Dipartimento di Farmacia
Corso di Laurea Magistrale in Farmacia
Tesi di Laurea
NUTRACEUTICAL APPROACH TO THE CARDIOMETABOLIC RISK:
EFFECTS OF ERUCA SATIVA MILL. IN AN EXPERIMENTAL MODEL
OF DYSLIPIDEMIA
Relatore:
Prof. Vincenzo Calderone
Prof.ssa Lara Testai
Correlatore: Candidato:
Dott.ssa Eugenia Piragine Marjan Nastarantehrani
INDEX
CHAPTER 1
INTRODUCTION
1.1 Brassicaceae...1
1.1.1 Eruca Sativa Mill. ...1
1.2 Brassicaceae Compounds ...4
1.2.1 Ascorbic Acid ………..…4
1.2.2 Phenolics……….4
1.2.3 Flavonoids………..5
1.2.4 Carotenoids………..…6
1.2.5 Glucosinolates……….7
1.2.5.1 Glucosinolate’s Bioavailibility………..8
1.2.5.2 Isothiocynates...9
1.2.5.2.1 isothiocynates as H₂S-donors...10
1.3 Brassicaceae and Health...11
1.3.1 Metabolic Syndrome...11
1.3.1.1 Obesity………..12
1.3.1.1.1 Citrate Synthase……….15
1.3.1.2 Dyslipidemia………..15
1.3.1.3 Diabetes Mellitus………15
1.3.2 Cardiovascular Diseases……….17
1.3.4 Carcinogenesis Protection……….19
1.3.5 Protection against Helicobacter pylori Infection………..20
1.4 Hydrogen Sulfide……….20
1.4.1 Mechanism of H₂S biological Action……….….21
1.4.2 The Possible role of H₂S in the Biological Activity of Isothiocinate………..23
CHAPTER 2
2.1 Purpose of the Research………24
CHAPTER 3
3.1 Materials and Methods………24
3.1.1 Animal Experiments………..24
3.1.2 Subchronical Experiments……….24
3.2 Experimental Protocol……….25
3.3 Enzymatic Test………26
3.3.1 Citrate synthase………26
3.3.2 Experimental Protocol of C.S. in Adipose Tissue………27
3.4 Compositions………..29
3.4.1 Diet Compositions………..29
3.4.2 Buffer Composition………31
3.4.2.1 STE……….31
CHAPTER 4
4.1 Results……….32
CHAPTER 1
1.INTRODUCTION
1.1 Brassicaceae
The Brassicaceae family consists of about 3500 worldwide species and includes 350 genera, such as Brassica, Camelina, Crambe, Sinapis and Thlaspi. In particular, the genus Brassica includes some edible species such as Brassica oleracea L., Brassica rapa L. and Brassica napus [Sasaki, Takahashi, 2002].
Brassicaceae are characterised by short cycle and wide adaptability; for this reason they are suited for cultivation in different seasons and in a variety of environments. As regards the nutritional profile, the Brassicaceae have a low caloric value (24–34 kcal/100 g) depending on the low content of protein (1.44–2.82/100 g) and fat (0.12–0.37/100 g) and an average content of fibre of 2.5/100 g. These vegetables are rich in potassium, calcium, magnesium and phosphorus, vitamins C, E, K and carotenoids [Heimler et al., 2006].
Turnip or Brassica rapa L. has been traditionally used to treat some diseases, such as hepatitis, jaundice, sore throats, and some cancer [Krtistal 2002] as well as to make Kimchi, a Korean traditional fermented vegetable food [Jeon et al., 2013].
1.1.1 Eruca Sativa Mill.
Eruca sativa Mill. is another member of this family. It is called with different names in different nations, such as rocket salad, roquette, rucola, rucoli, rugula, arugula and colewort. Eruca Sativa(E.S.) is a fast-growing and cool-season crop, already known for its medical properties [Khoobchandani et al. 2010, Sarawak et al. 2007], native to the Mediterranean region, from Morocco and Portugal in the west, to Syria, Lebanon and Turky in the east (Figure 1).
Figure 1. World wide distribution areas of E. Sativa shown in brown.
E.S. is an annual plant growing from 20 to 100 cm tall. The pinnate leaves have from 4 to 10 small, deep, lateral lobes and a large terminal lobe. The flowers are from 2 to 4 cm in diameter, arranged in a corymb typical of Brassicaceae, with creamy white petals veined in purple, and yellow stamens; the sepals are shed soon after the flower opens. The fruit is a siliqua (pod) from 12 to 35 millimetres long with an apical beak, and containing several edible seeds. The species has a chromosome number of 2n = 22. [Blamey and Grey-Wilson 1989, Huxley 1992] The leaves can be cut after 20 days and are sequentially harvested from re-growth [Jaks et al., 2013].
The responsible bioactive compounds present in these plants are Phenolics, Glucosinolates,
Carotenoids, Tocopherols and Ascorbic acid.
In particular, broccoli, white cabbage and cauliflower are rich in Glucosinolates, specially in the seeds, while shoots have the lowest Glucosinolates concentration [Bhandari et al., 2015]. Glucoraphanin is one of the most interesting and studied glucosinolate products which is transformed by myrosinase into sulforaphane, an isothiocynates largely investigated because its anticancer properties (Figure 2).
Instead, the major glucosinolate found in rocket seeds is glucoerucin that represents 95% of total glucosinolates. It is not only in seeds, but also in sprouts and mature leafs. Glucoerucin possess both direct and indirect antioxidant activities [Barillari et al., 2005].
Figure 2. Hydrolysis of Glucoraphanin to Sulforaphane by Myrosinase.
E.S. is considered an excellent source of antioxidants, such as phenolic compounds, carotenoids and glucosinolates, too. [Villatoro-Pulido et al. 2012]. The seeds contain approximately 45% erucic acid and about 9% gadoleic acid (C20:1) [Lazzeri et al., 2004].
Furthermore, E.S. possesses anti-secretory, anti-inflammatory, antimicrobical, antiplatelet, cytoprotective and also anti-ulcer activity. The anti-ulcer effect is possibly due to prostaglandin-mediated activity and/or through anti-secretory and antioxidant properties [Aliqasoumi et al., 2009 Khan H. and Khan M.A., 2014].
1.2 Brassicaceae compounds
1.2.1 Ascorbic Acid
Ascorbic acid (AsA) and dehydroascorbic acid are known to reduce and neutralize reactive oxygen species (ROS) [Padayatty et al., 2003]. (Figure 3)
In addition, AsA is able to protect the myocardium when associated to ferulic acid [Yogeeta et al., 2006], while, in association with vitamin E, it can prevent ox-LDL-induced (oxidized-Low Density Lipoprotein) and overexpression of vascular endothelial growth factor (VEGF), responsible for atherosclerotic plaque formation [Rodriguez et al., 2005].
Figure 3. Ascorbic acid known as Vitamin C.
1.2.2 Phenolics
Phenolic compounds have been studied for their ability to chelate redox-active metal ions, to inibit LDL cholesterol oxidation and to neutralize other processes involving ROS, since they are efficient free radical scavengers [Gallo et al., 2013].
Antioxidant activity is directly linked to the particular structure of phenols. In fact, alterations in the arrangement of the hydroxyl groups and degree of substitution by glycosylation decrease the antioxidant activity [Rice-Evans et al., 1997].
Moreover, dietary polyphenols may inibit the growth of adipose tissue by modulating adipocyte metabolism [Herranz-Lopez et al., 2012]. It seems that they are able to enhance glucose uptake in adipocytes and muscle cells by GLUT4, a glucose transporter that exerts its action through the
1.2.3 Flavonoids
Flavonoids are a large family of polyphenolic plant compounds that carry out important functions in plants, such as attracting pollinating insects, combating environmental stresses (such as micro iCal infection), and regulating cell growth [Kumar and Pandey 2013].
During and after intestinal absorption, flavonoids are rapidly and extensively metabolized in intestinal and liver cells, so that they are likely to appear as metabolites in the bloodstream and urine [Rothwell et al., 2016].
Their bioavailability and biological activities in humans appear to be strongly influenced by their chemical structure (bound to one or more sugar molecules [Williamson et al., 2004]) (Figure 4), interaction with food matrix (the binding affinity and potential non covalent interactions of flavonoids with food proteins, carbohydrates, and fats are directly associated with the physiochemical properties of flavonoids [Gonzales et al., 2015]), composition of gut microbiota (in the large intestine, gut microbial enzymes transform flavonoids through deglycosylation, ring fission, dehydroxylation, demethylation, etc. into metabolities that can be absorbed or excreted [Monagas et al., 2010. Roowi et al., 2010]) and the detoxification pathway (as they are recognized as xenobiotics by the body, that they undergo extensive modifications first in the intestinal mucosa and then in the liver).
The antiradical activity of flavonoids has been determined in the case of seven local edible Brassicaceae: Italian kale (cavolo nero), broccoli, Savoy cabbage, white cabbage, cauliflower, green cauliflower and Brussels sprouts, while Broccoli and Italian kale exhibit the highest content of both total phenolics and flavonoids [Heimler et al., 2006].
Flavonoids can normalize blood glucose levels and promote ß-cell regeneration in islets of alloxan-treated rats [Vessal et al. 2003], instead, Epicatechin and Quercetin can stimulate insulin production in isolated rat islets [Tabatabaei-Malazy et al. 2013].
Figure 4. General structure of Flavonoid class.
1.2.4 Carotenoids
Also known as Tetraterpenoids, they are produced by plants and algae, as well as several bacteria and fungi (Figure 5). Carotenoids level in Brassicaceae family largely differ from 0.26 mg/100 g FW (Fresh Weight) in white cabbage to 6mg/100 g FW in Brussels sprouts [Podsędek, 2007]. Carotenoids are pigments precursors of vitamin A (i.e., carotene, γ-carotene, and β-cryptoxanthin), which are characterized by the presence of conjugated double bonds responsible for the radical scaveng and quenching of singlet oxygen (dioxidene) [Podsędek, 2007].
A negative correlation between the ß-carotene serum level and rates of cancer, metabolic syndrome factors, cardiovascular diseases and myocardial infarction has been demonstrated. Moreover, a negative correlation between the ß-cryptoxanthin and metabolic syndrome factors has been demonstrated, too [Suzuki et al., 2011].
1.2.5 Glucosinolates
Glucosinolates represent a group of phytochemicals found in 15 botanical families, as secondary metabolites of the order of Capparales and are very abundant in Brassicaceae [Barbieri et al., 2009]. Glucosinolates constitute a natural class of organic compounds containing sulfur and nitrogen, and are derived from glucose and an amino acid (Figure 6). In different broccoli extracts have been found a very different profile of Glucosinolates [Padilla et al., 2007]. For example, in a recent paper, the most abundant Glucosinolates found in different broccoli samples were glucobrassicin and neoglucobrassicin, followed by glucoraphanin. Curiously, in the ecotype “Friariello“ from the Campania region the complete absece of glucoraphin has been shown [Barbieri et al., 2009]. Another item which may evidently change in different broccoli varieties, is the quantity of Glucosinolates. An example, is the study on a collection of 113 varieties of turnip greens (Brassica rapa L.), cultivated in two different sites of Spain, which has demonstrated the range of Glucosinolates differ from 12 to 70 macromol/g DW (Dry Weight) at one site, and from 7 to 60 micromol/g DW at the other site [Padilla et al., 2007-32]. The mentioned condition is affected by different parameters such as genetic background, climatic condition, crop management strategies, time, and other condition of storage, characterizing the time from harvest to initial processing in the industry or retailer, the methods of cooking and consumption at home [Francisco et al., 2017].
Glucosinolates are thioglycosides that differ in the structure of the aglycone side chain; they are relatively biologically inactive, but following tissue disruption they undergo hydrolysis to form a broad range of structurally diverse hydrolysis products possessing varying biological activities [Holst and Fenwick, 2003].
Some of the hydrolysis products as thiocyanate ion, several isothiocyanates and nitrile may have antinutritional or toxic effects; others, especially isothiocyanates as sulforaphane, are considered to be responsible for the protective, anticarcinogenic effects of a cruciferous-rich diet. These adverse and beneficial effects are highly dose-dependent and the physiological range is relatively narrow [Holst and Fenwick, 2003].
Glucosinolates are studied for their potential to affect human health, their biological effects are related to their ability to inhibit the invasive potential of human cancer cell line in vitro [Rose et al., 2006] and to regulate the phase I and/or phase II detoxification enzyme activity [Das et al., 2000].
Figure 6. General structure of Glucosinolate.
1.2.5.1 Glucosinolate’s Bioavailibility
The bioavailibility of Glucosinolates is linked to the function of a ß-thioglucosidase enzyme, called myrosinase.
Interestingly, mammalian tissues do not support this hydrolising process because they do not contain myrosinases, instead this enzyme exists in bacterial microflora of the gastrointestinal tract, where the bioavailability of Glucosinolates still occurs. Reduction of the bowel microflora by cleansing and antibiotic treatment almost eliminates this conversion [Shapiro et al., 1998]. As shown in figure 7, the hydrolysis of glucosinolates is catalyzed by myrosinase to give unstable aglucones and liberate glucose. Depending on the reaction conditions and the structure of the glucosinolate side chain (R), a series of products can be formed, including nitriles, thiocyanites, epithionitriles, oxazolidine-2-thiones, and isothiocyanates.
Figure 7. The main pathway of Glucosinolate`s hydrolysis catalyzed by Myrosinase.
1.2.5.2 Isothiocyanates
Isothiocynates are one the most important products of hydrolysis process of glucosinolates. During the hydrolising of inactive Glucosinolates by myrosinases, the isothiocynates are associated to important protecting effects [Mukherjee et al., 2008]. One of the most extensively studied isothiocyanates, sulforaphane, was isolated from extracts of broccoli as a potent inducer of cytoprotective enzymes [Zhang et al., 1992] (Figure 8). The glucosinolate precursor of sulforaphane, glucoraphanin, is most abundant specially in seeds, in particular, the 3-day-old broccoli sprouts contain from 20 to 50 fold higher levels than mature broccoli [Fahey et al., 1997].
In humans, the isothiocyanates are metabolized via the mercapturic acid pathway (Figure 9). Initially, they are conjugated with glutathione in a glutathione transferase (GST)-catalyzed reaction. The glutathione conjugates undergo sequential cleavage reactions catalyzed by g- glutamyltranspeptidase (g-GT), cysteinylglycinase (CGase), and N-acetyltransferase (AT) to give N-acetylcysteine conjugates (mercapturic acids). The isothiocyanates and all their glutathione-derived conjugates, collectively known as dithiocarbamates, are detected by the cyclocondensation reaction with 1,2-benzenedithiol.
Figure 9. Metabolism of isothiocyanates by mercapturic acid.
1.2.5.2.1 Isothiocynates as H2S-donors
Isothiocyanates, are receiving greatest scientific interest for their numerous biological and pharmacological effects and useful applications in many aspects of human health [Dinkova-Kostova and [Dinkova-Kostova 2012]. It is worthy to note that there is an interesting overlap between many biological effects attributed to some Brassicaceae and those exhibited by the gasotransmitter H2S [Citi et al., 2014].
1.3 Brassicaceae and Health
The Mediterranean diet, which is characterized by a high consumption of plant-based foods, has been associated with a lower risk of cardiovascular disease and mortality, low grade inflammation and metabolic syndrome in different epidemiological studies.
In the last few years, several studies, both in vitro and in vivo, have focused on the effect of Brassicaceae on chronic diseases and on the bioactive compounds of these plants that may be responsible for the mentioned effects [Jeon et al., 2013, Shah et al., 2016].
1.3.1 Metabolic Syndrome
Also known as syndrome X or dysmetabolic syndrome, refers to a cluster of metabolic conditions that can lead to C.V. diseases.
The main features of metabolic syndrome include insulin resistance, hypertension (high blood pressure), abnormal ematic level of cholesterol, and an increased risk for clotting. People diagnosed with metabolic syndrome are usually overweight or obese.
Approximately 20%-30% of the population in industrialized countries has metabolic syndrome. Both genetics and the environment play important roles in the development of metabolic syndrome. Genetic factors influence each component of the syndrome, and the syndrome itself. Environmental issues such as low activity, sedentary lifestyle and progressive weight gain also contribute significantly to the risk of developing metabolic syndrome.
Metabolic syndrome is present in about 5% of people with normal body weight, 22% of those who are overweight and 60% of those considered obese. Adults who continue to gain 5 or more pounds per year raise their risk of developing metabolic syndrome by up to 45%.
While obesity itself is likely to be the greatest risk factor for developing metabolic syndrome, other factors include:
• Postmenopausal age
• Smoking
• High intake of carbohydrates
[National institute of diabetes and digestive and kidney diseases].
Bioactive compounds in Brassicaceae are positively effective on metabolic syndrome. In a study on obese mouse model exposed to Brassica rapa extracts, it has been demonstrate that these compounds are able to induce the expression of lipolysis related genes in white adipocytes [An et al., 2010], while in another study on overweight subjects with the same extracts, they can reduce the total cholesterol/HDL-cholesterol ratio, free fatty acid and adipsin* levels [Jeon et al., 2013]. In rats fed with Brassica oleracea extract, has been seen the improvement in body weight, water and food intake, that is consider as an antidiabetic potential effect of Brassica oleracea in type 2 diabete [Shah et al., 2016] (Figure 10).
Figure 10. Main effects of Brassicaceae compounds on metabolic syndromes.
*Adipsin is an adipokine that promotes ß cell function in diabetes.
1.3.1.1 OBESITY
Obesity is the most common nutritional disorder, considered as a characteristic feature of metabolic syndrome, and is a risk factor for the genesis and development of various diseases, including coronary heart disease, hypertension, type 2 diabetes mellitus, cancer, respiratory complications and osteoarthritis [Kopelman 2000].
Overweight is generally defined as a Body Mass Index (BMI) of 25 or more, the health risks associated with obesity occur at a lower BMI, although the prevalence of obesity is lower than that in Europe and in some Asian populations, thus, lower cut off points for Asians were suggested for overweight (BMI ≥ 23.0) and obesity (BMI ≥ 25.0) [Jeon et al., 2013].
A study on an obese mouse model, has submitted that the ethanol extracts from Brassica rapa enhanced the expression of lipolysis-related genes in white adipocytes, the activation of cyclic AMP-dependent protein kinase, and in the induction of extracellular signal-regulated kinases, suggesting that Brassicaceae extracts may be used as safe and effective “anti-obesity agents” [An et al.,2010].
The ß3-adrenergic receptor (ß3-AR) is located on the surface of both white and brown adipocytes. The ß3-AR has attracted interest as a potential treatment of obesity because agonists stimulate lipolysis and thermogenesis in rodent and human white adipose tissues (WATs), influencing fuel supply and concomitantly increasing body energy expenditure via stimulation of uncoupling protein (UCP) [Lafontaine et al., 2007]. Furthermore, ß3-AR agonists normalize secretion of adipocytokines from adipocytes, which influence insulin sensitivity through the action of adiponectin and tumor necrosis factor-α (TNF- α) [ Zhang et al., 2002]. Adipocyte lipolysis is stringently regulated by hormones, neurotransmitters, and other effector molecules and by hormone-sensitive lipase (HSL) [Holm 2003]. HSL is an enzyme that hydrolyzes intracellular triacylglycerol and diacylglycerol [Carmen and Vıctor 2006], and its activity is controlled by phosphorylation of specific serine residues [Watt et al., 2006]. The binding of agonists to ß3-ARs coupled with adenylate cyclase via the stimulatory G-protein leads to an increased production of cyclic AMP and activation of cyclic AMP-dependent protein kinase (PKA). It has been demonstrated that ethanolic extract of Brassica rapa treatment induced the expression in white adipocytes of lipolysis-related genes, including ß-3 AR, hormone-sensitive lipase (HSL), adipose triglyceride lipase, and uncoupling protein 2. The lipolytic effect of Brassica rapa involves ß3-AR modulation, as inferred from the inhibition by the ß3-AR antagonist propranolol. According to the mentioned observations, ethanolic extract of Brassica rapa may
have potential as a safe and effective anti-obesity agent via the inhibition of adipocyte lipid accumulation and the stimulation of ß3-AR-dependent lipolysis [An et al., 2010].
Lipid storage and mobilization in adipocytes are tightly linked to the functional state of the mitochondria [Ernster and Schwartz 1981]. It has been demonstrated that the gene transcript of about 50% of the nuclear-encoded mitochondrial proteins is decreased in obesity [Wilson-Frisch et al., 2004].
A modest weight loss in obese or overweight individuals is reported to be associated with a decreased risk of cardiovascular disease and/or type 2 diabetes risk and of their mortality [Bosello et al., 1997, Van Gaal et al., 1997]. The significant reduction in the plasma Free Faty Acid (FFA) concentration by Brassica rapa ethanol extract (BREE) supplementation, partly, corresponded with previous studies that the BREE supplement significantly reduced total cholesterol and FFA concentrations in the db/db mice (db/db mice is a genetically muted mouse that is extremely obese and has many metabolic defects, as shown in figure 11). According to several studies, FFA levels are a strong independent predictor of sudden cardiac death and other cardiovascular deaths [Jouven et al., 1999, Pliz et al., 2006].
An interesting investigation based on enzymatic assay and determination of protein levels revealed that the development of obesity is associated with a significant impact on citrate synthase in mitochondria of human omental adipose tissue, the enzymatic activities of citrate synthase and its protein levels were significantly reduced in obesity. The state of obesity appears to affect mitochondrial function in human omental adipose tissue by limiting this key enzyme of the tricarboxylic acid cycle rather than by limiting the activities of respiratory chain enzymes [Christe et al., 2013].
1.3.1.1.1 CITRATE SYNTHASE
Citrate synthase is a Krebs tricarboxylic acid (TCA) cycle enzyme that catalyzes the synthesis of citrate from oxaloacetate and acetyl coenzyme A (Figure 12). It is nuclear encoded and found in the mitochondrial matrix where it is the rate-limiting enzyme of the TCA. In the past, citrate synthase activities or protein levels were considered to be stable, and thus the enzyme has often been used as a mitochondrial marker. However, developmental or aging studies indicated that citrate synthase cannot be regarded as a stable marker [Drahota et al., 2004. Marin-Garcia et al., 1998].
Figure 12. Citrate synthesis reaction catalyzed by Citrate synthase.
1.3.1.2 DISLIPIDEMY
Dislipidemy is a disorder linked to an abnormal amount of fatty substances such as triglycerides, cholesterol and/or fat phospholipids, in the blood. A very common form of dislipidemy is the one frequently referred to as "high cholesterol". Prolonged elevation of insulin levels can also lead to dyslipidemia. The National Cholesterol Education Program Expert Panel (NCEP) has suggested that non-HDL-C may be a better predictor of cardiovascular risk than low-density lipoprotein– cholesterol [NCEP].
However, dislipidemy is a term that refers to all health problems related to lipids, due to the excess or shortage of certain kinds of lipids.
The danger is that when lipids start to build up inside the artery walls, scar tissue and other debris begin thickening and hardening the walls. Some arteries literally become clogged. This condition is called atherosclerosis. (Figure 13)
Figure 13. A normal artery versus an artery narrowed by atherosclerotic plaque.
1.3.1.3 DIABETES MELLITUS
It is a group of metabolic disorders caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. Such a deficiency results in increased concentrations of glucose in the blood, which in turn damage many of body’s systems, in particular the blood vessels and nerves.
There are two principle forms of diabetes:
• Type 1, known as insulin-dependent: the pancreas fails to produce insulin which is essential for survival. This form of diabet develops most frequently in children and adolescents, but is being increasingly noted later in life.
• Type 2, known as non-insulin-dependent: results from the body’s inability to respond properly to the action of insulin produced by the pancreas. This type of diabete is much more common and accounts for around 90% of all diabetes cases worldwide. It occurs most frequently in adults, but is being increasingly in adolescents as well [WHO].
Epidemiologic studies and clinical trials have demonstrated that diabetic individuals are at increased risk for CVD [Haffner et al., 1998. Kookiness et al., 1992].
1.3.2 CARDIOVASCULAR DISEASEs
Epidemiological studies indicate that individuals with higher intakes of fruits and vegetables tend to have a lower occurrence of CVD. Therefore, increased consumption of fruits and vegetables has been recommended as a key component of a healthy diet for the prevention of chronic diseases, including CVD [Hung et al., 2004, Slavin and LIoyd 2012].
Moreover, there is growing evidence that certain groups of vegetables, such as cruciferous vegetables, may be particularly beneficial for human health [Slavin and LIoyd 2012, Munich and Bland 2007].
Cardiovascular disease (CVD) is a type of diseases that involve the heart and/or blood vessels, it includes coronary artery diseases (CAD) such as angina and myocardial infarction (heart attack). Other CVDs include stroke, heart failure, hypertensive, rheumatic, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis [Shanthi Mendis et al., 2011].
Cardiovascular diseases are the main cause of death globally. Coronary artery disease, stroke, and peripheral artery disease involve atherosclerosis. This may be caused by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive alcohol consumption, among others. High blood pressure results in 13% of CVD deaths, while tobacco results in 9%, diabetes 6%, lack of exercise 6% and obesity 5% [Shanthi Mendis et al., 2011].
In the spontaneously hypertensive stroke-prone rats feeding either with dried broccoli sprouts or sulforaphane, decreased oxidative stress in cardiovascular and kidney tissues, as demonstrated by lower protein nitrosation and increased glutathione, glutathione reductase, and glutathione peroxidase levels [Wu et al., 2009. Senanayake et al., 2012]. In addition, the endothelial-dependent relaxation of the aorta was improved, the number of infiltrating activated macrophages was reduced, and the blood pressure decreased. In an ischemia/reperfusion model, feeding rats with broccoli extracts improved postischemic ventricular function, reduced myocardial infarct size, and decreased cardiomyocyte apoptosis
[Mukherjee et al., 2008]. The ability of isothiocyanates to induce cytoprotective proteins and prevent chronic inflammation underlies these protective effects.
Sulforaphane can induce phase II enzymes through the activation of the Keap1/Nrf2 antioxidant response pathway [Nestle 1997]. Most importantly, sulforaphane can also induce redox-regulated protein thioredoxin through the antioxidant-responsive element [Tanito et al., 2005]. Because thioredoxin has recently been implicated in cardioprotection, it seems likely that broccoli, in addition to serving as a chemoprotective vegetable, may also function to protect the cardiovascular system.
There is a significant cardioprotection with broccoli, which induced the expression of several genes and proteins of the thioredoxin (Trx) superfamily including Trx1, Trx2, thioredoxin reductase, Grx1, Grx2, and peroxiredoxin (Prdx) as well as caused the activation of the antioxidant response pathway and several antideath proteins of the survival pathway [Mukherjee et al., 2008].
In mice fed high fat diets supplementation with RCMG (Red Cabbage MicroGreen) or RC (Red Cabbage) had a beneficial effect against lipid risk factors for the development of CVD [Huang et al., 2016].
1.3.3 NEUROPROTECTIVE
Brassicaceae have shown neuroprotective effects, mediated by antioxidant and anti inflammatory activities [Dinkova‐Kostova and Kostov 2012. Orhan et al., 2015].
Inflammation acts as a defensive response when a tissue is damaged by toxins or infective agents in order to repair the tissue and eliminate the causative factors, but, when it is prolonged, it may be detrimental. One of the most sensitive tissues to inflammation is nervous system, given its limited regeneration capacity. In fact, neuroinflammation has been associated to different disorders, including neurodegenerative diseases [Kempuraj et al., 2016. Chen Zhang and Huang 2016]. During neuroinflammation, different mechanisms cause neuronal degeneration and death, including the release of different cytokines by activated microglia and the infiltration of immune cells from periphery.
Recent studies demonstrate the ability of dietary phytochemicals widely found in fruits and vegetables to reduce the neuronal death occurring in neurodegenerative diseases through several adaptive mechanisms. These mechanisms belong to the phenomenon called hormesis, including the activation of the nuclear factor (erythroid- derived 2)-like 2 (Nrf2), a master regulator of the antioxidant network and cytoprotective genes [Zhang et al., 2013, Qin and Hou 2016]. An increase of glutathione (GSH) through activation of Nrf2 in the dopaminergic neurons may be a promising neuroprotective strategy, among Nrf2 activators, the isothiocyanate sulforaphane has gained attention as a potential neuroprotective compound [Marroni et al., 2018].
1.3.4 CARCINOGENESIS PROTECTION
The prevention of chronic disease is reported as a vantage of isothiocynates. Morever, there is a strong correlation between the consumption of cruciferous vegetables and the reduced risk of different types of cancer[Barbieri et al., 2009]. Several researches reported that BREE protects against some cancers and cisplatin-induced nephrotoxicity [Van Popped et al., 1999, Kim et al., 2006]. In addition, it has been shown that extracts of broccoli and watercress can inhibit the invasive potential of human breast cancer cell lines in vitro [Barbieri et al., 2009]. The explanation of this effect may be the ability of isothiocynates to regulate the phase I and/or phase II detoxification enzymes activity [Das et al., 2000].
It has been recognized that the mechanism(s) of action of isothiocyanates, are multiple and include at least the following items: alterations of carcinogen metabolism due to changes in the activities of drugs-metabolizing enzymes, induction of cell cycle arrest and apoptosis, inhibition of angiogenesis and metastasis, changes in histon acetylation status, and antioxidant, anti inflammatory, and immunomodulatory activities [Dinkova-Kostova and Kostova 2012]
1.3.5 Protection against Helicobacter pylori infection
Sulforaphane also protects against H. pylori infections, which are strongly associated with the development of gastric cancer [Fahey et al., 2002]. Sulforaphane-rich broccoli sprouts administrated to mice infected with H. pylori and maintained on a high-salt diet, reduced gastric bacterial colonization, attenuated mucosal inflammation, and prevented high-induced gastric corpus atrophy [Yanaka et al., 2009]. These findings promoted a placebo-controlled intervention study in which 48 subjects infected with H. pylori were randomly assigned to consuming either broccoli sprouts or an equal weight of alfalfa sprouts (as placebo) for two months [ Yanaka et al., 2009]. Intervention with broccoli sprouts, but not placebo, decreased markers of colonization (urease measured by the urea breath test and H. pylori stool antigen) and inflammation (serum pepsinogens I and II).
1.4 HYDROGEN SULFIDE
Hydrogen sulfide (H₂S) is a colorless, poisonous, corrosive and inflammable gas with characteristic foul odor of rotten eggs [Greenwood 1997] (Figure 14). H₂S is an important endogenous modulator, biosynthesized in mammalian tissues by cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) [Martelli et al., 2010]. In the last decade, H₂S has been recognized as an important pleiotropic gasotransmitter and has been defined as the new nitric oxide [Sanderson 2009], since it is endowed with pivotal regulatory roles in almost all physiological systems in the human body and particularly in the cardiovascular one [Citi et al., 2014]. In the CV system, H₂S production is mainly ensured by the CSE, starting from the aminoacid L-Cysteine [Hosoki et al., 1997].
1.4.1 Mechanism of H₂S Biological Action
It has been demonstrated that a low concentration of H₂S (<20μM) is able to donate electrons to the electron transport chain at the level of ubiquinone [Goubern et al., 2007]. These electrons, donated by H₂S, following the respiratory chain, pass to complex III, cytochrome oxidase, and finally to oxygen, on the other word, H₂S exhibiting the antioxidant properties of inorganic and organic sulfites, acts as a scavenger of at least four different reactive oxygen species: superoxide radical anion [Mitsuhashi et al., 2005], hydrogen peroxide [Geng et al., 2004], peroxynitrite [Whiteman et al., 2004] and hypochlorite [Whiteman et al., 2005]. These compounds are highly reactive and can damage the proteins and lipids, so their neutralization by H₂S, protects organism from damages induced by such aggressive molecules [Whiteman et al., 2004, Whiteman et al., 2005]. Such an oxidation of sulphide is catalyzed by sulphide-quino-oxidoreductase enzyme, that is located in the inner mitochondrial membrane [Theissen and Martin 2008].
The suppression of ROS production, reduction of upregulated cleaved caspase-3 expression, prevention of glutathione (GSH) loss and mitochondrial membrane potential loss in rat cardiomyoblasts, are the nonspecific cytoprotective role of H₂S [Chen et al., 2009]. Also increasing the production of GSH by enhancing cystine/cysteine transporters and redistributes GSH to mitochondria, are two antioxidant mechanisms observed in cultured neuroblastoma cells, mediated by H₂S [Kimura et al., 2010]. H₂S can also exert its effects through specific molecular targets such as the activation of ATP-sensitive potassium channel (K-ATP) [Zhao et al., 2001] and calcium-activated potassium channels (K-Ca) [Martelli et al., 2010]. K-ATP channels are almost ubiquitous and their role is particularly important in the regulation of biological functions in several tissues/systems, such as pancreatic cells, neurons, myocardial, skeletal and smooth muscle cells [Martelli et al., 2010]. K-ATP channels are efficient biological systems able to link the metabolic status of the cells with their excitability [Nichols et al., 2006], because the intracellular levels of ATP and ADP are the key factors determining channel inhibition and activation, respectively [Martelli et al., 2010]. It is currently acknowledged that H₂S relaxed
blood vessels mainly (not exclusively) by opening the K-ATP channel of vascular smooth muscle cells [Martelli et al., 2010].
New findings have demonstrated that at low concentrations, H₂S stimulates oxidative phosphorylation and increases ATP biosynthesis, and high levels of ATP are the prevalent inhibitory factors on channel activity [Martelli et al., 2010]. Three subtypes of K-Ca are classified in accordance with single channel conductance, as SK (small conductance, 2-25pS), IK (intermediate conductance, 25-100pS), and BK (big conductance, 100-300 pS) [Martelli et al., 2010]. A role of H₂S as an activator of SK channel has been suggested in dorsal raphe serotoninergic neurons, where the possible neuroprotective action of H₂S seems to be mediated through the SK activation [Kombian et al., 1993].
Interestingly, CSE inhibitors reduce the K-ATP channel current, suggesting a constant stimulation of the channel by basal levels of endogenous H₂S [Martelli et al., 2010]. It has been demonstrated that, the genetic deletion of CSE in mice, markedly reduces H₂S levels in the serum, heart, and aorta, and developed marked hypertension and a decreased endothelium-dependent vasorelaxant effect [Yang et al., 2008].
Also, it has been observed that the deficiency of endogenous H₂S contributes to the pathogenesis of hypertension [Martelli et al., 2010]. Indeed, CSE expression/activity is lower in spontaneously hypertensive rats (SHRs) [Martelli et al., 2010]. In addition to the vasorelaxing activity, H₂S is endowed with a wide range of additional biological roles, which are relevant for a polyedric control of CV system [Martelli et al., 2010]. H₂S inhibits platelet aggretion/adhesion induced by ADP, collagen, epinephrine, arachidonic acid, thromboxane mimetic U46619, and thrombin. Such an effect does not seem to be related to the cAMP/cGMP generation, NO release, or potassium-channel opening [Zagli et al., 2007]. H₂S can also affect the vascular inflammatory reaction, which plays an important role in atherosclerotic plaque destabilization and rupture [Martelli et al., 2010]. Moreover, recent evidence has shown a direct interplay between H₂S and cyclooxygenase-drived vasoactive prostaglandins in the regulation of vascular tone [Koenitzer et al., 2007].
The protective role of H₂S on heart diseases has been further confirmed by an articulated work on sudden cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in mice, in which the administration of Na₂S before CPR markedly improved survival rate at 24hr, prevented CA/CPR-induced oxidative stress, and ameliorated left ventricular dysfunction. By contrast, a delayed administration of Na₂S, 10min after CPR, failed to improve the outcome after CA/CPR. These data have been supported by a cardioprotective effect of Na₂S on isolated-perfused mouse hearts subjected to global ischemia and reperfusion, and by the observation that a cardiomyocyte-specific overexpression of CSE significantly improved the outcome of CA/CPR [Martelli et al., 2010].
1.4.2 The Possible Role of H₂S in the Biological Activity of Isotiocinate
In a recent study, it has been investigated, the possible H₂S releasing activity of some important isothiocinates such as: allyl isothiocyanate (AITC, highly present in black mustard, B. nigra), 4-hydroxybenzyl isothiocyanate (HBITC, highly present in white mustard, S. alba), benzyl isothiocyanate (BITC, highly present in garden cress, Lepidium sativum L.) and erucin (ERU, present in different species such as broccoli, B. oleracea L., and rocket, Eruca vesicaria L.), by a reliable amperometric approach [Citi et al., 2014]. In the mentioned study, it has been demonstrated that the mentioned natural isothiocyanates behave as slow H₂S-releasing agent [Citi et al., 2014].
Also, in another recent study, it has been demonstrated that the isothiocyanate functional group can be viewed as an original and suitable slow H₂S- releasing moiety, endowed with vascular effects, typical of this gasotransmitter, thus it can be also viewed as a novel and versatile chemotype of H₂S-donor, for the development of promising cardiovascular drugs [Martelli et al., 2014].
CHAPTER 2
2.1 PURPOSE OF THE RESEARCH
Cardiovascular disease are the main cause of death globally [Shanthi Mendis et al., 2011]. Among the risk factors, dyslipidemia is one of the main factors that leads to CVDs, and so is one the main cause of death globally.
Asian populations habitually consume a large amount of cruciferous vegetables and other plant-based foods [Zhang et al., 2011]. The beneficial role of the Mediterranean diet in the prevention of chronic diseases, including cardiovascular diseases is well-recognized [Raiola et al., 2017]. In a recent clinical research has been demonstrated that there is an indirect relation between the prolonged consumption of cruciferous vegetables and total and cardiovascular disease mortality [Zhang et al., 2011].
In this study, we evaluate on in vivo dyslipidemia and hyperglycemia models to prove the hypothesis of the beneficial role of Brassicaceae, in particular Eruca Sativa Mill., in the prevention of CVDs.
CHAPTER 3
3.1 MATERIALS AND METHODS
3.1.1 Animal Experiments
BALB-C male mice were used and have been kept under monitored conditions with 12 hours light/dark cycle, temperature of 22°C, with abundance food and water.
Experimental was according to the community legislation (directive CEE 750/2013) and Italian legislation (DL n° 26/2014).
3.1.2 Subchronical Experiments
We divided the mice in 4 groups and every group contained 5 mice. The duration of this experiment was 10 weeks (70 days) and during this period, we monitored and registered the food and water intake every day, and the body weight of each mouse, twice a week, to make sure of
their health state and also to evaluate the eventual weight increase. We provided necessary food and water every day.
Every group was fed as below:
The first group named Std., used as the control group, fed with 6 gr per mouse of pulverized standard diet (Figure 15).
The second group, HF, fed with 6 gr per mouse of pulverized High Fat (HF) diet (Figure 16).
The third group, HF+E.S., fed with 6 gr per mouse of pulverized High Fat diet enriched with an Eruca Sativa seed extract (0.75% p/p), titled in
glucoerucin and glucoraphanine (400 µmol/g di GLS).
The fourth group, Std.+E.S., Fed with 6 gr per mouse of Pulverized Standard diet enriched with the same extract mentioned for the third group.
We used to pulverize their food to make possible the enrichment of their diet with Eruca Sativa seed extract, and we did it for all 4 groups to make uniform the condition of experimental for all mice in experiment.
3.2 EXPERIMENTAL PROTOCOL
At the end of this chronic experimental, day 71, we measured the glycemia from caudal vein of each fasted mouse (24h), using auto-controlled strips (Glucocard G meter, Menarini Diagnostics®). We measured their waist circumference, height, weight and we calculated the body mass index (BMI g/cm2) of each mouse.
Using an overdose of carbamate (uretano) 30% p/p, all the mice have been anesthetized.
Then we collected the venous blood of each mouse and immediately transferred to the blood collection tubes containing EDTA as anticoagulant (BD Vacutainer®), then we used their blood to evaluate the lipid panel (cholesterelo, HDL, LDL) and glycated hemoglobin levels, using Cobas b101 tool (Roche Diagnostics), the rest of blood has been centrifuged at 2010 RCF for 10 minutes to obtain the plasma.
Subsequently, every mouse has been euthanized and have been collected, heart, liver, adipose tissue, femur and brain.
Heart has been washed in PBS 1X (pH 7.4), weighed and 2/3 have been conserved in paraformaldehyde for further fibrosis analysis, instead the rest 1/3 has been conserved at -80°C for further histological and functional analysis.
Liver has been washed in PBS 1X (pH 7.4), weighed and 1/4 has been conserved in paraformaldehyde for further fibrosis analysis, 2/4 have been conserved at -80°C for further histological and functional analysis, 1/4 has been conserved at -80°C for CBS/CSE analysis. Brain has been washed in PBS 1X (pH 7.4), weighed and divided in 2 eppendorfs and both have been conserved at -80°C for further histological and functional analysis.
The left femur has been conserved at -20°C for further histological and functional analysis. Adipose tissue has been washed in PBS 1X (pH 7.4), weighed and collected approximately 0.3 g to be analyzed by an enzymatic test immediately and the rest of tissue has been conserved at -80°C for further histological and functional analysis.
The obtained information has been elaborated with GraphPad Prisma 6.0 program. The t student analysis method has been used to evaluate the statistical differences between the values obtained from the control group and the other groups fed with different nutraceutical contribution.
3.3 ENZYMATIC TEST
3.3.1 Citrate Synthase
Citrate synthase (C.S.) is an enzyme active in all examined cells, where it is most often responsible for catalyzing the first reaction of the citrate acid cycle that is a Krebs cycle (tricarboxylic acid cycle). In fact, the C.S. catalyze the condensation of acetyl-Coenzyme A (Acetyl-CoA) and Oxaloacetate (OAA) to form Citrate. The hydrolysis of the thioester of Acetyl-CoA leads to the formation of CoA with a thiol group (CoA-SH):
Acetyl-CoA + Ossalacetate → Citrate + CoA-SH + H* + H2O
3.3.2 Experimental Protocol of C.S. in Adipose Tissue
In this study we measured the activity of C.S. in adipose tissue, since this tissue is the target of dyslipidemia and obesity. The following analysis was carried out for every mouse separately in a separate signed eppendorf tube. Approximately 0.3 g of visceral adipose tissue of each mouse was collected and washed in PBS 1X (pH 7.4) and transferred in a tube where was added 500 µL of STE and 10 µL of Triton 1%, then it was homogenized by Ultra-Turax, keeping the temperature at 4°C using an ice box (to prevent enzymatic damage by the released heat).
To evaluate the concentration of total proteins in our collected samples, it has been used a calibration curve with bovine albumin (BSA). The BSA 10 mg/ml as the initial concentration (mother) has been diluted with bidistilled water to obtain 1 mg/ml, 100 µg/ml, 20 µg /ml, 10 µg /ml and 2 µg /ml concentrations. Subsequently, we prepared the dilutions of our supernatant (containing the proteins) to obtain the following dilutions: 1:10, 1:100, 1:200, 1:500 and 1:1000. Later, they have been collected 200 µl from the dilutions 20, 10 and 2 µg/ml of BSA and have been plated tripled and subsequently, they have been added 200 µl of Bradford reactive in every cockpit, obtaining 10, 5 and 1 µg /ml concentrations (dilution 1:2). The same method has been used with the dilutions 1:100, 1:200, 1:500 and 1:1000, obtained from our supernatant. Then, they have been read at 595 nm (λ where absorb the blue color formed from the reaction between Bradford and the proteins), then using GraphPad Prisma 6.0, the data have been interpolated to evaluate the concentration of total proteins in samples. Once we obtained the yield, we evaluated the activity of C.S. present in our samples, using the following reactions:
Acetyl-CoA + Ossalacetate → Citrate + CoA-SH + H* + H2O
We built a calibration curve for C.S. using 0 U/ml, 0.3 U/ml, 0.6 U/ml and 0.9 U/ml concentrations. In every cockpit we obtained a final volume of 200 µl, consisting of:
• 2 µl of DTNB 10mM (100 µM/well) • 2 µl of Acetyl-CoA 10 mM (100 µM/well) • 2 µl of C.S. (30 U/ml, 60 U/ml, 90 U/ml) • 174 µl of Tris Buffeter 0.1 M
Subsequently, we prepared an eppendorf where we used 2 µl of Tris buffer instead of C.S. (used as control group) and another eppendorf where we used 2 µl of our sample instead of C.S. Then we prepared an eppendorf containing 1 mg/ml of proteins, collecting an appropriate quantity of supernatant according to the obtained calibration curve, before. And from this last eppendorf, we collected 2 µl to obtain a dilution of 1:100, once added to cockpits (10 µl/ml).
It has been carried a baseline reading at 412 nm and subsequently, we added 20 µl of Oxaloacetic acid 50 mM in every cockpit to trigger the reaction, and it has been followed for 3 minutes (reading at 15-seconds intervals) at 412 nm (CS protocol, Perkin Elmer).
A linear regression has been performed from the calibration curve obtained from the different concentrations of C.S. (0.3 U/ml, 0.6 U/ml and 0.9 U/ml), that this linear regression allows us to measure the slope (corresponding to the reaction speed) for every concentraion. So, with 3 obtained slope values, we build a curve, which is used to interpolate the activity of enzyme in incognito sample.
3.4 COMPOSITIONS
3.4.1 Diet Composition
Here has been reported the composition of different diets used to feed the mouses during this experiment. (Figure 15 and 16)
3.4.2 Swabs Composition 3.4.2.1 STE COMPONENTS QUANTITY Sucrose 250 mM Tris 5 mM EGTA 1mM 3.4.2.2 PBS 1% COMPONENTS QUANTITY NaH2PO4 x H2O 0.128 g NaH2PO4 x 12H2O 0.597 g NaCl 4.388 g H2O q. b 500 ml
CHAPTER 4
4.1 Results
The habitual consumption of vegetables of Brassicaceae family has been linked to the reduction of C.V. risk, which represents the main cause of morbidity and mortality in west world.
According to this epidemiological evidence, the goal of this thesis is to evaluate the protective effects of a plant that belongs to the Brassicaceae family, i.e. Eruca sativa Mill. (E.S.), commonly known as rucola, in an experimental model characterized by a high fat intake.
This plant has a high percentage of glucosinolate, similar to the other plants of Brassicaceae family. In particular, Eruca contains a high quantity of glucoerucin and a minimum quantity of glucoraphanine.
As it has been mentioned, glucosinolates, in a reaction catalyzed by myrosinase enzyme, can release their corresponding isotiocinates, which have been known as hydrogen sulfide (H₂S) donors [Martelli et al., 2010].
Therefore, a second goal of this thesis has been evaluating the role of H2S in protective effect of E.S.
Male BALB-C male mice have been fed for 10 weeks with a diet characterized by an high fat intake (HF). This type of diet has produced an higher body weight in mice respect than mice fed with standard diet (Std.) (Graphic 1).
On the other hand, the body weight of animals fed with enriched food with Eruca extract (Std. + E.S. and HF. + E.S.) has been increased perfectly in agreement with their correspondent references (Std. and HF). In other word, the Eruca extract has not influenced significantly the increase in body weight (Graphic 1).
Graphic 1. Increase in percentage in body weight vs the days of treatment.
The Body Mass Index (BMI) analysis demonstrated that the HF diet significantly increase this parameter respect than Std. diet. The presence of E.S. both in the Std. and HF diet reduced the BMI value, although it is not possible to say that it is significant (Graphic 2).
The ponderal analysis of visceral adipose tissue has demonstrated that feeding with the diet HF, increased this parameter (express as g of adipose tissue/kg of animal).
This data agreed with the literature, about the usage of HF diet. Interestingly it has been observed that adding the E.S. extract has decreased the visceral fat both in animals fed with Std. diet and HF diet.
The reduction is significant in animals fed with Std. diet, while the reduction in those fed with HF diet is evidence but data is not significant at the moment (Graphic 3).
The BMI value and the ponderal analysis of adipose tissue, suggested us to evaluate the metabolic and functional activity of this tissue. In literature, the activity of C.S. enzyme has been considered as an effective indicator of cell metabolism, therefore in this thesis, it has been evaluated the enzymatic activity of C.S. in visceral adipose tissue samples collected from treated animals, according to the mentioned protocol.
The C.S. enzyme demonstrated an improvement in its activity when the animal has been treated with E.S. extract. In fact, U/ml of C.S. results to be increased both in animals fed with Std. + E.S. vs those fed with Std. diet and animals fed with HF + E.S. vs those fed with HF diet. However, comparing the activity of C.S. enzyme in the animals fed with Std. and HF diet, has not been observed any significant difference (Graphic 4A).
Instead, when we evaluated the activity of C.S. enzyme respect to the total number of proteins present in adipose tissue, we observed that the supplement markedly influenced this parameter. The activity of C.S. measured in animals fed with HF diet is 1/5 less than the activity of this enzyme in the animals fed with Std. diet. This data is almost in agreement with literature [REF].
Again, the presence of E.S. extract demonstrated a significant improvement of C.S. activity (Graphic 4B).
The ponderal analysis of other organs, liver, heart and brain, does not highlight particular differences related to the treatments (Graphic 5).
Graphic 5. Ponderal analysis of A)Liver, B)Heart, B)Brain
The lipid profile analysis has highlighted an increase in total cholesterol and HDL levels of animals treated with HF diet respect than to those treated with Std. diet. In addition, the presence of E.S. extract in HF diet, has contributed to a significant reduction of total cholesterol and HDL levels, reporting values similar to those treated with Std. diet.
These are the only parameters that seem to be significantly by changed, while triglyceride and LDL parameters did not significantly change (Graph 6A).
Cardiovascular risk analysis, indicated as the ratio between the total cholesterol and HDL, has demonstrated that adding the E.S. extract, significantly has decreased the C.V. risk in the animals fed with Std. diet, while in this moment it is not possible to say if there is a protective effect in the animals fed with HF diet. (Graphic 6B)
Graphic 6. A) Total cholesterol, HDL, LDL and triglyceride levels. B) C.V. risk calculated from the relation between
total cholesterol and HDL.
At the end, the analysis of the glycemic parameters has demonstrated that feeding with HF diet does not produce hyperglycemia.
However, we observed that the presence of E.S. extract has contributed to the reduction of glycemic values both in animals treated with Std. diet, and specially significantly in animals treated with HF diet (Graphic 7A).
Similar consideration can be done about the glycated hemoglobin (HbA1C), where the HF diet produces a light increase respect than the Std. diet, and adding the E.S. extract, reduces them significantly (Graphic 7B).
Graphic 7. A) plasma levels of venous glycemia. B) Plasma levels of HbA1C.
In conclusion, taking together these results, suggest that adding E.S. extract to the diet, can have a positive effect on the metabolic activity of adipose tissue.
The future experiments would be addressed to reinforce preliminary data and to evaluate the role of H₂S in this benefit effect.
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