COMENIUS UNIVERSITY IN BRATISLAVA FACULTY OF MEDICINE

EFFECTS OF PYCNOGENOL IN TOXIC LIVER INJURY (HISTOLOGICAL CHANGES)

DIPLOMA THESIS

DECLARATION

I hereby declare that this is my original work and has been written by me based on information and sources that are cited in the literature.

Date:

Siganture:

ABBREVIATION AND SYMBOLS

carbon tetrachloride -CCL4cytochrome -CYPTrichloromethyl radical -CCl3*Trichloromethylperoxy radical -CCl3OO*Hepatic stellate cell -HSCTumor necrosis factor -TNFNitric oxide -NOTransforming growth factors -TGFHepatocyte growth factor -HGFInterleukin 6 - IL6Pycnogenol -PYCUltraviolet -UVAttention deficit hyperactivity disorder -ADHDN-Acetylcysteine-NACNon-alcoholic fatty liver disease-NAFLDNon-alcoholic steatohepatitis -NASH

PROLOGUE

In the last 10 years, the non-alcoholic fatty liver disease (NAFLD) prevalence is rapidly increasing all around the world, in contrast with the prevalence of the rest hepatopathies that stayed the same or even declined. This rapid increase lead NAFLD to become one of the most common liver disease worldwide. According to studies the prevalence of NAFLD, in the general population of Western countries, is around 20% to 30%.44,45,46 According to World Gastroenterology Organization Global Guidelines, NAFLD can be defined as a condition with excessive fat accumulation in the form of triglycerides (steatosis) in the liver (> 5% of hepatocytes histologically). A subgroup of NAFLD is the non-alcoholic steatohepatitis (NASH), where the patients have a combination of liver cell injury, inflammation and accumulation of excessive fat in the liver (steatohepatitis) and has the same histological picture with alcoholic liver disease. The progression of NAFLD to NASH dramatically increases the risks of cirrhosis, liver failure, and hepatocellular carcinoma (HCC).46 In the case of NASH, as in a variety of hepatic disorders, is believed that the main pathogenesis of liver damage is the oxidative stress and cytokines. 46

This increase in prevalence in addition to the almost pandemic rates of obesity and diabetes mellitus type 2, that are tightly connected with NAFLD, increase the need for find an efficient way to stop the oxidative stress in the liver in order to promote its regeneration. So apart from the obvious need for lifestyle changes the use of antioxidants and anti-fibrotic agents could prove a valuable ally in the treatment of NASH.The use of chemical antioxidants like N-Acetylcystein (NAC) with proved antioxidant function, is well documented in numerous studies, and are widely used in treatment protocols for multiple toxic and non-toxic induced liver injuries.40,41,42,43 At the same time a huge effort into the investigation and research of natural antioxidants, especially those ones that are from plant origin, is widely done in order to find a new and efficient ways to stop the oxidative stress damage and promote the spontaneous regeneration of liver. This also lead to an increase in advertising and use of, over the counter, natural supplements and medication, like pycnogenol (PYC) a maritime pine bark extract, but with not enough studies to support their efficiency. On the other hand the liver damage caused by carbon tetrachloride (CCl4) can act as an experimental model of free radical induced liver injury, and can be used in experimental animals in order to induce liver fibrosis and cirrhosis and thus use them to research the action of various antioxidants and their ability to halt or ever reverse the fibrotic and inflammatory changes on liver and promote the hepatic regeneration.47,7,8

In the light of all of the above we understand how important it is to study the regeneration of liver affected by various hepatopathies and also to study the effects of easily obtained over the counter, natural substances that are promoted as risk free remedies. This is going to be the purpose of this study; to study the effects of PYC in CCl4 induced toxic liver injury in rats and try to conclude if PYC can be an effective antioxidant for preventing the fibrosis and steatosis of human liver and thus reduce the hepatic injuries in various hepatopathies and especially in the rapidly increasing NAFLD and NASH.

REVIEW OF CURRENT STATE OF KNOWLEDGE

1.1 LIVER NORMAL ANATOMY AND HISTOLOGY

In order to have a better understanding of this thesis topic a review on liver normal and histopathological anatomy and findings is essential.

1.1.1 MACROSCOPICAL ANATOMY The liver is the second biggest organ, after the skin, in the human organism, weighting around the 2% of an adult’s body weight (on average of 1500g. ). It situated beneath the diaphragm in the right upper abdomen and midabdomen and extends up to the left upper abdomen. The liver has the general shape of a wedge, with its base situated to the right and its apex to the left. It is brownish-reddish in colour, normal liver has a soft consistency, and is highly vascular.Macroscopically we can divide the liver into two surfaces, a convex diaphragmatic and a visceral one.The diaphragmatic surface consists of the anterior part, which includes the right and left lobe that are separated by the falciform ligament, and the inferior border where the anterior surface converges with the visceral one, the superior part, where we have the attachment of liver to the diaphragm, in the bare area, and we can distinguish the coronary ligament, which is continues as left and right triangular ligament, and the fibrous appendix of liver.The visceral surface has a complex arrangement of structures which are subdivided by a set of H-shaped grooves. The portal hepatis forms the horizontal part, the fissure for round ligament the left part and the fossa for gall bladder and the groove for vena cava the right one. The left limb of H divides the right and left lobe while the right limb of H divides the right lobe into the caudate lobe behind and quadrate lobe in front.1,2

1.1.2 MICROSCOPICAL ANATOMY AND HISTOLOGICAL FEATURESMicroscopically we can distinguish that the liver is enclosed in a fibrous capsule that extends and accompanies the hepatic vessels and into the interior of the organ creating a supporting connective tissue framework known as Glisson's capsule. In the spaces of connective tissue framework we can find the hepatocytes that are the epithelial cells of liver grouped in interconnected plates. The Connective tissue together with the hepatocytes and the vessels form the architectonic structural unit of liver, known as lobules of liver.Hepatic lobules microscopic anatomy shows a polyhedral shape with three to six portal areas as its periphery and a venule, called a central vein in its center. The portal areas at the corners of the lobules consist of connective tissue where are embedded a venule, which is a branch of portal vein, an arteriole, which is a branch of hepatic artery and a duct of cuboidal epithelium, which is a part of bile duct system, this three

structures are collectively called as portal triad. Portal areas also contain nerve fibers and lymphatics. (figure 1)The interconnected plates of the hepatocytes, in the hepatic lobules, are arranged radially around the central vein. The plates of the hepatocytes are branch and anastomose freely leading to a sponge like structure. Between the plates we can find the liver sinusoids, which are an important microvascular component of the liver. The liver sinusoids are irregular and are separated by hepatocytes by a thin discontinuous basal lamina and a very narrow perisinusoidal space called space of Disse, where we have the micro-projection of hepatocytes villi necessary for exchanges between hepatocytes and plasma. By this villi we have the secretion of macromolecules into the plasma, which are produced in liver (eg. lipoproteins, albumin, fibrinogen), and also the take up of macromolecules from plasma for their catabolism by the liver. Around these sinusoids we can find thin and delicate sheaths of reticular fibers, which surround them and support them. (fingure 1)Histologically we have two more noteworthy cells associated with the sinusoids, those are the Kupffer cells or stellate macrophages, found between sinusoidal endothelial cells and within the sinusoid lumen, and the Ito cells or fat storing cells, found in perisinusoidal space. (figure1)Kuppfer cells function is to break down aged red blood cells and free heme for re-use, to remove bacteria and debris entered by the portal circulation and last but not least have the role of

antigen presenting cells in adaptive immunity.Ito cells have small lipid droplets containing vitamin A and the is main functions are to store vitamin A, produce the extracellular matrix components and act as regulators in local immunity. In chronic liver disease Ito cells proliferate and also acquire functions of myofibroblasts, accompanied with losing or not their lipid droplets.Those

Figure 1: Three-dimensional aspect of the normal liver. In the upper center is the central vein; in the lower center is the portal vein. Note the bile canaliculus, liver plates, Hering's canal, Kupffer cells, sinusoid, fat-storing cell, and sinusoid endothelial cells.Source:Basic Histology: Text & Atlas (Junqueira's Basic Histology). Junqueira,Luiz. Carneiro,Jose 11th Edition. McGraw-Hill Medical (2005)

cells are found in close proximity with the damaged hepatocytes and play a major role in liver fibrosis and fibrotic changes after alcoholic liver disease. Hepatocytes are the chief functional cells of the liver performing a number of metabolic, endocrine and secretory functions and they contribute around the 80% of the total mass of the liver.The hepatocytes have a polygonal shape and their sides can be in contact either with sinusoids, called as sinusoidal face, or neighbouring hepatocytes, called as lateral faces. A portion of the lateral faces of hepatocytes is modified to form bile canaliculi which is the first portion of the bile duct system, later on the bile canaliculi form a complex anastomosing network progressing along the plates of the hepatic lobules and terminate in the region of portal spaces where they are emptying into the bile ducts.Microvilli are present abundantly on the sinusoidal face and project sparsely into bile canaliculi.

The hepatocytes in H&E staining have eosinophilic cytoplasm, due to large amount of mitochondria up to 2000 per cell, with large spherical nuclei with nucleoli.(figure2) The cells are usually have more than one nucleus and are polyploid characterised by bigger size, always proportional to their ploidy.(figure 2) Hepatocytes have an active role in synthesis of protein and lipids for export. This leads to large quantities of both rough and smooth endoplasmic reticulum, as seen by electronic microscopy, something that cakes in contrast to most glandular epithelial cells which contain a single Golgi organelle where hepatocytes usually have many stacks of Golgi membranes. Golgi vesicles are in great number around the bile canaliculi, due to the transport of bile constituents into those channels. Also an other main function of hepatocytes is to synthesise and secrete very low density lipoproteins, which are seen in electron microscope as electron dense particles inside smooth endoplasmatic reticulum. The hepatocyte also frequently contains deposits of glycogen, which appears as coarse, electron dense granules in the cytosol, usually close to the smooth endoplasmatic reticulum. This glycogen depot is needed when glucose level in the blood falls below normal, in this way hepatocytes

Figure 2: a)hepatosites (H) and sinusoids (S) in H&E, b)reticular fibers (R) and hepatocytes treated with silver staining, c)radiation of hepatocyte’s plates to central vein (C) in mallory trichrome,d)peripheral portal areas Portal vein (PV) hepatic artery (HA) and bile canaliculi (BC)source: Anthony Mescher,Junqueira’s Basic Histology: Text and Atlas.13th Edition.McGraw-Hill Medical.2013.

maintain a steady level of blood glucose. Hepatocytes also store triglycerides in small lipid droplets. This metabolite storage is crucial because is needed for suppling to the body energy between meals. Hepatocytes are also responsible for converting the lipids and amino acids into glucose by the process of gluconeogenesis and also they are the place of amino acid deamination that results into production of urea which is transported into the blood and later excreted by kidneys. Last but not least, studies of liver micro anatomy, physiology, and pathology have given rise to three related ways to view the liver’s organisation according to different aspects of hepatocyte activity. The classic lobule which offer a basic understanding of the structure and function relationship in liver organisation and emphasises the endocrine function of the hepatocytes as the blood pass through them toward the central vein, which means that drains blood from portal vein and hepatic artery to hepatic or the central vein. The portal lobule emphasises the exocrine action of the hepatocytes and the flow of bile from the region of three classic lobules towards the bile duct in the center of the three classic lobules, so its function is to drain bile from hepatocytes to the bile duct. The liver acinus, the last of the three concept of liver organisation, which supplies oxygenated and nutrient contents of blood to hepatocytes.(figure 3) 1,3,4

1.1.3 REGULATION OF LIVER MASS- REGENERATIONThe liver is one organ with the ability to restore itself even after significant hepatic tissue loss from either acute liver injury or even by partial hepatectomy. Studies have shown that in partial hepatectomy where up to 70% of the liver has been removed, the remaining lobes enlarge and restore the the liver to its original size. This process is remarkably rapid and needs only up to 7 days in rats. The control of regeneration process remain poorly understood but factors such as hepatocyte growth factor (HGF), tumor necrosis factor (TNF), inteleukin 6 (IL6) and transforming growth factor(TGF) ,appear to be important factors. 10

With more details, the HGF is known that is produced by mesenchymal cells, but not the

hepatocytes and appears to play a major role in liver regeneration. The blood levels of HGF have been shown to rise more than 20 fold, after experimental partial hepatectomy, but the mitotic activity is only found in the liver. . The IL6, even though is induced by TNFα, has a clearly antiapoptotic effect and the TGFβ is a cytokine secreted by hepatic cells with potent inhibitory action on liver cell proliferation and has been proposed to be regarded as the major terminator of liver regeneration. 7,10

Last but not least according to literature experiments indicate that there mudt be an unknown signal that regulates the liver growth in relation to body size, in order a optimal metabolic function could be maintained.Liver injuries and diseases associated with fibrosis, hepatitis and viral infections lead to severe impairment of liver regenerative ability and deterioration of liver functions. 10

Figure 3: Concepts of hepatic lobule and function relationshipssource: Anthony Mescher,Junqueira’s Basic Histology: Text and Atlas.13th Edition.McGraw-Hill Medical.2013.

1.2 LIVER DRUG AND CHEMICAL TOXICITY

1.2.1 HEPATIC DRUG METABOLISMLiver id the main metabolic organ of the organism and plays a major role in metabolising a great number of organic and inorganic chemicals and drugs that enter the organism. Point of entry for this substances can be the respiratory tract, if entered by inhalation, the cardiovascular system, if entered directly to the blood flow by injection, or most commonly by gastrointestinal tract and especially via the intestinal tract.The main drug and chemical metabolism is take place in the microsomal fraction of the smooth endoplasmic reticulum of the liver cells by the P-450 cytochrome and the cytochrome reductase enzyme systems. Other steps which take place in the metabolism of tis substances are their conjugation with endogenous molecules, their active transport from the hepatocytes and at the end their excretion in the bile or by the urine, depending on their molecular weight.Predisposing factors for an individual to hepatic toxic injury are the pre-existing liver disease, ageing, female sex and genetic inability to perform a particular biotransformation. 5,6

1.2.2 HEPATOTOXICITY The toxic liver injury produced by drugs or chemicals may virtually appear as any form of natural occurring liver disease. This is very important in clinical praxis, because we need always to check for hepatic toxic injury in the case of patient presenting with liver disease or unexplained jaundice, in this cases we must always check the anamnesis of the patient for drug intake or exposure to chemicals and always bear in mind that iatrogenic related hepatotoxicity is the commonest form of toxic liver injury. The clinical symptoms become more and more obvious and serious if the exposure to the toxic factor is not discontinued. The exposure to the toxic compounds could be accidental, homicidal or suicidal ingestion.5

Toxic compounds can be inorganic or organic substances. Inorganic hepatotoxic compounds are arsenic, phosphorous, copper and iron ; the organic hepatotoxic compounds usually includes certain naturally occurring plant toxins such as pyrrolizidine alkaloids, mycotoxins, and bacterial toxins. The synthetic group of organic substances are usually a large number of medicinal agents.Drug reaction affecting the liver could be divided in two main classes, those are the direct, also called predictable, and the indirect, also called unpredictable or idiosyncratic.Direct reaction is when the drug-chemical or one of its metabolites is directly toxic to the liver or it may lower the host immune defence mechanisms. The toxic effects are occurring in most individuals who consume them and their hepatotoxicity is dose dependent in example carbon tetrachloride.Indirect reaction is when the drug-chemical or one of their metabolites acts as hapten thus inducing hypersensitivity in the host, in these instances can be associated with presence of autoantibodies, to liver-kidney microsomes ,directed against cytochrome P450 enzyme. The toxic effects in this group are not occurring in most individuals who consume them and their effect is not dose related, a great example of this category is the acetaminophen.The pathological changes induced by hepatotoxins can be summarised in the Table 1 and can be included in 2 broad categories, acute liver injury and chronic liver injury.In acute liver injury the main characteristics are the cholestasis, hepatocellular necrosis, fatty changes-steatosis, granulomatous reactions and/or vascular disease. On the other hand in

chronic liver disease the prominent features are variable degrees of fibrosis, cirrhosis and also of neoplasia. 5

Table 1 classification of hepatic drug reactionsACUTE LIVER DISEASE

Pathologic changes Agents

1.zonal necrosis carbon tetrachlorideacetaminophenhalothane

2.massive necrosis halothaneacetaminophenmethyldopa

3.fatty change tetracyclinesalicylatesmethotrexateethanol

4.hepatitits methyldopaisoniazid halotheneketoconazole

5.granuloma formation sulfanamidesmethyldopaquinidineallopurinol

6.cholestasis sex hormones (including oral contraceptives)chlorptomazinenitrofurantoin

7.veno-occlusive disease cytotoxic drugs

8.hepatic/portal vein thrombosis oral contraceptives

Chronic liver disease

Pathologic changes Agents

1.fibrosis-cirrhosis methotrexate

2.focal nodular hyperplasia vinyl chloridevitamine Asex hormone

3.adenoma sex hormones

4.hepatocellular carcinoma sex hormones

Table sources:MOHAN,harsh.textbook of pathology.6th edition.jaypee brothers medical publishers.2010.933p.ISBN:978-81-8448-702-2

1.2.3 CARBON TETRACHLORIDE HEPATOTOXICITYCarbon tetrachloride (CCL4) belongs to the halogenated alkanes, together with other substances such as chloroform and iodoform, the use of those substances have been banned or severely restricted due to their high toxicity. CCl4 is widely used, even nowadays, as a model substance to elucidate the mechanisms of action of hepatotoxic effects. The CCl4 administration lead to hepatocyte injury, necrosis, inflammation, and fibrosis, this spreads to link the vascular structures that feed into and drain the hepatic sinusoid like the portal tract and central vein respectively. It also activates the hepatic stellate cell (HSC) leading to apoptosis of the hepatocytes and also zone III necrosis.9 Studies have shown that ingestion of carbon tetrachloride with single doses around 90-180 mg kg-1 of body weight can cause mild hepatotoxicity, for example fatty liver with macro and micro steatosis but ingestion of 670 mg kg-1 lead to marked hepatotoxicity with severe zonal necrosis and also nephrotoxicity. Also continuous administration of CCl4 can provide moderate cell necrosis and fatty infiltration in about four weeks.7,8 According to several factors such as, dose, exposure time, age of the affected organism, regeneration of liver is possible leading to full recovery from liver damage.CCl4 is activated by cytochrome (CYP)2E1, CYP2B1 or CYP2B2, and possibly CYP3A, to form the trichloromethyl radical, CCl3*. This radical is able to bind to nucleic acids, proteins and lipids leading to impairing cellular processes like lipid metabolism. The impaired lipid metabolism may lead to fatty liver degeneration (steatosis). In addition CCl3* and DNA adduct formation is thought to function as initiator of hepatic cancer. CCl3* has also the ability to react with oxygen and form the trichloromethylperoxy radical CCl3OO* which is highly reactive and can initiate the chain reaction of lipid peroxidation, which attacks and destroys polyunsaturated fatty acids, in particular those associated with phospholipids. This attack and destruction of phospholipids affects the permeability of mitochondrial, endoplasmic reticulum, and plasma membranes, leading to loss of intracellular calcium sequestration and homeostasis, something that heavily adds to cell damage. Additionally by the degradation of the fatty acids we have the production of reactive aldehydes, especially 4-hydroxynonenal, which has the ability to easily bind to functional groups of proteins leading to inhibition of important enzyme activities. Together with all of the above the CCl4 intoxication also leads to hypo-methylation of cell components, that in the case of RNA the outcome is the inhibition of protein synthesis and in the case of phospholipids it may play a major role in the inhibition of lipoprotein secretion. Here it is important to note that none of the above processes per se can be considered the ultimate cause of CCl4-induced cell death. It is by cooperation of all of the above that lead to a fatal outcome always provided that the CCL4 acts in a high single dose, or over longer periods of time at low doses. 7

On molecular level CCl4 have the ability to activate tumor necrosis factor alpha, nitric oxide, and transforming growth factors alpha and beta intracellular; this appear to direct the cell toward apoptosis (self destruction) or fibrosis. TNFα direct toward apoptosis, whereas the TGFα and TGFβ appear to pushes to fibrosis.7

Several of the above processes can be interrupted by the use of antioxidants and mitogens, respectively, by restoring cellular methylation, or by preserving calcium sequestration. Chemicals that induce cytochromes that metabolize CCl4, or delay tissue regeneration when co-administered with CCl4 will potentiate its toxicity thoroughly, while appropriate CYP450 inhibitors will alleviate much of the toxicity. Oxygen partial pressure can also direct the course of CCl4 hepatotoxicity. Pressures between 5 and 35 mmHg favor lipid peroxidation, whereas absence of oxygen, as well as a partial pressure above 100 mmHg, both prevent lipid peroxidation entirely. Consequently, the location of CCl4-induced damage mirrors the oxygen gradient across the liver lobule.7

1.3 POLYPHENOLS

1.3.1 POLYPHENOLS FUNCTIONPolyphenols can be found in food products of plant origin such as fruits, vegetables, fresh beverages and are one of the main sources of phenolic compounds in human diet. Polyphenols are directly related to specific characteristics of foods like flavour and colour. The polyphenols are metabolized both in tissues and also by the colonic normal microbial flora and normally, the circulating polyphenols are glucuronidated and/or sulphated and no free aglycones are found in plasma. The presence of phenolic compounds in the diet has been shown to be beneficial to health due to their antioxidant (with rationale that are able to prevent the free radical damage to macromolecules), anti-inflammatory, and vasodilating properties. The health effects of polyphenols are in direct relation to the amount consumed and also their bioavailability. Moreover, polyphenols are able to exterminate or halt the growth of microorganisms like bacteria, fungi, or protozoans and also some dietary polyphenols have shown that they may have a significant effect on the colonic flora providing a prebiotic effect. All of the above can make the polyphenols a potential functional food.11

However, newer studies have shown that the reality is much more complex than this and that several issues, notably content in food, bioavailability, or in vivo antioxidant activity need to be resolved in order to widely use the polyphenols compounds in clinical praxis.12

According to a study realised on 2005, performed by Gary Williamson and Claudine Manach, review 93 studies related to polyphenols and the is activity in human organism, taking into consideration the bioavailability and metabolism factors, has shown that for some classes of dietary polyphenols, there are now sufficient studies to indicate the type and magnitude of effects among humans also in vivo, on the basis of short-term changes in biomarkers.12

According to their findings Isoflavones (genistein and daidzein, found in soy) have significant effects on bone health in postmenopausal women, together with some weak hormonal effects. Monomeric catechins (found at especially high concentrations in tea) have effects on plasma antioxidant biomarkers and energy metabolism. Procyanidins (oligomeric catechins found at high concentrations in red wine, grapes, cocoa, cranberries, apples, and some supplements such as Pycnogenol) have pronounced effects on the vascular system, including but not limited to plasma antioxidant activity. Quercetin (the main representative of the flavonol class, found at high concentrations in onions, apples, red wine, broccoli, tea, and Ginkgo biloba) influences some carcinogenesis markers and has small effects on plasma antioxidant biomarkers in vivo, although some studies failed to find this effect. Compared with the effects of polyphenols in vitro, the effects in vivo, although significant, are more limited. The reasons for this are 1) lack of validated in vivo biomarkers, especially in the area of carcinogenesis; 2) lack of long-term studies; and 3) lack of understanding or consideration of bioavailability in the in vitro studies (The range of concentrations required for having an effect in vitro is from <0.1 μmol/L to >100 μmol/L but the physiologic concentrations rarely exceed the 10 μmol/L. So the effects of polyphenols in vitro at concentrations of >10 μmol/L should generally consider invalid), which are subsequently used for the design of in vivo experiments.12

1.3.2 PROCYADINSProcyanidins are oligomeric catechins, covalently linked together with the commonest be dimers and trimers . Procyanidins, as shown in studies13, can be found in high concentrations in cocoa, grapes (and grapes products such as wine), apples and are in many other fruits, like blackberries, cherries, sour cherries, figs, and plums.

Purified procyanidins have been found to be weak bioactive in vitro but on the other hand they exhibit numerous effects in vivo in intervention studies. Procyanidins usually occur together with monomeric (+)-catechin and (−)-epicatechin; therefore, it is not clear whether the observed effects are attributable to the procyanidin component, the monomeric component, or both.12 Their main effects are on the vascular system and include increase in plasma antioxidant activity, decreased platelet aggregation, decreased plasma concentrations of lipid peroxide, reduced LDL cholesterol concentration together with increased HDL cholesterol concentration in addition to decreased susceptibility of LDL to oxidation. Also endothelium-dependent blood vessel dilation with decreased blood pressure, beneficial effects on capillary fragility and permeability together with increased platelet-derived nitric oxide production, decreased superoxide release and many others.12

1.4 PYCNOGENOL

1.4.1 CHEMICAL COMPOSITION AND FUNTION OF PYCNOGENOLPycnogenol (PYC), is a standardized extract of French maritime pine bark that is composed primarily by procyadins and phenolics acids. According to literature14, PYC include a variety of procyanidins that can be from the monomeric catechin and taxifolin to oligomers with 7 or even more flavonoid subunits. The phenolic acids are derivatives of benzoic and cinnamic acids. The ferulic acid and taxifolin components are rapidly absorbed and excreted as glucuronides or sulphates in men, whereas procyanidins are absorbed slowly and metabolized to valerolactones which are excreted as glucuronides. PYC has shown low levels of acute or chronic toxicity and very few and mild unwanted effects occurring only on a small portion of patients after oral administration.12,14

Pycnogenol has been reported to have many benefits range from cardiovascular such as vaso-relaxant activity, angiotensin-converting enzyme inhibiting activity, and ability to enchase the microcirculation by increasing capillary permeability. Moreover, effects on the immune system and modulation of nitrogen monoxide metabolism have been reported.15

Below is cited a table with a complication of studies/trials on pycnogenol activity in various medical problems mentioning only the main finding regarding the pycnogenol effects. (Table 2)

Table 2: Studies/trials of Pycnogenol with summary of their resultsEFFECTS PYCNOGENOL

ADMINISTRATION RESULTS

REFFERENCE

Antioxidant and free radical scavenging activities

1) Decreasing in the stand cleavage in pBR322 plasmid DNA caused by hydroxyl radicals produced by iron/ascorbic acid.

2) Synergetic antioxidant effects with vitamins C, E, and also lutein for prevention of lipid peroxidation in liposome and porcine retinal homogenate.

1) 18

2) 16,17

Table 2: Studies/trials of Pycnogenol with summary of their resultsAnti-inflammatory effects 1) Protective effects against UV-

light skin damages.

2) The inhibition of NF-kappaB activation in lipopolysaccharide -stimulated monocytes.

3) Decrease of symptoms of osteoarthritis, in more detail, lower pain and stiffness.

1) 19

2) 20

3) 21,22

cardiovascular effects 1) Could help to reduce the dose of the calcium antagonist nifedipine and to improve endothelial function of hypertensive patients.

2) Decrease in serum thromboxane concentration and the systolic blood pressure.

3) Reduce smoke-enhanced thromboxane B formation and also platelet aggregation in response to cigarette smoking.

1) 23

2) 24

3) 25

venous system effects 1) Reducing the lower limb circumference and improving the symptoms of chronic venous insufficiency.

2) Venous ulcers improvement and much faster healing with topical use.

1) 26

2) 27

cholesterol lowering effects 1) Significantly lowering of LDL and increasing of HDL

1) 15,26

antidiabetics effects 1) Reduce the risk of microangiopathy and diabetic retinopathy.

1) 28,29,30

asthma related effects 1) Reduction of asthma related symptoms and improvement of lung functions, able to act as adjunct in the management of d asthma

1) 31,32

anti-microbial and antiviral effects

1) Pycnogenol inhibited the growth

of 23 tested microorganisms

(both prokaryotic and eukaryotic

organisms) with minimum

concentrations dosage ranging

from 20 to 250 μg/mL

1) 33

Table 2: Studies/trials of Pycnogenol with summary of their resultsDysmenorrhea and menstrual pain

1) Decrease the need for analgestic medication in dysmenorrhea, analgesic effect on menstrual pain

1) 34,35

Attention deficit hyperactivity disorder

1) decrease of ADHD symptoms, normalise the catecholamine concentrations, to reduced oxidative stress and reducing hyperactivity, improves attention, visual-motoric coordination and concentration

1) 36,37

Table sources:16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,33,33,34,35,36,37

1.5 N-ACETYLCYSTEINE (NAC)

1.5.1 FUNCTIONS OF NAC AND MOLECULAR MECHANISMS OF ACTION (A BRIEF REVIEW OF LITERATURE)

N-acetylcysteine (NAC) is a precursor of glutathione and its role in treating acetaminophen induced acute liver failure has been established through many studies.38,39 According to literature the antidote action of N-Acetylcystein in reducing or even can stop the toxicity is related by its ability to reduce the formation and accumulation of N-acetyl-p-benzoquinoneimine and acting as glutathione substitute together with enhancement of nontoxic sulfate conjugation.40 But also studies have shown that NAC has also antioxidant function, according to published articles, NAC antioxidant function is due to specific molecular mechanisms such as activation of c-Jun N-terminal kinase, p38 MAP kinase and redox-sensitive activating protein-1 and nuclear factor kappa B transcription factor activities regulating expression of numerous genes. Also it seems to be able to prevent the apoptosis and promote cell survival by activating extracellular signal regulated kinase pathway.41 All of the above seems to prevent the reactive oxygen species related damage and also, due to the fact, that NAC is able to prevent apoptosis it may have a positive effect in degenerative diseases.41NAC also show multiple medical functions such as anti-inflammatory, inotropic, and vasodilating effects that according to studies can improve the micro-circulatory flow of blood and oxygen to organs.42,43

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