i

RESVERATROL: ANTIOXIDANT EFFECTS AND DRUG INTERACTIONS

BY Puja Agrawal

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Major in Chemistry South Dakota State University 2003

UMI Number: 1417435

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UMI Microform 1417435 Copyright 2004 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ____________________________________________________________ ProQuest Information and Learning Company 300 North Zeeb Road PO Box 1346 Ann Arbor, MI 48106-1346

ii RESVERATROL: ANTIOXIDANT EFFECTS AND DRUG INTERACTIONS

This thesis is approved as a creditable and independent investigation by a candidate for the Master of Science degree and is acceptable for meeting the thesis requirements for this degree. Acceptance of this thesis does not imply that the conclusions reached by the candidate are necessarily the conclusions of the major department.

Dr. Fathi T. Halaweish Advisor

Date

Dr. Chandradhar Dwivedi Co-Advisor

Date

Dr. James Rice Department Head, Chemistry and Biochemistry

Date

iii ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. Fathi T. Halaweish and Dr. Chandradhar Dwivedi for their guidance and advice throughout this study and course work. I am also grateful for the work experience I have gained in the Chemistry Labs and Pharmaceutical Research Labs, which will be invaluable for my future career. I would like to thank Dr. James Rice, Dr. Igor Sergeev, Dr. Timothy Wittig and Dr. Manisha Sonee for serving in my advisory committee. I also would like to thank the staff and graduate students who offered help during the graduate study program.

iv ABSTRACT

RESVERATROL: ANTIOXIDANT EFFECTS AND DRUG INTERACTIONS PUJA AGRAWAL July 14th 2003

Resveratrol is a phytoalexin found in grapes. Epidemiological and experimental studies suggest that the consumption of wine, particularly red wine reduces the incidence and morbidity from coronary heart diseases. The purpose of this investigation is to study the in vitro antiperoxidant effect of resveratrol and compare with vitamin E, a known antioxidant. People using cardiovascular drugs may also consume resveratrol containing products for reducing the risk of disease. Thus, a potential interaction of resveratrol with the drugs is possible. Therefore, effects of resveratrol on the blood level of nifedipine, niacin, lovastatin in rats. IC50 values for resveratrol and vitamin E were 1.5x10-5M and 1x10-3 M (NADPHinduced) and 1.5x10-4 M, 1x10-3 M (Ascorbate-induced) respectively. These results indicate that resveratrol has higher antiperoxidant effect than vitamin E. For the drug interaction experiments, nifedipine (antihypertensive), niacin (antilipemic) or lovastatin (antilipemic) were co-administered with resveratrol and serum prepared from blood obtained after one hour or one week was assayed using highperformance liquid chromatography.

v Resveratrol pretreatment caused a 65% decrease in the concentration of nifedipine in both one hour and one week experiments. A modest increase (approximately 20%) in the level of niacin was observed in the presence of resveratrol at one hour and one week experiment. A decrease of 35% and 14.6% in the level of lovastatin was observed in both one hour experiment and one week experiment respectively. Inorder to elucidate the possible mechanism of action of these drug interaction, effects of resveratrol on rat liver microsomal drug metabolizing enzymes was investigated. Resveratrol caused a 22% decrease in cytochrome P450 3A in enzyme activity. This decrease may explain increased niacin levels but not the nifedipine and lovastatin. Nifedipine and lovastatin may be metabolized by different cytochrome P450 enzyme systems.

vi TABLE OF CONTENTS

Page Acknowledgements... iii Abstract.. iv List of Tables.... vii List of Figures...... viii Introduction..1 Literature Review.3 Methodology..... 27 Results... 39 Discussion..53 Conclusion.57 References..58

vii LIST OF TABLES Page 1. Antioxidant values of resveratrol and Vitamin E......... 41

viii LIST OF FIGURES Page 1. Structure of resveratrol....5 2. Structure of nifedipine...17 3. Structure of niacin......20 4. Structure of lovastatin24 5. Separation of resveratrol by HPLC40 6. Effects of resveratrol on ascorbate-induced lipid peroxidation.42 7. Effects of resveratrol on NADPH-induced lipid peroxidation..42 8. Effects of resveratrol pretreatment (1 hour) on nifedipine44 9. Effects of resveratrol pretreatment (1 week) on nifedipine...44 10. Determination of nifedipine by HPLC...45 11. Effects of resveratrol pretreatment (1 hour) on niacin...46 12. Effects of resveratrol pretreatment (1 week) on niacin..46 13. Determination of niacin by HPLC.48 14. Effects of resveratrol pretreatment (1 hour) on lovastatin.49 15. Effects of resveratrol pretreatment (1 week) on lovastatin49 16. Determination of lovastatin by HPLC...50 17. Effect of resveratrol on hydroxylation of testosterone by rat liver microsomal metabolizing enzyme52

1 INTRODUCTION The first known use of grape extracts for the benefit of human health can be dated back over thousand years. Darakchasava, an Ayurvedic medicine wherein the main constituent is Vitis vinifera L, is prescribed as cardiotonic in India (1). A significant portion of this preparation is resveratrol (trans-3,4,5-trihydroxystilbene), a polyphenolic phytoalexin that is generated in response to stress in specific plants and is found in grape skins, peanuts and red wine, as well as host of nonedible plants (2). This compound is abundant in grape vines compared with other flowering plants, but is relatively low in fruits and vegetables (3). Resveratrol is produced preferentially in response to fungal infections as seen in mature vine berries (4). In the recent years the possible beneficial role of mild to moderate red wine consumption in the prevention of heart disease has gained attention in the scientific community as well as in the media. Several studies suggest that the consumption of wine, particularly red wine in French population imparts a greater benefit in prevention of coronary heart diseases than that obtained from drinking other alcoholic beverages (5,6). These reports gave rise to what is now popularly termed the French paradox a lower incidence of cardiovascular disease in France despite a diet rich in saturated fats (2,7). Structurally, resveratrol is a stilbene, the parent skeleton structure of a family of compounds including the cis- and trans-isomers (8). Resveratrol, which reduces the risk of carcinogenesis, has attracted attention of researchers towards its role in prevention of cancer and heart diseases. Resveratrol has been reported to have numerous biological

2 effects including anti-inflammatory, antiplatelet, and anticarcinogenic effects in humans (9,10). The effects have been attributed to its chemical structure (6,7) and possible antiperoxidant effects (11,12). Resveratrol containing products such as red wine are consumed by the people who have or may be at higher risk to acquire cardiovascular diseases (e.g. hypertension, myocardial infarction, stroke etc.). A substantial number of resveratrol consumers may be also taking various drugs (antihypertensive, antilipemic) to treat or prevent the risk of cardiovascular diseases. Studies on the interaction of resveratrol and cardiovascular drugs during concurrent use are lacking. The objective of this study was to investigate the effects of resveratrol 1. to isolate resveratrol from grape extract, 2. on the blood level of niacin, nifedipine, lovastatin in rats, and 3. on liver microsomal lipid peroxidation and compare it with Vitamin E, a well known antioxidant, and liver microsomal metabolizing enzymes.

3 Literature Review

Nature has been a source of medicinal agents for thousands of years, and an impressive number of modern drugs have been isolated from natural sources, many based on their use as traditional medicines (13). The use of natural substances, particularly plants, to control diseases is a century old practice that has lead to the discovery of half of the modern pharmaceuticals (14). Documentation of the use of natural substances for medicinal purposes can be found as far back as 78 A.D (15). The use of natural products as an alternative to prescription medicines has become increasingly popular in the last decade, 120 active compounds have been currently isolated from higher plants and are widely used in modern medicine today (16). The drugs used by ancient civilizations were extracts of plants or animal products, plants in medicinal use have been estimated very highly, e.g. in India where the Ayurveda gave a broad access to the variety of medicines from plants reported since 1000 B.C (16). Natural products have a vast molecular diversity and functionality and they currently account for $20 billion annual sales in pharmaceutical and agrochemical markets (17). The plant-based systems of many other cultures has been extensively documented and play an essential role in health care, and it has been estimated by the World health Organization that approximately 80% of the world inhabitants rely mainly on traditional medicines for their primary health care (18). The use of herbal supplements in the US has become increasingly popular in recent years. In a survey conducted in 1999, 49% of adult Americans were estimated to

4 have used herbal products during the previous year (19). Of these, about 24% had used these products on a regular basis. These medications fall into the category of alternative/complementary medicines and, as such, are not regulated by the Food and Drug Administration (FDA) with the same scrutiny as conventional drugs. Several factors are believed to contribute to the increasing trend of herbal supplement utilization in this country e.g. ease of accessibility, the desire for self-medication, and the perceptions that the herbs are safer, gentler and less costly than conventional drugs. There are related aspects of herbal supplements that have been reviewed in some details recently (20-22). As the use of herbal supplements continues to grow under the prevailing scenario, some concerns have become apparent regarding the safety of these products. Of particular safety concern is potential interaction of these products with conventional drugs. It has been documented that as many as 31% of the patients who use herbal supplements do so in conjunction with prescribed drugs (19). A survey of the literature shows possible adverse interactions that may occur between some of the popular herbal supplements in the market and analgesic drugs (19). Although people usually take supplements in an effort to improve their health, some people may actually endanger their health by using these products, especially if they take prescription drugs. Some dietary supplements may interact in dangerous ways with prescription or over the counter medicines. Drugs and herbal supplements may interact in a variety of ways. These interactions may be additive, synergistic, whereby the herbal product increases or potentiates the action of the drug. By contrast, the herb may also be antagonistic to the

5 action of the drug. While herb-drug interactions may involve pharmacodynamic and pharmacokinetic mechanisms, they may result in either beneficial or adverse effects (90).

Resveratrol Resveratrol (trans-3,4,5-trihydroxystilbene) (Figure1), is a natural phytoalexin synthesized by plants including, Vitis vinifera, in response to injury or fungal attack (4).

Figure 1.

OH

H HO

H OH

Structure of trans- Resveratrol

Resveratrol has been reported to have numerous biological effects, including antiinflammatory, antiplatelet, and anticarcinogenic effects in humans. Recent studies have shown that resveratrol could be one of the active ingredients responsible for the reduced coronary heart diseases (24-25). Resveratrol is abundant in grapes and red wine. Resveratrol is unique as it is virtually absent in most fruits and vegetables that form a

6 major portion of the human diet. Wine may be considered the predominant bioavailable dietary source (1), but obviously ingestion of grapes or peanuts may be relevant. Resveratrol bears a simple chemical structure that may interact with a variety of receptors and enzymes thus acting as an activator or inhibitor in a number of pathways (4). The cancer preventive and therapeutic potential of resveratrol has also been studied in recent years. Significantly the activity has been much less debated than its estrogenic or cardioprotective properties (26). Systemic studies in a wide range of cell lines and carcinogenesis checkpoints have established its utility as a chemopreventive agent. It has shown to affect 3 major stages of carcinogenesis (4), and inhibit the formation of preneoplastic lesions in a mouse mammary organ culture model (27). The wide array of biological activities mediated by resveratrol suggests its mode of action may be complex, and this has been of great interest to scientists working in the areas of basic biology and pharmacology (9).

Cardiovascular diseases Cardiovascular Disease (CVD) includes dysfunctional conditions of the heart, arteries, and veins that supply oxygen to vital life-sustaining areas of the body like the brain, the heart itself, and other vital organs. Many risk factors are associated with cardiovascular diseases, most of them can be managed, but some cannot. Many people with cardiovascular disease have elevated or high cholesterol levels (84). Low high density lipoprotein (HDL) cholesterol (known as the good cholesterol) and high low density lipoprotein (LDL) cholesterol (known as the bad cholesterol) are more

7 specifically linked to cardiovascular disease than is total cholesterol (85). Hypertension (high blood pressure) is a major risk factor for cardiovascular diseases, and the risk increases, as blood pressure rises (86). Overweight individuals are more likely to have additional risk factors related to heart disease, specifically hypertension, high blood sugar levels, high cholesterol, high triglycerides, and diabetes (87). Alcohol is used throughout the world and it has long been known that heavy alcohol consumption is hazardous to various body organs including cardiovascular system (82). There is now substantial evidence that the intake of light or moderate amounts of alcohol is associated with reduced morbidity and mortality from several cardiovascular conditions, particularly coronary heart disease (83).

ANTIOXIDANT ACTIVITY Antioxidants An antioxidant may be defined as a substance that when present at low concentrations compared with those of an oxidizable substrate such as fats, proteins, carbohydrates, or DNA, significantly delays or prevents the oxidation of the substance.(45). Antioxidants include some vitamins, minerals and other compounds that occur naturally in food. Numbers of researchers have believed that foods rich in antioxidants may afford protection against some forms of cancer and other chronic illnesses such as heart diseases.

8 Over the past few years, free radical mediated reactions such as lipid peroxidation (32) has received considerable attention, following the identification of these processes in various disease states (28,30,33) and in aging (34). Lipid peroxidation has been implicated in post-ischemic diseases (28-30), head injury and strokes (35,36), inflammation (30), and carcinogenesis (37,38).

Lipid peroxidation Autoxidation of unsaturated lipids, like those found in biological membranes, is generally termed as lipid peroxidation. It is often suggested that lipid peroxidation is responsible for numerous effects observed in biological systems (32,36,39,40). Lipid peroxidation is known to be a free radical initiated and propagated event (48). Although cells are known to have natural defense mechanisms to protect them from free radicalmediated damage (31), under certain pathological conditions, oxygen derived free radicals can initiate a cascade of events, causing peroxidative damage to biological membranes (32). Lipid peroxidation has shown to be involved in certain disease states (28-30,35-38) and drug induces toxicities (41-46). Lipid peroxides are known to be involved in homogenates of different tissues given the availability of appropriate electron source (49-53). Various cell fractions such as lysosomes, mitochondria and microsomes form lipid peroxides in suitable incubation systems. However, the rate and amount of lipid peroxides formed is the highest in the microsomal fraction (52).

9 Nonenzymic lipid peroxidation Free radicals are the initiators of lipid peroxidation that can initiate a chain reaction in bulk lipid. These have sufficient energy to abstract a hydrogen atom from a methylene carbon of an unsaturated fatty acid (LH). The resulting carbon center radical (L.) reacts rapidly with molecular oxygen to form a peroxy radical (LOO.), the peroxy radical itself can abstract a hydrogen atom from an unsaturated fatty acid leaving a carbon centered radical and a lipid hydroperoxide. The peroxide products of a chain reaction (LOOH) are complex mixtures of isomers. Unless mediated by other oxidants or enzyme systems, oxidation proceeds through a free radical chain reaction mechanism involving three stages.

1. Initiation The lipid peroxidation reaction is initiated by the oxidation of an unsaturated fatty acid (LH): -H. LH L.

It is possible that reactive free radicals such as OH initiate this reaction (29). For example: LH + HO. The lipid radical (L) is oxidized to a peroxy radical: L. + O2 LOO. H2O + L.

10 2. Propagation The initiation is followed by various chain reactions resulting in the formation of monohydroperoxides of lipids (LOOH) and other products:

LOO. + LH

LOOH + L.

The peroxides can break down by metal catalysis and initiate other chain reactions:

LOOH + Fe2+

LOO. + Fe3+ + OH-

LOOH + Fe3+

LOO. + Fe3+ + H+

These lipid-derived radicals can now react with unsaturated fatty acids and propagate the chain (32). An important end product of lipid peroxidation is malonaldehyde; this compound is commonly used for the lipid peroxidation assay. At least 3 isolated double bonds of unsaturated fatty acids are required for malonaldehyde formation. Various intermediate products can undergo cyclic rearrangements and free radicals such as alkyl radicals can be generated for further chain propagation. The cyclic endoperoxy-hydroperoxides can form monohydroperoxides: It is important to note that several end products of the lipid peroxidation reactions, such as malonaldehyde and alkanes have been identified in biological systems, both in vivo and in vitro (32,42,54).

11 It is clear from above discussion that the lipid peroxidation process consists of a complex chain of free radical mediated reactions. Since the chain reactions are selfpropagating, it is essential for the cells to break the reaction chain in order to protect themselves against extensive peroxidative damage. A variety of primary and secondary defense mechanisms have been identified in biological systems (31,32). These include chain breaking antioxidants such as -tocopherol also known as vitamin E and enzymatic defense mechanisms such as superoxide dismutase (SOD) and glutathione peroxidase (42,47,55).

3. Chain termination steps The chain termination step proceeds by -:

LOO. + L.

nonradical products

L. + L.

nonradical products

Enzymic lipid peroxidation In enzymic lipid peroxidation the generation of lipid peroxidation products takes place at an active site of an enzyme. The hydroperoxides and endoperoxides produced are stereospecific and have important biological functions (32). The enzymes cyclooxygenase and lipoxygenase catalyze enzymic peroxidation. Free radicals are probably important intermediates in the reaction, but are localized to active centers of

12 proteins. During the formation of endoperoxides by cyclooxygenase, a powerful oxidant (Ox) is generated, which is amenable to scavenging by antioxidants (33). Although lipid peroxidation has been described in different cellular components, the endoplasmic reticulum (microsomal fraction) is the most susceptible (32,49,50,52). The endoplasmic reticulum is rich in various enzymes, is known to sequester, and has high lipid content. The microsomes originating from different organs such as the heart, liver, kidney, etc., vary in their susceptibility to lipid peroxidation. This may be due to different levels of enzymes responsible for propagation as well as termination of the lipid peroxidation reaction (54,56-58). Physiologically, enzyme-catalyzed lipid peroxidation is believed to be more important (32). It should be mentioned however, that lipid peroxidation of microsomes is also possible in non-enzymatic systems, given the availability of a suitable cofactor such as ascorbate and metal catalyst (52). Iron catalyzed, enzymatic microsomal lipid peroxidation depends primarily on NADPHcytochrome P-450 reductase (5). Molecular oxygen can be enzymatically reduced to O2-. and the chain reaction described earlier can lead to the peroxidation of microsomal lipids (29).

DRUG-DRUG INTERACTIONS The great variation in chemical structures, physical properties, and pharmacological effects of the numerous compounds used as therapeutic agents suggest that any two drugs administered concurrently might interact in a number of ways (86).

13 However experimental findings and clinical experience have shown that the great majority of interactions occur by a small number of basic mechanisms (101). A drug interaction is defined as to occur whenever the effects of one drug are modified in or on the body by the prior or concurrent administration of another pharmacologically active substance. When drug interactions occur, it may result in an antagonistic, synergistic, additive or unexpected response. The increase in the potency and number of new drugs has contributed immeasurably to modern drug therapy, but this has also created new problems. A matter of increasing concern is the greater incidence of adverse effects when two or more drugs are given concurrently. Serious crisis, and even death, has been reported in some interactions. Most drug interaction in humans that result in increased toxicity or decreased therapeutic effects are detected by qualitative observations during the clinical use of several drugs concurrently. Drugs interact with each other through a variety of mechanisms, but most can be classified as pharmacokinetic, pharmacodynamic, or combined toxicity. Drugs and herbal supplements may interact in a variety of ways. These interactions may be additive, synergistic, whereby the herbal product increases or potentiates the action of the drug. By contrast, the herb may also be antagonistic to the action of the drug. While herb-drug interactions may involve pharmacodynamic and pharmacokinetic mechanisms, they may result in either beneficial or adverse effects (90).

14 Interactions at site of action Multiple drug therapy is widely practiced either to treat a medical disorder to treat several concurrently existing ailments in the same patient. Pharmacokinetic drug interaction alteration of the pharmacokinetic profile of a drug by co-administered drugs can have pharmacological and / or toxicological consequences. Whole pharmacokinetic drug interactions can occur by during absorption, metabolism, disposition, and elimination phases after initial drug administration, interference with drug metabolism appears to be the predominant mechanism. The interference can occur via inhibition or induction of the metabolism of one drug by a co-administered drug. Inhibition of drug metabolism results in an increase in plasma/tissue drug concentration, which can lead to toxicity. Conversely, induction of drug metabolism can result in a decrease in drug concentration to a level, which is no longer efficacious (87).

Pharmacokinetic mechanisms: Drugs may affect the absorption, distribution, metabolism or renal excretion of other drugs (101). Absorption Drug absorption may be inhibited by, drugs with large surface area, binding resins, drugs which effect gastrointestinal motility, drugs that alter the gastrointestinal pH. In few cases, absorption may be reduced to the extent that therapeutic response is impaired, but in many other instances only small reductions in serum concentrations of the object drug occur.

15 Distribution Drugs may compete with each other for binding sites on plasma proteins or tissues. When this occurs, the unbound or free serum concentration of one or both drugs may increase, this may theoretically increase drug response. Metabolism Drug metabolism can be enhanced or reduced by the concurrent administration of other drugs. Alterations in metabolism of one drug by another probably cause more clinically important drug interactions than any other type of pharmacokinetic mechanism. Inhibition of drug metabolism can increase plasma drug concentrations and drug response with possible toxicity. Renal Excretion Most renal excretion drug interactions involve reduced excretion of one drug by another thereby increasing the plasma concentration of the drug.

Pharmacodynamic mechanisms The majority of the known pharmacodynamic drug interactions, some-times called pharmacologic drug interactions, occur when drugs with additive or antagonistic response are given together (101). Although such drug combinations often used therapeutically, in such situations the additive or antagonistic response may produce adverse effects (eg, patient receiving several drugs with anticholinergic effects). Some pharmacodynamic drug interactions involve mechanisms other than additive or antagonistic pharmacologic effects. For example, tricyclic antidepressants tend to reduce

16 blood pressure when given alone, but are likely to increase blood pressure in patients stabilized on clonidine. Pharmacodynamic drug interactions occur commonly in clinical medicine and, when anticipated, usually do not cause serious clinical difficulties. Nevertheless, it is difficult to anticipate all such interactions in a patient receiving upto 10-15 drugs, each of which may have two or three pharmacologic effects. Drug interactions can take various forms, occurring immediately or over several weeks. Some drugs simply should not be used together, while others can be combined only if accompanied by careful monitoring to detect emergency problems. Interactions can occur when one therapy affects how another is absorbed, metabolized, distributed or excreted. Interactions can also occur when one therapy alters the effect of another. In some prevention regimens, drug interactions may even cause more harm than good. For example, one drug might reduce blood levels of another drug, leading to the development of drug resistance. In other words, drug interactions could result in the development of a disease unresponsive to standard treatment. Resveratrol containing products such as red wine are consumed by the people who have or may be at higher risk to acquire cardiovascular diseases (e.g. hypertension, myocardial infarction, stroke etc.). Various drugs (such as antihypertensive, antilipemic) are also taken by people who also take resveratrol through various food sources or as dietary supplements. There may be a possibility that resveratrol interacts with these drugs when taken concomitantly.

17 NIFEDIPINE Nifedipine is a calcium (Ca2+) channel blocker that inhibits the transmembrane influx of calcium into cardiac muscle cells and vascular smooth muscle through specific ion channels. It induces relaxation of smooth muscle and decreases peripheral resistance (62,63) for which it is widely used for the treatment of hypertension, angina pectoris and other cardiovascular disorders (64,65). After oral administration, nifedipine is rapidly or almost completely absorbed but undergoes extensive first pass metabolism in humans (66,67). The structure of nifedipine is given in Figure 2. Figure 2.

NO2 H3COOC H3C N H COOCH3 CH3

Structure of Nifedipine Pharmacological effect All the calcium channel blockers that have been approved for clinical use decrease coronary vascular resistance and increase coronary blood flow. The dihydropyridines are more potent vasodilators in vivo and in vitro than is verapamil, which is more potent than diltiazem. Nifedipine given intravenously increases forearm blood flow with little effect on venous pooling, this indicates selective dilation of arterial

18 resistance vessels. The decrease in arterial blood pressure elicits sympathetic reflexes, with resultant tachycardia and positive inotropy. However, Nifedipine relaxes vascular smooth muscle at significantly lower concentration than those required for prominent direct effects on the heart. Thus, arteriolar resistance and blood pressure is lowered, contractility and segmental ventricular function are improved, and heart rate and cardiac output are increased modestly. After oral administration of nifedipine, arterial dilation increases peripheral blood flow while venous tone does not change (91-92).

Mechanism of action Nifedipine works by blocking the movement of calcium through the cell walls. Calcium is vital for the proper functioning of certain cells in the body. Nifedipine prevents calcium entering a cell and changes the way it works. In the case of then heart, blocking calcium reduces the contraction of the heart muscle and lowers the amount of electrical impulses generated within the heart to regulate the heartbeat. The mechanism by which nifedipine reduces arterial blood pressure involves peripheral arterial vasodilatation and consequently, a reduction in peripheral vascular resistance (92). The binding of nifedipine to voltage-dependent and possibly receptor-operated channels in vascular smooth muscle results in an inhibition of calcium influx through these channels (68). Stores of intracellular calcium in vascular smooth muscle are limited and thus dependent upon the influx of extracellular calcium for contraction to occur (69). The increased peripheral vascular resistance that is an underlying cause of hypertension results from an increase in active tension in the vascular smooth muscle. Studies have

19 demonstrated that the increase in active tension reflects an increase in cytosolic free calcium (70). Pharmacokinetics Nifedipine is rapidly and almost completely absorbed from the gastro-intestinal tract, and undergoes extensive first pass metabolism resulting in bioavailability between 45 - 75%. Peak plasma concentrations occur after 30-120 min, with a half-life of 2-5 hrs. Nifedipine is about 92-98% bound to plasma proteins. It is extensively metabolized in liver and 70-80% of the dose is excreted in urine almost entirely as inactive metabolites (92). The minimal effective concentration of nifedipine in humans is about 15 ng/ml. With convention formulations, plasma concentrations reach 100-200 ng/ml 1-2 h after drug administration and fall to about 5-10 ng/ml after 6-8 hr (71). Drug interactions Experience in over 1400 patients in a non-comparative clinical trial has shown that concomitant administration of nifedipine and beta-blocking agents is usually well tolerated, but there have been literature reports that suggest the combination may increase the likelihood of congestive heart failure, severe hypotension or exacerbation of angina (92). Interactions of nifedipine with many drugs have been reported. The effects of acetazolamide, amphotericin B, corticosteroids, dichlorphenamide, diuretics, methazolamide, beta-blockers like acebutolol, atenolol, betaxolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, timolol with nifedipine may increase and for carbamazepine, cyclosporine, procainamide, quinidine,

20 digitalis glycosides has been reported to increase when administered together with nifedipine (92-92).

NIACIN Niacin is a very important water-soluble vitamin. Deficiency of niacin causes a severe disease, called pellagra. Niacin is the generic word used for nicotinic acid, and also for derivatives used for exhibiting qualitatively the biological activity of niacinamide (73). Niacin is available in wide variety of foods, especially in meat and yeast extracts. Niacin is one of the oldest drugs used to treat dyslipidemia and is the most versatile in that it favorably affects virtually all lipid parameters. Niacin is a water-soluble Bcomplex vitamin that functions as a vitamin only after its conversion to nicotinamide adenine dinucleotide that acts as a component of other coenzymes (74). Niacin is the best agent available for increasing HDL-C (increments of 30% - 40%), its also lowers triglycerides by 35% to 45% and reduces LDL-C levels by 20%-30%. Niacin is also the only lipid-lowering drug that reduces Lp (a) levels significantly by about 40%. However adequate control of other lipid abnormalities renders an elevation of Lp (a) harmless (94). The structure of niacin is given in Figure 3. Figure 3.

COOH N
Structure of Niacin

21 Pharmacological actions Niacin is not directly converted to nicotinamide, which arises only from metabolism of NAD, the pharmacological effects and toxicity of niacin in man including flushing, pruritus, gastrointestinal distress, hepatotoxicity, and activation of peptic ulcer disease. Larger doses of niacin (2-6 gms per day) are sometimes used in the treatment of hyperlipoproteinemia. The important toxic effects of niacin are only seen with these doses (94).

Mechanism of action Niacin has various effects on lipoprotein metabolism. In adipose tissue, niacin inhibits the lipolysis of triglycerides by hormonesensitive lipase, which reduces transport of free fattyacids to the liver and decrease hepatic triglyceride synthesis. In the liver, niacin reduces triglyceride synthesis by inhibiting both the synthesis and esterification of fatty acids, effects that increase apoB degradation. Reduction of triglyceride synthesis reduces hepatic very low density lipoprotein (VLDL) production, which accounts for the reduced LDL levels. Niacin also enhances LPL activity, which promoted the clearance of chylomicrons and VLDL triglycerides. Niacin raises HDL-C levels by decreasing the fractional clearance of apoA-I in HDL rather than by enhancing HDL synthesis (94). Pharmacokinetic effect Niacin is rapidly and extensively absorbed (at least 60%-75% of the dose) when administered orally. Studies using radiolabel niacin in mice shoe that niacin and its

22 metabolites concentrate in the liver, kidney and the adipose tissue. The pharmacokinetic profile of niacin is complicated due to rapid and first pass metabolism, which is species and dose rate specific. In humans, one pathway is through a simple conjugation step with glycine to form nicotinuric acid (NUA). NUA is excreted in the urine, although there may be small amount of reversible metabolism back to niacin. The other pathway is formation of nicotinamide adenine dinucleotide (NAD). Nicotinamide is further metabolized to at least N-methylnicotinamide (MNA). Niacin and its metabolites are rapidly eliminated in the urine (94).

Drug interactions Niacin binds bile-acid sequestrants (cholesterol-lowering medications such as colestipol and cholestyramine) and may decrease their effectiveness; therefore, niacin and these medications should be taken at different times of the day. When niacin is taken at the same time as another class of cholesterol-lowering medications, called HMG-CoA reductase inhibitors or statins, the likelihood for serious side effects, such as muscle inflammation or liver toxicity is increased. Doses of niacin that are high enough to reduce cholesterol levels may raise blood sugar and lead to a loss of blood sugar control. However, one study suggests that niacin may actually benefit patients with recent onset of Type I diabetes. People taking insulin, metformin, glyburide, glipizide, or other similar medications used to treat high blood sugar levels should monitor their blood sugar levels closely.

23 Niacin has been reported to interact with tetracycline, an antibiotic, because it interferes with the absorption and effectiveness of this medication. When niacin is taken with certain blood pressure medications (such as prazosin, doxazosin, and guanabenz), the likelihood of side effects from these medications is increased. Niacin may interact with many drugs being administered concomitantly (95).

LOVASTATIN Lovastatin is cholesterol reducing agent isolated from strains of Aspergillus terreus, and is a member of a group of compounds known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are known as monacolins. This family of natural products includes nuclear variants of compactin that lack the 2methyl-1-oxobutyl groups. The active form of all these compounds is the hydroxy acid structure. Lovastatin is called monacolin K and has also been called mevinolin. This family of compounds has been studied in great detail for their influence on serum cholesterol levels (74,78). After oral ingestion, lovastatin, which is an inactive lactone, is hydrolysed to the corresponding -hydroxy acid form, which reduces cholesterol levels by inhibiting HMG-CoA reductase. This enzyme is responsible for catalyzing the conversion of hydroxy-methylglutaryl-CoA to mevalonate, the rate-determining step in de novo cholesterol biosynthesis in the human liver (80). The chemical structure of lovastatin is presented in Figure 4.

24 Figure 4.

OH O H O O H

Structure of Lovastatin Clinical pharmacology Lovastatin, the precursor of cholesterol produced by HMG-CoA reductase, is also essential in the production of molecules important for vital cell functions such as membrane integrity, cell signaling, protein synthesis and cell cycle progression (76). Lovastatin has been demonstrated to have antitumor activity in vitro against a variety of human cancer cells including melanomas, adenocarcinomas, and neuroblastoma (77-79).

Mechanism of action Lovastatin functions by inhibiting the enzyme HMG-CoA, 3-hydroxy-3methylglutaryl-coenzyme A reductase, that is required for the conversion of 3-hydroxy3-methylglutaryl-coenzyme A to mevalonic acid. Lovastatin exerts its major effect on

25 the reduction of LDL levels by competitively inhibiting HMG-CoA reductase, by product inhibition. Lovastatin affects blood cholesterol level by inhibiting cholesterogenesis in the liver, which results in increased expression of LDL receptor gene. In response to the reduced free cholesterol content within hepatocytes, membrane bound sterol regulatory element binding protein (SREBP) are cleaved by protease and traslocated to the nucleus. The transcription factors are then bound by the sterol-responsive element of the LDL receptor gene, enhancing transcription and ultimately increasing the synthesis of LDL receptors. Degradation of LDL receptors is also reduced. The greater number of LDL receptors on the surface of hepatocytes result in increased removal of LDL from the blood, thereby lowering the LDL-C level (97).

Pharmacokinetic All the statins are administered as the active -hydroxy acid form except for lovastatin and simvastatin, which are administered in the lactone form and must be hydrolysed in vivo to the corresponding -hydroxy acid in the liver. Lovastatin has been used for many years at doses of 20-80 mg/d to treat elevated serum cholesterol. Pharmacokinetic studies of lovastatin at these dose levels have found that 10% of the dose was excreted in urine and 83% in faces. The bioavailability of lovastatin was approximately 30%, likely because of extensive hepatic extraction. The maximum plasma concentration after a single oral dose was reached within approximately 3 hours, suggesting that the rate of absorption is relatively slow (74). Absorption of lovastatin appeared better when the drug was administered with food. Lovastatin is highly protein

26 bound (>90%) and is eliminated by metabolism via cytochrome P-450 in the liver. The elimination half-life is 2-4 hours (75).

Drug interaction Gemfibrozil and other fibrates, and niacin increase the risk of myopathy when given concomitantly with lovastatin. Lovastatin has not been reported to have too many drug interactions (97).

27 MATERIALS AND METHOD The following part of this thesis describes various reagents materials used and their preparation. Materials Trans resveratrol, ADP, NADP, sodium ascorbate, lovastatin (mevinolin), arabic gum, NADPH (tetrasodium salt), hydrocortisone acetate, testosterone, -tocopherol were purchased from Sigma Chemical Company (St Louis, MO) and glucose-6-phosphate dehydrogenase was obtained from Boeringer, the 2-thiobarbituric acid (TBA) was obtained from Eastman Organic Chemicals (Rochester, NY), nifedipine from ICN Biochemicals Inc. (Aura, OH) and niacin (nicotinic acid) from Acros Organics (New Jersey). Dimethyl sulfoxide (DMSO) was obtained from Fisher Scientific Comp. (Fairlawn, N.J). Instruments A homogenizer (model 5000) was purchased from Omni International (Waterbury, CT). A spectrophotometer (Du-64) was purchased from Beckman Instruments, Inc. (Irvine, CA). The ultracentrifuge (model LE80K) was obtained from Beckman Coulter (Fullerton, CA). The HPLC instrument used in this research was Varian Prostar, model 210 equipped with PDA detector, 25ml pumpheads for the solvent delivery system and rhodyne injection port with 20l loop. Type of column- Alltech C18, 4.6 x 150 mm, 5u ID 4.6mm. Alltech Associates, Inc (Deerfield, IL).

28 Solvents Acetonitrile, methanol (HPLC grade) was purchased from Pharmeo (Brookfield, OH), glacial acetic acid from Fisher Scientific Co. (Fairlawn, N.J).

Animals Female Sprague-Dawley rats (obtained from Charles-River, Wilmington MA) weighing 190-240 gms were maintained on commercial rat chow (Wayne Pet Food Division, Chicago, IL) and housed in the College of Pharmacy environmentally controlled animal house (temp.22 1C, humidity 40-60%, light 6am- 6pm) at least 4 weeks before use when their weight was approximately 150 gms so that enough blood could be obtained. The rats were randomly divided into groups and were housed three per cage.

Statistical analysis The INSTAT software (Graph Pad, San Diego, CA) was used to analyze the data. Students test was conducted and significance was considered at P<0.05.

EXTRACTION AND DETERMINATION OF RESVERATROL Grape extract (327gms) was dissolved in methanol (1.5L). It was then mixed in a grinder and then centrifuged for 10 min at 10000g. The grape extract was then rotaevaporated at 42C to. Water was added to the rotaevaporated extract and freezedried. The extract was then stored in cool place as the sample absorbed moisture.

29 The sample collected was dissolved in 3% NaHCO3 and filtered through Watmann filter paper under vacuum. The extract was partitioned 4 times against ethyl acetate (10ml). This ethyl acetate fraction was then washed with deionized water. Aqueous residue was removed from the organic phase by adding MgSO4 (5g). The ethyl acetate fraction was evaporated (which is the filtrate). The residue was resuspended in a small volume of ethyl acetate. It was then transferred in a vial and dried under nitrogen at room temperature. Further separation of resveratrol from other compounds was done using HPLC. Different fractions obtained were then rotaevaporated and dried under nitrogen. Dried resveratrol was then weighed.

HPLC analysis of resveratrol The samples were first analyzed by analytical column for which the flow rate was maintained at 1 ml/min and 20 l was the injection volume. Resveratrol was then separated from the other constituents of the grape extract by preparative column where the flow rate was 7ml/min and injection volume being 100 l. Mobile phase solvent A - water and acetonitrile (7:3), solvent B- water and methanol (1:1). Mode of solvent delivery system was gradient elution. The run time was 15 min.

30 ANTIOXIDANT ACTIVITY Method The rats were fasted overnight and were sacrificed under ether anesthesia next morning. The livers were taken out and were washed and homogenized with 1.15% KCl. The microsomes were prepared by differential centrifugation. The liver homogenate was first centrifuged at 10,000 X g for 15 min. The supernatant thus obtained was then again centrifuged at 105,000 X g for 90 min in an ultracentrifuge. The microsomal pellet was washed twice with 1.15% KCl and was resuspended in 1.15% KCl (89). The microsomal protein was determined by the Bradford method using a Bio-Rad protein assay kit. Microsomal lipid peroxidation was induced both enzymatically and non-enzymatically. The enzymatic was NADPH-induced and the non-enzymatic was ascorbate-FeCl3 induced (89).

Enzymic lipid peroxidation The microsomal suspensions were incubated with freshly prepared NADPH generating system (glucose-6-phosphate, 5 mM; NADP, 0.3 mM; 0.5 units of D-glucose6-phosphate dehydrogenase) in phosphate buffer (15 mM, pH 7.4) in the presence or absence of resveratrol. Total incubation volume was 2ml (89).

Nonenzymic lipid peroxidation For induction of nonenzymic lipid peroxidation the NADPH generating system was replaced by ascorbate (0.5 mM) and ADP-Fe complex (0.012 mM FeCl3, 0.04 mM

31 ADP). The incubation was carried aerobically at 37C. The total incubation volume was 2ml. Different concentrations of resveratrol and vitamin E were dissolved in DMSO and added to the incubation mixture for both enzymatically and nonenzymatically induced lipid peroxidation. A blank was run with DMSO alone (89).

Lipid peroxidation assay All the experiments were done in triplicate. The thiobarbituric acid assay for malonaldehyde was used to measure lipid peroxidation (89). Reactions were stopped after 20 min of incubation by adding 1 ml of 35% trichloroacetic acid followed by addition of 1 ml thiobarbituric acid solution (1.5%). The reaction mixture was heated for 20 min in boiling water and then was allowed to cool for 20 min, followed by centrifugation. The absorbance of the filtrate was read at 532nm using a Beckman DU64 spectrophotometer. Malonaldehyde values were calculated using a molar extinction coefficient of 1.56 X 105 M-1 cm-1 at 532nm.

Protein assay Protein was assayed in the liver microsome using Bio-Rad protein Assay kit (BioRad, Richmond, CA) (104).

32 RESVERATROL AND DRUG INTERACTION Short term experiment Rats were divided in two groups having three rats in each group. The first group received resveratrol (10 mg/kg, intraperitoneally (i.p.) whereas the second group was treated only with vehicle (gum acacia). One hour after the administration of resveratrol rats in both groups received appropriate dose of various drugs (nifedipine, niacin, lovastatin). Rats from both groups were sacrificed under anesthesia one hour after administration of the drug. The blood was drawn by cardiac puncture and was allowed to stand at room temperature (25C) for one hour so that the serum separates out. Serum was separated by centrifugation at 2000 X g. Serum was used for the determination of various drugs after appropriate extractions. The samples were then injected in the HPLC to determine the level of drug present in the serum.

Long term experiment Rats were divided in two groups having three rats in each group. First group received resveratrol (10 mg/kg, i.p) whereas second group was treated with vehicle (gum acacia) for one week. After 7 days, one hour after the administration of resveratrol in rats, both groups received appropriate dose of various drugs (nifedipine, niacin, lovastatin). Rats from both groups were sacrificed under anesthesia after one hour of administration of the drug. The blood was drawn by cardiac puncture and was allowed to stand at room temperature (25C) for one hour so that the serum separates out. Serum

33 was separated by centrifugation at 2000 X g. Serum was used for the determination of various drugs after appropriate extractions. The samples were then injected in the HPLC to determine the level of drug present in the serum.

Nifedipine experiment A dosage of 10 mg/kg and 20 mg/kg, i.p., was used for resveratrol and nifedipine respectively. Both resveratrol and nifedipine were suspended in gum acacia (5%). Extraction of nifedipine from serum 1. Acetonitrile (2 ml) was added to 0.5 ml of serum and was mixed in vortex for 30 min. 2. After centrifugation at 4000 rpm for 20 min, 2 ml of supernatant was added into test tube containing 1 ml of distilled water. 3. The reaction mixture was combined with 4.5 ml of acetone chloroform mixture (1:1 v/v) and mixed in vortex for 1 hr (this is to ensure complete extraction of nifedipine in the organic phase. 4. The resulting mixture was centrifuged at 4000 rpm for 20 min to separate organic and aqueous phase. 5. The aqueous phase was discarded. 6. The organic phase (5ml) was transferred to another test tube and evaporated under nitrogen. 7. The residue was dissolved in 100L of methanol. 8. The sample was then injected in HPLC for determination (98).

34 Standard preparation of nifedipine The standard stock solution of 1mg/ml concentration of nifedipine was prepared in methanol. Nifedipine is soluble in methanol and does not decompose. Dilutions were made to the desired concentrations of 0.1 mg/ml, 0.2 mg/ml, 0.01 mg/ml, 0.02 mg/ml, 0.08 mg/ml, and 0.02 mg/ml.

Determination of nifedipine Mobile phase water, acetonitrile, and methanol (50:25:25). Flow rate- 0.75ml/min. Injection volume- 20L. An isocratic solvent delivery was used. The run analysis was conducted for 30 min. Data acquisition was started after 6 min.

Niacin experiment A dosage of 10 mg/kg and 20 mg/kg, i.p., was used for resveratrol and niacin respectively. Both resveratrol and niacin were suspended in gum acacia (5%).

Extraction of niacin 1. Methanol (2 ml) was added to 0.5 ml of serum and was mixed in vortex for 30 min. 2. After centrifugation at 4000 rpm for 20 min, 2 ml of supernatant was added into test tube containing 1 ml of distilled water.

35 3. The reaction mixture was combined with 4.5 ml of acetone chloroform mixture (1:1 v/v) and mixed in vortex for 1 hr (this is to ensure complete extraction of niacin in the organic phase. 4. The resulting mixture was centrifuged at 4000 rpm for 20 min to separate organic and aqueous phase. 5. The aqueous phase is discarded. 6. The organic phase (5ml) was transferred to another test tube and evaporated under nitrogen. 7. The residue was dissolved in 100L of methanol. 8. The sample was then injected in HPLC for determination (99).

Standard preparation of niacin The standard stock solution of 1 mg/ml concentration of niacin was prepared in methanol. Niacin is soluble in methanol and does not decompose. Dilutions were made to the desired concentrations of, 0.1 mg/ml, 0.2 mg/ml, 0.01 mg/ml, 0.02 mg/ml, 0.08 mg/ml, and 0.02 mg/ml.

Determination of niacin Mobile phase methanol, acetonitrile, glacial acetic acid (75:20:5). Flow rate- 1.30 ml/min. Injection volume- 20L. An isocratic solvent delivery system was used. The runtime was 30 min.

36 Lovastatin experiment A dosage of 10 mg/kg and 4 mg/kg, i.p., was used for resveratrol and lovastatin respectively. Both resveratrol and lovastatin were suspended in gum acacia (5%).

Extraction of lovastatin 1. Acetonitrile (2 ml) was added to 0.5 ml of serum and was mixed in vortex for 30 min. 2. After centrifugation at 4000 rpm for 20 min, 2 ml of supernatant was added into test tube containing 1 ml of distilled water. 3. The reaction mixture was combined with 2 ml ethyl acetate and mixed in vortex for 1 hr (this is to ensure complete extraction of niacin in the organic phase). 4. The resulting mixture was centrifuged at 4000 rpm for 20 min to separate organic and aqueous phase. 5. The aqueous phase is discarded. 6. The organic phase (2ml) was transferred to another test tube and evaporated under nitrogen. 7. The residue was dissolved in 100L of acetonitrile. 8. The sample was then injected in HPLC for determination.

Standard preparation of lovastatin The standard stock solution of 1mg/ml concentration of lovastatin was prepared in methanol. Lovastatin is soluble in acetonitrile and does not decompose. Dilutions

37 were made to the desired concentrations of, 0.1 mg/ml, 0.2 mg/ml, 0.01 mg/ml, 0.02 mg/ml, 0.08 mg/ml, and 0.02 mg/ml.

Determination of lovastatin Mobile phase acetonitrile and 1% Phosphoric acid (65:35). Flow rate- 1 ml/min. Injection volume- 20L. Isocratic solvent delivery system was used. The runtime was 15 min.

Liver microsomal and drug metabolizing assay To determine the reason behind the change in serum concentration of the drugs the drug metabolizing assay was conducted. Rats were divided in two groups having three rats in each group. The first group received normal saline whereas second group was treated with resveratrol (10 mg/kg, i.p). One hour after the administration of resveratrol rats in both groups were sacrificed under anesthesia. Microsomes were prepared as mentioned under antioxidant activity. Hydroxylation of testosterone by rat liver microsomal P450s Phosphate buffer (200 l) (pH 7.4, ~0.09 M), 35l of rat liver microsomes (1mg/ml) were taken in a microcentrifuge. It was gently shaken before taking the microsomes. 35 l of NADPH (0.5 mM), and 2.5 l of testosterone (2.5 mM) were mixed in 1 ml-microcentrifuge tube for 5 seconds. The mixture was incubated at 37C

38 for 40 min. Acetonitrile (400 l) was added to the incubation mixture to stop the enzymatic reactions followed by the internal standard (35 l, 50 g/ml). The mixture was left to stand for 5 min and centrifuged at 8000 rpm for 5 min. The supernatant was then transferred, with a disposable plastic pipette, into a 1 ml disposable syringe with a filter (0.2 m, 13 mm) and filtered into a glass tube. The organic solvents are removed by a steam of air and the remaining solution (~200 l) was transferred into a small vial. The sample was then injected into HPLC for analysis. HPLC analysis of - hydroxy testosterone Mobile phase A: Methanol, B: Water. Flow rate- 1.00 ml/min. Injection volume- 20 L. Gradient elution was the mode of solvent delivery system. The run time was 34 min.

39 RESULTS

Separation and identification of resveratrol from grape extract Resveratrol was isolated from the grape extract. Trials for HPLC separation was conducted according to published method (Figure 5). Resveratrol concentration seemed very low. Total resveratrol recovered after preparative separation was 100 g hence resveratrol was obtained from pure source. Antioxidant activity Effects of resveratrol on enzymatic and nonenzymatic rat liver microsomal lipid peroxidation were investigated in order to evaluate the antioxidant activity. Figure 6 shows the effects on ascorbate-FeCl3-induced lipid peroxidation of rat liver microsomes. Resveratrol inhibited ascorbate-FeCl3-induced lipid peroxidation in a concentration dependant manner. The concentration required to reduce lipid peroxidation by 50% was 1.5x10-4 M. These values were compared with those of Vitamin E, which is an established antioxidant (5), for which the IC50 was determined to be 1x10-3 M (Table1). Lipid peroxidation was also induced enzymatically by NADPH generating system on rat liver microsomes. As it can be seen in Figure 7, resveratrol inhibited NADPH-induced lipid peroxidation in a concentration dependant manner and IC50 was of 1.5x10-5 M. These values were then compared with Vitamin E for which the IC50 was determined to be at 1x10-3 M (Table1). These results indicate that resveratrol is a better antiperoxidant than Vitamin E under our experimental conditions.

40 Figure 5 Separation of resveratrol by HPLC

Resveratrol

41 Table 1. Antioxidant values of resveratrol and vitamin E

Sample Resveratrol Vitamin E

NADPH-induced lipid peroxidation 1.5 X 10-4 M 1 X 10-3 M

Ascorbate-induced lipid peroxidation 1.5 X 10-5 M 1.25 X 10-3 M

42 Figure 6.

Effects of Resveratrol on Ascorbate-Induced Lipid Peroxidation

nmoles of malonaldehyde/mg protein 120 100 80 60 40 20 0 0 0.5 1 1.5 2 2.5 3 3.5 Concentration of resveratrol ( mM)

Figure 7.

Effects of Resveratrol on NADPH-Induced Lipid Peroxidation

nmoles of malonaldehyde/mg protein
50 40 30 20 10 0 0 0.5 1 1.5 2 2.5 3 3.5

Concentration of resveratrol (mM)

43 Nifedipine experiment Figure 8 shows the results from the experiment in which rats were injected with either nifedipine (one dose only) or nifedipine (one dose only) after pretreated with resveratrol. The mean value of serum level concentration of nifedipine in nifedipine alone rats was 22 7.5 ng/ml whereas nifedipine level was 0.8.3 2.00 ng/ml in rats which were pretreated with resveratrol. Resveratrol pretreatment caused a decrease in the serum level of nifedipine by 63.30%. Similar results were also observed in which nifedipine alone was given for a week (Figure 9) or in resveratrol pretreated rats. For the one week experiment the serum level concentration for nifedipine alone rats was 4.7 0.43 ng/ml and for the resveratrol pretreated rats was 1.4 0.46 ng/ml. Again a decrease of 68.99% was observed with resveratrol pretreated. These results suggest that concomitant use of resveratrol with nifedipine decreased the blood level of nifedipine. Concentration of nifedipine was determined by comparing the values obtained from the serum with the standard curve (Figure 10).

Niacin experiment Figure 11 shows the results from the experiment in which rats were injected with either niacin or niacin in rats pretreated with resveratrol. The mean value of the serum level Figure 8. Concentration of niacin in niacin alone rats was 18 0.16 ng/ml whereas the niacin level was 23 1.5 ng/ml in resveratrol pretreated rats. Resveratrol pretreatment

44 Figure 8. Effect of Resveratrol Pretreatment (1 Hour) on Nifedipine

Concentration in ng/ml 30.00 25.00 20.00 15.00 10.00 5.00 0.00 Nifedipine Nifedipine + Resveratrol

Figure 9. Effect of Resveratrol Pretreatment (1 week) on Nifedipine

Concentration in ng/ml 6.00 5.00 4.00 3.00 2.00 1.00 0.00 Nifedipine Nifedipine + Resveratrol

45 Figure 10 Determination of nifedipine by HPLC

Nifedipine

46 Figure 11.

Effect of Resveratrol Pretreatment (1 hour) on Niacin

Concentration in ng/ml 30.00 25.00 20.00 15.00 10.00 5.00 0.00

Niacin

Niacin + Resveratrol

Figure 12.

Effect of Resveratrol Pretreatment (1 Week) on Niacin

Concentration in ng/ml 30.00 25.00 20.00 15.00 10.00 5.00 0.00

Niacin

Niacin + Resveratrol

47 caused an increase in the serum level by 21.3%. A similar experiment for one week (Figure 12) was found to be in agreement with the one-hour experiment. For the one week experiment the serum level concentration for niacin alone rats was 18 0.70 ng/ml and for the resveratrol pretreated rats was 23 0.20 ng/ml. Again an increase of 20% was observed with resveratrol pretreated. The concentration of niacin was determined by comparing the values obtained from the serum with the standard curve (Figure 13). These results suggest that concomitant use of resveratrol increase the blood level of niacin.

Lovastatin experiment Figure 14 shows the results from the experiment in which rats were injected with either lovastatin (one dose only) or lovastatin pretreated with resveratrol. The mean value of serum level concentration of lovastatin in lovastatin alone rats was 2.33 0.12 ng/ml whereas nifedipine level was 1.4 0.16 ng/ml in lovastatin combined with resveratrol. Resveratrol pretreatment caused a decrease in the serum level by 35.42%. A similar experiment for one week (Figure 15) was found to be in agreement with the onehour experiment. For the one week experiment the serum level concentration for nifedipine alone rats was 1.1 0.030 ng/ml and for the resveratrol pretreated rats was 0.93 0.14 ng/ml. Again a decrease of 14.6% was observed with resveratrol pretreated serum level. The concentration of lovastatin was determined by comparing the values obtained from the serum with the standard curve (Figure 16). These results suggest that the concomitant use of resveratrol decreases the blood level of lovastatin.

48 Figure 13 Determination of niacin by HPLC

Niacin

49 Figure 14.

Effect of Resveratrol Pretreatment (1 Hour) on Lovastatin

Concentration in ng/ml

2.50 2.00 1.50 1.00 0.50 0.00

Lovastatin

Lovastatin + Resveratrol

Figure15.

Effect of Resveratrol Pretreatment (1 Week) on Lovastatin

1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00
Concentration in ng/ml

Lovastatin

Lovastatin + Resveratrol

50 Figure 16. Determination of lovastatin by HPLC

Lovastatin

51 Effect of resveratrol on liver metabolizing enzymes Effects of resveratrol on the liver metabolizing enzyme using hydroxylation of testosterone is presented in Figure 17. The mean value for the three control rats which were injected with normal saline was determined to be 183.2 14.4 ng/ml and that for the resveratrol injected rats was 146 8.2 ng/ml. A decrease of 22% was observed in the formation of -hydroxy testosterone from testosterone for the resveratrol treated rats.

52 Figure 17.

0.25 Concentration in microgram/ml 0.2 0.15 0.1 0.05 0

Effect of Resveratrol on Hydroxylation of Testosterone by Rat Liver Microsomal Metabolizing Enzyme

Normal Saline

Resveratrol

53 DISCUSSION Extraction and determination of resveratrol The concentration of resveratrol obtained from grape extract was low. The possible reason behind this could be attributed to the low concentration of resveratrol in the grape extract. There may be several reasons behind the low concentration as the production of resveratrol in the grapes varies. Resveratrol is produced preferentially in response to fungal infections. Environmental factors are also responsible for the production of resveratrol in grapes. Antioxidant activity The antioxidant activity of resveratrol was compared with that of vitamin E. For the nonenzymic lipid peroxidation resveratrol was found to be fifteen fold more effective as an antioxidant than vitamin E. The IC50 of resveratrol in NADPH-induced lipid peroxidation was 150 fold less than Vitamin E. Hence it can be said that resveratrol is potent antioxidant when compared to Vitamin E under our experimental conditions. Resveratrol is a potent antioxidant in nature and is an effective inhibitor of lipid peroxidation, a free radical mediated phenomena. It seems that resveratrol modulates free radical dependant processes by quenching free radicals or by diverting electrons away from the biological pathway leading to peroxide formation. Resveratrol has been reported to have numerous biological effects including anticarcinogenic effects and reducing coronary heart diseases (4,24-25). Antiperoxidant effects of resveratrol may be responsible for decreasing the heart diseases and cancer.

54 Nifedipine experiment The blood serum level of nifedipine after coadministration with resveratrol compared with nifedipine alone for both the experiments was about 65% decrease in the concentration. Statistically the decrease in the level of nifedipine was significant as p<0.05 determined by Students t-test. Thus, resveratrol pretreated decreases the concentration of nifedipine. Resveratrol may be increasing the metabolism of nifedipine thus decreasing the blood levels. In the previous studies it has been shown that P 450 is the enzyme responsible for the metabolism of nifedipine. Hence in order to check the effect of resveratrol on the activity of P 450 enzyme, an experiment was conducted on the rat liver microsomes. There was a 22% decrease in the activity of the enzyme, which was a significant difference. These results indicate that resveratrol interacts with nifedipine and consumers that take nifedipine and resveratrol or products containing resveratrol concomitantly should be careful.

Niacin experiment A modest increase of approximately 20% in the level of niacin was observed in the presence of resveratrol at one hour and one week experiment. The increase in the level of niacin was significant as p<0.05 determined by Students t-test. The possible reason behind this increase can be attributed to decrease in the enzyme action or other pharmacokinetic factors responsible for metabolism of nifedipine caused by resveratrol.

55 Lovastatin experiment A decrease of 35% and 14.6% in the level of lovastatin was observed in both the one-hour experiment and the one week experiment, respectively. For both the experiments the difference was significant as the p<0.05 determined statistically. Again resveratrol may be inducing the enzyme involved in the metabolism of lovastatin.

In order to investigate the possible mechanism of action of the changes in blood level of nifedipine, niacin, and lovastatin, effects of resveratrol on rat liver microsomal drug metabolizing enzyme was determined. A single dose of resveratrol (10 mg) resulted in 20% decrease in the hydroxylation of testosterone. The difference was significant as p<0.05 determined statistically. Cytochrome P450 3A isoenzymes catalyze the hydroxylation of testosterone (102). Thus cytochrome P450 3A may not be the isoenzyme responsible for the metabolism of nifedipine and lovastatin, as we did not observe an increased level of nifedipine and lovastatin in rats pretreated with resveratrol. However this decrease in hydroxylation of testosterone does explain a modest increase in the blood level of niacin. This can be said because the decrease in level of - hydroxy testosterone shows the decrease in the level of P450 3A isoenzymes. This is the enzyme responsible for metabolizing niacin (103) and the decrease in the level of P450 3A explains the increase in the serum concentration of niacin. These studies suggest the possible interaction of resveratrol with nifedipine, niacin and lovastatin. However, further extensive studies are needed to completely understand the effects of resveratrol on the interaction with other drugs to maximize the

56 beneficial effects of resveratrol (or products containing it) without interfering with clinical efficacy of other drugs used concomitantly.

57 CONCLUSION

Resveratrol inhibits both enzymatic and nonenzymatic lipid peroxidation. Resveratrol is more potent antioxidant than vitamin E under our experimental conditions. From the drug interaction experiments it was seen that resveratrol interacted with all the three drugs under investigation. Resveratrol decreased the level of nifedipine and lovastatin and increased the level of niacin. Resveratrol (or products containing it) should be used with caution while other drugs are taken.

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