Selenium is an essential trace element, and it is also an antioxidant nutrient involved in the defense of the body from oxidative stress [Mason 2007]. Humans and animals require selenium for the function of a number of selenium-dependent enzymes that are essential in many metabolic functions in the body [Rayman 2000].
Mechanism of Action
During selenoprotein synthesis, selenocysteine is incorporated into a very specifi c location in the amino acid sequence to form a functioning protein. However, when selenium is present in the soil, plants incorporate it into compounds that contain sulfur [Rayman 2000].
Absorption
Most dietary selenium is in the form of selenomethionine or selenocysteine. Both these forms are well absorbed. Other forms of selenium include selenate and selenite, commonly found in supplements and fortifi ed foods. Selenium is destroyed when foods are refi ned or processed. Selenium in general is known to be well absorbed after intake; however, a signifi cant amount is known to be eliminated in the urine [Otten, Helwig, and Meyers 2006].
Distribution
Two pools of reserve selenium are present in the body. The fi rst is selenomethio- nine, which may have a similar function as methionine. The second reserve pool is the selenium found in liver glutathione [Otten, Helwig, and Meyers 2006].
Elimination
Selenite, selenate, and selenocysteine are metabolized to selenide in the body. These selenides can be metabolized further or be converted to excretory metabolite. Selenium is excreted through urine [Otten, Helwig, and Meyers 2006].
Bioavailability
Bioavailability of selenium from animal sources, and particularly fi sh, is compa- rable with plant sources such as yeast. Bioavailability from fortifi ed foods is lower than natural sources [Otten, Helwig, and Meyers 2006].
Uses
Selenium can possibly be used in cancer prevention. Selenium supplementation can increase sperm motility. Selenium has been found to have a positive effect on mood swings in some individuals. Patients with asthma, decreased immune function, and HIV infection may benefi t from selenium supplements [Mason 2007].
Deficiency
Insuffi cient selenium intake results in decreased activity of certain enzymes that are responsible for antioxidant activities in the body, such as glutathione peroxidases, thioredoxin reductase, and thyroid deiodinases. Obvious clinical illness is rare with selenium defi ciency, even in a more severe form. However, selenium-defi cient individuals appear to be more susceptible to additional physiological stresses. Muscle pain and tenderness have both been associated with selenium defi ciency [Burk and Levander 1999; Mason 2007].
Dietary Sources
Selenium content of the food greatly depends on the soil in which the animal was raised or the plant was grown. The richest food sources of selenium are organ meats and seafood, followed by muscle meats. Brazil nuts grown in areas of Brazil with selenium-rich soil may provide more than 100 mcg of selenium in one nut, whereas those grown in selenium-poor soil may provide 10 times less [Chang 1995].
Commercial Preparations
Selenium supplements are available in several forms. Sodium selenite and sodium selenate are inorganic forms of selenium, whereas selenomethionine represents the organic form, which is rich in most naturally occurring food [Panel on Dietary Antioxidants and Related Compounds et al. 2000].
Adverse Effects
High doses of selenium can be toxic. Acute and fatal toxicities have occurred with accidental or suicidal ingestion of gram quantities of selenium. Chronic selenium toxicity (selenosis) may occur with smaller doses of selenium over long periods of time. Hair and nail brittleness are known to be common in selenium toxicity. Gastrointestinal problems, skin rashes, garlic breath odor, fatigue, irritability, and nervous system problems have also been reported with selenium toxicity [Panel on Dietary Antioxidants and Related Compounds et al. 2000].
Interactions
There are no known food interactions affecting absorption and usage of selenium [Jelin et al. 2008]. Limited information is currently found on interaction between
selenium and medications. Medications such as valproic acid have been found to decrease selenium levels [Flodin 1990]. Clozapine, an antipsychotic drug, has also been found to decrease selenium levels [Mason 2007].
The effi cacy of cholesterol-lowering agents, such as simvastatin and niacin, has been reduced when used in conjunction with beta-carotene, vitamin C, vitamin E, and selenium [Jelin et al. 2008].
ZINC
Zinc is an essential trace element throughout the life process [Mason 2007].
Mechanism of Action
Zinc is the second most prevalent trace element in the body. It is known that the cell nucleus contains about 30% of zinc. It acts as a cofactor in many biological activities that take place in the body such as DNA, RNA, and protein synthesis in general [Jelin et al. 2008].
Absorption
The small intestine, particularly jejunum, acts as the most effective zinc absorp- tion site. Zinc defi ciency enhances absorption [Otten, Helwig, and Meyers 2006; Mason 2007; Jelin et al. 2008].
Distribution
Zinc is well distributed, and more than 85% is stored in skeletal muscle and bone. Other zinc storage tissues include the liver, kidney, pancreas, prostate gland, and retina [Otten, Helwig, and Meyers 2006; Mason 2007].
Elimination
Zinc is eliminated mainly through feces. Starvation and trauma tend to increase zinc losses through urine. Skin cell shedding, sweat, hair, semen, and menstruation are the other means of zinc loss through the body [Otten, Helwig, and Meyers 2006].
Bioavailability
Absorption of zinc can be impaired by phytate and calcium, whereas proteins have positive impact on zinc absorption [Lonnerdal 2000].
Uses
A zinc supplement is indicated for treatment and prevention of its defi ciency. It can also be used for managing the common cold, recurrent ear infection, and respiratory infection [Jelin et al. 2008].
Deficiency
There is a broad range of physiological signs of zinc defi ciency because of the multiple biological functions and multiple distribution sites in tissues. Clinical signs of frank zinc defi ciency may be seen in skin problems such as infl ammation [Van Wouwe 1989]. Diarrhea, impaired cognitive, behavioral problems, impaired mem- ory, learning disability, and neuronal atrophy can be seen in infants as indications of zinc defi ciency [Hambidge 1986]. Zinc defi ciency in pregnancy can lead to growth retardation in the unborn baby in addition to conditions such as congenital abnor- malities in fetus [Mason 2007].
Dietary Sources
Zinc occurs in a wide variety of foods but is found in highest concentrations in animal sources, particularly in red meat and seafood. Whole grains contain more zinc than refi ned grains [Otten, Helwig, and Meyers 2006].
Adverse Effects
High intakes of zinc are possible either through supplemental zinc or by con- tact with environmental zinc. Toxicity symptoms, such as nausea, vomiting, epi- gastric pain, diarrhea, and lethargy, may occur with acute high intakes [Fosmire 1990].
Interactions
Absorption of copper, iron, and folate can be decreased by zinc. Conversely, zinc absorption may be reduced by phytic acid, fi ber, calcium, and phosphates. Protein intake positively affects zinc absorption [Otten, Helwig, and Meyers 2006]
REFERENCES
American Nutraceutical Association. http://www.ana-jana.org.
Anderson R. A., N. Cheng, N. A. Bryden, M. M. Polansky, N. Cheng, J. Chi, and J. Feng. 1997. Elevated intakes of supplemental chromium improve glucose and insulin vari- ables with type 2 diabetes. Diabetes 46:1786–1791.
Burk, R. F. and O. A. Levander. 1999. Selenium. In Modern Nutrition in Health and Disease. 9th edition. Edited by Shils, M., J. A. Olson, M. Shike, and A. C. Ross. Baltimore, MD: Lippincott Williams and Wilkins, pp. 265–276, 600.
Calcium Monograph. http://kroger.naturalstandard.com. Accessed October 21, 2008.
Celotti, F. and A. Bignamini. 1999. Dietary calcium and mineral/vitamin supplementation: A controversial problem. J. Int. Med. Res. 27:1–14.
Centers for Disease Control. 2008. Calcium and Bone Health. http://www.cdc.gov/nutrition/ everyone/basics/vitamins/calcium.html. Accessed November 3, 2008.
Chang, J. C. 1995. Selenium content of brazil nuts from two geographic locations in Brazil.
Chemosphere 30:801–802.
Copper Monograph. http://kroger.naturalstandard.com. Accessed October 21, 2008.
Flodin, N. W. 1990. Micronutrient supplements: toxicity and drug interactions. Prog. Food
Nutr. Sci. 14:277–331.
Fosmire, G. J. 1990. Zinc toxicity. Am. J. Cin. Nutr. 51:225.
Glinsmann, W. H. and W. Mertz. 1966. Effect of trivalent chromium on glucose tolerance.
Metabolism 15:510–519.
Hambidge, K. M. 1986. Zinc defi ciency in the weanling: How important? Acta. Peadiatr.
Scand. Suppl. 323:52.
Healthnotes. 2004. Phosphorus for sports and fi tness. http://www.evitamins.com/healthnotes. asp?ContentID=3892007#Reference-List. Accessed November 12, 2008.
Heaney, R. P. 2001. Calcium needs of the elderly to reduce fracture risk. J. Am. Coll. Nutr. 20 (2 Suppl.):192S–197S.
International Molybdenum Association. 2008. Molybdenum in Human Health. http://www. imoa.info/HSE/environmental_data/human_health/molybdenum_therapeutic_uses. html. Accessed October 11, 2008.
Iodine Monograph. http//kroger.naturalstandard.com. Accessed October 21, 2008.
Jeejeebhoy, K. N., R. C. Chu, E. B. Marliss, G. R. Greenberg, and A. Bruce-Robertson. 1977. Chromium defi ciency, glucose intolerance, and neuropathy reversed by chromium sup- plementation in a patient receiving long-term total parenteral nutrition. Am. J. Clin. Nutr. 30:531–538.
Jelin, J. M., P. J. Gregory, F. Batz, et al. 2008. Pharmacist’s Letter/Prescriber’s Letter Natural
Medicines Comprehensive Database. 10th ed. Stockton, CA: Therapeutic Research
Faculty, pp. 278, 607, 844, 973, 990.
Knochel, J. P. 2006. Phosphorus. In Modern Nutrition in Health and Disease. 10th edition. Edited by Shils, M. E., M. Shike, A. C. Ross, B. Caballero, R. J. Cousins. Baltimore, MD: Lippincott Williams and Wilkins, pp. 211–222.
Lee, N. A. and C. A. Reasner. 1994. Benefi cial effect of chromium supplementation on serum triglyceride levels in NIDDM. Diabetes Care 17:1449–1451.
Lonnerdal, B. 2000. Dietary factors infl uencing zinc absorption. J. Nutr. 130:1378.
Manganese Monograph. 2008. http://online.lexi.com/crlsql/servlet/crlonline. Accessed November 11, 2008.
Mason, P. 2007. Dietary Supplements. 3rd edition. Grayslake, IL: Pharmaceutical Press. McEvoy, G. K., ed. 2008. AHFS Drug Information 2008. Bethesda, MD: American Society of
Health-System Pharmacists.
National Collegiate Athletic Association. 2007. List of banned drug classes 2004–2005. http://www.ncaa.org/wps/wcm/connect/resources/file/ebab6d4a1276fc7/checklist. pdf?MOD=AJPERES. Accessed October 2008.
Otten, J. J., J. P. Helwig, and L. D. Meyers. 2006. Dietary reference intakes: The essential guide
to nutrient requirements. Washington, D.C.: The National Academies Press, pp. 321–327.
Panel on Dietary Antioxidants and Related Compounds, et al. 2000. Selenium. In Dietary
Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. Washington,
D.C.: National Academy Press, 284–324. http://books.nap.edu/openbook.php?record_ id=9810&page=R1.
Panel on Micronutrients, et al. 2002. Dietary reference intakes for vitamin A, K, arsenic, boron, chromium, copper, iodine, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, D.C.: Academy Press. http://books.nap.edu/catalog.php?record_ id=10026. Accessed October 2008.
Pattanaungkul, S., B. L. Riggs, A. L. Yergey, N. E. Vieira, W. M. O’Fallon, S. Khosla. 2000. Relationship of intestinal calcium absorption to 1,25-dihydroxyvitamin D [1,25(OH)2D] levels in young versus elderly women: Evidence for age-related intestinal resistance to 1,25(OH)2D action. J. Clin. Endocrinol. Metab. 85:4023–4027.
Peterson, L. N. 1997. Potassium in nutrition. In Handbook of Nutritionally Essential Minerals. Edited by O’Dell, B. L. and R. A. Sunde. New York: Marcel Dekker, pp. 153–183. Power, M. L., R. P. Heaney, H. J. Kalkwarf, R. M. Pitkin, J. T. Repke, R. C. Tsang, and
J. Schulkin. 1999. The role of calcium in health and disease. Am. J. Obstet. Gynecol. 181:1560–1569.
Rayman, M. P. 2000. The importance of selenium to human health. Lancet 356:233–241. Sheng, H.-W. 2000. Sodium, chloride and potassium. In Physiological Aspects of Human Nutrition.
Edited by Stipanuk M. Philadelphia, PA: W. B. Saunders Company, pp. 686–710. Turnlund, J. R., W. R. Keyes, G. L. Peiffer, and G. Chiang. 1995. Molybdenum absorption,
excretion, and retention studied with stable isotopes in young men during depletion and repletion. Am. J. Clin. Nutr. 61:1102–1109.
Turnlund, J. R., C. M. Weaver, S. K. Kim, W. R. Keyes, Y. Gizaw, K. H. Thompson, and G. L. Peiffer. 1999. Molybdenum absorption and utilization in humans from soy and kale intrinsically labeled with stable isotopes of molybdenum. Am. J. Clin. Nutr. 69:1217–1223.
Van Wouwe, J. P. 1989. Clinical and laboratory diagnosis of acrodermatitis enteropathica. Eur.
J. Pediatr. 149:2.
Wester, P. O. 1971. Trace element balances in two cases of pancreatic insuffi ciency. Acta. Med.
Scand. 190:155–161.
Whitney, E., C. B. Cataldo, S. R. Rolfes, eds. 1998. Understanding normal and clinical nutri- tion. Belmont, CA: Walsworth.
Wuebbens, M. M., M. T. Liu, K. Rajagopalan, and H. Schindelin. 2000. Insights into molyb- denum cofactor defi ciency provided by the crystal structure of the molybdenum cofactor biosynthesis protein MoaC. Struct. Fold. Des. 8:709–718.
125
Physiochemical Characterization
of Nutraceuticals
Ajoy Koomer CONTENTS Introduction ... 125 Phytosterols ... 126 Fatty Acids ... 127 Carotenoids ... 128 Anthocyanins ... 128 Amino Acids ... 129 Water-Soluble Vitamins ... 130 References ... 130 INTRODUCTIONAccording to the American Nutraceutical Association, the term “nutraceutical” was derived by condensing the terms “pharmaceutical and nutrition” in 1989 by Stephen DeFelice, who was founding chairman of the Foundation for Innovation in Medicine [American Nutraceutical 2008]. DeFelice suggests that, “A nutraceutical is any substance that is a food or a part of a food and provides medical or health benefi ts, including the prevention and treatment of disease. Such products may range from isolated nutrients, dietary supplements and specifi c diets to genetically engineered designer foods, herbal products, and processed foods such as cereals, soups and beverages” (2008). Nutraceuticals and dietary supplements can be broadly classifi ed into fi ve categories, namely vitamins, minerals, botanical substances, herbal extracts, and miscellaneous or specialty components [Krull and Swartz 2001]. The vitamins category includes fat and water-soluble vitamins and nutritional factors [Krull and Swartz 2001]. The minerals category comprises mineral chelates, salts, single
and trace elements, and multiple minerals consisting of amino acids mixes [Krull and Swartz 2001]. Botanical substances include mixed and single whole herbs, essential oils, tea mixtures, and traditional formulas [Krull and Swartz 2001]. The specialty components include antioxidants, carotenoids, essential (omega-3) and omega-3 and -6 fatty acids, phytosterols, anthocyanins, fl avonoids, probiotics, lecithins, glandular, diet acids, and digestive acids [Krull and Swartz 2001]. As noted by specialists, nutraceuticals are gaining public acceptance because of escalating consumer market share for “wellness products” [Krull and Swartz 2001; Dureja, Kaushik, and Kumar 2003; Metha et al. 2007]. Because of a lack of standardization of active ingredients in nutraceutical-related products coupled with increased market interest, the United States has had increased attention “in the marketing, claims substantiation, manufacturing, and FDA-based regulations of nutraceuticals” [Krull and Swartz 2001; Dureja, Kaushik, and Kumar 2003; Mehta et al. 2007]. The tightening of the regulation apparatus is the driving force for the implementation of reliable analytical techniques for the reliable detection of active gradients in nutraceutical complex matrices and their physicochemical characterization for enhanced formulation and quality-control studies [Krull and Swartz 2001; Dureja, Kaushik, and Kumar 2003]. The nutraceutical testing methods available in industry include stability testing, dissolution testing, in vitro release rate testing, content uniformity testing, high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), mass spectrometric analysis, Fourier transform infrared spectroscopy, and ultraviolet/visible light (UV-vis) spectroscopy [Analytical Solutions 2008]. A major factor in physicochemical characterization is stress and stability testing. Stress testing is performed to identify all potential degradants. Generally, chromatographic techniques are used as methods of validation in stress testing, with detection being performed by UV-vis spectroscopy [Mehta et al. 2007]. In this chapter, we will focus on physicochemical characterization of phytosterols, fatty acids, caratenoids, amino acids, anthocyanins, and water-soluble vitamins.
PHYTOSTEROLS
Phytosterols are diphenolic compounds present in plant and animals that resemble the human sex hormone estrogen [Hurst 2002]. They can be classifi ed into three categories, which are coumestans, lignans, and isofl avonoids [Hurst 2002]. They can be detected and quantifi ed by TLC, HPLC, and gas chromatography-mass spectrometry (GC-MS) coupled with UV-vis spectroscopy [Hurst 2002]. However, before implementation of any methods for validation, sample preparation and extraction play a critical role. Usually, phytosterol matrix samples are spiked with 20% (w/v) of purifi ed isofl avones, such as daidzein, genistein, and biochanin A [Hurst 2002]. The spiked solution consists of 1.3% (w/v) of tertiary butyl hydroquinone in methanol [Hurst 2002]. The use of isofl avones as internal standards stem from the pros that it allows for accurate “determination of analyte recoveries and account for weight loss in the extraction procedure” [Hurst 2002]. After sample preparation, they are extracted with 80% methyl alcohol for 0.5 h, although other solvents, such as ethanol, acetonitrile, and acetone, can be used [Hurst 2002]. TLC has been used routinely in the identifi cation
of isofl avonoids [Hurst 2002]. Scientists have used precoated polyamide TLC plates for the separation of isofl avones and other diphenolic compounds from soya bean extracts [Hurst 2002]. As noted by scientists, the methanolic extracts spotted on 20 × 20 plates were developed with methanol/acetic acid/water mixture in proportions of 90:5:5 (weight/volume) [Hurst 2002]. After eluting the bands with ethanol, they were “rechromatographed on polyamide using chloroform-methanol-methyl ethyl ketone solvent system in the ratio 12:2:1” [Hurst 2002]. When viewed under a UV lamp at 366 nm, the analyzed fractions produced characteristics Rf values corresponding to daidzein, genistein, formononetin, and biochanin A [Hurst 2002]. It is to be noted that TLC is a qualitative procedure that cannot be used for quantifi cation of individual phytosterol fractions from complex plant and food matrices [Hurst 2002]. HPLC is the method of choice here. Although MS techniques can be used with improved sensitivity, they are not popular because of cumbersome sample preparation time and costs [Hurst 2002]. Generally, phytosterols can be purifi ed and quantifi ed by mixtures of “methanol or acetonitrile and aqueous acids or buffers” by the use of reverse-phase (RP) C18 separation columns [Hurst 2002]. The most frequently used HPLC techniques use either linear or nonlinear gradient elutions for isofl avonoid separation and “quantitative estimation from legume or soybean matrices” [Hurst 2002]. Generally, in gradient elution HPLC, “acetonitrile increases by 2.25% min,” enabling “the separation of isofl avone-β-glucoside conjugates and aglucones” in a single experiment [Hurst 2002]. The experimental runtime usually does not exceed 60 min, with equilibration between the cycles [Hurst 2002]. Recently, RP-HPLC has been used to separate isofl avones such as daidzein and genistein [Hurst 2002]. Isocratic elution conditions have been reported by researchers in the separation of phytosterols, although the method has been unsuccessful, except for genistein [Hurst 2002]. Because of the high concentration of isofl avonoids in soy or legume products, UV-vis spectroscopy coupled with RP-HPLC is used for detection [Hurst 2002]. However, the method suffers from challenges in quantifying isofl avonoids from sources with low concentration or if phytosterols other than isofl avonoids are involved [Hurst 2002]. In those cases, “fl uorometric detection, amperometric methods, or thermospray MS with SIMI” can be considered to increase sensitivity of common UV detection systems [Hurst 2002].