Hindawi Publishing Corporation Journal of Environmental and Public Health Volume 2012, Article ID 727630, 7 pages doi:10.1155/2012/727630
Review Article The Alkaline Diet: Is There Evidence That an Alkaline pH Diet Benefits Health?
Gerry K. Schwalfenberg
University of Alberta, Suite No. 301, 9509-156 Street, Edmonton, AB, Canada T5P 4J5
Correspondence should be addressed to Gerry K. Schwalfenberg, [email protected]
Received 3 July 2011; Accepted 8 August 2011
Academic Editor: Janette Hope
Copyright © 2012 Gerry K. Schwalfenberg. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine.
1. Background decrease in potassium (K) compared to sodium (Na) and an increase in chloride compared to bicarbonate found in Life on earth depends on appropriate pH levels in and the diet [6]. The ratio of potassium to sodium has reversed, around living organisms and cells. Human life requires a K/Na pre-viously was 10 to 1 whereas the modern diet has tightly controlled pH level in the serum of about 7.4 (a slight- aratioof1to3[7]. It is generally accepted that agricul- ly alkaline range of 7.35 to 7.45) to survive [1]. tural humans today have a diet poor in magnesium and As a comparison, in the past 100 years with increasing potassium as well as fiber and rich in saturated fat, simple industrialization, the pH of the ocean has dropped from sugars, sodium, and chloride as compared to the pre-agri- 8.2 to 8.1 because of increasing CO2 deposition. This has a cultural period [6]. This results in a diet that may induce negative impact on life in the ocean [1, 2]andmayleadto metabolic acidosis which is mismatched to the genetically the collapse of the coral reefs [3]. Even the pH of the soil determined nutritional requirements [8]. With aging, there in which plants are grown can have considerable influence is a gradual loss of renal acid-base regulatory function on the mineral content of the food we eat (as minerals are and a resultant increase in diet-induced metabolic acidosis ff used as bu ers to maintain pH). The ideal pH of soil for while on the modern diet [9]. A low-carbohydrate high- the best overall availability of essential nutrients is between 6 pro-tein diet with its increased acid load results in very and 7. Acidic soils below pH of 6 may have reduced calcium little change in blood chemistry, and pH, but results in and magnesium, and soil above pH 7 may result in chemi- many changes in urinary chemistry. Urinary magnesium cally unavailable iron, manganese, copper and zinc. Adding levels, urinary citrate and pH are decreased, urinary calcium, dolomite and manure are ways of raising pH in an acid soil undissociated uric acid, and phosphate are in-creased. All environment when the pH is below 6 [4]. of these result in an increased risk for kidney stones When it comes to the pH and net acid load in the [10]. human diet, there has been considerable change from the Much has been written in the lay literature as well as hunter gather civilization to the present [5]. With the agricul- many online sites expounding on the benefits of the alkaline tural revolution (last 10,000 years) and even more recently diet. This paper is an attempt to balance the evidence that is with industrialization (last 200 years), there has been an found in the scientific literature. 2 Journal of Environmental and Public Health
Table 1: Ph of selected fluids, organs, and membranes.
Organ, fluid or membrane pH Function of pH (1) Skin Natural pH is between 4 and 6.5 [17] Barrier protection from microbes (2) Urine 4.6 to 8.0 [18] Limit overgrowth of microbes (3) Gastric 1.35 to 3.5 Break down protein (4) Bile 7.6 to 8.8 Neutralize stomach acid, aid in digestion (5) Pancreatic fluid 8.8 Neutralize stomach acid, aid in digestion (6) Vaginal fluid <4.7 [13] Limit overgrowth of opportunistic microbes (7) Cerebrospinal fluid 7.3 Bathes the exterior of the brain (8) Intracellular fluid 6.0–7.2 [19] Due to acid production in cells (9) Serum venous 7.35 Tightly regulated (10) Serum arterial 7.4 Tightly regulated
2. The Role of pH in Various Cells, Organs, the urine with the modern diet over time could be as high and Membranes as almost 480 gm over 20 years or almost half the skeletal mass of calcium [21]. However, urinary losses of cal-cium ThepHinourbodymayvaryconsiderablyfromoneareato are not a direct measure of osteoporosis. There are many another with the highest acidity in the stomach (pH of 1.35 regulatory factors that may compensate for the urinary calci- to 3.5) to aid in digestion and protect against opportunistic um loss. When the arterial pH is in the normal range, a microbial organisms. But even in the stomach, the layer just mild reduction of plasma bicarbonate results in a negative outside the epithelium is quite basic to prevent mucosal calcium balance which could benefit from supplementing injury. It has been suggested that decreased gastric lining bicarbonate in the form of potassium bicarbonate [22]. It secretion of bicarbonates and a decrease in the alkaline/acid has been found that bicarbonate, which increases the alkali secretion in duodenal ulcer patients may play a significant content of a diet, but not potassium may attenuate bone role in duodenal ulcers [11]. The skin is quite acidic (pH loss in healthy older adults [23]. The bone minerals that are 4–6.5) to provide an acid mantle as a protective barrier to wasted in the urine may not have complete compensation the environment against microbial overgrowth. There is a through intestinal absorption, which is thought to result in gradient from the outer horny layer (pH 4) to the basal layer osteoporosis. However, adequate vitamin D with a 25(OH)D (pH 6.9) [12]. This is also seen in the vagina where a pH of level of >80 nmol/L may allow for appropriate intestinal less than 4.7 protects against microbial overgrowth [13]. absorption of calcium and magnesium and phosphate when The urine may have a variable pH from acid to alkaline needed [24]. Sadly, most populations are generally deficient depending on the need for balancing the internal environ- in vitamin D especially in northern climates [25]. In ment. Acid excretion in the urine can be estimated by a chronic renal failure, correction of metabolic acidosis with formula described by Remer (sulfate + chloride + 1.8x bicarbonate significantly improves parathyroid levels and phosphate + organic acids) minus (sodium + potassium + levels of the active form of vitamin D 1,25(OH)2D3 [26]. 2x calcium + 2x magnesium) mEq [14]. Foods can be cate- Recently, a study has shown the importance of phosphate gorized by the potential renal acid loads (PRALs) see Table 2. in Remer’s PRAL formula. According to the formula it Fruits, vegetables, fruit juices, potatoes, and alkali-rich and would be expected that an increase in phosphate should low phosphorus beverages (red and white wine, mineral soda result in an increase in urinary calcium loss and a negative waters) having a negative acid load. Whereas, grain products, calcium balance in bone [27]. It should be noted that meats, dairy products, fish, and alkali poor and low phos- supplementation with phosphate in patients with bed rest phorus beverages (e.g., pale beers, cocoa) have relatively high reduced urinary calcium excretion but did not prevent bone acid loads [15]. Measurement of pH of the urine (reviewed loss [28]. The most recent systematic review and meta- in a recent study with two morning specimens done over a analysis has shown that calcium balance is maintained and five-year span) did not predict bone fractures or loss of bone improved with phosphate which is quite contrary to the mineral density [16]. However, this may not be reflective of acid-ash hypothesis [29]. As well a recent study looking at being on an alkaline or acid diet throughout this time. For soda intake (which has a significant amount of phosphate) more details, see Table 1. and osteoporosis in postmenopausal American first nations women did not find a correlation [30]. It is quite possible 3. Chronic Acidosis and Bone Disease that the high acid content according to Remer’s classification needs to be looked at again in light of compensatory phos- Calcium in the form of phosphates and carbonates represents phate intake. There is online information promoting an alka- a large reservoir of base in our body. In response to an acid line diet for bone health as well as a number of books. How- load such as the modern diet these salts are released into ever, a recent systematic review of the literature looking for the systemic circulation to bring about pH homeostasis [7]. evidence supporting the alkaline diet for bone health found It has been estimated that the quantity of calcium lost in no protective role of dietary acid load in osteoporosis [31]. Journal of Environmental and Public Health 3
Table 2: Potential renal acid loads (PRALs) of selected foods [20].
Food or food group PRAL mEq of: Cl + P04 +SO4− Na − K − Ca − Mg Dairy Parmesan cheese 34.2 Processed cheese plain 28.7 Cheddar reduced fat 26.4 Hard cheese (average) 19.2 Fresh cheese (quark) 11.3 Cottage cheese plain 8.7 Yogurt whole milk 1.5 Ice Cream 0.8 Whole milk 0.7 Buttermilk 0.5 Eggs Eggs yolk 23.4 Eggs white 1.1 Eggs chicken whole 8.2 Meats Corned beef 13.2 Luncheon meat canned 10.2 Turkey 9.9 Veal 9.0 Lean beef 7.8 Frankfurters 6.7 Sugars Sugar white −0.1 Honey −0.3 Vegetables Cucumber −0.8 Broccoli −1.2 Tomato −3.1 Eggplant −3.4 Celery −5.2 Spinach −14.0 Fats and Oils Butter 0.6 Margarine −0.5 Olive oil 0.0 Fruits and nuts and fruit juices Peanuts 8.3 Walnuts 6.8 Grape juice unsweetened −1.0 Orange juice unsweetened −2.9 Apples or apple juice unsweetened −2.2 Apricots −4.8 Banana −5.5 Black currents −6.5 Raisins −21.0 Grains and grain products Brown Rice 12.5 Rolled Oats 10.7 Spaghetti whole meal 7.3 Spaghetti white 6.5 4 Journal of Environmental and Public Health
Table 2: Continued.
Food or food group PRAL mEq of: Cl + P04 +SO4− Na − K − Ca − Mg Cornflakes 6.0 Rice white 4.6 Bread rye flower 4.1 Bread whole wheat 1.8 Legumes Lentils green and brown 3.5 Green beans −3.1 Fish Trout brown 10.8 Cod fillets 7.1 Beverages Beer pale 0.9 Coca-Cola 0.4 Beer draft −0.2 Wine white −1.2 Coffee infusion −1.4 Wine red −2.4
Another element of the modern diet is the excess of sodi- prior to exhaustive exercise resulted in significantly less um in the diet. There is evidence that in healthy humans the acidosis in the blood than those that were not supplemented increased sodium in the diet can predict the degree of hyper- with sodium bicarbonate [42]. chloremic metabolic acidosis when consuming a net acid producing diet [32]. As well, there is evidence that there are adverse effects of sodium chloride in the aging population. 5. Alkaline Supplementation and A high sodium diet will exacerbate disuse-induced bone Growth Hormone and muscle loss during immobilization by increasing bone resorption and protein wasting [33]. Excess dietary sodium It has long been known that severe forms of metabolic has been shown to result in hypertension and osteoporosis in acidosis in children, such as renal tubular acidosis, are as- women [34, 35]. As well, dietary potassium which is lacking sociated with low levels of growth hormone with resultant in the modern diet would modulate pressor and hyper- short stature. Correction of the acidosis with bicarbonate calciuric effects of excess of sodium chloride [36]. [7] or potassium citrate [43] increases growth hormone sig- Excess dietary protein with high acid renal load may nificantly and improved growth. The use of enough pota- decrease bone density if not buffered by ingestion of supple- ssium bicarbonate in the diet to neutralize the daily net ments or foods that are alkali rich [37]. However, adequate acid load in postmenopausal women resulted in a significant protein is necessary for prevention of osteoporosis and increase in growth hormone and resultant osteocalcin [44]. sarcopenia; therefore, increasing the amount of fruit and veg- Improvinggrowthhormonelevelsmayimprovequalityof etables may be necessary rather than reducing protein [38]. life, reduce cardiovascular risk factors, improve body com- position, and even improve memory and cognition [45]. As well this results in a reduction of urinary calcium loss equi- 4. Alkaline Diets and Muscle valent to 5% of bone calcium content over a period of 3 years [46]. As we age, there is a loss of muscle mass, which may predis- pose to falls and fractures. A three-year study looking at a diet rich in potassium, such as fruits and vegetables, as well 6. Alkaline Diet and Back Pain as a reduced acid load, resulted in preservation of muscle mass in older men and women [39]. Conditions such as There is some evidence that chronic low back pain improves chronic renal failure that result in chronic metabolic acidosis with the supplementation of alkaline minerals [47]. With result in accelerated breakdown in skeletal muscle [40]. supplementation there was a slight but significant increase in Correction of acidosis may preserve muscle mass in condi- blood pH and intracellular magnesium. Ensuring that there tions where muscle wasting is common such as diabetic is enough intracellular magnesium allows for the proper ketosis, trauma, sepsis, chronic obstructive lung disease, and function of enzyme systems and also allows for activation of renal failure [41]. In situations that result in acute acidosis, vitamin D [48]. This in turn has been shown to improve back supplementing younger patients with sodium bicarbonate pain [49]. Journal of Environmental and Public Health 5
7. Alkalinity and Chemotherapy (2) The resultant increase in growth hormone with an alkaline diet may improve many outcomes from car- ff The e ectiveness of chemotherapeutic agents is markedly diovascular health to memory and cognition. influenced by pH. Numerous agents such as epirubicin and adriamycin require an alkaline media to be more effective. (3) An increase in intracellular magnesium, which is re- Others, such as cisplatin, mitomycin C, and thiotepa, are quired for the function of many enzyme systems, is more cytotoxic in an acid media [50]. Cell death correlates another added benefit of the alkaline diet. Available with acidosis and intracellular pH shifts higher (more alka- magnesium, which is required to activate vitamin line) after chemotherapy may reflect response to chemother- D, would result in numerous added benefits in the apy [51]. It has been suggested that inducing metabolic alka- vitamin D apocrine/exocrine systems. losis may be useful in enhancing some treatment regimes by (4) Alkalinity may result in added benefit for some using sodium bicarbonate, carbicab, and furosemide [52]. chemotherapeutic agents that require a higher pH. Extracellular alkalinization by using bicarbonate may result in improvements in therapeutic effectiveness [53]. There is From the evidence outlined above, it would be prudent to no scientific literature establishing the benefit of an alkaline consider an alkaline diet to reduce morbidity and mortality diet for the prevention of cancer at this time. of chronic disease that are plaguing our aging population. One of the first considerations in an alkaline diet, which in- cludes more fruits and vegetables, is to know what type of 8. Discussion soil they were grown in since this may significantly influence the mineral content. At this time, there are limited scientific The human body has an amazing ability to maintain a steady studies in this area, and many more studies are indicated in pH in the blood with the main compensatory mechanisms regards to muscle effects, growth hormone, and interaction being renal and respiratory. Many of the membranes in our with vitamin D. body require an acid pH to protect us and to help us digest food. It has been suggested that an alkaline diet may prevent a number of diseases and result in significant health benefits. References Looking at the above discussion on bone health alone, certain aspects have doubtful benefit. There does not seem to be [1] A. Waugh and A. Grant, Anatomy and Physiology in Healthand Illness, Churchill Livingstone Elsevier, Philadelphia, Pa, USA, enough evidence that milk or cheese may be as detrimental as 10th edition, 2007. 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J Assoc Physicians India. 2006 Apr;54:30919. Biological significance of essential fatty acids. Das UN1. Author information
Abstract Essential fatty acids (EFAs)linoleic acid (LA) and alphalinolenic acid (ALA) are critical for human survival. EFAs are readily available in the diet. But, to derive their full benefit, EFAs need to be metabolized to their respective longchain metabolites. EFAs not only form precursors to respective prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), but also give rise to lipoxins (LXs), resolvins, isoprostanes, and hydroxy and hydroperoxyeicosatetraenoates. Certain PGs, TXs, and LTs have proinflammatory actions whereas LXs and resolvins are antiinflammatory in nature. Furthermore, EFAs and their longchain metabolites modulate the activities of angiotensin converting and HMGCoA reductase enzymes, enhance acetylcholine levels in the brain, increase the synthesis of endothelial nitric oxide, augment diuresis, and enhance insulin action. Thus, EFAs and their metabolites may function as endogenous ACE and HMGCoA reductase inhibitors, nitric oxide enhancers, betablockers, diuretics, antihypertensive, and antiatherosclerotic molecules. In addition, EFAs and their longchain metabolites react with nitric oxide (NO) to yield respective nitroalkene derivatives that exert cellsignaling actions via ligation and activation of peroxisome proliferatoractivated receptors (PPARs). Thus, EFAs and their derivatives have varied biological actions that may have relevance to their involvement in several physiological and pathological processes.
PMID: 16944615 [Indexed for MEDLINE]
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https://www.ncbi.nlm.nih.gov/pubmed/16944615 1/2 8/30/2017 Biological significance of essential fatty acids. - PubMed - NCBI
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Linus Pauling Institute » Micronutrient Information Center
Other Nutrients » Essential Fatty Acids
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Contents Summary Linoleic acid (LA), an omega-6 fatty acid, and α- Summary linolenic acid (ALA), an omega-3 fatty acid, are Introduction considered essential fatty acids (EFA) because they cannot be synthesized by humans. (More Metabolism and Bioavailability information) Biological Activities The long-chain omega-3 fatty acids, Membrane structure and function eicosapentaenoic acid (EPA) and Vision docosahexaenoic acid (DHA), can be synthesized from ALA, but due to low conversion e ciency, it Nervous system is recommended to obtain EPA and DHA from Synthesis of lipid mediators additional sources. (More information)
Regulation of gene expression LA, arachidonic acid (AA), and DHA are the most common polyunsaturated fatty acids (PUFA) De ciency accumulating in tissues. (More information) Essential fatty acids Both omega-6 and omega-3 fatty acids are Omega-3 de ciency important structural components of cell Omega-3 index membranes, serve as precursors to bioactive lipid mediators, and provide a source of energy. Disease Prevention Long-chain omega-3 PUFA in particular exert Visual and neurological development anti-in ammatory e ects and it is recommended to increase their presence in the Gestation and pregnancy diet. (More information) Cardiovascular disease Both dietary intake and endogenous metabolism Alzheimer's disease in uence whole-body status of EFA. Genetic Disease Treatment polymorphisms in fatty acid synthesizing enzymes can have a signi cant impact on fatty Coronary heart disease acid levels in the body. (More information) Diabetes mellitus DHA is important for visual and neurological In ammatory diseases development. Feeding infants formula enriched Neuropsychiatric disorders with DHA and AA appears to have no signi cant e ect on cognitive development and a very Alzheimer's disease and dementia modest e ect on visual acuity. (More Sources information)
Food Replacing saturated fat in the diet with a mixture of PUFA (both omega-6 and omega-3) is Biosynthesis of EPA and DHA associated with bene cial e ects on the Supplements cardiovascular system. (More information)
Infant formula A large body of scienti c research suggests that Safety higher dietary omega-3 fatty acid intakes are associated with reductions in cardiovascular Adverse e ects disease risk. Thus, the American Heart Infant formula Association recommends that all adults eat sh, particularly oily sh, at least twice weekly. (More Pregnancy and lactation information) Contaminants in sh The results of prospective cohort studies and Contaminants in supplements randomized controlled trials indicate that sh Drug interactions and sh oil consumption decreases the risk of coronary heart disease (CHD) mortality, Nutrient interactions including fatal myocardial infarction (heart Intake Recommendations attack) and sudden cardiac death. (More information) US Institute of Medicine Low DHA status may be a risk factor for International recommendations Alzheimer's disease and other types of AHA recommendation dementia, but it is not yet known whether DHA LPI Recommendation supplementation can help prevent or treat such cognitive disorders. (More information) Authors and Reviewers Increasing EPA and DHA intake may be bene cial References in individuals with type 2 diabetes, especially those with elevated serum triglycerides. (More information)
Randomized controlled trials have found that sh oil supplementation reduces the requirement for anti-in ammatory medication in patients with rheumatoid arthritis. (More information) Although some data suggest that omega-3 fatty acid supplementation may be a bene cial adjunct in the therapy of depression, bipolar disorder, and schizophrenia, great heterogeneity between trials prevents conclusive determination of therapeutic e cacy. (More information) Introduction Omega-6 and omega-3 fatty acids are polyunsaturated fatty acids (PUFA), meaning they contain more than one cis double bond (1). In all omega-6 fatty acids, the rst double bond is located between the sixth and seventh carbon atom from the methyl end of the fatty acid (n-6). Similarly, in all omega-3 fatty acids, the rst double bond is located between the third and fourth carbon atom counting from the methyl end of the fatty acid (n-3). Scienti c abbreviations for fatty acids tell the reader something about their chemical structure. One scienti c abbreviation for α-linolenic acid (ALA) is 18:3n-3. The rst part (18:3) tells the reader that ALA is an 18-carbon fatty acid with three double bonds, while the second part (n-3) tells the reader that the rst double bond is in the n-3 position, which de nes it as an omega-3 fatty acid (Figures 1a and 1b). Double bonds introduce kinks in the hydrocarbon chain that in uence the structure and physical properties of the fatty acid molecule (Figure 1c). Although humans and other mammals can synthesize saturated fatty acids and some monounsaturated fatty acids from carbon groups in carbohydrates and proteins, they lack the enzymes necessary to insert a cis double bond at the n-6 or the n-3 position of a fatty acid (1). Consequently, omega-6 and omega-3 fatty acids are essential nutrients. The parent fatty acid of the omega-6 series is linoleic acid (LA; 18:2n-6), and the parent fatty acid of the omega-3 series is ALA (Table 1 and Figure 2). Humans can synthesize long-chain (20 carbons or more) omega-6 fatty acids, such as dihomo-γ-linolenic acid (DGLA; 20:3n-6) and arachidonic acid (AA; 20:4n-6), from LA and long-chain omega-3 fatty acids, such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), from ALA (see Metabolism and Bioavailability).
Table 1. Names and Abbreviations of the Omega-6 and Omega-3 Fatty Acids Omega-6 Fatty Acids Omega-3 Fatty Acids
Linoleic acid LA 18:2n-6 α-Linolenic acid ALA 18:3n-3
γ-Linolenic acid GLA 18:3n-6 Stearadonic acid SDA 18:4n-3
Dihomo-γ-linolenic acid DGLA 20:3n-6 Eicosatetraienoic acid ETA 20:4n-3
Arachidonic acid AA 20:4n-6 Eicosapentaenoic acid EPA 20:5n-3
Adrenic acid 22:4n-6 Docosapentaenoic acid DPA (n-3) 22:5n-3
Tetracosatetraenoic acid 24:4n-6 Tetracosapentaenoic acid 24:5n-3
Tetracosapentaienoic acid 24:5n-6 Tetracosahexaenoic acid 24:6n-3
Docosapentaenoic acid DPA (n-6) 22:5n-6 Docosahexaenoic acid DHA 22:6n-3
Metabolism and Bioavailability Prior to absorption in the small intestine, fatty acids must be hydrolyzed from dietary fats (triglycerides and phospholipids) by pancreatic enzymes (2). Bile salts must also be present in the small intestine to allow for the incorporation of fatty acids and other fat digestion products into mixed micelles. Fat absorption from mixed micelles occurs throughout the small intestine and is 85-95% e cient under normal conditions.
Blood concentrations of fatty acids re ect both dietary intake and biological processes (3). Humans can synthesize longer omega-6 and omega-3 fatty acids from the essential fatty acids LA and ALA, respectively, through a series of desaturation (addition of a double bond) and elongation (addition of two carbon atoms) reactions (Figure 3) (4, 5). LA and ALA compete for the same elongase and desaturase enzymes in the synthesis of longer polyunsaturated fatty acids, such as AA and EPA. The capacity to generate DHA from ALA is higher in women than men. Studies of ALA metabolism in healthy young men indicate that approximately 8% of dietary ALA is converted to EPA and 0-4% is converted to DHA (6). In healthy young women, approximately 21% of dietary ALA is converted to EPA and 9% is converted to DHA (7). The better conversion e ciency of young women compared to men appears to be related to the e ects of estrogen (8, 9). Although ALA is considered the essential omega-3 fatty acid because it cannot be synthesized by humans, evidence that human conversion of EPA and, particularly, DHA is relatively ine cient suggests that EPA and DHA may be considered conditionally essential nutrients.
In addition to gender di erences, genetic variability in enzymes involved in fatty acid metabolism in uences one's ability to generate long-chain polyunsaturated fatty acids (LC-PUFA). Two key enzymes in fatty acid metabolism are delta-6 desaturase (FADS2) and delta-5 desaturase (FADS1) (see Figure 3 above) (10). Two common haplotypes (a cluster of polymorphisms) in the FADS genes di er dramatically in their ability to generate LC-PUFA: haplotype D is associated with increased FADS activity (both FADS1 and FADS2) and is more e cient in converting fatty acid precursors (LA and ALA) to LC-PUFA (EPA, GLA, DHA, and AA) (11). These FADS polymorphisms are relatively common in the population and may explain up to 30% of the variability in blood levels of omega-3 and omega-6 fatty acids among individuals (3).
Finally, DHA is retroconverted to EPA at a low basal rate and following supplementation (see Figure 3 above) (12). After supplementing omnivores (n=8) and vegetarians (n=12) for six weeks with an EPA-free preparation of DHA (1.62 g/day), both EPA and DHA levels increased in serum and platelet phospholipids (13). Based on the measured changes, the estimated percent retroconversion of DHA to EPA was 7.4-11.4% (based on serum phospholipid data) and 12.3-13.8% (based on the platelet phospholipid data), with no signi cant di erence between omnivores and vegetarians. Due to this nontrivial retroconversion e ciency, DHA supplementation represents an alternative to sh oil to increase blood and tissue levels of EPA, DPA, and DHA (5) (see Supplements). Biological Activities Membrane structure and function Omega-6 and omega-3 PUFA are important structural components of cell membranes. When incorporated into phospholipids, they a ect cell membrane properties, such as uidity, exibility, permeability, and the activity of membrane-bound enzymes (14). In addition to endogenous metabolism, dietary consumption of fatty acids can modify the composition and molecular structure of cellular membranes. Thus, increasing omega-3 fatty acid intake increases the omega-3 content of red blood cells, immune cells (15), atherosclerotic plaques (16), cardiac tissue (17), and other cell types throughout the body.
DHA is selectively incorporated into retinal cell membranes and postsynaptic neuronal cell membranes, suggesting it plays important roles in vision and nervous system function. Vision DHA is found at very high concentrations in the cell membranes of the retina; the retina conserves and recycles DHA even when omega-3 fatty acid intake is low (18). Animal studies indicate that DHA is required for the normal development and function of the retina. Moreover, these studies suggest that there is a critical period during retinal development when inadequate DHA will result in permanent abnormalities in retinal function. Research indicates that DHA plays an important role in the regeneration of the visual pigment rhodopsin, which plays a critical role in the visual transduction system that converts light hitting the retina to visual images in the brain (19). Nervous system The phospholipids of the brain’s gray matter contain high proportions of DHA and AA, suggesting they are important to central nervous system function (20). Brain DHA content may be particularly important, since animal studies have shown that depletion of DHA in the brain can result in learning de cits. It is not clear how DHA a ects brain function, but changes in DHA content of neuronal cell membranes could alter the function of ion channels or membrane-associated receptors, as well as the availability of neurotransmitters (21). Synthesis of lipid mediators Eicosanoids
Eicosanoids are potent chemical messengers that play critical roles in immune and in ammatory responses. The term 'eicosanoid' encompasses numerous bioactive lipid mediators that are derived from 20-carbon LC-PUFA. Following stimulation by hormones, cytokines, and other stimuli, DGLA, AA, and EPA are released from cell membranes and become substrates for eicosanoid production (Figure 4). Eicosanoid synthesis relies primarily on three families of enzymes: cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome p450 mono-oxygenases (P450s) (22). From 20-carbon lipid precursors, COX enzymes produce prostaglandins, prostacyclins, and thromboxanes (collectively known as prostanoids); LOX produces leukotrienes and hydroxy fatty acids; and P450s produce hydroxyeicosatetraenoic acids ("HETEs") and epoxides (Figure 5).
Physiological responses to AA-derived eicosanoids di er from responses to EPA-derived eicosanoids. In general, eicosanoids derived from EPA are less potent inducers of in ammation, blood vessel constriction, and coagulation than eicosanoids derived from AA (23). Nonetheless, it is an oversimpli cation to label all AA-derived eicosanoids as pro-in ammatory. AA-derived prostaglandins induce in ammation but also inhibit pro-in ammatory leukotrienes and cytokines and induce anti-in ammatory lipoxins, thereby modulating the intensity and duration of the in ammatory response via negative feedback (see Figure 5 above) (16).
Pro-resolving mediators
A separate class of PUFA-derived bioactive lipids, specialized pro-resolving mediators (SPMs), has been recently identi ed (reviewed in 24). These molecules function as local mediators of the resolution phase of in ammation, actively turning o the in ammatory response. SPMs are derived from both omega-6 and omega-3 PUFA (see Figure 5 above) (25). The S-series of SPMs results from the LOX-mediated oxygenation of EPA and DHA, giving rise to S-resolvins, S-protectins, and S- maresins. A second class of SPMs, the R-series, is generated from the aspirin-dependent acetylation of COX-2 and subsequent generation of aspirin-triggered SPMs from AA, EPA, and DHA. It appears that these mediators may explain many of the anti-in ammatory actions of omega-3 fatty acids that have been described (15, 26).
Isoprostanes
Isoprostanes are prostaglandin-like compounds that are formed by non-enzymatic, free radical- induced oxidation of any PUFA with three or more double bonds (see Figure 5 above) (22). Because they are produced upon exposure to free radicals, isoprostanes are often used as markers for oxidative stress. In contrast to prostanoids, isoprostanes are synthesized from esteri ed PUFA precursors and remain bound to the membrane phospholipid until cleaved by PLA2 and released into circulation. In addition to being used as markers of oxidative stress, isoprostanes may also function as in ammatory mediators, exerting both pro- and anti-in ammatory e ects (22). Regulation of gene expression The results of cell culture and animal studies indicate that omega-6 and omega-3 fatty acids can modulate the expression of a number of genes, including those involved with fatty acid metabolism and in ammation (27, 28). Omega-6 and omega-3 fatty acids regulate gene expression by interacting with speci c transcription factors, such as peroxisome proliferator-activated receptors (PPARs) (29). In many cases, PUFA act like hydrophobic hormones (e.g., steroid hormones) to control gene expression and bind directly to receptors like PPARs. These ligand-activated receptors then bind to the promoters of genes and function to increase/decrease transcription.
In other cases, PUFA regulate the abundance of transcription factors inside the cell's nucleus (30). Two examples include NFκB and SREBP-1. NFκB is a transcription factor involved in regulating the expression of multiple genes involved in in ammation. Omega-3 PUFA suppress NFκB nuclear content, thus inhibiting the production of in ammatory eicosanoids and cytokines. SREBP-1 is a major transcription factor controlling fatty acid synthesis, both de novo lipogenesis and PUFA synthesis. Dietary PUFA can suppress SREBP-1, which decreases the expression of enzymes involved in fatty acid synthesis and PUFA synthesis. In this way, dietary PUFA function as feedback inhibitors of all fatty acid synthesis. Deficiency Essential fatty acid de ciency Clinical signs of essential fatty acid de ciency include a dry scaly rash, decreased growth in infants and children, increased susceptibility to infection, and poor wound healing (31). Omega-3, omega- 6, and omega-9 fatty acids compete for the same desaturase enzymes. The desaturase enzymes show preference for the di erent series of fatty acids in the following order: omega-3 > omega-6 > omega-9. Consequently, synthesis of the omega-9 fatty acid eicosatrienoic acid (20:3n-9, mead acid, or 5,8,11-eicosatrienoic acid) increases only when dietary intakes of omega-3 and omega-6 fatty acids are very low; therefore, mead acid is one marker of essential fatty acid de ciency (32). A plasma eicosatrienoic acid:arachidonic acid (triene:tetraene) ratio greater than 0.2 is generally considered indicative of essential fatty acid de ciency (31, 33). In patients who were given total parenteral nutrition containing fat-free, glucose-amino acid mixtures, biochemical signs of essential fatty acid de ciency developed in as little as 7 to 10 days (34). In these cases, the continuous glucose infusion resulted in high circulating insulin levels, which inhibited the release of essential fatty acids stored in adipose tissue. When glucose-free amino acid solutions were used, parenteral nutrition up to 14 days did not result in biochemical signs of essential fatty acid de ciency. Essential fatty acid de ciency has also been found to occur in patients with chronic fat malabsorption (35) and in patients with cystic brosis (36). Recently, it has been proposed that essential fatty acid de ciency may play a role in the pathology of protein-energy malnutrition (32). Omega-3 fatty acid de ciency At least one case of isolated omega-3 fatty acid de ciency has been reported. A young girl who received intravenous lipid emulsions with very little ALA developed visual problems and sensory neuropathy; these conditions were resolved when she was administered an emulsion containing more ALA (37). Isolated omega-3 fatty acid de ciency does not result in increased plasma triene:tetraene ratios, and skin atrophy and dermatitis are absent (1). Plasma DHA concentrations decrease when omega-3 fatty acid intake is insu cient, but no accepted plasma omega-3 fatty acid or eicosanoid concentrations indicative of impaired health status have been de ned (1). Studies in rodents have revealed signi cant impairment of n-3 PUFA de ciency on learning and memory (38, 39) prompting research in humans to assess the impact of omega-3 PUFA on cognitive development and cognitive decline (see Visual and neurological development and Alzheimer's disease). Omega-3 index The omega-3 index is de ned as the amount of EPA plus DHA in red blood cell (RBC) membranes expressed as the percent of total RBC membrane fatty acids (40). The EPA + DHA content of RBCs correlates with that of cardiac muscle cells (41, 42), and several observational studies indicate that a lower omega-3 index is associated with an increased risk of coronary heart disease (CHD) mortality (43). It is therefore proposed that the omega-3 index be used as a biomarker for cardiovascular disease risk, with proposed zones being high risk, <4%; intermediate risk, 4-8%; and low risk, >8% (44).
Supplementation with EPA + DHA from sh oil capsules for approximately ve months dose- dependently increased the omega-3 index in 115 healthy, young adults (20-45 years of age), validating the use of the omega-3 index as a biomarker of EPA + DHA intake (45). Before the omega-3 index can be used in routine clinical evaluation, however, clinical reference values in the population must be established (46). Additionally, fatty acid metabolism may be altered in certain disease states, potentially making the omega-3 index less relevant for some cardiovascular conditions (5).
Disease Prevention Disease Prevention Visual and neurological development The last trimester of pregnancy and rst six months of postnatal life are critical periods for the accumulation of DHA in the brain and retina (47). Human milk contains a mixture of saturated fatty acids (~46%), monounsaturated fatty acids (~41%), omega-6 PUFA (~12%), and omega-3 PUFA (~1.3%) (48). Although human milk contains DHA in addition to ALA and EPA, ALA was the only omega-3 fatty acid present in conventional infant formulas until the year 2001.
Infant formulas Although infants can synthesize DHA from ALA, they generally cannot synthesize enough to prevent declines in plasma and cellular DHA concentrations without additional dietary intake. Therefore, it was proposed that infant formulas be supplemented with enough DHA to bring plasma and cellular DHA levels of formula-fed infants up to those of breast-fed infants (49). Although formulas enriched with DHA raise plasma and red blood cell DHA concentrations in preterm and term infants, the results of randomized controlled trials (RCTs) examining measures of visual acuity and neurological development in infants fed formulas with or without added DHA have been mixed (50, 51). A 2012 meta-analysis of RCTs (12 trials, 1,902 infants) testing LC-PUFA supplemented versus unsupplemented formula, started within one month of birth, found no e ect of LC-PUFA supplementation on infant cognition assessed at approximately one year of age (52). A lack of e ect was observed regardless of the dose of LC-PUFA or the prematurity status of the infant. With respect to visual acuity, a 2013 meta-analysis of RCTs (19 trials, 1,949 infants) found a bene cial e ect of LC-PUFA-supplemented formula, started within one month of birth, on infant visual acuity up to 12 months of age (53). Notably, two di erent types of visual acuity assessment were evaluated in the meta-analysis. Visual acuity assessed by using the visually evoked potential (VEP) (10 trials, 852 infants) showed a signi cant positive e ect of LC-PUFA supplemented formula at 2, 4, and 12 months of age. When assessed by the behavioral method (BM) (12 trials, 1,095 infants), a signi cant bene t of LC-PUFA-supplemented formula on visual acuity was found only at the age of two months. No moderating e ects of dose or prematurity status were observed.
Maternal supplementation (placental transfer and breast milk)
The e ect of maternal omega-3 LC-PUFA supplementation on early childhood cognitive and visual development was evaluated in a 2013 systematic review and meta-analysis (54). Included in this assessment were 11 RCTs (a total of 5,272 participants) that supplemented maternal diet with omega-3 LC-PUFA during pregnancy or during pregnancy and lactation. Visual outcomes (eight trials) could not be evaluated in the meta-analysis due to variability in assessments; overall, four of six trials had null ndings and the remaining two trials had very high rates of attrition. Cognitive outcomes (nine trials) included the Developmental Standard Score (DSS; in infants, toddlers, and preschoolers) or Intelligence Quotient (IQ; in children) and other aspects of neurodevelopment, such as language, behavior, and motor function. No di erences were found between DHA and control groups for cognition measured with standardized psychometric scales in infants (<12 months), toddlers (12-24 months), and school aged children (5-12 years); preschool children (2-5 years) in the DHA treatment group had a 3.92 point increase in DSS compared to controls. The authors note that many of the trials of LC-PUFA supplementation in pregnancy had methodological weaknesses (e.g., high rates of attrition, small sample sizes, high risk of bias, multiple comparisons) limiting the con dence and interpretation of the pooled results.
Although epidemiological investigations have demonstrated that higher intakes of omega-3 LC- PUFA from sh and seafood during pregnancy are associated with improved developmental outcomes in o spring (54), trial evidence does not conclusively support or refute this relationship. At present, the potential bene ts associated with obtaining long-chain omega-3 fatty acids through moderate consumption of sh (e.g., 1-2 servings weekly) during pregnancy and lactation outweigh any risks of contaminant exposure, though sh with high levels of methylmercury should be avoided (55). For information about contaminants in sh and guidelines for sh consumption by women of childbearing age, see Contaminants in sh. Gestation and pregnancy The results of randomized controlled trials (RCTs) during pregnancy suggest that omega-3 fatty acid supplementation does not decrease the incidence of gestational diabetes, pregnancy-induced hypertension, or preeclampsia (56-58) but may result in modest increases in length of gestation, especially in women with low omega-3 fatty acid consumption. A 2006 meta-analysis of six randomized controlled trials in women with low-risk pregnancies found that omega-3 PUFA supplementation during pregnancy resulted in an increased length of pregnancy by 1.6 days (59). A 2007 meta-analysis of randomized controlled trials in women with high-risk pregnancies found that supplementation with long-chain PUFA did not a ect pregnancy duration or the incidence of premature births but decreased the incidence of early premature births (<34 weeks of gestation; 2 trials, N=291; Relative Risk [RR]: 0.39 (95% CI: 0.18-0.84) (60).
Because maternal dietary intake of LC-PUFA determines the DHA status of the newborn, several expert panels in the US recommend that pregnant and lactating women consume at least 200 mg DHA per day, close to the amount recommended for adults in general (250 mg/day) (47, 61). The European Food and Safety Authority (EFSA) recommends that pregnant and lactating women consume an additional 100-200 mg of preformed DHA on top of the 250 mg/day EPA plus DHA recommended for healthy adults (62). Cardiovascular disease Omega-6 fatty acids: linoleic acid LA is the most abundant dietary PUFA and accounts for approximately 90% of dietary omega-6 PUFA intake (63). Taking into consideration the results from RCTs and observational cohort studies, a 2009 American Heart Association scienti c advisory concluded that obtaining at least 5-10% of total caloric intake from omega-6 PUFA is associated with a reduced risk of coronary heart disease (CHD) relative to lower intakes (64, 65). A pooled analysis of 11 cohort studies, encompassing 344,696 individuals followed for 4 to 10 years, found that replacing 5% of energy from saturated fatty acids (SFAs) with PUFA was associated with a 13% lower risk of coronary events (95% CI: 0.77, 0.97) and a 26% lower risk of coronary deaths (95% CI: 0.61, 0.89) (66). A 2012 meta-analysis of seven RCTs corroborated this bene cial e ect, with an estimated 10% reduction in CHD risk (RR: 0.90, 95% CI: 0.83-0.97) for each 5% energy increase in PUFA consumption (67).
In controlled feeding trials, replacing dietary SFA with PUFA consistently lowers serum total and LDL cholesterol concentrations (68, 69). In fact, LA has been shown to be the most potent fatty acid for lowering serum total and LDL cholesterol when substituted for dietary SFA (70). The mechanisms by which linoleic acid lowers blood cholesterol include (1) the upregulation of LDL receptor and redistribution of LDL-C from plasma to tissue, (2) increased bile acid production and cholesterol catabolism, and (3) decreased conversion of VLDL to LDL (71).
Although dietary LA lowers blood cholesterol levels, supplementation with concentrated sources of LA may have adverse cardiovascular e ects in individuals with preexisting CHD (see Disease Treatment).
Omega-3 fatty acids: α-linolenic acid
Several prospective cohort studies have examined the relationship between dietary ALA intake and cardiovascular disease (CVD). A 2012 meta-analysis of observational studies evaluated the risk of incident CVD related to dietary consumption or biomarkers of ALA (72). The analysis included 27 studies, 251,049 individuals and 15,327 CVD events (fatal coronary heart disease [CHD], nonfatal CHD, total CHD, and stroke). Overall, the pooled analysis found a moderately lower risk of CVD with higher ALA exposure (Relative Risk [RR]: 0.86; 95% CI: 0.77, 0.97).
Unlike LA, the cardioprotective e ects of higher ALA intakes do not appear to be related to changes in serum lipid pro les. A meta-analysis of 14 randomized controlled trials concluded that ALA supplementation had no e ect on total cholesterol, LDL cholesterol, or triglyceride levels (73). However, several controlled clinical trials have found that increasing ALA intake decreased serum concentrations of C-reactive protein (CRP), a marker of in ammation that is strongly associated with the risk of cardiovascular events, such as MI and stroke (74-76).
Long-chain omega-3 fatty acids: eicosapentaenoic acid and docosahexaenoic acid
Evidence is accumulating that increasing intakes of long-chain omega-3 fatty acids (EPA and DHA) can decrease the risk of cardiovascular disease by (1) preventing arrhythmias that can lead to sudden cardiac death, (2) decreasing the risk of thrombosis (a clot) that can lead to myocardial infarction (MI) or stroke, (3) decreasing serum triglyceride levels, (4) slowing the growth of atherosclerotic plaque, (5) improving vascular endothelial function, (6) lowering blood pressure slightly, and (7) decreasing in ammation (77). In spite of these possible biological e ects, clinical trials have not shown a signi cant e ect of long- chain omega-3 supplementation on major cardiovascular events. A 2006 systematic review and meta-analysis of randomized controlled trials and prospective cohort studies concluded that long- chain omega-3 fatty acids do not signi cantly reduce the risk of total mortality or cardiovascular events (78). Likewise, a 2012 meta-analysis of secondary prevention trials (20 RCTs, including 68,680 patients) found no signi cant e ect of omega-3 supplements (~1.5 g/day of EPA + DHA for a median of 2 years) on all-cause mortality, cardiac death, sudden death, myocardial infarction, or stroke (79). The same lack of e ect was observed in a 2012 systematic review and meta-analysis of RCTs investigating the impact of omega-3 supplementation on in ammatory biomarkers in both healthy and ill individuals (80).
Although supplementation trials have not demonstrated a clear clinical bene t of omega-3 supplements, a recent multicenter, prospective, observational cohort study found a strong relationship between plasma phospholipid omega-3 PUFA levels (a biomarker of omega-3 status) and cardiovascular mortality (81). The Cardiovascular Health Study (CHS) related circulating levels of total and individual LC-PUFA (EPA, DPA, and DHA) to risk of total and CVD-speci c mortality in 2,692 older (≥65 years) US adults. Higher levels of individual and combined total omega-3 PUFA in plasma phospholipids were associated with lower total mortality (HR for total omega-3: 0.73, 95% CI: 0.61-0.86). Looking more closely at cause-speci c mortality, the observed reduction in risk was attributed mainly to fewer arrhythmic cardiac deaths (HR: 0.52, 95% CI: 0.31-0.86) and speci cally with higher circulating DHA content (45% lower risk). Only EPA was associated with nonfatal MI (28% lower risk), while DPA was most strongly associated with stroke death (47% lower risk).
Coronary heart disease: A 2012 meta-analysis of 17 cohort studies with 315,812 participants and an average follow-up of 15.9 years calculated the pooled e ect of sh consumption on coronary heart disease (CHD) mortality (82). Low (1 serving/week) or moderate sh consumption (2-4 servings/week) had a signi cant bene cial e ect on the prevention of CHD mortality. Speci cally, compared with the lowest sh consumption (<1 serving/month or 1-3 servings/month), consumption of 1 serving of sh per week and 2-4 servings/week was associated with a 16% (RR: 0.84, 95% CI: 0.75, 0.95) and 21% (RR: 0.79, 95% CI: 0.67,0.92) lower risk of fatal CHD, respectively.
Overall, among the various CVD outcomes (Figure 6), ndings from prospective cohort studies and RCTs consistently indicate that consumption of sh or sh oil signi cantly reduces CHD mortality, including fatal myocardial infarction and sudden cardiac death (77, 83, 84). There is little evidence that these e ects di er by sex, age, or race/ethnicity (77). Sudden cardiac death: Sudden cardiac death (SCD) is the result of a fatal ventricular arrhythmia, which usually occurs in people with CHD. Studies in cell culture indicate that long-chain omega-3 fatty acids decrease the excitability of cardiac muscle cells (myocytes) by modulating ion channel conductance (85). The results of epidemiological studies suggest that regular sh consumption is inversely associated with the risk of SCD. A 2011 systematic review and meta-analysis of eight prospective cohort studies evaluated the impact of consuming <250 mg versus ≥250 mg omega-3 PUFA per day on various CHD outcomes (86). Consumption of ≥250 mg omega-3 PUFA per day was associated with a signi cant, 35% reduction in the risk of SCD (RR: 0.65; 95% CI: 0.54, 0.79).
A meta-analysis of nine randomized controlled trials found no signi cant e ect of omega-3 supplements on SCD or ventricular arrhythmias in patients with previous MI compared to those taking placebo (87). Notably, although the pooled analysis reported no signi cant e ect, the included trials reported either a protective e ect (six trials) or null e ect (three trials), with no harmful outcomes reported. Stroke: Ischemic strokes are the result of insu cient blood ow to an area of the brain, which may occur when an artery supplying the brain becomes occluded by a clot. Hemorrhagic strokes occur when a blood vessel ruptures and bleeds into the brain. In the United States, 87% of strokes are ischemic strokes (88). A 2012 meta-analysis of 16 prospective studies, encompassing 402,127 individuals for a mean of 12.8 years, found that increased sh intake was associated with a decreased risk of ischemic stroke, but not hemorrhagic stroke (89). According to this analysis, consuming sh even once per week may signi cantly reduce the risk of ischemic stroke. In a separate dose-response meta-analysis of these prospective studies, a 3-servings/week increase in sh consumption was associated with a 6% decreased risk of total stroke (95% CI: 0.89-0.99) (90). Again, the association remained signi cant only for ischemic stroke (RR: 0.90, 95% CI: 0.84-0.97).
Although the protective e ect of sh intake could be attributed to many things (e.g., the displacement of red meat, a marker of an overall healthier lifestyle and dietary pattern, nutrient interactions (91, 92)), its high content of omega-3 PUFA may be a major contributing factor. A meta- analysis of eight prospective studies that assessed the association between omega-3 PUFA intake on stroke risk found evidence of a nonlinear relationship between LC-PUFA intake and stroke risk, with only moderate intakes of 200-400 mg/day omega-3 PUFA associated with signi cantly reduced risk of total stroke (93). Additionally, when analyzed by stroke type, the risk for ischemic stoke was lower in the highest versus lowest category of long-chain omega-3 PUFA intake (RR: 0.82, 95% CI: 0.71-0.94).
Another 2012 systematic review and meta-analysis assessed both prospective cohort studies and RCTs that investigated sh consumption or omega-3 supplementation on cerebrovascular disease (any fatal or non-fatal ischemic stroke, hemorrhagic stroke, cerebrovascular accident, or transient ischemic attack) (91). From 26 prospective cohort studies, the pooled relative risk (RR) for cerebrovascular disease for 2-4 versus ≤1 serving of sh per week was 0.94 (95% CI: 0.90-0.98); for >5 servings versus ≤1 serving per week, the RR was 0.88 (95% CI: 0.81-0.96). No signi cant association was found between long-chain omega-3 biomarkers and risk of cerebrovascular disease. From the 12 RCTs analyzed, 10 of which recruited subjects with preexisting cardiovascular disease at baseline, no signi cant e ect of omega-3 supplementation (mean dose of 1.8 g/day for a mean duration of 3 years) on cerebrovascular disease outcomes was observed. The same lack of e ect of omega-3 supplementation on total stroke risk was observed in a second meta-analysis of RCTs (nine trials) (79).
Serum triglycerides: A meta-analysis of 17 prospective studies found hypertriglyceridemia (serum triglycerides >200 mg/dL) to be an independent risk factor for cardiovascular disease (94). Numerous controlled clinical trials have demonstrated that increasing intakes of EPA and DHA signi cantly lower serum triglyceride concentrations (95). The triglyceride-lowering e ects of EPA and DHA increase with dose (96), but clinically meaningful reductions in serum triglyceride concentrations have been demonstrated at doses of 2 g/day of EPA + DHA (97). In its recommendations regarding omega-3 fatty acids and cardiovascular disease (see Intake Recommendations), the American Heart Association indicates that an EPA + DHA supplement may be useful in patients with hypertriglyceridemia (23).
A 2011 meta-analysis of RCTs compared the e ect of EPA alone (10 trials), DHA alone (17 trials), or EPA versus DHA (6 trials) on serum lipids (98). Although both EPA and DHA reduce triglyceride levels, they have di erent e ects on LDL and HDL levels. DHA raises LDL and HDL, whereas EPA has no signi cant e ect. Importantly, DHA may increase LDL via increased conversion of VLDL to LDL and by producing larger, more buoyant LDL particles (3).
Summary: omega-3 and omega-6 PUFA and cardiovascular disease prevention
The results of observational studies and randomized controlled trials suggest that replacing dietary SFA with omega-6 and omega-3 PUFA (from both plant and marine sources) lowers LDL cholesterol and decreases cardiovascular disease risk. Additionally, the results of epidemiological studies provide consistent evidence that increasing dietary omega-3 fatty intake is associated with signi cant reductions in cardiovascular disease risk through mechanisms other than lowering LDL cholesterol. In particular, increasing sh consumption to at least two servings of oily sh per week has been associated with signi cant reductions in fatal myocardial infarction and sudden cardiac death (77). This amount would provide about 400-500 mg/day of EPA + DHA (23). Alzheimer's disease Alzheimer's disease is the most common cause of dementia in older adults. Alzheimer's disease is characterized by the formation of amyloid plaque in the brain and nerve cell degeneration. Disease symptoms, including memory loss and confusion, worsen over time (99). Some epidemiological studies have associated high intake of sh with lower risks of impaired cognitive function (100), dementia (101), and Alzheimer's disease (101, 102). Proposed mechanisms for a protective e ect of long-chain omega-3 fatty acids in the brain and vascular system include (1) the mitigation of in ammation, (2) improved cerebral blood ow, and (3) reduced amyloid aggregation (103).
A 2009 systematic review reported on the association between eating sh (as a source of long- chain omega-3 fatty acids) or taking an omega-3 supplement and the risk of cognitive decline or Alzheimer's disease (103). Out of 11 observational studies, three reported a signi cant bene t of omega-3 fatty acids on cognitive decline; four of eight observational studies reported positive ndings on incident Alzheimer's disease or dementia. The four small clinical trials reviewed showed no evidence for prevention or treatment of any form of dementia (103).
DHA, the major omega-3 fatty acid in the brain, appears to be protective against Alzheimer's disease (104). Observational studies have found that lower DHA status is associated with increased risk of Alzheimer's disease (105-107), as well as other types of dementia (106). The relationship between DHA status and cognitive decline may be dependent on apolipoprotein E (APOE) genotype. Of three common APOE alleles (epsilon 2 [ε2], ε3, and ε4), the presence of the APOE ε4 (E4) allele is associated with increased risk and earlier onset of Alzheimer's disease (108). A protective e ect of consumption of fatty sh on the risk for dementia and AD may not apply to carriers of the E4 allele (109, 110). EPA + DHA supplementation did not increase plasma levels of these omega-3 PUFA to the same extent in E4 carriers compared to non-carriers (111), and [13C]DHA tracer studies indicate that DHA metabolism di ers in E4 carriers, with greater oxidation and lower plasma levels in E4 positive versus negative individuals (112).
Overall, the data favor a role for diets rich in long-chain omega-3 fatty acids in slowing cognitive decline but not for supplementation in the prevention or treatment of any type of dementia. The e cacy of omega-3 supplementation may depend on the underlying pathology of AD (i.e., the involvement of a vascular issue) (103) or the presence of the APOE4 allele (110, 111). Additionally, consistency in outcome measures and diagnostic criteria, and longer duration trials may be necessary to see a consistent e ect. Disease Treatment Coronary heart disease Dietary intervention trials
Omega-6 fatty acids (linoleic acid): In a reanalysis of the Sydney Heart Health Study (SHHS), a single- blind, RCT in 458 men (ages 30-59 years) with a recent coronary event, the replacement of dietary saturated fat with omega-6 linoleic acid led to higher rates of death from all-causes, cardiovascular disease, and coronary heart disease (CHD) compared to controls (113). Furthermore, a meta- analysis that included the SHHS and two other secondary prevention trials revealed an increased risk of mortality when saturated fat is replaced with concentrated sources of linoleic acid. There are some important limitations and considerations with the SHHS to keep in mind: (1) LA intake went from 6% to 15% of total energy for the study participants; in US adults, the average intake of LA is approximately 7% of total energy (114); (2) there may have been displacement of monounsaturated fatty acids and other PUFA in addition to saturated fats in the experimental intervention; and (3) the experimental formulation of sa ower oil margarine may have provided trans fat. Regardless of these issues, substituting dietary saturated fat with mixed PUFA (both omega-6 and omega-3) rather than linoleic acid alone reduces CVD risk (113) and is recommended for both the primary and secondary prevention of CVD (65, 67).
Omega-3 fatty acids: In the Diet and Reinfarction Trial (DART), total mortality and fatal MI decreased by 29% in male MI survivors advised to increase their weekly intake of oily sh to 200-400 g (7-14 oz)—an amount estimated to provide an additional 500-800 mg/day of long-chain omega-3 fatty acids (EPA + DHA) (115). The Diet and Reinfarction Trial 2 (DART-2) administered similar dietary advice but to a di erent cohort of high-risk individuals: those with stable angina (116). In this case, advice to eat oily sh or sh oil did not a ect all-cause mortality but was associated with an increased risk of sudden cardiac death. This increased risk was con ned to the use of sh oil capsules rather than dietary sh intake. Though the results of the DART trials seem to contradict each other, there are important di erences that o er explanations, namely the timing of the intervention (shortly after rst MI in the rst trial) and the stage of CHD (early versus stable) in the study population. These trials suggest that sh oil may reduce mortality during recovery from MI but perhaps not during later stages of the disease.
The Alpha Omega Trial tested if low doses of EPA + DHA (400 mg per day), ALA (2 g per day), or both in margarines reduced the risk of cardiovascular (CV) events among 4,837 patients (78% male; mean age, 69 years) who had a MI in the previous 10 years (117). After 40 months, none of the omega-3 PUFA treatments signi cantly a ected the rate of major CV events compared to placebo. Notably, medication use in the study population was high: antithrombotic agents (97.5%), antihypertensive drugs (89.7%), and statins (85%).
Supplementation trials
In the largest randomized controlled trial of supplemental omega-3 fatty acids to date, the GISSI- Prevenzione Trial, CHD patients who received supplements providing 850 mg/day of EPA + DHA for 3.5 years had a risk of sudden death that was 45% lower than those who did not take supplements; supplement users also experienced a 20% lower risk of death from all causes compared to non- supplement users (118). Interestingly, it took only three months of supplementation to demonstrate a signi cant decrease in total mortality and four months to demonstrate a signi cant decrease in sudden death (119).
The results of a meta-analysis that pooled the ndings of 29 randomized controlled trials of dietary or supplementary omega-3 fatty acids indicated that omega-3 fatty acids were not associated with a statistically signi cant reduction in all-cause mortality or risk of restenosis in high-risk cardiovascular patients (120). Heterogeneity in trial size and follow-up time limited the analysis, and the authors note that the probability of bene t from omega-3 fatty acids still remains high for both endpoints.
Summary
The results of randomized controlled trials in individuals with documented CHD suggest a bene cial e ect of dietary and supplemental omega-3 fatty acids. Based on the results of these trials, the American Heart Association recommends that individuals with documented CHD consume approximately 1 g/day of EPA + DHA, preferably by consumption of oily sh (see Intake Recommendations) (121). Diabetes mellitus Cardiovascular disease are the leading causes of death in individuals with diabetes mellitus (DM). The dyslipidemia typically associated with diabetes is characterized by a combination of hypertriglyceridemia (serum triglycerides >200 mg/dL), low HDL-C, and abnormal LDL composition (122). A 2009 meta-analysis of 23 randomized controlled trials (RCTs), including 1,075 individuals with type 2 diabetes, found that omega-3 fatty acid supplementation (mean dose, 3.5 g/day) lowered serum triglyceride levels by 0.45 mmol/L, lowered VLDL-C by 0.07 mmol/L, but raised LDL-C by 0.11 mmol/L (123). No signi cant changes in total cholesterol, HDL-C, HbA1c, fasting glucose, fasting insulin, or body weight were observed.
Since 2009, the results of two additional trials of omega-3 supplementation in diabetic patients have been published. The Alpha Omega Trial evaluated the e ect of low-dose supplementation with omega-3 fatty acids on ventricular arrhythmias and fatal CHD in stable, post-MI patients (117). While the main analysis found no e ect of supplementation, a secondary analysis of 1,014 diabetic participants found that low-dose supplementation of combined omega-3 fatty acids (~400 mg EPA + DHA and 2 g ALA per day in an experimental margarine spread for 40 months) resulted in fewer ventricular arrhythmia-related events (HR 0.16, 95% CI 0.04-0.69) compared to placebo margarine (124). In the ORIGIN trial, 1 g/day of omega-3 fatty acids (465 mg EPA + 375 mg DHA per day for 6 years) had no e ect on rates of major vascular events, all-cause mortality, cardiovascular mortality, or death from arrhythmia in 12,536 dysglycemic patients at high risk for cardiovascular events compared to placebo (125). Triglyceride levels were reduced by 14.5 mg/dL (0.16 mmol/L), but no other blood lipids were a ected by supplementation. In both of these trials, a high proportion of study participants used cardiovascular medications.
Summary
Increasing EPA and DHA intakes may be bene cial to diabetic individuals, especially those with elevated serum triglycerides or with a history of MI (126). There is no compelling evidence that daily EPA + DHA intakes of less than 3 g/day adversely a ect long-term glycemic control in diabetics (127-129). The American Diabetes Association recommends that diabetic individuals increase omega-3 fatty acid consumption by consuming two to three 3-oz servings of sh weekly (121). In ammatory diseases Rheumatoid arthritis
A 2012 meta-analysis of 10 randomized controlled trials, involving 270 rheumatoid arthritis (RA) patients, assessed the e cacy of high-dose omega-3 PUFA supplementation on clinical outcomes of RA (130). Omega-3 consumption of ≥2.7 g/day for a minimum of three months reduced nonsteroidal anti-in ammatory drug (NSAID) use but had no signi cant e ect on tender joint count, swollen joint count, morning sti ness, or physical function compared to placebo. On the other hand, a 2012 systematic review, which included 23 trials investigating omega-3 supplementation (mainly as sh oil) in RA patients, concluded that there appears to be a consistent, though modest, bene t of marine-derived omega-3 PUFA (average intake ~3g/day) on some clinical symptoms of RA (131). Thus, high-dose supplementation of long-chain omega-3 PUFA spares the need for anti-in ammatory medications and may reduce joint pain and swelling in some RA patients.
In ammatory bowel disease In ammatory bowel disease
Crohn’s disease: A 2013 systematic review evaluated the e cacy of omega-3 supplementation in Crohn’s disease (CD) patients, considering the evidence base from both short-term (9 to 24 weeks) and long-term (1 year) trials (132). Among ve trials that evaluated the e cacy of omega-3 supplementation on CD relapse rates, con icting outcomes were reported. Most trials were limited by small sample sizes and short duration; up to three years may be necessary to see an e ect on CD relapse rates given the natural relapsing-remitting course of the disease. The two largest and most recent trials (EPIC-1 and EPIC-2) showed no signi cant e ect of omega-3 supplementation on symptoms of CD remission compared to placebo (133). Two additional systematic reviews and a meta-analysis reached similar conclusions (134, 135). Three short-term trials showed positive e ects of omega-3 supplementation on plasma biochemical parameters (e.g., reduced in ammatory cytokine expression, increased plasma EPA and DHA levels) compared to controls (132). In spite of its impact on biochemical changes in the short-term, however, the ability of omega-3 supplementation to maintain remission or e ect clinically meaningful changes in CD is not supported by the current evidence.
Ulcerative colitis: Seven randomized controlled trials of sh oil supplementation in active ulcerative colitis (UC) patients reported signi cant improvement in at least one outcome measure, such as decreased corticosteroid use, improved disease activity scores, or improved histology scores (135). In inactive UC patients, omega-3 supplementation had no e ect on relapse rates compared to placebo in four separate trials (134, 135).
While no serious side e ects were reported in any trials of sh oil supplementation for the maintenance or remission of in ammatory bowel disease, diarrhea and upper gastrointestinal symptoms occurred more frequently with omega-3 treatment (134, 135).
Asthma
In ammatory eicosanoids (leukotrienes) derived from arachidonic acid (AA; 20:4n-6) are thought to play an important role in the pathology of asthma (28). Since increasing omega-3 fatty acid intake has been found to decrease the formation of AA-derived leukotrienes, a number of clinical trials have examined the e ects of long-chain omega-3 fatty acid supplementation on asthma. Although there is some evidence that omega-3 fatty acid supplementation can decrease the production of in ammatory mediators in asthmatic patients (136, 137), evidence that omega-3 fatty acid supplementation decreases the clinical severity of asthma in controlled trials has been inconsistent (138). Three systematic reviews of randomized controlled trials of long-chain omega-3 fatty acid supplementation in asthmatic adults and children found no consistent e ects on clinical outcome measures, including pulmonary function tests, asthmatic symptoms, medication use, or bronchial hyperreactivity (139-141).
Immunoglobulin A nephropathy Immunoglobulin A (IgA) nephropathy is a kidney disorder that results from the deposition of IgA in the glomeruli of the kidney. The cause of IgA nephropathy is not clear, but progressive renal failure may eventually develop in 15-40% of patients (142). Since glomerular IgA deposition results in increased production of in ammatory mediators, omega-3 fatty acid supplementation could potentially modulate the in ammatory response and preserve renal function.
A 2012 meta-analysis assessed the e cacy of omega-3 fatty acid treatment on adult IgA nephropathy (143). Five randomized controlled trials were included in an analysis involving 239 patients (mean age range, 37 to 41 years) who received placebo or supplemental EPA + DHA at doses of 1.4 to 5.1 g/day for 6 to 24 months. Compared with control groups, omega-3 treatment had no signi cant e ect on urine protein excretion or glomerular ltration rate. Only two trials measured changes in serum creatinine, a marker of renal function, and end-stage renal disease; in both trials, omega-3 treatment had a bene cial e ect on these two parameters. No adverse events associated with omega-3 treatment were reported in any of the trials. Neuropsychiatric disorders Major depression and bipolar disorder
Data from ecologic studies across di erent countries suggest an inverse association between seafood consumption and national rates of major depression (144) and bipolar disorder (145). Several small studies have found omega-3 fatty acid concentrations to be lower in the plasma (146- 148) and adipose tissue (fat) (149) of individuals su ering from depression compared to controls. Although it is not known how omega-3 fatty acid intake a ects the incidence of depression, modulation of neuronal signaling pathways and eicosanoid production have been proposed as possible mechanisms (150).
There may be some bene t of omega-3 PUFA supplementation on depressive disorders, but it is di cult to compare studies and draw conclusions due to great heterogeneity among the trials (151, 152). Small sample sizes, lack of standardization of therapeutic doses, type of omega-3 PUFA administered, co-treatment with pharmacological agents, and diagnostic criteria vary among the trials.
A 2012 systematic review of all published RCTs investigated the e ect of omega-3 PUFA supplementation on the prevention and treatment of several types of depression and other neuropsychiatric disorders (151). With respect to major depression, a majority of studies reported a positive e ect for omega-3 supplements on depressive symptoms, though e cacy is still considered inconclusive given the great variability among trials. A few themes emerged from this review: more trials reported positive e ect for omega-3 PUFA supplements as an adjunct to pharmacological treatment; in monotherapy trials, EPA alone was more e ective than DHA alone; and in combination trials, positive e ects were more likely if an EPA:DHA ratio of >1.5–2.0 was administered. Unipolar depression and bipolar disorder are considered distinct psychiatric conditions, although major depression occurs in both. As with major depression, the 2012 review of RCTs indicated that omega-3 supplementation may have a positive e ect as an adjunct to therapy in patients with bipolar disorder (151).
While there is some promising evidence for the use of omega-3 fatty acids for major depression and bipolar disorder, additional trials that account for dietary omega-3 intake, changes in red blood cell PUFA levels, the ratio of EPA:DHA provided, co-treatment with medications, and of su cient duration are necessary.
Schizophrenia
A 2013 meta-analysis of 18 studies compared the PUFA composition of red blood cell (RBC) membranes in schizophrenic patients to those of normal controls (153). The majority of studies investigated medicated patients, though the authors separated the analysis into three groups of patients at time of measurement in order to account for possible confounding from pharmacologic agents: antipsychotic-medicated, antipsychotic-naïve, and antipsychotic-free. Overall, decreased levels of DPA, DHA, and AA in RBC membranes were associated with the schizophrenic state. Several mechanisms may account for the altered PUFA levels in schizophrenia, such as altered lipid metabolism, increased oxidative stress, or changes in diet consequent to disease-related behavior.
The use of long-chain omega-3 fatty acid supplements to alleviate symptoms of schizophrenia or to mitigate adverse e ects of antipsychotic medications has been investigated in a number of clinical trials (154). Overall, there was no e ect of sh oil or LC-PUFA supplements on symptoms of schizophrenia. However, given the high safety pro le of sh oil supplements and some evidence of a positive e ect of EPA supplementation in a subset of trials, some clinicians may consider EPA a useful adjunct to antipsychotic therapy in schizophrenic patients. Alzheimer's disease and dementia Some epidemiological studies have associated decreased DHA status with Alzheimer’s disease and other types of dementia (see above). Although results of studies in animal models have been promising (reviewed in 155), the existing clinical trial data do not support a recommendation for DHA supplementation in the treatment of Alzheimer's disease in humans. A randomized controlled trial (RCT) was conducted to determine if supplementation with DHA slows cognitive and functional decline in individuals with Alzheimer disease (156). Two hundred ninety- ve individuals with mild to moderate Alzheimer's disease (Mini-Mental State Examination [MMSE] scores, 14-26) received algal DHA supplement (1 g twice daily) or placebo (corn or soy oil) for 18 months. Although DHA supplementation signi cantly increased levels of DHA in plasma phospholipids and cerebrospinal uid, it had no e ect on the rate of change on the cognitive subscale of the Alzheimer Disease Assessment Scale (ADAS-cog), the Clinical Dementia Rating (CDR) sum of boxes, or brain atrophy compared to placebo. No adverse e ects were associated with DHA treatment in this trial.
Sources Sources Food sources Omega-6 fatty acids
Linoleic acid: Food sources of LA include vegetable oils, such as soybean, sa ower, and corn oil; nuts; seeds; and some vegetables. Dietary surveys in the US indicate that the average adult intake of LA ranges from 17-20 g/day for men and 12-13 g/day for women (63). Some foods that are rich in LA are listed in Table 2.
Table 2. Food Sources of Linoleic Acid (18:2n-6) (157)
Food Serving Linoleic Acid (g)
Sa ower oil 1 tablespoon 10.1
Sun ower seeds, oil roasted 1 oz 9.7
Pine nuts 1 oz 9.4
Sun ower oil 1 tablespoon 8.9
Corn oil 1 tablespoon 7.3
Soybean oil 1 tablespoon 6.9
Pecans, oil roasted 1 oz 6.4
Brazil nuts 1 oz 5.8
Sesame oil 1 tablespoon 5.6
Arachidonic acid: Animals, but not plants, can convert LA to AA. Therefore, AA is present in small amounts in meat, poultry, and eggs.
Omega-3 fatty acids
α-Linolenic acid (ALA): Flaxseeds, walnuts, and their oils are among the richest dietary sources of ALA. Canola oil is also an excellent source of ALA. Dietary surveys in the US indicate that average adult intakes for ALA range from 1.8-2.0 g/day for men and from 1.4-1.5 g/day for women (63). Some foods that are rich in ALA are listed in Table 3.
Table 3. Food Sources of α-Linolenic Acid (18:3n-3) (157)
Food Serving α-Linolenic acid (g)
Flaxseed oil 1 tablespoon 7.3 Chia seeds, dried 1 oz 5.1
Walnuts, English 1 oz 2.6
Flaxseeds, ground 1 tablespoon 1.6
Walnut oil 1 tablespoon 1.4
Canola oil 1 tablespoon 1.3
Soybean oil 1 tablespoon 0.9
Mustard oil 1 tablespoon 0.8
Walnuts, black 1 oz 0.6
Tofu, rm ½ cup 0.2
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA): Oily sh are the major dietary source of EPA and DHA. Dietary surveys in the US indicate that average adult intakes of EPA range from 0.03-0.06 g/day, and average adult intakes of DHA range from 0.05-0.10 g/day (63). Omega-3 fatty acid-enriched eggs are also available in the US. Some foods that are rich in EPA and DHA are listed in Table 4.
Table 4. Food Sources of EPA (20:5n-3) and DHA (22:6n-3) (97)
Amount Providing Food Serving EPA (g) DHA (g) 1 g of EPA + DHA
Herring, Paci c 3 oz* 1.06 0.75 1.5 oz
Salmon, chinook 3 oz 0.86 0.62 2 oz
Sardines, Paci c 3 oz 0.45 0.74 2.5 oz
Salmon, Atlantic 3 oz 0.28 0.95 2.5 oz
Oysters, Paci c 3 oz 0.75 0.43 2.5 oz
Salmon, sockeye 3 oz 0.45 0.60 3 oz
Trout, rainbow 3 oz 0.40 0.44 3.5 oz
Tuna, canned, white 3 oz 0.20 0.54 4 oz
Crab, Dungeness 3 oz 0.24 0.10 9 oz
Tuna, canned, light 3 oz 0.04 0.19 12 oz
*A three-ounce serving of sh is about the size of a deck of cards.
Biosynthesis of EPA and DHA 8/30/2017 Mango Supplementation Prevents Gut Microbial Dysbiosis and Modulates Short Chain Fatty Acid Production Independent of Body Weight Reduction in C57B…
The FASEB Journal www.fasebj.org
April 2016 The FASEB Journal vol. 30 no. 1 Supplement 1166.6 Mango Supplementation Prevents Gut Microbial Dysbiosis and Modulates Short Chain Fatty Acid Production Independent of Body Weight Reduction in C57BL/6 Mice Fed a High Fat Diet
Babajide Ojo1, Guadalupe ElRassi2, Penelope PerkinsVeazie3, Stephen Clarke1, Brenda J Smith1 and Edralin A Lucas1
+ Author Affiliations
Abstract
Fermentable nondigestible carbohydrates and fiber from plant food sources are suggested to prevent high fat (HF) dietinduced gut microbial dysbiosis and other obesityrelated outcomes. Various parts of mango have been studied for their antiobesogenic, immunomodulatory and gastroprotective properties. This study investigated the effects of 12week freezedried mango pulp supplementation on the gut microbiota and their fermentation products, and its impact on body composition, glucose homeostasis and gut inflammatory markers in C57BL/6 mice fed a HF diet. Sixweek old male C57BL/6 mice were randomly assigned to four dietary treatment groups: Control (AIN93M, 10% kcal from fat), HF (60% kcal from fat), and HF+1% or 10% mango. Cecal content analyses using 16S rDNA sequencing showed that HF feeding reduced Bifidobacteria and Akkermansia while mango supplementation prevented the loss of these populations in a dosedependent manner. In contrast to previous studies, mango supplementation did not reduce body weight or fasting blood glucose. Interestingly, both mango doses lowered HFinduced hypertriglyceridemia. The HF+10% mango significantly lowered plasma nonesterified fatty acids, but increased plasma total cholesterol. In comparison to the HF group, a dose dependent increase in microbial fermentation was observed with mango supplementation, as evident in increased fecal and cecal acetic and butyric acid, but not propionic acid. Furthermore, mango supplementation modulated gut inflammation, as observed with an increase in ileal and colonic interleukin (IL)10 gene expression compared to the HF group. These findings demonstrated that mango supplementation in high fat feeding modulated some of the adverse effects that accompanies high fat dietinduced obesity.
Support or Funding Information
This study was funded, in part, by the National Mango Board
Footnotes
This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
http://www.fasebj.org/content/30/1_Supplement/1166.6.short 1/2 8/30/2017 Mango Supplementation Prevents Gut Microbial Dysbiosis and Modulates Short Chain Fatty Acid Production Independent of Body Weight Reduction in C57B…
http://www.fasebj.org/content/30/1_Supplement/1166.6.short 2/2 8/30/2017 In vitro and in vivo evaluation of the prebiotic activity of water-soluble blueberry extracts | SpringerLink
In vitro and in vivo evaluation of the prebiotic activity of watersoluble blueberry extracts
World Journal of Microbiology and Biotechnology
July 2009, Volume 25, Issue 7, pp 1243–1249
Abdul Lateef Molan (1) Email author ([email protected]) Mary Ann Lila (2) John Mawson (1) Shampa De (1)
1. Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand 2. College of Agriculture, Consumer and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana-Champaign, USA
Original Paper
First Online: 12 March 2009
Received: 04 December 2008 Accepted: 24 February 2009
1 Shares 659 Downloads
Abstract
The prebiotic effects of water extracts of two blueberry (BBE) cultivars (‘Centurion’ and ‘Maru’) were studied using pure and mixed cultures of human faecal bacteria. The results demonstrated for the first time that addition of BBE from both cultivars to broth media containing pure cultures of Lactobacillusrhamnosus and Bifidobacteriumbreve resulted in a significant increase (P < 0.05–0.0001) in the population size of these strains. Batch fermentation system was used to monitor the effect of BBE addition on the mixed faecal bacterial populations (obtained from healthy human donors). Addition of BBE from both cultivars to batch cultures inoculated with mixed human faecal cultures resulted in a significant increase in the number of lactobacilli (P < 0.01–0.0001) and bifidobacteria (P < 0.05–0.0001). Furthermore, a significant influence on the population size of
https://link.springer.com/article/10.1007/s11274-009-0011-9 1/8 8/30/2017 In vitro and in vivo evaluation of the prebiotic activity of water-soluble blueberry extracts | SpringerLink
lactobacilli and bifidobacteria was observed after administration of extracts from both cultivars to rats daily for 6 days in comparison with the control group. In rats gavaged orally with 4 ml kg−1 day−1 of BBE for 6 days, the population size of lactobacilli (P < 0.05) and bifidobacteria (P < 0.05–0.01) was increased significantly. We hypothesize that BBE could modify the bacterial profile by increasing the numbers of beneficial bacteria and thereby improving gut health.
Keywords
Blueberry extract Prebiotic activity Lactobacilli Bifidobacteria
References
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Cite this article as: Molan, A.L., Lila, M.A., Mawson, J. et al. World J Microbiol Biotechnol (2009) 25: 1243. https://doi.org/10.1007/s11274-009-0011-9
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https://link.springer.com/article/10.1007/s11274-009-0011-9 8/8 Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 479505, 13 pages http://dx.doi.org/10.1155/2013/479505
Research Article Berry and Citrus Phenolic Compounds Inhibit Dipeptidyl Peptidase IV: Implications in Diabetes Management
Junfeng Fan,1 Michelle H. Johnson,2 Mary Ann Lila,3 Gad Yousef,3 and Elvira Gonzalez de Mejia2,4
1 College of Bioscience and Biotechnology, Beijing Forestry University, Beijing 100083, China 2 Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 3 Plants for Human Health Institute, NC Research Campus, North Carolina State University, Kannapolis, NC 28081, USA 4 Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Drive, Urbana, IL 61801, USA
Correspondence should be addressed to Elvira Gonzalez de Mejia; [email protected]
Received 30 March 2013; Revised 1 July 2013; Accepted 1 July 2013
Academic Editor: Mohd Roslan Sulaiman
Copyright © 2013 Junfeng Fan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Beneficial health effects of fruits and vegetables in the diet have been attributed to their high flavonoid content. Dipeptidyl peptidase IV (DPP-IV) is a serine aminopeptidase that is a novel target for type 2 diabetes therapy due to its incretin hormone regulatory effects. In this study, well-characterized anthocyanins (ANC) isolated from berry wine blends and twenty-seven other phenolic compounds commonly present in citrus, berry, grape, and soybean, were individually investigated for their inhibitory effects on DPP-IV by using a luminescence assay and computational modeling. ANC from blueberry-blackberry wine blends strongly inhibited DPP-IV activity (IC50, 0.07 ± 0.02 to >300 𝜇M). Of the twenty-seven phenolics tested, the most potent DPP-IV inhibitors were resveratrol (IC50, 0.6 ± 0.4 nM), luteolin (0.12 ± 0.01 𝜇M), apigenin (0.14 ± 0.02 𝜇M), and flavone (0.17 ± 0.01 𝜇M), with IC50 values lower than diprotin A (4.21 ± 2.01 𝜇M), a reference standard inhibitory compound. Analyses of computational modeling showed that resveratrol and flavone were competitive inhibitors which could dock directly into all three active sites of DPP-IV,while luteolin and apigenin docked in a noncompetitive manner. Hydrogen bonding was the main binding mode of all tested phenolic compounds with DPP-IV. These results indicate that flavonoids, particularly luteolin, apigenin, and flavone, and the stilbenoid resveratrol can act as naturally occurring DPP-IV inhibitors.
1. Introduction incretin hormones after secretion by cleavage of the penul- timate proline or alanine at N-terminus, and thus forms their Type 2 diabetes is characterized by excessive blood glucose inactive metabolites [5–7]. Both hormones have very short and insulin resistance due to an improper insulin response half-lives (approximately 2 min) due to the rapid degradation ofthebodytomanageglucosefromthediet[1]. Dipeptidyl by DPP-IV [8]. Among the several peptide substrates of DPP- peptidase IV (DPP-IV, EC 3.4.14.5), a serine peptidase, is IV, GLP-1 is one of the well-characterized physiological and one of the newest pharmaceutical targets for type 2 diabetes pharmacological substrates of the enzyme. GLP-1, which is treatment [1]. On the other hand, incretin-based therapy secreted in a nutrient-dependent manner, stimulates glucose- has several potential sites of action for the treatment of dependent insulin secretion and regulates glycemia. How- type 2 diabetes ranging from increasing insulin secretion, ever, the actions of GLP-1 do not last long due to degradation reducing glucagon secretion, and regulating glucose control by DPP-IV. For this reason, DPP-IV inhibition is expected [2]. It is well known that glucagon-like peptide-1 (GLP- to result in elevated plasma insulin levels by inhibiting the 1) and glucose-dependent insulinotropic polypeptide (GIP) degradation of active GLP-1 after oral glucose intake. This are major human incretin hormones that stimulate insulin in turn leads to the suppression of blood glucose elevation. release in a glucose-dependent manner in healthy individuals Therefore, development of DPP-IV inhibitors is being actively [3, 4]. However, DPP-IV rapidly transforms these two gut conducted worldwide, and control of blood glucose levels 2 Evidence-Based Complementary and Alternative Medicine by enhancement of GLP-1 action is a new option for the using Saccharomyces bayanus as previously described [17]. treatment of diabetes. After the fermentation, blends ranging from 100% blueberry In recent years, protein-ligand docking has become a to 100% blackberry were made using room temperature powerful tool for drug development, and is also a method fermented wines. Blends were prepared with different ratios to be able to identify binding modes with high accuracy. of % blueberry :%blackberry.Theratioswere100:0,75:25, For DPP-IV, computational docking analyses have been 25 : 75, and 0 : 100 of blueberry: blackberry wine blends, commonly used for designing inhibitors [9], screening of respectively. potential inhibitors [10], and explaining the differences in All solvents used for phenolic extraction were HPLC- activity of drugs with different structures11 [ ]. However, most grade and were purchased from Fisher Scientific (Pittsburg, of the previously investigated inhibitors of DPP-IV have been PA). Amberlite XAD-7 was purchased from Sigma-Aldrich synthetically derived. As for naturally occurring flavonoids, (St. Louis, MO). Sephadex LH-20 was purchased from the binding modes with DPP-IV are still not yet established. GE Life Sciences (Buckinghamshire, UK). Porcine kidney Phenolic compounds, such as flavonoids, widely abun- DPP-IV enzyme (88% sequence homology with human; dant in fruits and vegetables, have been suggested as impor- both are homodimers with a subunit molecular mass of tant compounds for diabetes reduction [9, 10]. However, ∼30 kDa) and diprotin A were purchased from Sigma- so far only a few phenolic compounds have been investi- Aldrich. DPP-IV GloTM Protease Assay kits were purchased gated to inhibit DPP-IV activity. These include procyanidin from Promega (Madison, WI). Flavonoids with high purities from grape seeds [12] and naringin from orange peel [13]. that were purchased from Sigma-Aldrich included luteolin Therefore, it is necessary to further elucidate the modulating (>98%), apigenin (>95%), quercetin (>98%), kaempferol effect on DPP-IV activity of phenolic compounds from other (>97%),rutinhydrate(>94%), naringenin (>95%), neo- natural sources. hesperidin (>90%), flavone> ( 97%), naringin (>90%), hes- In epidemiological studies, berries were the most impor- peridin (>80%), cyanidin-3-glucoside (>95%), cyanidin tant contributors to lowering risk for type 2 diabetes [14]. (>95%), malvidin (>95%), resveratrol (>99%), protocate- Additionally, an inverse relationship between intake of chuic acid (>97%), catechin (>98%), epicatechin (>90%), flavonoids, specifically those from berries, and risk of type 2 epigallocatechin gallate (EGCG, >95%), gallic acid (>97.5%), diabetes was found [15]. However, there is lack of evidence caffeic acid> ( 98%), and chlorogenic acid (>95%). Hesperetin for the role of specific phenolics in clinical trials, and there (>95%) was purchased from Sigma-Aldrich (Wicklow, Ire- is not yet sufficient data to confirm that anthocyanins have land) and limonin (>90%) from MP BioMedicals (Solon, a protective effect against the risk of type 2 diabetes16 [ ]. OH). Narirutin (>93.9%) and eriocitrin (>97.4%) were pur- Additionally, anthocyanins found in berries have been found chased from Chromadex (Irvine, CA). Genistein (>90%) and to have a beneficial effect on glucose metabolism; however, genistin (>90%) were kindly donated by Dr. Mark Berhow, stronger scientific evidence is needed. USDA. All other reagents were of analytical grade. Anthocyanins (ANC) from blueberry-blackberry wine blends have been evaluated for DPP-IV and carbohydrate- utilizing enzymes inhibitor studies in our laboratory, and they 2.2. Phenolic Extraction and Preparation of ANC Fractions. have exhibited potent DPP-IV and 𝛼-glucosidase inhibitory Phenolic extraction and preparation of ANC fractions were activities [17]. Thus, the aim of the present study was to conducted as previously described [17]. Briefly, each wine was further characterize the ANC-rich fractions from blueberry- firstly acidified, dealcoholized, and then mixed with amber- blackberry wine blends by HPLC and analyze their DPP-IV lite XAD-7 resin to remove sugars and phenolic acids. After inhibitory effect in vitro. Furthermore, a variety of other phe- nonpolar compounds were further removed from the crude nolic compounds commonly present in berries, citrus, and polyphenolics, the polar eluate was loaded onto a Sephadex other plant foods were studied for their DPP-IV inhibitory LH-20 column to generate ANC-enriched fractions. With an activity. We hypothesized that berry and citrus phenolics isocratic elution using water : methanol (80 : 20, containing couldbindtotheactivesitesofDPP-IV,thusinhibitingDPP- 0.1% TFA) and then 50% methanol, five anthocyanin-rich IV enzyme activity. For the most potent compounds, kinetic fractions (ANC 1–5) were obtained. ANC 2–5 from each andcomputationaldockinganalyseswereusedtoelucidate blend of blueberry and blackberry were analyzed by HPLC the binding modes with the DPP-IV enzyme. to determine their ANC composition.
2. Materials and Methods 2.3. Anthocyanin Analysis. ANC analyses were conducted as previously published [17] using a 1200 HPLC (Agilent 2.1. Materials. Wines were produced from highbush blue- Technologies, Santa Clara, CA) with a Supelcosil LC-18 RP berry (Vaccinium corymbosum)cultivarsBlueChip,Blue- column (250 × 4.6 mm, 5 𝜇M) (Supelco, Bellefonte, PA). ANC crop, Blue Haven, Blue Jay, Blueray, Bluetta, Collins, Coville, were detected at 520 nm using a diode array detector (DAD). Darrow, Earliblue, Elliot, Jersey, Late Blue, and Spartan and Specific anthocyanins were identified based on comparison blackberry (Rubus fruticosus) cultivars A-1937, A-2215, A- to our previously published data [18, 19]. A previously well- 2241 Natchez, A-2315, APF 27, APF 40, APF 41, and Prime characterized blueberry extract [19] was included with each Jan, collected from Dixon Springs Agricultural Center in sample run to verify compound separation and identification. Simpson, IL, USA during the ripening season of 2010. Blue- Using the peak areas as measured by HPLC at 520 nm, total berry wine and blackberry wine were separately fermented ANC were quantified from a standard curve generated from Evidence-Based Complementary and Alternative Medicine 3
0.125, 0.25, 0.5, and 1.0 mg/mL of cyanidin-3-glucoside (C3G) structure was prepared using Accelrys Discovery Studio 3.5 and ANC amounts are presented as C3G equivalents. program (Accelrys Software Inc., San Diego, CA). For the computational docking study, the energies of diprotin A 2.4. DPP-IV Inhibition. Measurement of the activity and and flavonoids were minimized by applying a CHARM22 potential inhibition of DPP-IV, a type II membrane glyco- force field, using the Accelrys Discovery Studio 3.5 program. protein, was done using the DPP-IV GloTM Protease Assay After removing water molecules and adding all the hydro- following the manufacture’s protocol (Promega, Madison, gen atoms, Gasteiger-Huckle¨ charges were assigned to the enzyme. The ligand conformers were treated as flexible and WI). Briefly, 50 𝜇L of DPP-IV GloTM reagent was added to a protein structures were treated as rigid during the docking white-walled 96-well plate containing 50 𝜇Lofblank,positive process. The docking was carried for 100 genetic algorithm control, or treatment. The blank contained the vehicle only runs, which was optimum to validate the crystal structure of while positive control contained the vehicle and purified the ligand. Most of the other genetic algorithm parameters DPP-IV enzyme (at a final concentration of 1 ng/mL). Treat- such as the population size were maintained at their default mentsusedwereenrichedANCfractions(0.5,5,20,and values. The best docking results were considered to be the 40 𝜇g/mL), phenolic compounds (0.5, 5, 20 and 40 𝜇g/mL) conformation having the lowest binding energy (Δ𝐺)using: orknowninhibitor,diprotinA(1,2,12,24,125,and250𝜇M), and the purified DPP-IV enzyme at a final concentration of Δ𝐺 = Δ𝐺 (intermolecular) +Δ𝐺 (internal) 1 ng/mL. The content of the wells was gently mixed using (1) an Ultra Microplate Reader (Biotek Instruments, Winooski, +Δ𝐺 (tor) −Δ𝐺 (unbound extended) , VT) at medium intensity for 4 s. DPP-IV cleavage of the Δ𝐺 provided Gly-Pro-amino methyl coumarin (AMC) substrate where (intermolecular) denotes the sum (kcal/mol) of Van der Waals energy, hydrogen bond energy, electrostatic generated a luminescent signal by luciferase reaction, with Δ𝐺 the amount of DPP-IV enzyme available to bind Gly-Pro- energy, and desolvation energy; (internal) is the final total internal energy (kcal/mol); Δ𝐺 (tor) denotes torsional AMC proportional to relative light units (RLU) produced. Δ𝐺 This signal in RLU was measured after 30 min in the Ultra free energy (kcal/mol); and (unbound extended) is the MicroplateReaderandthencomparedtotheblank.Diprotin unbound system’s energy (kcal/mol). 2 𝐾 Alinearstandardcurve(𝑦 = 41.936𝑥 +27.294,𝑅 = 0.91), In the context of Autodocking, inhibition constant ( 𝑖)is where 𝑦 was the % inhibitory activity of diprotin A and directly related to the binding energy: 𝑥 was the log10 of the concentration (𝜇M) of known inhibitor [Δ𝐺/(𝑅𝑇)] 𝐾𝑖 =𝑒 , (2) diprotin A, was used to calculate IC50 value: the concentration needed to decrease the activity of the enzyme by 50% of its where 𝑒 isthebasenumberofnaturallogarithm(approxi- original activity. IC50 values were calculated based on the mately equals 2.72), 𝑅 is the gas constant (kcal/mol), and 𝑇 is molecularmassofeachcompoundorC3Gastheequivalent the absolute temperature. Smaller 𝐾𝑖 and more negative Δ𝐺 for ANC-enriched fractions. mean tighter binding.
2.5. Inhibitory Kinetics Study. Porcine kidney DPP-IV activ- 2.7. Statistical Analyses. Data were expressed as means of ity was measured at various concentrations of three flavon- independent duplicates with at least three replicates. The oids (5 and 10 mg/mL for luteolin, apigenin, and flavone; and dose-response analysis of each compound on DPP-IV activity 0.25 and 0.5 mg/mL for resveratrol). Each concentration was was performed using nonlinear or linear regression (curve fit) evaluated in the presence of various concentrations of Gly- using EXCEL Microsoft (e.g., see Supplementary Information Pro-AMC (0–60 𝜇M). DPP-IV activity was measured using available online at http://dx.doi.org/10.1155/2013/479505). the DPP-IV Glo Protease Assay as mentioned above. The Statistical analysis was conducted using the proc GLM pro- inhibitionpatternwasevaluatedutilizingtheLineweaver- cedures of SAS version 9.3 (SAS Inst. Inc., Cary, NC, 2009). Burk plot. Enzyme-inhibition constant 𝐾𝑖 was determined by Group mean comparisons were conducted using Duncan plotting the reciprocal of the initial luminescence versus the means and were considered to be significant at 𝑃 < 0.05 reciprocal of the initial substrate concentration. based on the least significant differences (LSD) from one-way analysisofvariance(ANOVA)withalpha=0.05.Correlations 2.6. Molecular Modeling and Computational Docking Study. were made using Pearson’s correlation values with 𝑃 < 0.05. The DPP-IV enzyme exists as a dimer in the crystal form, and each monomer consists of 726 amino acids [20]. The 3. Results docking studies were conducted with the monomeric unit of the enzyme, as the active site of the enzyme resides 3.1. Blackberry Wine Presented High Concentrations of Del- deep within each monomer of the receptor protein and phinidin-3-arabinoside. Anthocyanin relative distributions not on the enzyme surface [21]. The molecular docking in the extracts of blueberry-blackberry wine blends are analysis of flavonoids was carried out using AUTODOCK shown in Table 1. Chromatographic analyses revealed up to 4.2 (CCDC, UK; http://www.ccdc.cam.ac.uk/products/csd/) seventeen ANC present in blueberry-blackberry wine blends. [22]. The crystal structure of the DPP-IV enzyme (Protein Malvidin-3-galactoside and cyanidin-3-glucoside were the Data Bank (PDB) ID: 2I03) was obtained from the pro- main ANC present in the blueberry wine, while delphinidin- tein data bank (http://www.rcsb.org/pdb), and the protein 3-arabinoside was the predominant ANC present in the 4 Evidence-Based Complementary and Alternative Medicine 7. 0 37.1 6.5 7.5 nd 30.6 nd 12.2 6.6 9.6 nd 12.2 7.2 100.9 1079.3 716.1 30.1 16.8 1244.7 794.1 35.8 17.8 29.7 9.3 10.2 12.2 nd nd 10.1 6.5 6.1 nd 24.5 9.2 44.7 150.2 32.7 14.5 6.4 38.4 nd 15.5 nd 6.7 6.3 15.9 nd 69.3 18.3 7.4 49.6 11.8 11.2 6.4 6.4 11.7 10.7 10.4 127.2 564.1 266.1 266.3 Anthocyanins (mg C3G equivalents/L) per blend (% Blueberry : % Blackberry) nd 9.0 51.0 7.7 nd 12.3 17.7 nd nd 8.8 nd nd nd nd nd nd nd nd 19.7 8.3 91.5 13.2 6.2 45.5 30.9 100 : 0 75 : 25 25 : 75 0 : 100 11.1 19.0 6.1 292.1 278.9 ∗ 63.1 74.8 ANC2 ANC3 ANC4 ANC5 ANC2 ANC3 ANC4 ANC5 ANC2 ANC3 ANC4 ANC5 ANC2 ANC3 ANC4 ANC5 Table 1: Anthocyanin (ANC) identification and quantificationHPLC by at maximum absorption of 520 nm. Total ANC 1653.8 2241.7 2907.5 3267.8 Sub-total ANC 190.5 1113.7 275.6 74.0 864.0 1112.3 183.8 81.6 1448.2 1073.3 239.2 146.8 1550.4 1204.8 302.8 229.8 Bold numbers indicate the dominant flavonoids in that particular ANC fraction. RT (min)24.4425.98 ANC Delphinidin-3-galactoside ID Delphinidin-3-glucoside 6.3 nd 10.8 26.8 10.0 nd 6.6 11.2 nd nd 6.9 7.0 nd nd nd nd nd nd 35.1937.14 Malvidin-3-glucoside Malvidin-3-arabinoside 5.9 39.0340.55 Delphinidin-6-acetyl-3-glucoside41.7042.25 nd Cyanidin-6-acetyl-3-glucoside44.40 Malvidin-6-acetyl-galactoside Petunidin-6-acetyl-3-glucoside nd 7.3 Malvidin-6-acetyl-3-glucoside nd nd 17.9 7.8 nd 26.7 6.7 nd 6.7 10.8 7.1 6.3 12.9 nd 10.5 nd 12.6 6.0 8.9 nd nd 11.4 nd nd 11.5 13.6 6.4 11.9 6.6 19.4 nd nd nd 8.4 nd nd nd nd nd 6.1 7.7 nd nd nd nd nd 6.8 nd 8.9 7.3 6.3 nd nd 8.1 6.9 nd 7.2 nd 6.4 43.9 nd nd nd nd nd nd 6.2 nd nd 14.2 7.2 9.3 6.9 7.1 6.6 5.9 nd 27.3328.86 Cyanidin-3-galactoside Delphinidin-3-arabinoside nd 6.1 11.9 6.5 nd nd nd 6.3 nd nd 17.7 nd nd 6.5 23.8 nd 6.9 7.0 29.69 Cyanidin-3-glucoside 6.4 23.4 nd nd 60.6 30.8231.62 Cyanidin-3-arabinoside Petunidin-3-glucoside nd 9.7 140.2 7.4 7.3 nd 24.9 10.5 11.0 56.1 16.7 79.6 8.0 13.3 7.2 6.7 nd 20.2 10.0 nd 116.8 nd 10.8 7.0 32.6933.9534.39 Peonidin-3-glucoside Petunidin-3-arabinoside Malvidin-3-galactoside 6.0 5.9 178.5 39.8 22.5 nd nd nd nd 8.3 34.5 6.7 9.1 nd nd 8.0 14.3 8.4 8.3 nd 10.4 13.9 6.5 7.0 9.7 10.1 nd 7.1 9.7 18.8 9.9 8.0 ∗ nd: peak not detected. Evidence-Based Complementary and Alternative Medicine 5 blackberry wine. Total ANC ranged from 1653.8 mg C3G Table 2: Anthocyanin (ANC) concentration (𝜇M) from blueberry- equivalents/L for blueberry wine to 3267.8 mg C3G equiva- blackberry wine blends needed to inhibit DPP-IV enzyme activity 1,2 lents/L for blackberry wine. It was also observed that there by 50% . was an obvious difference between ANC amounts of different Blend ratio Fraction IC (𝜇M) fractions generated as ANC 2–5. (% blueberry : % blackberry) 50 > 3.2. Anthocyanins from Blackberry Wine Potently Inhibited ANC1 300 DPP-IV. ANC-enriched fractions (ANC 1–5) isolated from ANC2 4.67 ± 0.63a blueberry-blackberry wine blends were analyzed for their 100% Blueberry ANC3 0.64 ± 0.33bc DPP-IV inhibitory effect. Table 2 shows the IC50 values ANC4 1.37 ± 0.58abc of ANC from blueberry-blackberry wine blends needed to ANC5 0.72 ± 0.25bc inhibit DPP-IV enzyme. Compared to a standard curve ANC1 NA3 of diprotin A (IC50, 4.21 ± 2.01 𝜇M), a known DPP-IV ± ab inhibitor with an Ile-Pro-Ile sequence, ANC 2–5 tested at ANC2 2.02 0.56 concentrations of 0.5, 5, 20, and 40 𝜇MinC3Gequivalents 75% : 25% ANC3 0.41 ± 0.11c c obtained from each blend had IC50 values ranging from ANC4 0.22 ± 0.05 2.64 ± 1.40 𝜇 0.07 ± c MinANC2fromblueberrywineto ANC5 0.36 ± 0.16 0.02 𝜇 MinANC3fromblackberrywine(Table 2). Table 2 ANC1 NA also shows that ANC from blackberry wine were the most ± c effective of the blends at reducing the activity of DPP-IV (with ANC2 0.34 0.10 25% : 75% ± c IC50 values of no more than 0.22 𝜇MC3G). ANC3 0.33 0.08 ANC4 0.52 ± 0.18c 3.3. Resveratrol, a Stilbenoid, Luteolin, Apigenin, and Flavone, ANC5 0.20 ± 0.10c Flavonoids Commonly Present in Fruits, Have Strong DPP- ANC1 NA IV Inhibitory Activity. Twenty-seven phenolic compounds ANC2 0.22 ± 0.03c commonly present in citrus, berries, grape, soybeans, and c 100% Blackberry ANC3 0.07 ± 0.02 other plants were tested for DPP-IV inhibitory effect ± c (Table 3). Sixteen phenolic compounds demonstrated DPP- ANC4 0.18 0.07 ± c IV inhibitory activity with IC50 values ranging from 0.6 ± ANC5 0.20 0.09 0.4 10.36 ± 0.09 𝜇 1 nM (resveratrol) to M (eriocitrin). Eleven IC50 values were determined from at least two independent duplicates done compounds did not have DPP-IV inhibitory activity includ- in triplicate and calculated in C3G equivalents. Values are means ± SEM. ing rutin, narirutin, naringin, hesperidin, limonin, neohes- Means with different letters are significantly different (𝑃 < 0.05). 2The positive control of inhibition for DPP-IV was diprotin A (Ile-Pro-Ile) peridin, genistin, catechin, epicatechin, chlorogenic acid, and ± 𝜇 with an IC50 value of 4.21 2.01 M. protocatechuic acid (data not shown). 3NA: No activity detected at >300 𝜇M. Of the sixteen effective phenolic compounds, three had IC50 values higher than diprotin A (4.21±2.01 𝜇M) including eriocitrin (IC50 value of 10.36 ± 0.09 𝜇M), EGCG (10.21 ± 0.75 𝜇M), and gallic acid (4.65 ± 0.1 𝜇M). However, IC50 theenzyme,wetestedtheenzymekinetics.Theinhibitory values of the other thirteen compounds were lower than that manner of the flavonoids was determined through generating of diprotin A, indicating that less of these compounds was aLineweaver-Burkplot(Figure 1). As noted in Figures 1(a) 𝑥 needed to inhibit DPP-IV. These thirteen phenolics could be and 1(d), both the slope and the -intercept were changed divided into three categories according to the results of statis- by the addition of inhibitors, but there was no effect on 𝑦 tical differences on their DPP-IV inhibitory effect: less active the -intercept. This is the definition of linear competitive with high IC50 values (1.31–3.37 𝜇M), intermediate activity inhibition. Therefore, resveratrolFigure ( 1(a)) and flavone with IC50 values of 0.24–0.74 𝜇M,andveryhighactivitywith (Figure 1(d)) inhibited DPP-IV activity in a competitive 𝐾 0.2 ± 0.01 𝜇 low IC50 values (0.0006–0.17 𝜇M). The phenolic compounds manner. The 𝑖 values were calculated to be M 18.6 ± 0.3 𝜇 with high IC50 values were cyanidin, quercetin, and caffeic for resveratrol and M for flavone. As for luteolin 𝑦 acid; the ones with intermediate activity were naringenin, andapigenin(Figures1(b) and 1(c)), both the slope and the - hesperetin, cyanidin-3-glucoside, kaempferol, and malvidin. intercept were changed by the added inhibitors, but there was 𝑥 The four phenolics with very high activity included resvera- no effect on the -intercept. Therefore, luteolin and apigenin 𝐾 trol,luteolin,apigenin,andflavone.IC50 value of resveratrol noncompetitively inhibited the enzyme, with 𝑖 values at 4.9 ± 0.2 𝜇 7.9 ± 1.4 𝜇 had the highest DPP-IV inhibitory activity among all of the Mand M, respectively. compounds tested (𝑃 < 0.05). 3.5. Diprotin A and Natural Phenolic Compounds Inhibit DPP- 3.4. Resveratrol and Flavone Inhibited DPP-IV Activity in a IVActivitybyBindingTightlyintotheActiveSiteofthe Competitive Manner, While Luteolin and Apigenin Inhibited Enzyme. Binding pose of diprotin A, resveratrol and flavone Noncompetitively. To examine whether the most potent phe- in the DPP-IV active site is indicated in Figures 2 and 3, nolic compounds, resveratrol, luteolin, apigenin and flavone, showing that these three compounds interact closely with key inhibited DPP-IV through interaction with the active site of residues of sites S1, S2 and S3 within the active pocket. 6 Evidence-Based Complementary and Alternative Medicine
1 𝐾 2 Table 3: DPP-IV inhibition by flavonoids (IC50), their number of hydroxyl groups (OH), binding energy, inhibition constant ( 𝑖) ,Hbonds involved, and 𝜋 interactions.
Number of Binding energy 3 Flavonoids IC (𝜇M) 𝐾𝑖 (𝜇M) H Bonds 𝜋 interactions 50 OH groups (kcal/mol) TYR547:HH-UNK:O22 UNK:H28-TYR666:OH Positive control 𝜋-cation 4.21 ± 2.01bc 0 −7.31 4.42 UNK:H11-GLU206:OE1 Diprotin A TYR666-UNK:N13 UNK:H11-GLU206:OE2 UNK:H12-GLU205:OE2 𝜋-𝜋 TRP563:HN-UNK:O15 UNK-B:TRP629 Berry flavonoids ALA564:HN-UNK:O18 1.41 ± 0.25e 5 −5.95 43.43 UNK-B:TRP629 Cyanidin UNK:H7-TYR48:OH UNK-B:TRP629 UNK:H11-GLY741:O UNK-B:TRP629 PHE357:HN-UNK:O20 ARG358:HH22-UNK:O17 ARG358:HE-UNK:O27 𝜋-cation GLU361:HN-UNK:O9 UNK-HIS126:NE2 Cyanidin-3-glucoside 0.42 ± 0.09ef 8 −6.35 22.33 ARG669:HH21-UNK:O31 UNK-ARG358:NH1 UNK:H1-PHE208:O UNK-ARG358:NH2 UNK:H6-GLU361:OE1 UNK:ARG358:NH1 UNK:H20-GLU206:OE1 UNK:H20-UNK:O26 𝜋-𝜋 ARG356:HH11-UNK:O13 UNK-PHE357 ARG356:HH11-UNK:O17 UNK-PHE357 ARG358:HE-UNK:O11 Malvidin 1.41 ± 0.44ef 3 −6.36 21.64 𝜋-cation ARG585:HH-UNK:O14 UNK-ARG669:NH2 UNK:H6-ILE405:O UNK-ARG669:NH1 UNK:H14-GLU206:O UNK-ARG669:NH2 ARG358:HE-UNK:O10 GLU361:NH-UNK:O1 𝜋-cation Citrus flavonoids UNK:H1-GLU361:OE1 UNK-ARG669:NH2 0.12 ± 0.01f 4 −6.26 25.83 Luteolin UNK:H6-GLU205:O 𝜋-sigma UNK:H7-SER209:OG UNK-PHE357:CB UNK:H9-UNK:O7 ARG356:HH11-UNK:O10 ARG358:HE-UNK:O5 GLU361:HN-UNK:O14 𝜋-cation Apigenin 0.14 ± 0.02f 3 −6.14 31.77 UNK:H4-UNK:O8 UNK-ARG669:NH2 UNK:H5-GLU205:O UNK:H10-GLU205:O UNK:H10-SER209:OG ARG356:HN-UNK:O5 𝜋-cation ARG356:HN-UNK:O3 unk-ARG358:NH2 UNK:H2-UNK:O4 Quercetin 2.92 ± 0.68d 5 −6.33 23.03 UNK-ARG358:NH2 UNK:H3-ARG358:O UNK-ARG669:NH1 UNK:H4-GLU206:O UNK-ARG669:NH2 UNK:H5-SER209:OG SER209:HG-UNK:O21 ARG356:HN-UNK:O8 𝜋-cation PHE357:HN-UNK:O13 UNK-ARG356:NH1 ARG358:HN-UNK:O13 Kaempferol 0.49 ± 0.02ef 4 −6.62 13.99 UNK-ARG358:NH1 UNK:H3-UNK:O8 UNK-ARG358:NH2 UNK:H4-ARG358:O UNK:H5-GLU361:OE2 UNK:H10-SER209:OG Flavone 0.17 ± 0.01f 0 −6.64 13.57 No hydrogen bonds No 𝜋 interactions Evidence-Based Complementary and Alternative Medicine 7
Table 3: Continued.
Number of Binding energy 3 Flavonoids IC (𝜇M) 𝐾𝑖 (𝜇M) H Bonds 𝜋 interactions 50 OH groups (kcal/mol) ARG358:HH22-UNK:O2 ARG669:HH21-UNK:O5 𝜋-cation Hesperetin 0.28 ± 0.07ef 3 −6.85 9.57 UNK:H2-GLU206:OE1 UNK-ARG358:NH1 UNK:H1-UNK:O2 UNK:H3-ARG358:O ARG356:HH11-UNK:O20 ARG358:HE-UNK:O10 𝜋-cation GLU361:HN-UNK:O19 Naringenin 0.24 ± 0.03ef 3 −6.83 9.90 UNK-arg358:NH1 UNK:H11-GLU361:OE1 UNK-ARG358:NH2 UNK:H12-UNK:O11 UNK:H10-SER209:OG ARG356:HH11-UNK:O8 PHE357:HN-UNK:O10 ARG358:HN-UNK:O10 ARG358:HE-UNK:O14 ARG429:HH22-UNK:O23 ARG669:HH21-UNK:O30 𝜋-cation Eriocitrin 10.36 ± 0.09a 9 −9.07 225.96 UNK:H5-ARG358:O UNK-ARG356:NH1 UNK:H12-TYR585:OH UNK:H28-TYR585:OH UNK:H32-CYS551:O UNK:H20-GLU206:O UNK:H15-GLU206:O ARG356:HN-UNK:O12 𝜋-𝜋 PHE357:HN-UNK:O10 PHE357-UNK ARG358:HN-UNK:O10 𝜋-cation Soy isoflavone 0.48 ± 0.04ef 3 −6.5 17.31 UNK:H4-ARG358:O UNK-ARG356:NH1 Genistein UNK:H4-UNK:O10 UNK-ARG358:NH1 UNK:H5-GLU36:OE1 UNK-ARG358:NH1 UNK:H10-GLU206:O UNK-ARG669:NH1 ARG669:HH21-UNK:O7 Grape stilbenoid UNK:H12-SER630:OG 0.0006 ± 0.0004g 3 −6.54 15.96 Resveratrol UNK:H5-SER209:OG UNK:H4-GLU206:O GLN553:HN-UNK:O32 𝜋-𝜋 UNK:H8-GLU206:OE1 PHE357-UNK Other flavonoids 10.21 ± 0.75a 8 −4.39 604.91 UNK:H11-TYR666:OH PHE357-UNK EGCG UNK:H15-TYR662:OH 𝜋-cation UNK:H15-TYR585:OH UNK-HIS740:NE2 ARG356:HH11-UNK:O11 PHE357:HN-UNK:O7 ARG358:HN-UNK:O7 𝜋-cation Gallic acid 4.65 ± 0.99b 3 −3.96 1.25 TRY585:HH-UNK:O10 UNK-ARG356:NH2 UNK:H3-ARG358:O UNK:H5-ILE405:O UNK:H6- ILE405:O ARG358:HE-UNK:O13 ARG358:HH22-UNK:O12 𝜋-𝜋 Caffeic acid 3.37 ± 0.14cd 3 −5.23 147.66 ARG669:HH21-UNK:O9 PHE357-UNK UNK1:H6-GLU206:OE1 UNK:H7-GLU206:OE1 1 𝜇 IC50 values were determined from at least two independent duplicates done in triplicate for each of the concentrations tested. Concentrations ( M) were calculated based on the molecular mass of each pure compound. Values are means ± SEM. Means with different letters in each column are significantly different for DPP-IV (𝑃 < 0.05). 2 𝐾𝑖 values were obtained from computational docking as indicated in Materials and Methods section. 3UNK refers to phenolic compound or diprotin A. 8 Evidence-Based Complementary and Alternative Medicine
4 7 6
3 5
4 2 3 (1/luminescence) (1/luminescence)
V 2 V 1 Ki = 0.2 ± 0.01 𝜇M 1/ 1/
1 Ki = 4.9 ± 0.2 𝜇M 0 0 −0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 −0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 1/S (1/𝜇M) 1/S (1/𝜇M) 2 2 Resveratrol = 0.44 𝜇M R = 0.999 Luteolin =6.99𝜇M R = 0.999 2 2 Resveratrol = 0.88 𝜇M R = 0.997 Luteolin =13.97𝜇M R = 0.999 2 2 Control R = 0.995 Control R = 0.995 (a) (b) 12 4
10 3 8
6 2 (1/luminescence) (1/luminescence) 4 V V K = 7.9 ± 1.4 𝜇 i M 1 K = 18.6 ± 0.3 𝜇 1/ 1/ i M 2
0 0 −0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 −0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 1/S (1/𝜇M) 1/S (1/𝜇M) 2 2 Apigenin =7.40𝜇M R = 0.999 Flavone = 9.0 𝜇M R = 0.999 2 2 Apigenin = 14.80 𝜇M R = 0.998 Flavone = 18.0 𝜇M R = 0.999 2 2 Control R = 0.995 Control R = 0.995 (c) (d)
Figure 1: Inhibition kinetics of porcine dipeptidyl peptidase-IV (DPP-IV) by resveratrol (a), luteolin (b), apigenin (c), and flavone (d). Different concentrations of the flavonoids (0, 5, and 10 𝜇g/mL for luteolin, apigenin, and flavone and 0, 0.25, and 0.5 𝜇g/mL for resveratrol) were incubated in the presence of various concentrations of Gly-Pro-AMC (0–60 𝜇M) as substrate. Initial rates of the reaction were measured, and the results are expressed as a Lineweaver-Burk plot. Data are expressed as the mean of four independent experiments.
Diprotin A is a potent DPP-IV inhibitor with Ile-Pro-Ile the reported results obtained from X-ray crystal structure sequence commonly used as a reference compound. Figure 2 complex of DPP-IV and diprotin A [20]. shows the binding mode of diprotin A with DPP-IV. The The overlay of binding poses of resveratrol (green) and binding site of diprotin A is located at the S1, S2 and S3 flavone (yellow) in the DPP-IV active site is shown in sites (Figure 2(A1)). In the S2 site (Figure 2(A2)), the N- Figure 3(A). As observed in Figure 3(A1), resveratrol and terminal amino group of diprotin A is hydrogen-bonded flavone dock very well into all three active sites S1, S2, and to the carboxyl oxygens of two Glu residues (Glu205 and S3 of DPP-IV. Resveratrol showed hydrogen bonding of 4 - Glu206). Furthermore, the N-terminal amino group forms a 𝜋 interaction to the Tyr666. The carbonyl oxygen of OH-, 3 -OH-, and 5 -OH-group with hydroxyl of side chain of Ser630 (S1 pocket) and Ser209 (S3 pocket). Hydrogen Ile-1 of diprotin A forms an electrostatic interaction with Tyr662, Arg125, and Asn710 residues. Pro-2 of diprotin A is bonds were also seen between 5 -OH of resveratrol, NH2- located in the S1 site and forms a hydrophobic interaction group of side chain of Arg 669 residues, and C=O groups of with the phenol rings of Tyr666, and Tyr547. The carbonyl side chains of Glu206 (S2 pocket) (Figure 3(B2)). At the same oxygen of Ile-3 of diprotin A also forms double hydrogen time, electrostatic interactions were also observed between bonds to Tyr547 and Tyr666. In the S3 site, Van der Waals resveratrol and S1 pocket (His740, Tyr631, Ser630, His125), S2 interactions are also seen between diprotin A and Ser209 and pocket (Glu205, Glu206), S3 pocket (Ser209), and Arg669 of Phe357 residues of DPP-IV. These observations agree with DPP-IV. Evidence-Based Complementary and Alternative Medicine 9
TYR B: 631 HIS B: 740
SER B: 630 TYR SER B: 666 B: 552 TYR Pi Diprotin A B: 662 TYR B: 547 SLY B: 549 H O GLU + CYS B: 206 O B: 551 O H S3 N H N GLN PRO B: 553 N B: 550 GLU B: 205 O S1 PHE S2 Diprotin A B: 357 ASN B: 710
SER B: 209
(A 1)(A 2)
Figure 2: Key interactions of diprotin A (A1,A2) with active sites of DPP-IV enzyme. Binding of diprotin A (A1, grey) in the DPP-IV active site is indicated (surface view: blue), wherein it interacts closely with key residues of active sites S1, S2, and S3. Residues with pink circles indicate hydrogen bond, or ionic or polar interactions; residues with green circles indicate Van der Waals interactions. The arrows indicate hydrogen bonds to side chain residues in blue and backbone residues in green.
ARG B: 125 VAL (A1) (A2) TYR (A3) B: 656 ARG TYR B: 666 B: 669 B: 547 GLU TYR B: 206 TYR B: 662 B: 666 SER B: 630 Flavone H VAL S3 GLU B: 656 O TYR B: 205 O ASN B: 631 H B: 710 O GLU ARG B: 205 B: 669 TYR B: 631 PHE VAL B: 357 B: 711 PHE B: 357 O ASN SER B: 710 GLU B: 630 H O B: 206 TYR S1 Resveratrol VAL B: 662 S2 HIS B: 711 B: 740 Resveratrol: green SER B: 209 HIS HIS B: 126 ARG Flavone: yellow B: 125 B: 740
TYR ARG B: 585 B: 356 (B1)(B2)(BPHE 3) B: 357 PHE Sigma B: 357
O ARG H Pi GLU B: 356 B: 205 Apigenin PRO O O O B: 359 VAL O B: 207 Pi H O GLU GLU + B: 206 B: 206 S1 ARG SER O H GLU H ARG B: 361 + B: 669B: 360 SER O B: 669 Pi S3 H O ARG B: 209 B: 358 S2 H ARG O GLU Luteolin B: 358 GLU B: 361 B: 205 Luteolin: green VAL B: 207 SER SER B: 360 B: 209 Apigenin: yellow PHE B: 208 GLU B: 204
GLU B: 669 VAL PHE ARG B: 207 B: 208 B: 669 (C1) + GLU (C3) Quercetin: green (C2) B: 205 + SER B: 209 GLU GLU B: 206 B: 205 HIS B: 363 Genistein: yellow H Genistein O O H Pi Pi PHE SER B: 357 B: 209 H Pi + GLU O ARG B: 206 Pi H B: 358 + Pi O O VAL GLU Pi Pi Pi Pi B: 361 O B: 207 O H PHE O B: 357 + H O SER + B: 360 O + SER S3 B: 360 ARG O H Quercetin ARG GLY B: 358 ARG B: 355 B: 356 PRO B: 356 S1 B: 359
S2 TYR B: 585 GLY ILE B: 355 B: 405
Figure 3: Key interactions of resveratrol (A1,A2), flavone 1(A ,A3), luteolin (B1,B2), apigenin (B1,B3), quercetin (C1,C2), and genistein (C1,C3) with active sites of DPP-IV enzyme. Binding pose of resveratrol (A1, green) and flavone (A2, yellow) in the DPP-IV active site is indicated (surface view: blue), wherein two compounds interact closely with key residues of active sites S1, S2, and S3. Binding pose of luteolin (B1, green), apigenin (B1,yellow),quercetin(C1, green) and genistein (C1, yellow) in the DPP-IV binding site is indicated, wherein these flavonoids interact closely with the key residues of sites S2, and S3. Residues with pink circles indicate hydrogen-bond, or ionic orpolar interactions, residues with green circles indicate Van der Waals interactions. The arrows indicate hydrogen bonds to side chain residues in blue and backbone residues in green. 10 Evidence-Based Complementary and Alternative Medicine
No hydrogen bonds were seen between flavone and correlation existed between 𝐾𝑖 values and IC50 values (𝑟= amino acids in the pockets of DPP-IV (Figure 3(A3)). How- 0.82, 𝑃 = 0.0002). Significant correlations were also found ever, electrostatic interactions between flavone and amino between 𝐾𝑖 values and binding energies (𝑟 = 0.56, 𝑃 < 0.05), acid residues in S1 pocket (Tyr547, Ser630, Asn710, and and between 𝐾𝑖 valuesandnumberofhydroxylgroup(𝑟= His740), S2 pocket (Arg125, Glu205), and Van der Waals 0.56, 𝑃 < 0.05). interactions between flavone and amino acid residues in S1 pocket (Tyr631, Val656, Tyr666, Val711), S2 pocket (Glu206) 4. Discussion and Conclusions andS3pocket(Phe357),allowedflavonetoanchorinthe active sites of DPP-IV. This study showed that ANC from berry wine and a variety The overlay of binding poses of luteolin (green) and of other phenolic compounds commonly present in fruits apigenin (yellow) in the DPP-IV active site is also shown in and vegetables had strong DPP-IV inhibitory effect in vitro Figure 3(B). As shown in Figure 3(B1), luteolin and apigenin and in silico. Computational docking analyses also showed had almost identical binding modes with the active sites of for the first time that these natural phenolics could inhibit DPP-IV with each having ring B and C docked into sites S2 DPP-IV activity by binding tightly into the active sites of and S3. Three common features of binding with DPP-IV exist the enzyme. The biological activities, stability, and bioavail- between both flavonoids (Figures 3(B2)and3(B3)). Firstly, ability of anthocyanins depend on their chemical structures. hydrogen bonds and 𝜋-interactions played important roles in Blends were created to generate a mixture of potentially docking both the flavonoids into the active pockets S2 and S3 bioactive compounds commonly present in both blueberries of DPP-IV enzyme. In the S2 pocket, the B ring 5 -hydroxyl and blackberries after fermentation, which can be optimized of luteolin formed a hydrogen bond with hydroxyl group based on the characterization and potential benefit. of side chain of Ser209 (S3 pocket), while the 4 -hydroxyl Previous studies on wine compounds and biological group on B ring of apigenin forms a similar hydrogen bond activity indicated that it is not the presence of a single com- within the S3 pocket. Luteolin also showed H-bonding by B pound that is responsible for beneficial effects such as antiox- ring 4 -hydroxyl with C=O groups of side chains of Glu205 idant capacity or ability to reduce inflammation, but rather (S2 pocket). Secondly, in the S3 pocket, both compounds involves several phenolic compounds. Major contributions formed a hydrogen bond of the C ring 1 -oxygen with the are from compounds such as transresveratrol as well as minor NH of Arg358’s guanidine side chain. H-bonding of the A contributions from cinnamic and hydroxycinnamic acids, ring 8 -hydroxyl with C=O groups of side chains of Glu361 cyanidin, and some phenolic acids [23]. The combination favors strong binding of both flavonoids to the DPP-IV active of these phenolic compounds within the blends produced site. A third common feature of both flavonoids was shown from fermented blueberry and blackberry provided a unique by 𝜋-cation interactions of ring B and the NH2 of Arg669. potential for inhibition of DPP-IV. Therefore, while the Additional features of each flavonoid added to their unique inhibitory effects demonstrated by the anthocyanin-enriched docking within the active site of DPP-IV. A hydrogen bond blends are primarily due to the major anthocyanin compo- between the A ring 5 -hydroxyl of apigenin and the NH of nents, the presence of other compounds also influenced the Arg361’s guanidine side chain also enhanced the docking of demonstrated potency. apigenin and DPP-IV.For luteolin, a 𝜋-sigma interaction was In general, anthocyanins may protect beta-cells, increase also seen between the side chain of Phe357 and C ring of the secretion of insulin, reduce the digestion of sugars in the luteolin (S3 pocket). small intestine, and thereby have multiple and simultaneous Quercetin and genistein had a comparable binding posi- antidiabetic effects. Inhibitors of DPP-IV have been found to tion to luteolin and apigenin (Figure 3(c)). Hydroxyl groups prevent pancreatic beta cell destruction in mice [24]. Extracts in A and B rings were also important for quercetin and enriched in flavonoids have been seen to inhibit plasma DPP- genisteintobindintotheS2andS3sites(Figures3(C2) IV [25]. and 3(C3)).Atthesametime,𝜋-interactions between these The primary anthocyanin in the blackberry blends was flavonoids and Arg358 and Arg669 also contributed to delphinidin, which has previously shown potency to inhibit the tethering of the two flavonoids to the active sites. All enzymatic activity of a glyoxalase I, which is being investi- hydrogen bonds formed between phenolic compounds and gatedasatargetforpreventionofcancer.Comparedtoother DPP-IV are indicated in Table 3. anthocyanins found in berries, (cyanidin and pelargonidin), The binding energies obtained by computational dock- delphinidin had the most potent DPP-IV inhibitory effect, ing analyses were compared among the compounds tested suggesting the importance of interactions of the hydroxy (Table 3). Gallic acid had the highest binding energy groups on the B ring of anthocyanins. Further, binding modes (−3.96 kcal/mol), while diprotin A had the lowest binding indicated that the hydroxyl groups located at the R1 position energy (−7. 31 k c a l / m o l ) . IC 50 values of the phenolic com- greatly contribute to inhibitory potency and specificity to the pounds that were found to inhibit DPP-IV activity correlated binding site [26]. This previous study, along with the results with their binding energies (𝑟 = 0.67, 𝑃 < 0.05). Both a from our research, indicates that the anthocyanin delphinidin lower IC50 value and lower binding energy indicate stronger can form several hydrogen bonds to several amino acids due inhibitory potency. The inhibition constant (𝐾𝑖)obtainedby to its hydroxyl groups at R1 position. computational docking analyses is also shown in Table 3.The Our previous study also showed that the blueberry- 𝐾𝑖 values of these phenolic compounds varied from 1.25 𝜇M blackberry wine contained high amounts of total antho- forgallicacidto604.91𝜇MforEGCG.Ahighlysignificant cyanin [17]. However, correlation was not seen between Evidence-Based Complementary and Alternative Medicine 11
DPP-IV inhibitory effect and anthocyanin concentration in chains of catalytic triad (Ser630, Asn710, and His740), which ANC fractions (P > 0.05) from berry wines. For example, are involved in strong hydrophobic interactions [10]. The ANC3, ANC4, and ANC5 from blackberry wine were of cavity near Glu205, Glu206 and Tyr662 residues is referred similar IC50 values to inhibit DPP-IV, while the anthocyanin to as the S2 pocket. The S3 pocket of DPP-IV consists of concentration was almost 4 times higher in ANC3 than in Ser209, Arg358, and Phe357 [21]. The outside position of ANC4 and ANC5. Additionally, ANC4 and ANC5 had the theS3pocketinDPP-IVallowslargergroupsaccesstothe same IC50 values and ANC concentration, but their antho- site; on the other hand, the inside position of the S3 pocket cyanin compositions differed. These results indicate that favors smaller groups [28]. The four most potent compounds, delphinidin-3-arabinoside, as the major anthocyanin iden- resveratrol, luteolin, apigenin and flavone, had low 𝐾𝑖 values tified in blackberry blends, could contribute to the DPPIV to inhibit DPP-IV,which indicated that they had high affinity inhibition, however, other ANC may also play an important to the active sites of DPP-IV. The kinetic analysis showed role in DPP-IV inhibition. Therefore, DPP-IV inhibitory that resveratrol and flavone inhibited DPP-IV activity in a effect of ANC could depend on not only the concentration competitive manner, while luteolin and apigenin were in but the composition and structures of flavonoids present. a noncompetitive manner. Further computational docking More research should be conducted to clarify the relationship analyses are consistent with the tested inhibitory manner between DPP-IV inhibitory effect and anthocyanin structure of the phenolic compounds. Docking analysis showed that of ANC from berries. resveratrol and flavone bound well into all the three sites S1, S2 Phenolic compounds are widely recognized for their andS3ofDPP-IV,whileluteolinandapigenincouldonlybind ability to improve diabetic conditions by decreasing blood into S2 and S3 pockets. Although luteolin and apigenin could glucose levels [27].ItisinterestingthatmostoftheANC dock into S2 and S3 pockets, the kinetic analysis showed fractions showed potent DPP-IV inhibitory activity, with the that they inhibited DPP-IV in a noncompetitive manner. We lowest IC50 value from blackberry wine. Grape seed-derived presume that the binding of luteolin and apigenin into S2 and procyanidins (GSPE) were also able to inhibit recombinant S3 may lead to DPP-IV conformational changes, or changes human DPP-IV activity, achieving around 70% inhibition at in the side chain of amino acid residues of DPP-IV, and the 200 mg/L of GSPE [12]. In order to compare with the GSPE, catalytic activity will be decreased when the substrate is also thepercentageinhibitionwasgiveninthisstudyasIC50 bound. values. The concentrations of all ANC from blackberry wine We found that apigenin had a similar effect as resveratrol forachievingthesameinhibitoryeffectonDPP-IVwereless to directly inhibit DPP-IV activity, and genistein also exhib- than 200 mg/L. Especially for ANC3 from blackberry wine, ited a potent DPP-IV inhibitory effect. In the present study, the concentration of 41.9 mg/L could lead to around 70% most of the glycosylated flavonoids with two sugar groups, inhibition of DPP-IV activity. These results suggest that ANC including naringin, rutin, narirutin, hesperidin, and neohes- from blueberry and blackberry wine have strong DPP-IV peridin, had no DPP-IV inhibitory effect. One explanation inhibitory activity. The efficacy of the ANC to inhibit DPP-IV is that conjugation of bulky sugar groups to the flavonoid enzyme activity at a rate comparable to diprotin A and GSPE core structure could sterically hinder binding to the active indicated that ANC may be able to act as naturally occurring sites within DPP-IV, thus resulting in no inhibitory capacity DPP-IV inhibitors. of the tested flavonoids. The computational docking analyses Many kinds of natural flavonoids exist in plants but only further supported this phenomenon. However, cyanidin-3- afewhavebeenreportedforDPP-IVinhibitoryeffect[12, 13]. glucoside, which has been identified as the major ANC In the present study of twenty-seven phenolic compounds in different blackberry species29 [ ], showed no statistical commonly present in berries, citrus, soybeans, and other difference (𝑃 > 0.05) on DPP-IV inhibitory activity (IC50, plant commodities, most flavonoids were determined to have 0.42 ± 0.09 𝜇M) than cyanidin (IC50, 1.31 ± 0.34)and DPP-IV inhibitory effect. It is interesting that most of the malvidin (IC50, 0.74 ± 0.16). Considering ANC-enriched flavonoids tested in the present study showed lower50 IC fractions from blueberry and blackberry wines contain a values and therefore were more potent than the reference mixture of flavonoids with only one sugar group, flavonoids inhibitor standard diprotin A. Resveratrol, luteolin, apigenin with monosugar groups may have better DPP-IV inhibitory and flavone showed the most potent DPP-IV inhibitory activ- effects than flavonoids with more sugar groups due to less ity due to their lowest IC50 values. In particular, this study steric hindrance. demonstrated that resveratrol was the most potent DPP-IV Flavone, luteolin, and apigenin have the same flavone inhibitor with IC50 value at 0.6 nM exhibiting even lower core structure. However, flavone could dock into all three values than sitagliptin (18 nM) and vildagliptin (3.5 nM) active sites of DPP-IV, while luteolin and apigenin could [10], two current pharmacologic drug inhibitors of DPP- dock into only two of them. Computational docking showed IV. A summary of current foods and food components in comparably strong binding of luteolin and apigenin due to the prevention of diabetes by Thomas and Pfeiffer16 [ ]has hydrogen bonds of ring B hydroxyls with residue Ser209 indicated that the potential evidence for phenolic compounds in the S3 pocket, for ring C 1 -oxygens with the NH of is not conclusive; however, resveratrol was found to have a guanidine side chain of Arg358 in the S3 pocket, and for beneficial effect on protecting beta cells, which may be dueto ring A 8 -hydroxyls with C=O groups of side chains of its ability to modulate the activity of DPP-IV. Glu361. These features also exist in the binding of other citrus DPP-IV has three binding pockets/active sites (S1, S2 flavonoids (including kaempferol, quercetin, hesperetin, and and S3). The specificity pocket S1 is composed of the side naringenin) to DPP-IV, which have the same flavone core 12 Evidence-Based Complementary and Alternative Medicine structure. Even the binding of genistein, a soy isoflavone, to Conflict of Interests DPP-IV also had these features. Therefore, hydroxyls in these flavonoids are important to dock into active sites of DPP-IV The authors have declared no conflict of interest. with the same binding modes. Furthermore, the formation of 𝜋-interaction between A or B ring of citrus flavonoids and Acknowledgments Arg669 or Arg358 also favors the binding of citrus flavonoids into S2 and S3 sites. Flavone has no hydroxyl residues capable Thanks are due to Dixon Springs Agricultural Center in of hydrogen bonding with residues in S2 and S3 pockets with Simpson, IL, USA, for providing the berries for the fermen- thesamebindingmodesasflavonoidslikeluteolin.Therefore, tation of the wines. Special thanks are due to the National although it could dock into all the three pockets of DPP-IV, Natural Science Foundation of China (Grant no. 31071524). flavone had a higher 𝐾𝑖 value due to absence of hydroxyl The Office of Research at the University of Illinois provided groups. funds for this research. Significant correlations were seen between50 IC values of 𝐾 these flavonoids and their binding energies and 𝑖 values References determined computationally in the present study. In the docking studies, if a compound shows lower binding energy [1] M.A.Nauck,T.Vilsbøll,B.Gallwitz,A.Garber,andS.Madsbad, compared to the standard, it proves that the compound “Incretin-based therapies: viewpoints on the way to consensus,” has higher activity [30]. These results indicated that more Diabetes Care, vol. 32, supplement 2, pp. S223–S231, 2009. negative binding energy and smaller 𝐾𝑖 result in tighter [2] J. J. Holst and J. Gromada, “Role of incretin hormones in binding, and then more potent inhibitory effect. Meanwhile, the regulation of insulin secretion in diabetic and nondiabetic a significant correlation also exists between the 𝐾𝑖 values humans,” American Journal of Physiology. Endocrinology and determined in silico and the number of hydroxyl groups of Metabolism,vol.287,no.2,pp.E199–E206,2004. flavonoids𝑟 ( = 0.56, 𝑃 < 0.05), which indicates that more [3]J.F.Gautier,S.Fetita,E.Sobngwi,andC.Salaun-Martin,¨ “Bio- hydroxyl groups of flavonoids can result in higher inhibition logical actions of the incretins GIP and GLP-1 and therapeutic constant and therefore higher IC50 value, indicating less affin- perspectives in patients with type 2 diabetes,” Diabetes and ity to bind the active site. This could explain why quercetin Metabolism,vol.31,no.3,pp.233–242,2005. with five hydroxyls has a higher IC50 value (less potent) than [4] L. L. Baggio and D. J. Drucker, “Biology of incretins: GLP-1 and the other citrus compounds, despite sharing the same flavone GIP,” Gastroenterology,vol.132,no.6,pp.2131–2157,2007. core structure. IC50 values of citrus compounds were also [5]J.J.Holst,T.Vilsbøll,andC.F.Deacon,“Theincretinsystem foundtobesignificantlycorrelatedwiththeirnumbersof and its role in type 2 diabetes mellitus,” Molecular and Cellular hydroxyls. Endocrinology,vol.297,no.1-2,pp.127–136,2009. We obtained 𝐾𝑖 values using the computational analyses [6] C. F. Deacon, “Circulation and degradation of GIP and GLP-1,” as well as experimentally. 𝐾𝑖 values determined with the com- Hormone and Metabolic Research,vol.36,no.11-12,pp.761–765, putational analyses were calculated from the binding energy. 2004. However, the binding energy is designed to score and rank [7] R. Mentlein, “Dipeptidyl-peptidase IV (CD26)-role in the in- conformations of ligand and protein and not designed to give activation of regulatory peptides,” Regulatory Peptides,vol.85, no.1,pp.9–24,1999. accurate binding energy. Therefore, 𝐾𝑖 values generated from autodock correlated with free binding energies significantly [8] C. F. Deacon, M. A. Nauck, J. Meier, K. Hucking,¨ and J. J. Holst, “Degradation of endogenous and exogenous gastric inhibitory (𝑟 = 0.56, 𝑃 < 0.05) but differed from the experimental 𝐾𝑖 values. polypeptide in healthy and in type 2 diabetic subjects as revealed using a new assay for the intact peptide,” Journal of Clinical In conclusion, our study demonstrated that ANC isolated Endocrinology and Metabolism,vol.85,no.10,pp.3575–3581, from blueberry-blackberry wine blends and a variety of other 2000. phenolic compounds commonly present in citrus, berry, [9] E. P.Semighini, J. A. 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Nutr Cancer. 2014;66(8):130414. doi: 10.1080/01635581.2014.956246. Epub 2014 Sep 29. Cytotoxic effects of ellagitannins isolated from walnuts in human cancer cells. Le V1, Esposito D, Grace MH, Ha D, Pham A, Bortolazzo A, Bevens Z, Kim J, Okuda R, Komarnytsky S, Lila MA, White JB. Author information
Abstract Walnuts contain many bioactive components that may slow cancer growth. A previous report showed that a diet supplemented with walnuts decreased the tumor size formed by MDAMB231 human cancer cells injected into nude mice. However, the mechanism of action was never determined. We characterized the effects of a methanol extract prepared from walnuts on human MDAMB231, MCF7, and HeLa cells. The extract was cytotoxic to all cancer cells. We identified compounds from the methanol extract that induced this cytotoxicity. The predominant compounds were Tellimagrandin I and Tellimagrandin II, members of the ellagitannin family. We also show a walnut extract decreases the intracellular pH, depolarizes the mitochondrial membrane with release of cytochrome c and phosphatidylserine flipping. The antimitogenic effects of walnut extract were associated with a twofold reduction of mitochondria respiration. These results suggest impairment of mitochondrial function and apoptosis as relevant mechanism of anticancer effects of the walnut extract.
PMID: 25264855 DOI: 10.1080/01635581.2014.956246 [Indexed for MEDLINE]
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https://www.ncbi.nlm.nih.gov/pubmed/25264855 1/2 8/30/2017 Cytotoxic effects of ellagitannins isolated from walnuts in human cancer cells. - PubMed - NCBI
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