation and inactivation of GSK-3,

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leading to an increase of glycogen synthesis,63 as described before. Also, OA increased IR and

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insulin receptor substrate-1 (IRS-1) mRNA and upregulated total IRS-1 protein expression in

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adipose tissue insulin resistance rats, suggesting that this compound improves insulin resistance

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via the IRS-1/PI3k/Akt pathway.81 Regarding UA (10-20 µM), this molecule was described to

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increase insulin sensitivity, by promoting GLUT-4 translocation in 3T3-L1 adipocytes,80,

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probably by activating the PI3K pathway.82 In a similar way, in vivo studies showed that

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treatment with UA or BA (10 mg/kg) increases glycogen content and glucose uptake in muscle

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by acting as insulin secretagogue and insulinomimetic agents via PI3K and MAPK, stimulating

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GLUT-4 synthesis and translocation in muscle.72, 73 All these effects are summarized in Table 1.

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TGR5 receptors: Accumulating evidence suggest that OA,83 UA and BA84 act in vitro as

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selective TGR5 agonists (EC50 1.04-2.25 µM). TGR5 is a G-protein-coupled receptor identified

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as the first cell surface receptor involved in energy homeostasis and which is activated by bile

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acids. Its activity ameliorates insulin resistance.94 Indeed, OA (10-30 µM) activated TGR5 in

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enteroendocrine cells, leading to glucagon-like peptide-1 (GLP-1) release.85 GLP-1 is a potent

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glucose-dependent insulin-stimulating hormone,95 that stimulates β-cell proliferation and pro-

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insulin gene expression,96 and which inhibits glucagon expression.97 Oleanolic acid (10-50 µM)

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also activated TGR5 in pancreatic β-cells, leading to TGR5-mediated glucose-stimulated insulin

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release 86 (Table 1). Contradictorily, a human phase I clinical trial showed that the hypoglycemic

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effect of intraduodenal OA perfusions (1 mM) was not accompanied by the release of GLP-1 in

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healthy male volunteers (University Hospital, Basel, Switzerland, coded NCT01674946).

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Improvement of β-cell survival: Current evidence shows that decreased function of β-cells

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always precedes the development of T2DM and induces profound metabolic dysfunction.

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Therefore, β-cells function preservation or recovery can be an effective therapeutic strategy to

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prevent or treat T2DM.98 It is known that UA (0.05%, w/w) preserved the integrity of β-cells,68

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and islet architecture69 in the pancreas, contributing to prevent the metabolic dysfunction of β-

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cells and consequently the development of T2DM. Oleanolic acid (0.5 mg/day during 2 days)

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prolonged the survival of transplanted islets due to its anti-inflammatory, immune-regulatory and

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anti-oxidant properties. Beneficial effects were suggested to be mediated by decreased γ-

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interferon-inducible protein 10 and IL-4 in serum, the number of T cells secreting γ-interferon,

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IL-4, IL-7, and IL-2, and the infiltration of CD4 and CD8 cells, as well as decreased reactive

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oxygen species (ROS) production,87 supporting the notion that the anti-inflammatory and anti-


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oxidant effects of these compounds can also contribute to preserve the integrity of β-cells and

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consequently the development of T2DM.

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Anti-inflammatory and anti-oxidant properties:

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Different studies demonstrated that OA,99 UA30 and BA31 down-regulate nuclear factor kappa B

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(NF-κB) signaling pathway in different cell models, which may decrease several inflammatory

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cytokines, and consequently improve insulin response in diabetic patients100, supporting the

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notion which the anti-inflammatory effects of these compounds may in turn prevent T2DM.

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Similarly, it was also described that the reduction of inflammatory cytokines tumor necrosis

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factor -α (TNF-α) and IL-6 mediated by reduction of NF-kB promoted by OA, may in turn

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down-regulate the expression of IRS-1 and GLUT-4 in HepG2 cell line,27 evidencing the crucial

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role between the reduction of oxidative stress and the suppression of inflammatory response in

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the prevention of T2DM. Another study also suggested that OA (20 mg/kg/day during 2 weeks)

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improves hepatic insulin resistance by acting as anti-inflammatory, down-regulating IL-1β, IL-6

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and TNF-α1, and as anti-oxidant by increasing superoxide dismutase (SOD), catalase (CAT)

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protein contents and mitochondrial reduced glutathione pool in diabetic mice. In the same study,

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OA also increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression18, which

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promoted the transcriptional induction of several anti-oxidant genes and suppression of

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inflammatory responses101, contributing thus to the improvement of insulin resistance102. The

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authors suggested that this increase of Nrf2 may in turn be responsible for reducing

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mitochondrial redox balance, also improving mitochondrial dysfunction, and also promoting the

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activation of AMPK signaling in diabetic mice18 . Although this study suggested that Nrf-2

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mediated protection may be a key mechanism in improving insulin resistance induced by OA, no



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more studies showed this effect in T2DM. However, the induction of Nrf2-dependent genes was

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detected when OA (22.5-90 mg/kg/day for 3 or 4 days)103-105 and UA (25 and 50 mg/kg/daily for

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one week)41 were administered as hepatoprotective agents against different hepatotoxicants in

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vivo studies. This supported the notion the two TAs suppressed inflammatory and oxidative

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response through the Nrf2 pathway, and perhaps contributed to reduce insulin resistance through

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that same mechanism. Another study also reported that UA (0.125%, 0.25%, 0.5% during 6

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weeks) promoted anti-inflammatory effects by decreasing TNF-α, monocyte chemotactic

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protein-1 (CCL2/MCP-1), IL-1β, IL-2, IL-6 and IL-8, and anti-oxidant effects, mediated by

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increasing SOD, CAT, and glutathione peroxidase (GPx) activities, and by decreasing

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malondialdehyde (MDA), which also indirectly contributed to improve insulin resistance.28 This

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study supported the notion that UA contributed to improve insulin resistance due to its anti-

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inflammatory28,

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inflammatory31 and anti-oxidant properties29, 31 are extensively described there are no studies that

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suggest the direct contribution of those effects in the improvement of insulin resistance. Instead a

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recent study indicated that BA was not able to activate Nrf2 pathway in Chinese hamster ovary

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cells66. Altogether, the data supports the notion that the anti-inflammatory and anti-oxidant

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properties of these compounds contribute, at least in part, to prevent development of T2DM,

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although doubts still exist on the role of Nrf2 regulation.

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30

and anti-oxidant properties28,

40, 41

. Regarding BA, despite its anti-

ANTI-OBESITY EFFECTS AND THEIR MECHANISMS OF ACTION

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In association with improved glucose tolerance and insulin sensitivity, OA, UA and BA are also

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anti-obesity promoters (Table 2).

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Several studies demonstrated the improvement of glucose tolerance being often accompanied by

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alterations in the lipid profile, by decreasing total cholesterol, triglycerides (TG) LDLc contents 10 ACS Paragon Plus Environment

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and by increasing the levels of HDLc in streptozotocin-106 and alloxan-21 -diabetic rats treated

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with OA (60-200 mg/kg/ day, during 40 days). The use of UA and BA (10 mg/kg/day during 15

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days) was associated with decreased glucose, total cholesterol and TG contents in HFD-induced

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obese mice.32, 48, 49 Additionally, treatment with OA, BA (5-20 mg/kg/ day, during 15 weeks) and

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UA (5-10 mg/kg/ day or 0.05% w/w during 5-15 weeks) also reduced body weight, visceral

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adiposity32,

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adipocyte size20 in obese diabetic rodents. Accumulating evidence suggest that the three TAs act

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as anti-obesity agents by reducing lipid absorption,107, 108 improving lipid homeostasis109-114 and

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promoting food intake, and body weight regulation.32,

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humans places into question the true hypolipidemic effects of these compounds, clearly

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highlighting the need for future human studies.

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Lipid absorption:

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Post-prandial hyperlipidemia in diabetes is a risk factor for the development of hyperlipidemia

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complications. Hence, the absorption of dietary lipids by the small intestine plays an important

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role in the regulation of post-prandial hyperlipidemia.

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Ursolic107 and betulinic acids107, 108 exert hyperlipidemic effects, at least in part, by inhibiting

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pancreatic lipase (IC50 values between 15.83 to 21.10 µM), which prevent the absorption of

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lipids from the small intestine. Other studies showed that OA and BA have inhibitory activities

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on human cholesterol acyltransferase-2 (hACAT-2),115 an isoenzyme responsible for cholesterol

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absorption in the intestine.116 When comparing OA and BA, the latter exhibited more potent

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hACAT-2 inhibitory activity, with an IC50 value of 28.8 µM.115 Taken together, these results

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suggest that OA and BA also decrease the lipid absorption process in intestinal mucosal cells

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(Table 2).

48, 49

, free fatty acids22 and hepatic lipid accumulation,20,

48, 49

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restoring the normal

However, the lack of studies in



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Lipogenesis and fatty acid β-oxidation:

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The inhibition of free fatty acids (FFA) synthesis (lipogenesis)117 and increasing fatty acid β-

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oxidation118 are two potential pathways which contribute to the reduction of hepatic fat

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accumulation in T2DM patients. Compounds which inhibit the transcription of sterol-regulatory-

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element-binding protein-1c (SREBP-1c), and consequently mediate the down- regulation of

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hepatic genes involved in lipogenesis, such as acetyl-coA carboxylase (ACC) and fatty acid

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synthase (FAS),119 result in a reduction of FFA synthesis. Additionally, agents that stimulate the

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up-regulation of carnitine palmitoyltransferase-1 (CPT-1),120 acyl-CoA oxidase (ACO)121 and

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peroxisome proliferator activated receptor-α (PPAR-α),122 increase fatty acid β-oxidation. An in

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vivo study showed that OA (25-50 mg/kg/ day) reduced the expression of lipogenic genes

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including ACC,109, 110 glycerol-3-phosphate acyltransferase,110 and FAS,109 thus inhibiting lipid

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synthesis, and consequently the retention of lipids in hepatocytes. Treatment with UA (5

263

mg/kg/day) down-regulated SREBP-1c, FAS and ACC transcripts, while promoting the

264

inhibition of lipogenesis in liver, and increased fatty acid oxidation (by upregulating CPT-1 and

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PPAR-α).22 Other studies described that the hypolipidemic effects of UA in vitro and in vivo are

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primarily achieved by the activation of hepatic PPAR-α, which acts as an antagonist. Once

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activated, PARP-α promotes fatty acid uptake and fatty acid β-oxidation, and decreases

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lipogenesis. Ursolic acid also increased the expression of fatty acid transport protein 4, enhances

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hepatic fatty acid uptake and induced acyl-CoA synthetase-1, CPT-1, and ACO1, improving

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hepatic fatty acid β-oxidation.23, 111 Besides that, UA decreased FAS and SREBP-1c transcripts,

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while inhibiting lipogenesis.23 Interestingly, Jia et al. showed that UA induces hepatic autophagy,

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which could also contribute to the degradation of lipid droplets in hepatocytes, and consequently

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regulate hypolipidemic effects of UA.23 It was also proposed that UA also reduces intracellular 12 ACS Paragon Plus Environment

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fat storage in skeletal muscle cells, by increasing FFA uptake and β-oxidation via activation of

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AMPK pathway and upregulation of uncoupling protein (UCP)-3

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membrane protein involved in mitochondrial uncoupling and fatty acid metabolism124,

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contributing thus to explain also the anti-obesity effects induced by this compound. Interestingly,

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BA (20-40 µM) decreased lipogenesis and lipid accumulation, suppressing SREBP-1, FAS and

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SCD-1 mRNA expression, and SREBP-1 nuclear translocation through the AMPK pathway125.

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This strengthens the notion that the activation of AMPK may be involved in hypoglycemic and

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hypolipidemic effects of these compounds. hypolypidemic effects of the compounds are

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described in Table 2.

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Adipogenesis and lipolysis:

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Several studies demonstrated that OA112, 114, 126, UA113, 127, 128 and BA108, 129 regulate adipogenesis

285

by inhibiting the expression and/or activation of different adipogenic transcriptional factors,

286

which may in turn reduce obesity and ultimately prevent the insulin resistance in T2DM.

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Oleanolic acid (1-25 µM) attenuated adipogenesis by reducing protein and, mRNA expression of

288

peroxisome proliferator receptor-γ (PPAR-γ) and CCAAT element binding protein α (C/EBP-

289

α).112 Regarding UA (2.5–10 µM), this molecule was shown to decrease protein expression of

290

C/EBP-α and -β, PPAR-γ, and SREBP-1c, promoting the increase of ACC phosphorylation, as

291

well as CPT-1 protein expression, and decrease protein contents of FAS and fatty-acid-binding

292

protein-4, through liver kinase B1/AMPK pathway. Therefore, these results also suggested that

293

UA reduces adipogenesis by stimulating fatty acid oxidation, and promoting also the inhibition

294

of fatty acids synthesis.113 Other studies reported that OA (31.7 µM)114 and BA (IC50 9.6 µM)129

295

decrease adiposity by inhibiting the activity of diacylglycerol acetyltransferase, involved in TG

296

deposition in adipocytes. However, OA and UA (25–100 µM) also decreased adiposity by 13 ACS Paragon Plus Environment

123

, an inner mitochondrial


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increasing lipolysis in fat cells, by promoting hormone-sensitive lipase translocation,126, 127 the

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rate-limiting enzyme in adipose tissue lipolysis, that hydrolyzes stored triglycerides into

299

monoglycerides and FFAs.130 In vitro studies demonstrated that UA (1–1000 µM) and BA (2.5–

300

10 µM) also promote lipolysis by inhibiting phosphodiesterase activity,108, 128 thus preventing or

301

reversing obesity and insulin resistance in T2DM.

302

Regulation of food intake and body weight:

303

Adipocytes and the gastrointestinal tract release a wide range of hormones, such as adiponectin,

304

resistin, leptin and ghrelin, which regulate appetite and energy metabolism. Altered production

305

or secretion of these hormones caused by excess adipose tissue and body weight contribute to

306

obesity and insulin resistance.131 Therefore, the regulation of adiponectin, resistin, leptin and

307

ghrelin contribute to avoid obesity and to improve insulin sensitivity in T2DM. Oleanolic,

308

ursolic and betulinic acids (5-20 mg/kg/day during 15 days) increased leptin and also decreased

309

the amount of ghrelin synthetized on HFD-induced obesity in mice,32,

310

appetite in this animal model. In contrast, Jia et al. showed that UA (50 and 200 mg/kg)

311

decreases leptin levels on HFD-induced obese mice, as a consequence of the reduction in

312

adipocyte tissue observed in this study. In the same work, it was also reported that UA increases

313

adiponectin,23 regulating lipid and glucose metabolism. Oleanolic acid (1-25 µM) suppressed the

314

production of resistin during adipogenic differentiation of 3T3-L1 adipocytes,132 suggests that

315

this is a mechanism which contributes to the improvement of obesity and insulin resistance in

316

T2DM (Table 2).

317

48, 49

thus regulating

COMORBIDITIES


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318

T2DM leads to the progression of macrovascular and microvascular comorbidities, including

319

atherosclerosis, cardiovascular and hepatic diseases, as well as nephropathy, which are the main

320

risk factors for death among diabetic patients.3 Therefore, the prevention of these related

321

complications may reduce the patients’ mortality risk.

322

Hepatic Diseases:

323

End-stage liver disease is one of the main causes of death in T2DM patients. Among liver

324

diseases in diabetes, non-alcoholic fatty liver disease (NAFLD) is an initial stage of a continuing

325

spectrum of liver alterations, which ultimately can be fatal.133-135 Considering that OA109, 110 and

326

UA22, 23, 111 decrease lipogenesis and stimulate fatty acid β-oxidation in hepatocytes (Table 2), it

327

is possible that both OA and UA provide effective treatment options for NAFLD, thus avoiding

328

progression of disease. In agreement with this, Zhou et al. reported that OA administration (100

329

mg/kg day during 4 weeks) activates AMPK,136 an energy sensor which regulates fatty acid

330

oxidation,137 and reduces lipogenesis and lipid accumulation in the liver.138 Once activated by

331

OA, AMPK induces the inhibition of SREBP and also leads to decrease hepatic ACC, FAS and

332

stearoyl-CoA desaturase-1 (SCD-1) in T2DM mice (Table 3), thus normalizing hepatic lipid

333

metabolism.136 Betulinic acid activates AMPK, which in turn leads to suppression of mRNA

334

expression and nuclear translocation of SREBP-1, repression of FAS and SCD-1 gene

335

expression, and increases PPAR-α and CD36 gene levels in human HepG2 hepatoma cells,

336

primary hepatocytes, and liver tissue from HFD-fed mice. These results suggest that BA can also

337

effectively improve intracellular lipid accumulation in liver cells, suppressing de novo

338

lipogenesis and increasing lipolysis through AMPK pathway activation.125 However, since UA

339

promotes FFA β-oxidation in muscle by activation of AMPK pathway123, a similar effect is

340

expected in the liver. Nonetheless, it is known that UA improves HFD-induced hepatic steatosis 15 ACS Paragon Plus Environment


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341

by regulating lipid metabolism through PPAR-α pathway, decreasing mRNA/protein content for

342

FAT/CD36, SREBP-1c, ACC, and FAS and increasing mRNA/protein content of CPT-1. It was

343

suggested that UA improves insulin resistance by decreasing insulin level and improving the

344

homeostasis model assessment of insulin resistance index (HOMA-IR), by decreasing

345

inflammatory signaling by down-regulating TNF-α, CCL2/MCP-1, IL-1β, IL-2, IL-6 and IL-8,

346

and stimulating the oxidative stress network, increasing SOD, CAT and GPx activities, resulting

347

in decreased MDA content,28 as described in Table 3. Taken all together, those effects may

348

contribute to the improvement of hepatic steatosis and reduction of metabolic dysfunctions on

349

NAFLD.

350

Additionally to hypolipidemic effects, the anti-oxidant activity of TAs may also prevent hepatic

351

dysfunction in diabetes. It has been proposed that the hepatoprotective effects of OA,139 and

352

UA10 are also mediated by a direct ROS scavenging role, i.e. decreasing hydroxyl and

353

superoxide radicals and by increasing the expression/activity of anti-oxidant enzymes, including

354

SOD, CAT, glutathione reductase, GPx and thioredoxin peroxidase (TPx), overall resulting in

355

lipid peroxidation inhibition. Although the mechanisms of anti-oxidant effects are still not

356

clarified, it has been proposed that anti-oxidant and anti-inflammatory effects of OA and UA are

357

mediated, to a great extent, through the activation of Nrf2 transcription, as described above.

358

Nevertheless, the known role of Nrf2 on the regulation of hepatic lipogenesis123, associated to the

359

expression of anti-inflammatory genes, lead us to propose that the activation of Nrf2 pathway by

360

OA and UA may in turn prevent the development of NAFLD by promoting anti-inflammatory

361

and anti-oxidant pathways reducing, at the same time, hepatic lipid accumulation.


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362

Although BA is unable to activate the Nrf2 pathway, it has been evidenced that its anti-oxidant

363

mechanisms responsible for the hepatoprotective activities may also be related with an

364

improvement of the tissue redox system, thus decreasing lipid peroxidation.29

365

Cardiovascular diseases:

366

Diabetes is associated with increased atherosclerosis and the development of cardiovascular

367

disease (CVD). Several studies suggested that OA, UA and BA are potential therapeutic agents

368

for diabetic-related CVD, mainly to prevent the development of atherosclerotic lesions and

369

myocardial dysfunction. Besides their hypolipidemic effects, these TAs have also anti-

370

atherogenic properties. Oleanolic and ursolic acids protected against low density lipoprotein

371

oxidation,140 and reduce pro-inflammatory cytokine production, including NF-kB, TNF-α and

372

IL-1β.24,

373

pathway by OA promoted the reduction of low density lipoprotein oxidation, contributing thus to

374

prevent atherosclerosis123. Other studies suggested that UA exert at least some of its anti-

375

atherogenic effects by preventing the accumulation of inflammatory monocytes in the blood,

376

reducing in vivo monocyte chemotactic activity, thus decreasing plaque size, and macrophage

377

content in atherosclerotic lesions in diabetic mice.25, 26 It was previously shown that BA vascular

378

protection occurs by inhibition of ROS and NF- kB, which consequently reduces the expression

379

of cell adhesion molecules such as vascular cell adhesion molecule-1, intracellular adhesion

380

molecule-1, and endothelial-selectin, contributing to suppression inflammatory responses31,

381

suggesting that the anti-oxidants and anti-inflammatory effects of these compounds are crucial to

382

prevent the development of atherosclerosis. Relevant data on anti-atherogenic effects are

383

summarized in Table 3.

141

According with this, Jiang et al. showed that the activation of Nrf2 signaling



Page 18 of 69

384

Another potential mechanism that may delay the development of atherosclerosis may involve a

385

decrease in blood pressure level. The anti-hypertensive effects of OA and UA may be mainly

386

attributed to their potent anti-hyperlipidemic and anti-oxidant effects, combined with diuretic,

387

natriuretic and saluretic effects, mostly due to the inhibition of Na+ and K+ reabsorption in the

388

early portion of the distal tubule.142 However, it was also reported that the anti-hypertensive and

389

anti-atherogenic properties of these compounds may also result from their effects on endothelium

390

dependent vasorelaxation. Impaired endothelial function, due to decreased bioavailability of NO

391

is a hallmark of atherosclerosis. In opposition, an increase in NO induces vasorelaxation,

392

preventing the development of atherosclerosis.143 In this context, UA and BA (0.1-100 µM)

393

displayed vasorelaxing properties, mediated by an increase of NO release and relaxation by

394

activation of endothelial nitric oxide synthase (eNOS).144-146 It was previously described that BA

395

increased the production of NO and the activation of eNOS mediated by the AMPK signaling

396

pathway147, suggesting that this compound used the same pathway to regulate also hepatic

397

glucose production19 (Table 1) and intracellular lipid accumulation125 (Table 2). Nevertheless, it

398

was also supposed that UA and BA (1–10 µM) upregulates eNOS expression, and

399

simultaneously, reduces nicotinamide adenine dinucleotide phosphate (NADPH) oxidase

400

expression in human endothelial cells.148, 149 As NADPH oxidase represents a major source of

401

ROS in CVD,150 this decreased expression may limit vascular oxidative stress, and increase

402

bioactive NO, which contributes to prevent the development of CVD. In opposition, Messner et

403

al. showed that UA (3-50 µM) induced pro-atherogenic effects, promoting DNA-damage, p53-

404

mediated mitochondrial- and caspase-dependent human endothelial cell apoptosis, accelerating

405

atherosclerotic plaque formation in vivo (Table 3). These authors supposed that pro-atherogenic

406

activities observed in this study are exerted at least in part by UA metabolite effects, instead


Page 19 of 69


407

directly by UA.11 However, further studies are needed to identify their metabolites and clarify

408

these effects.

409

Other studies also evidenced that OA12 and UA151 exhibit cardioprotective effects against

410

hyperglycemia-induced myocardial damage, at least in part due to their anti-oxidant and anti-

411

apoptotic properties. Although the precise mechanisms for these TAs-mediated anti-oxidant

412

effects in the heart are unclear, earlier data described that OA (0.6–1.2 mmol/kg/day during 3

413

days ≈ 274–550 mg/kg/day)152 and UA (35 mg/kg/ day during 8 weeks)151 increase the

414

expression of several enzymatic and non-enzymatic anti-oxidants, including α-tocopherol, GPx,

415

CAT, TPx and SOD in the heart tissue. Oleanolic and ursolic acids are also believed to act as

416

direct free radical scavengers and to reduce lipid peroxidation.153 Thus, it is possible that TAs

417

anti-oxidant effects may prevent cardio-metabolic complications in diabetes. In agreement,

418

Mapanga et al. proposed that OA12 anti-oxidant effects attenuate myocardial apoptosis and

419

thereby leading to improvement in contractile functional recovery following ischemia-

420

reperfusion of hyperglycemic rat hearts. In vitro, OA is a potent cardioprotective agent against

421

high-glucose-induced injury, acting as anti-oxidant, anti-apoptotic and anti-inflammatory

422

agent.13 Although there are no studies concerning UA and BA cardioprotective effects in

423

diabetes, it is known that BA14 and UA154 prevent cardiomyocyte apoptosis.

424

Nephropathy:

425

Diabetic renal injury, or the so-called diabetic nephropathy, is an important diabetic

426

complication, which exacerbate the severity and the mortality of T2DM. Non-enzymatic

427

glycation with formation of Maillard reaction products, also known as advanced glycation end-

428

products (AGEs), including glycated hemoglobin, Nε-(carboxymethyl)lysine (CML) and 19 ACS Paragon Plus Environment


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429

glycated albumin have been implicated in the pathogenesis of diabetic nephropathy.155,

430

Particularly, it is suggested that the accumulation of CML and glycated albumin contributes to

431

the deterioration of diabetic nephropathy.155 Oleanolic acid treatment (100 mg/kg/day during 2

432

weeks) on HFD- and streptozotocin-induced diabetes in mice decreases the loss of glucose in

433

urine and improves kidney structure.157 Other studies also indicated that OA and UA (0.01-0.2%

434

for 10 – 12 weeks) improved kidney function in diabetic mice,158,

435

structure and by inhibiting the polyol pathway. The inhibition of this pathway probably occurred

436

by decreasing renal sorbitol and fructose concentrations,159 and by decreasing renal aldose

437

reductase and renal sorbitol dehydrogenase activities,17, 59, 159 thereby inhibiting AGEs formation

438

and preventing kidney and liver injury. Besides that, these two TAs also decrease plasma HbA1c,

439

pentosidine and CML, as well as urinary glycated albumin in streptozotocin-diabetic mice.159

440

These results were different from those observed in vitro, where OA inhibited formation of

441

pentosidine and CML, but UA only suppressed CML, without affect pentosidine160 (Table 3).

442

Considering also that the oxidative and inflammatory reactions contribute to glycative

443

processes161, the anti-inflammatory and anti-oxidant properties of these three TAs can also

444

contribute to reduce AGE production, although there are no studies evidencing these effects.

445

Despite that, UA was found to decrease the receptor for AGE (RAGE) expression in brain of

446

aging mice162, which subsequently interact with AGEs in the circulation and tissues to form

447

AGE-RAGE complex. As it was described that AGE-RAGE complex generate pro-

448

inflammatory, pro-thrombotic molecules and ROS (reviewed in161), it is expected that the

449

reduction of AGE-RAGE complex contributes to reduce the glycative stress, and delays the

450

development of CVD and diabetic renal injury. It is clear that human studies are needed to

159

156

by preserving kidney


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451

further confirm the anti-glycative effect of these TAs in the progression of development of

452

diabetic co-morbidities.

453

PRESENT AND FUTURE PRESPECTIVES

454

Oleanolic, ursolic and betulinic acids are three TAs that display a wide range of promising anti-

455

diabetic properties, without demonstrating apparent hepatotoxic effects at the dosages

456

administered, contrarily to most anti-diabetic agents currently used. A large number of studies

457

evidenced that OA, UA and BA act as hypoglycemic and anti-obesity agents mainly through (i)

458

reducing the absorption of glucose; (ii) decreasing endogenous glucose production and

459

increasing glycogen synthesis; (iii) increasing insulin sensitivity; (iv) improving lipid

460

homeostasis; and (v) promoting body weight regulation. Besides these promising beneficial

461

effects, OA, UA and BA may also contribute to protect against diabetes comorbidities, such as

462

kidney injury, cardio-metabolic and hepatic diseases (Figure 2). It is interesting to note that many

463

of their multiple anti-diabetic effects occur by mediation of common signaling pathways, which

464

may in turn result in different outcomes. Among multiple effects, it should be noted that AMPK

465

and Nrf2 pathways are involved in many mechanisms of action of these compounds, being

466

AMPK signaling responsible by hypoglycemic, hypolipidemic and anti-atherosclerotic

467

mechanisms of these TAs, while Nrf2 may modulate many of anti-oxidant, anti-inflammatory

468

and hypolipidemic effects of OA and UA, making them important action targets of these

469

compounds.

470

Although the high potential of these three TAs in the treatment and prevention of T2DM is very

471

promising, clinical trials with OA, UA and BA are practically non-existent, with the exception of

472

a 2005 Phase II clinical trial with UA which at the University of Guadalajara, México

473

(#NCT02337933). The trial was performed to evaluate its effects on insulin sensitivity and 21 ACS Paragon Plus Environment


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474

metabolic syndrome, as characterized by overweight/obesity, insulin resistance, hyperglycemia,

475

dyslipidemia and hypertension, and by an inflammatory state, which together increases the risk

476

for CVD or T2DM (University of Guadalajara, México, coded NCT02337933).The results of

477

that clinical trial are still undisclosed. The very limited number of clinical puts into question the

478

true effect of these compounds in humans, highlighting the need to clarify the true efficacy,

479

safety and tolerability of these compounds and the doses required to achieve these anti-diabetic

480

effects of these TAs, before they can be used in T2DM management.

481

Another major limitation in the use of these compounds is their low bioavailability, supported by

482

the fact that the peak plasma concentration after administration of OA163, UA164 and BA165 (10-

483

100 mg/kg) never exceed 1% of the oral dose administered in rats, possibly due to their poor

484

gastrointestinal absorption, limiting their human applications. To improve the bioavailability and

485

efficacy of these compounds, some strategies have been used, either by developing new systems

486

for drug delivery in order to increase the bioavailability of these compounds, or through the

487

developing also of new derivative structures. Numerous efforts have been made to improve the

488

bioavailability of these compounds by developing non-covalent complexes with hydrophilic

489

cyclodextrins, as well as the use of nanosuspensions166. Ursolic acid liposomes (UALs) was until

490

recently the only system approved to be used in a phase I clinical trial (# 2009L00634, China) to

491

improve bioavailability in patients with advanced solid tumors167. This strategy may increase the

492

therapeutic use of these compounds, although its use with OA and BA, as well as their effects in

493

diabetic patients still remains unstudied.

494

Another strategy has been the development of OA168, UA169 and BA84 derivatives to improve anti-

495

hypoglicemic, anti-inflammatory, anti-oxidant effects of these natural compounds. However, the

496

only derivative so far studied in clinical trials was the semi-synthetic OA derivative, bardoxolone


Page 23 of 69


497

methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid), also designated as

498

CDDO-Me or RTA 402. Bardoxolone methyl successfully completed Phase II clinical trials

499

(coded NCT01574365) for the treatment of patients with type 2 diabetes-associated chronic

500

kidney disease.170,

501

chronic kidney disease were halted due to a higher incidence of cardiovascular events.172 Despite

502

this, development of new TAs derivatives may eventually represent an excellent strategy to treat

503

diabetes in the near future.

504

Apart from these strategies, a very recent study reported also the ability of OA (100 mg/kg day,

505

during 4 weeks) to synergistically potentiate anti-diabetic effects of metformin (250 mg/kg day,

506

during 4 weeks), by improving glucose and insulin homeostasis in diabetic mice.173 It is

507

reasonable to speculate that these compounds may be used to increase the anti-diabetic effects

508

and possibly to counteract hepatotoxicity derived from commonly used therapeutics. However,

509

OA possible hepatotoxic effect should be confirmed, since it has been reported that OA, at

510

higher concentrations (≥ 90 mg/kg/ day), caused cholestatic liver injury in mice.174, 175 Regarding

511

UA and BA, there are no studies reporting their synergistic effects when combined with anti-

512

diabetic agents, neither their potential hepatotoxicity. In this regard, the highly promising effect

513

of these compounds should push clinical assays in order to confirm the efficacy, safety and

514

tolerability of OA, UA and BA individually or in combination with anti-diabetic agents.

515

In summary, this review provides a unique and holistic view on the anti-diabetic properties of

516

OA, UA and BA in the management of T2DM, highlighting the strengths and weaknesses to the

517

future application these compounds in humans. A strong point of the three compounds is their

518

global anti-diabetic effects mediated by specific mechanisms of action, a peculiar characteristic

519

of these compounds which makes them a true asset for treatment and prevention of T2DM.

171

However, Phase III trials (# NCT01351675) with patients in end-stage



Page 24 of 69

520

Potential weaknesses involve their low bioavailability, and the very limited number of clinical

521

trials. More strategies should be designed to increase the bioavailability of these natural

522

compounds, testing also their effects in clinical trials with T2DM patients. In this perspective, a

523

forcing need is also the clarification of the signaling pathways involved in anti-diabetic effects,

524

searching new therapeutic targets that are involved in Nrf2 and AMPK signaling pathways so

525

that new derivatives with selective anti-diabetic effects can explore those signaling pathways.

526

Therefore, by merging all the information described above, we expected that the development of

527

new strategies to improve the bioavailability and efficacy of these TAs in managing T2DM. An

528

optimal future scenario would be that these compounds could be used as pharmaceutical agents

529

and/or as food supplements for the prevention and/or management of T2DM.

530 531

AUTHOR INFORMATION

532

Corresponding Author

533

*Tel: (+351) 284314399, [email protected]

534 535

Funding sources

536

This work is funded by FEDER funds through the Operational Programme Competitiveness

537

Factors - COMPETE and national funds by FCT - Foundation for Science and Technology under

538

the projects NEucBark– New valorization strategies for Eucalyptus spp. Bark Extracts

539

(PTDC/AGR-FOR/3187/2012), and strategic project UID/NEU/04539/2013 (CNC).

540 541

Notes

542

The authors declare no competing financial interest.

543 24 ACS Paragon Plus Environment

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544

ABBREVIATIONS

545

ACC, acetyl-coA carboxylase; ACO, acyl-CoA oxidase; AGEs, advanced glycation end-

546

products; AMPK, 5′ AMP-activated protein kinase; BA, betulinic acid; CAT, catalase; C/EBP-α

547

and –β, CCAAT element binding protein α and β; CCL2/MCP-1, monocyte chemotactic protein-

548

1;

549

cardiovascular disease; eNOS, endothelial nitric oxide synthase; FAS, fatty acid synthase; FFA,

550

free fatty acid; GLP-1, glucagon-like peptide-1; GLUT-2 and -4, glucose transporter -2 and -4;

551

GP, glycogen phosphorylase; G6Pase, glucose-6-phosphatase; GPx, glutathione peroxidase;

552

GSK-3, glycogen synthase kinase-3; hACAT-2, human cholesterol acyltransferase -2; HFD,

553

high-fat diet; HOMA-IR, homeostasis model assessment of insulin resistance index; IR, insulin

554

receptor; IRS-1, insulin receptor substrate -1; MDA, malondialdehyde; NADPH, nicotinamide

555

adenine dinucleotide phosphate; NAFLD, non-alcoholic fatty liver disease; NF-κB, factor

556

nuclear kappa B; Nrf2, nuclear factor erythroid 2-related factor 2; OA, oleanolic acid; PEPCK,

557

phosphoenolpyruvate carboxykinase; PI3KB, phosphatidylinositol 3-kinase-dependent; PPAR-α

558

and γ, peroxisome proliferator activated receptor-α and γ; PTP-1B, protein tyrosine phosphatase

559

1B; RAGE, receptor for AGE; ROS, reactive oxygen species; SCD-1, stearoyl-CoA desaturase-

560

1; SGLT-1, sodium-dependent glucose co-transporter; SOD, superoxide dismutase; SREBP-1c,

561

sterol-regulatory-element-binding protein-1c; TAs, triterpenic acids; T2DM, type 2 diabetes

562

mellitus; TG, triglycerides; TNF-α1, tumor necrosis factor –α1; TPx, thioredoxin peroxidase;

563

UA, ursolic acid, UCP-3, uncoupling protein-3.

CML,

Nε-(carboxymethyl)lysine;

CPT-1,

carnitine

palmitoyltransferase-1;

CVD,

564



565

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566

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1086

Page 50 of 69

FIGURES AND LEGEND

1087 1088

Figure 1. Chemical structures of some bioactive triterpenic acids: oleanolic, ursolic and betulinic

1089

acids. Adapted from4.

1090


Page 51 of 69


1091 1092

Figure 2. Mechanisms of oleanolic (OA), ursolic (UA) and betulinic (BA) acids on diabetes type 2 and

1093

related comorbidities. The different hypoglycemic (orange continuous arrow) and anti-obesity (red

1094

dash arrows) properties of these TAs are schematized. These TAs promote the reduction of glucose

1095

absorption (1) by inhibition of pancreatic α-amylase and α-glucosidase or by down-regulation of

1096

sodium-dependent glucose co-transporter (SGLT-1) and glucose transporter 2 (GLUT2); as well as

1097

reducing the endogenous glucose production (EGP) and increase in glycogen synthesis (2), as

1098

consequence of the reduction of gluconeogenesis and glycogenolysis, and stimulation of glycogenesis.

1099

The improvement of insulin signaling (3) by OA, UA and BA is highlighted and mediated i) by

1100

inhibition of protein tyrosine phosphatase (PTP), ii) by improvement of β-cell survival and direct

1101

stimulation of phosphatidylinositol 3-kinase-dependent (PI3KB)/protein kinase Akt pathway, iii) by

1102

translocation of GLUT-4, iv) by activation TGR5 and glucagon-like peptide-1 (GLP-1) release, and

1103

also v) by inhibition of ROS production and suppression of inflammatory processes. Anti-obesity 51 ACS Paragon Plus Environment


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1104

effects occur due to the reduction of lipid absorption (4) by inhibition of pancreatic α-lipase or human

1105

cholesterol acyltransferase -2 (hACAT-2); regulation of lipid metabolism (5) by reduction of

1106

lipogenesis and stimulation of fatty acid β-oxidation; and also by promoting reduction adipogenesis,

1107

stimulating the lipolysis in adipose tissue. The regulation of appetite (6) is another effect of these

1108

compounds, which occur as consequence of the reduction of ghrelin and resistin and the increase of

1109

adiponectin and leptin levels, although the effect of UA on leptin remain controversial. The protective

1110

properties of OA, UA and BA against diabetes-related comorbidities (blue dash arrows) may act on

1111

preventing the development of non-alcoholic fatty liver disease (NAFLD) (7), cardiovascular disease

1112

(CVD) (8) or diabetic nephropathy (9), as consequence of their hypolipidemic, anti-atherogenic,

1113

hypotensive, anti-inflammatory and anti-oxidant properties, and also by preserving kidney structure

1114

and promoting the inhibition of polyol pathway. ↕= regulates, ↑= enhances/stimulates/promotes, (+):

1115

up-regulates/activates, ↗= improves, ↓= reduces, (-) = downregulates,

= inhibits.

1116


Page 53 of 69


1117

TABLES

Biological action

TAs dosage and biological system used OA and UA: 5 µg/mL (~ 10 µM) Extracted from six Malaysian plants BA: 50 µg/mL (~100 µM) Extracted from leaves of Dillenia indica OA: 5-20 mg/kg/day during 15 weeks In vivo: HFD fed mice UA: 10 mg/kg/day during 15 weeks In vivo: HFD fed mice

Biological outcome to TAs

Reference

↓↓pancreatic α-amylase activity

46

↓↓pancreatic ߙ-amylase and ߙ-glucosidase activities

47

32 ↓↓pancreatic α-amylase activity, ↓blood glucose levels

BA: 10 mg/kg/day during 15 weeks In vivo: HFD fed mice

Absorption and uptake of glucose

48

49

OA: IC50 35 µM, UA: IC50 39 µM Extracted from Punica granatum L. flowers

54

OA: IC50 231.3 µM Extracted from Phlomis stewartii

50

UA: IC50 0.119 µM Extracted from Stereospermum colais

51 ↓↓α-glucosidase activity

UA: IC50 16 µM Extracted from stem barks of Uncaria laevigata

52

OA: IC50 49,5 µM, UA: IC50 47,6 µM, BA: IC50 14,0 µM Extracted from gold-red apple

53

OA: IC50 0.1 mg/mL (~200 µM) In vivo: male GK/Jcl type 2 diabetic rats and healthy human adult volunteers Extracted from olive leaf

↓↓pancreatic and salivary α-amylase activity, ↓blood glucose levels

55

OA: 80 mg/kg twice daily during 5 weeks (in vivo) 4.37–21.9 µM (in vitro) In vivo: STZ-DM rats

In vivo:↓blood glucose levels, ↓SGLT-1 and GLUT-2 expressions In vitro:↓↓α-amylase (IC50 of 3.60 µM) and α-glucosidase ( IC50 of 12.40 µM) activities

16



Table 1. Summary of studies reporting the hypoglycemic effects of OA, UA and BA. Endogenous glucose production and synthesis of glycogen

OA: IC50 14.9 µM UA: IC50 9.0 µM BA: IC50 43.0 µM UA: 0.01-0.05% (w/w) during 4 weeks

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↓↓muscle GP activity

57

↓blood glucose level, ↓↓hepatic G6Pase and ↑↑GK activities, ↑GK/G6Pase ratio, ↑GLUT-2

17

1118


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In vivo: STZ/NA-DM mice UA: 5 mg/kg/day during 5 weeks In vivo: HFD C57BL/6J mice UA: 0.05% (w/w) during 4 weeks In vivo: HFD and STZ-DM mice

mRNA levels ↓glucose, HbA1c and insulin levels, ↑glycogen content, ↑↑GK and ↓↓G6Pase activities ↓blood glucose level, ↑hepatic glycogen content, ↑↑GK and ↓↓G6Pase activities

UA: 2 mg/kg/day during 7 days In vivo: healthy rats

↓ plasma glucose concentration, ↑liver glycogen levels, ↑phospho-GSK-3

62

↑glucose uptake, ↑glycogen synthesis ↑phospho-AMPK, ↑ phospho-GSK-3β

64

↑blood glucose levels, ↑insulin signaling, ↓ G6P and PEPCK protein expression, ↑PGC-1α mRNA expression, ↑phospho-AMPK, ↑mitochondrial density, ↓structural injury of mitochondria, ↑∆Ѱm, ↑ATP content

18

BA: 10-20 µM and 5-10 mg/kg during 3 weeks In vitro: Human HepG2 hepatoma cells In vivo: HFD ICR mice

In vitro: ↓hepatic glucose, ↓PGC-1α, PEPCK, and G6Pase gene expression,↑AMPK, ↓phospho-CREB, ↑phospho-CAMKK, In vivo: ↓plasma glucose, ↓insulin resistance index

19

BA: 10 µM In vitro: differentiated 3T3-L1 adipocytes, C2C12 myotubes, HUVEC, RAW264.7 macrophages, MEF and CHO cells

↑ glucose uptake, ~LDH, ATP levels, cell viability, ↑ higher extracellular acidification rate and ↑extracellular lactate, ↑mitochondrial content and mitochondrial ROS production, ↓oxygen consumption rate, ↑UCP-1 and -2 protein expression, ↑GLUT1 and GLUT2 protein expression, phosphor-AMPK ↑glucose levels, ↑ insulin and c-peptide, → β-cells,↑proliferation T lymphocytes, ↑IL-2 and IFN-γ, ↓TNF-α

68

OA, UA and BA: 10 µM In vitro: Human HepG2 hepatoma cells OA: 20 mg/kg/day during 2 weeks In vivo: Lep(db)(/)(db) obese DM mice

UA: 0.05% (w/w) during 4 weeks In vivo: HFD STZ-DM mice

20

59

69

Insulin resistance UA: 0.05% (w/w) during 8 weeks In vivo: HFD mice

↓glucose levels, ↑insulin level, →islet architecture

70

1119



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OA: 30 and 50 µM In vitro: pancreatic β-cells (INS-1 832/13 cells) and rat islets

↑insulin secretion, ↑acute glucose-stimulated insulin secretion, ↑insulin protein and mRNA levels, ~ somastatin, glucagon, cellular Ca2+ and cAMP levels

71

OA: 5–50 µg/mL (~10 – 100 µM) In vitro: pancreatic β-cells Extracted from Cabernet Sauvignon grape skin

↑Insulin production

73

↗ glycemia, ↑insulin secretion, ↑glycogen content, ↑glucose uptake in muscle ↑GLUT-4 protein expression, ↑localization GLUT-4 at the plasma membrane ↗ glycemia, ↑insulin vesicle translocation, ↑insulin secretion, ↑glycogen content in muscle, ↑glucose uptake in muscle tissue ↑GLUT-4 protein and mRNA expression

74

BA: 0.1, 1 and 10 mg/kg In vivo: Male wistar rats

UA: 0.1, 1 and 10 mg/kg In vivo: Male wistar rats

OA: IC50 3.37-3.40 µM

↓↓PTP-1B (IC50 3.37 µM), ↓↓TCPTP (IC50 3.40 µM)

OA: IC50 9.50 µM, UA: IC50 2.30 µM

75

76

79 78

UA: IC50 3.8 µM Extracted from S. paniculata BA: IC50 0.7 µg/mL Extracted from roots of Saussurea lappa C.B.Clarke

↓↓PTP-1B

BA: IC50 3.5 µM Extracted from the fruits Sorbus pohuashanensis UA: IC50 2.73-3.33 µM

77

71

↓↓PTP-1B (IC50 3.08 µM), ↓↓TCPTP (IC50 3.33 µM), ↓↓SHP-2 (IC50 2,73 µM)

77

1120


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UA: 0.01–100 µg/mL (~ 0.02–200 µM) In vitro: CHO/IR cells and 3T3-L1 primary adipocytes

OA: 5 and 25 mg/kg/day during 10 weeks In vivo: Adipo-IR rats

UA: 2.5–10 µM In vitro: 3T3-L1 pre-adipocytes

OA: EC50 1.42 µM and 100 mg/kg/day during 7 and 14 days In vitro: CHO-TGR5 cell line In vivo: HFD C57BL/6J mouse Extracted from Olea europaea

OA: EC50 2.25 µM, UA: EC50 1.43 µM, BA: EC50 1.04 µM In vitro: CHO cells OA: 10-30 µM In vitro: Murine enteroendocrine cells (STC-1) OA: 10–50 µM In vitro: pancreatic β cell line MIN6 and also in mouse and human pancreatic islets

↑phospho-IR β-subunit in CHO/IR and adipocytes, ↑IR in CHO/IRs, ↑phospho-Akt and phospho -ERK in CHO/IRs, ↑phospho-Akt and phospho GSK-3 in adipocytes, ↑translocation of GLUT-4 in adipocytes insulin-mimetic agent (>50 µg/mL) insulin-sensitizer (

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