Polyphenols

2 Polyphenols

Polyphenols are a group of compounds belonging to the phytochemicals (plant secondary metabolites) which are important for normal growth development of the plant, defence against infection and injury (Chakravartula and Guttarla 2007), the prevention of UV damage and the formation of plant colour (Crozier et al. 2009). Polyphenols can reach high concentrations in the plant tissues (Crozier et al. 2009). The last decade the interest in polyphenols of both the general public and the scientific community has increased tremendously. Polyphenols gained much popularity as natural dietary antioxidants, but more recently it was discovered that their role as antioxidants is not as large as previously thought. However, by mechanisms not completely defined, polyphenols exert protective roles in inflammatory diseases like cardiovascular disease (CVD), obesity and diabetes type II (DBII) (Rice-Evans et al. 1996). The health effects of polyphenols will be discussed in more detail in chapter....

Phenolics are characterized by having at least one aromatic ring with one or more hydroxyl groups attached. There are more than 8000 polyphenols known today, and theys can be classified based on their molecular structure (Crozier et al. 2009). The four major classes of polyphenols are flavonoids, lignans, phenolic acids and stilbenes. The flavonoids and the phenolic acids are divided in subclasses. An overview of the major classes and the subclasses is shown in table 1. In LoGiCane only flavonoids (mainly catechins and quercetin) and phenolic acids (such as syringic and ferulic acid) occur in detectable concentrations (see table 2). In this chapter, flavonoids and phenolic acids will be described shortly.

Table 1. Overview of major polyphenol classes and subclasses.

Class

Subclass

Example

Flavonoids

Anthocyanins

Cyanidin

Chalcones

Xanthohumol

Dihydrochalcones

Phloretin

Dihydroflavonols

Dihydroquercetin

Flavanols

(+)-Catechin

Flavanones

Naringin

Flavones

Apigenin

Flavonols

Kaempferol

Isoflavonoids

Genistein

Lignans

Lignans

Sesamol

Phenolic acids

Hydroxybenzoic acids

Galllic acid

Hydroxycinnamic acids

Caffeic acid

Hydroxyphenylacetic acids

Homovanillic acid

Hydroxyphenylpropanoic acids

Dihydrocaffeic acid

Stilbenes

Stilbenes

resveratrol

In table 1 an overview of polyphenol classes and subclasses is shown. Of each subclass, one example is mentioned. Polyphenols occuring in LoGiCane are marked in yellow.

Table 2. Polyphenol composition of LoGiCane UF powder.

Class

Subclass

Compound

Content (mg catechin equivalent/kg)

phenolic acids

hydroxycinnamic acid

Caffeic acid

12

Chlorogenic acid

26.3

p-Coumaric acid

30.9

Ferulic acid

34.7

hydroxybenzoic acid

Gallic acid

4.6

Syringic acid

85.5

Vanillic acid

2.7

flavonoids

flavones

Apigenin

1.7

Diosmin

2.3

Luteolin

0.7

flavanols

(+)-Catechin

1

(-)-Catechin Gallate

5.9

(-)-Epicatechin

7.8

flavonols

Kaempferol

0.3

Quercetin

4.7

Rutin

1.8

In this table the polyphenols occuring in LoGiCane are shown. Polyphenols with a concentration of 10 mg catechin equivalent/kg or higher are marked yellow.

2.1 Flavonoids

Flavonoids are polyphenols consisting of fifteen carbons, with two aromatic rings connected by a three carbon bridge, C6-C3-C6. They are present in high concentrations in fruits, like strawberries and in tea (both black and green tea) (Crozier et al. 2009). The main subclasses occurring in LoGiCane are flavones (i.e. diosmin), flavanols (i.e. (-)-epicatechin) and flavonols (i.e. quercetin). Some examples of structures of flavonoids are shown in 1. The structures are shown as aglycones (the basic polyphenol skeleton without extra side groups). However, in plants flavonoids are most often conjugated to side groups. Sugars are very common, in fact, most flavonoids exist in planta as glycosides. Side groups can change the properties of the polyphenols, like function and bioavailability (Crozier et al. 2009). Quercetin for example has stronger anti-inflammatory actions then many of its metabolites (Kim, 2005). Below some aglycone structures of flavonoids are shown ( 1).

2.2 Phenolic acids

Phenolic acid are polyphenols with one aromatic ring connected to a carbon group, C6-C1. They are present in high concentrations in fruits, like apples, strawberries and blackberries, and to a lesser amount also in coffee and tea. Like flavonoids, they can either occur as aglycones or as conjugates, which will alter their properties (Crozier et al. 2009). Hydroxycinnamic (i.e. ferulic acid) and hydroxybenzoic acids (i.e. syringic acid) are the main phenolic acids occuring in LoGiCane. Some examples of structures of phenolic acids are shown in 2.

3 Bioavailability and ADME

Research on bioavailability and ADME (absorption, distribution, metabolism and excretion, for overview see 3) of polyphenols is quite complicated. Polyphenols are extensively metabolized after ingestion, giving rise to numerous metabolites. Often only a few of these metabolites are monitored in urine and plasma, leading to an underestimation of the absorption and bioavailability of polyphenols. Some polyphenols, like rutin, are not absorbed in the small intestine but are metabolized by microflora in the large intestine. The bioavailability of these kinds of polyphenols is probably underestimated, because the metabolism by the gut microflora yields many metabolites (often small phenolic acids), the formation of which is also very variable between individuals (Crozier 2009). Detecting all the metabolites and monitoring them in blood and urine is very difficult.

The studies that have examined the bioavailability of polyphenols often administered the polyphenols as a pure compound. Because the food matrix and other polyphenols greatly influence the absorption of phenolic compounds, these studies often over- or underestimate the bioavailability (Crozier 2009, Scheepens 2009).

This chapter summarises the scientific literature on bioavailability and ADME of polyphenols, and starts with a general overview of the absorption and metabolism of polyphenols.

3.1 Absorption and metabolism

The pathways for polyphenols absorption and metabolism explained in this chapter have been discovered in research with flavonoids. The same pathways have been proposed in the absorption of phenolic acids, in addition to passive diffusion (Karakaya 2004). More research however would have to proof whether these pathways are indeed implicated in the absorption of phenolic acids. An overview of polyphenol absorption and metabolism is given in 4.

When dietary flavonoids are ingested some, but not all, flavonoids are absorbed in the small intestine into the circulatory system. The brush-border enzyme lactase phloridizin hydrolase (LPH) hydrolyses the flavonoid, releasing the aglycone (the flavonoid without attached side groups). After hydrolysis, the aglycone is often more lipophilic and is able enter the epithelial cell via passive diffusion. Before passage into the blood stream, the aglycones are metabolized by sulfotransferases (SULT), uridine-5char_2032-diphosphate glucuronosyltransferases (UGTs) and catechol-O-methyltransferases (COMT). Metabolism by these enzymes leads to respectively sulphated, glucuronidated and methylated metabolites. Some polyphenols are transported back into the lumen of the small intestine, mediated by multidrug resistance protein and P-glycoprotein, transporters that bind a broad spectrum of xenobiotics and transport them out of cells. Other polyphenols are transported into the bloodstream. Once in the bloodstream, metabolites are transported to the liver where phase II metabolism (conjugation reactions which often deactivate xenobiotics) takes place. Via the liver, polyphenols can be secreted into the bile and back into the small intestine (Crozier et al. 2009). In conclusion, the human body treats polyphenols as xenobiotics. Few receptors take up polyphenols, metabolism takes place in order to make them less active, and they are transported back to the small intestine.

The flavonoids that are not absorbed in the small intestine reach the large intestine, where they are metabolized by colonic microflora. Metabolism by microflora leads to the production of numerous metabolites, of which many are not identified yet. Polyphenols are first cleaved to release the aglycone, and then extensively metabolized to form small phenolic acids and hydroxycinnamics. These polyphenols are often quite bioavailable and can be absorbed into the bloodstream (Crozier et al. 2009). In conclusion, for many polyphenols the absorption is the small intestine is low. In the large intestine numerous more readily absorbable metabolites are formed. Because of the wide range of metabolites it is very difficult to monitor these polyphenolic metabolites in the body. In the next subchapters, an overview of absorption, metabolism, excretion and bioavailability of flavonoids and phenolic acids is presented.

3.1.1 Absorption, metabolism, bioavailability and excretion of flavonoids

There are large differences in the absorption and thus bioavailability of flavonoids. Some flavonoids, like catechins, are well absorbed and readily bioavailable compared to other polyphenols (for example rutin) (Manach, 2005). In general, polyphenols have quite low bioavailability, as shown by the maximum concentrations achieved in plasma and urinary recovery data (see below).

There are several factors important for the bioavailability of polyphenols. The nature of the side groups of the polyphenol greatly influences its bioavailability. Many polyphenols for example are more bioavailable when sugar side groups are present, because of the affinity of LPH for sugar groups. To make matters more complicated, the presence of other polyphenols/phytochemicals also influences the absorption, and the food matrix also has to be taken into consideration (Manach 2005 Crozier 2009). Furthermore, metabolism of polyphenols yields many metabolites, which are not all known, and thus concentrations of polyphenols in blood and urine are often underestimated. For these reasons, the true bioavailability of polyphenols is difficult to assess.

In this subchapter some studies concerning the absorption, bioavailability and excretion of flavonoids are discussed. The flavonoids discussed are quercetin, rutin and the catechins. For other flavonoids present in LoGiCane no reliable in vivo research has been performed.

Onion quercetin-O-glucosides

In an acute human feeding study fried onions containing quercetin-O-glucosides were fed to human volunteers. Plasma and urine samples were collected for analysis by HPLC-MS. Five quercetin metabolites were found in plasma, all of them were either sulphated or glucuronidated. (Crozier et al. 2009).

The two major metabolites, quercetin-3-O-sulfate (maximum plasma concentration, Cmax 665 nmol/L) and quercetin-3-O-glucuronide (Cmax 351 nmol/L) appeared in plasma within 30 min of the ingestion of onions, both had Tmax (time to reach maximum plasma concentration) values of less than 1 h and T1/2 (time needed to eliminate half the compound) values of 1.71 and 2.33 h respectively. This T1/2 is much shorter than values obtained in earlier flavonol absorption studies, which is almost certainly a consequence of the enhanced accuracy of analytical data available with the advent of HPLC-MS (Crozier et al. 2009).

Most urinary excretion of quercetin metabolites took place within 8 h after consumption of the onions. After 24 h of collection 4.7% of the intake was recovered in urine (Crozier et al. 2009).

The data obtained in this investigation suggests that quercetin is absorbed in the proximal part of the small intestine (Crozier et al. 2009). Based on the metabolites found in plasma and urine, it is clear that quercetin is sulphated, glucuronidated and methylated. The metabolites in plasma were not the same as the metabolites found in urine, indicating that quercetin is subjected to phase II metabolism and rapid turnover. Quercetin is rapidly excreted form the body via the kidneys (Crozier et al. 2009).

Tomato juice quercetin-3-O-rutinoside (rutin)

The bioavailability of rutin was investigated by feeding tomato juice to human volunteers. In this research only two metabolites were detected in plasma, quercetin-3-O-glucuronide and isorhamnetin-3-O-glucuronide. They were present in ca. 25-fold lower quantities than in the onion study, with respective Cmax values of 12 and 4.3 nmol/L. The Tmax times extended to ca. 5 h, which suggest that absorption takes place in the large intestine instead of the small intestine. A total of nine methylated and glucuronidated quercetin metabolites were detected in urine but some volunteers excreted only 3-4 metabolites. The level of metabolite excretion in urine ranged from 0.02 to 2.8% of rutin intake. The variation is probably contributable to the variations in the colonic microflora of the individual volunteers. Absorption in the large intestine was confirmed in a different feeding study with subjects who had an ileostomy (removal of the colon). In contrast with the healthy subjects with a functioning colon, neither plasma nor urinary metabolites were detected. Fluid collected in the last part of the small intestine after tomato juice consumption contained 86% of the ingested rutin. (Crozier et al. 2009).

Green tea catechins

Green tea is an extremely rich source of catechins. Green tea was given as an acute supplement to ten human volunteers. Plasma and urine were collected for 24 h. The tea contained mainly (?)-epigallocatechin and (?)-epigallocatechin-3-O-gallate, and lesser amounts of (?)-epicatechin, (?)-epicatechin-3-O-gallate and (+)-gallocatechin.

Two unmetabolised green tea flavanols, (?)-epicatechin-3-O-gallate and (?)-epigallocatechin-3-O-gallate were identified by HPLC-MS in plasma. Metabolised flavanols were also detected, namely glucuronide, methyl-glucuronide and methyl-sulfate metabolites of (epi)gallocatechin and glucuronide, sulfate and methyl-sulfate metabolites of (epi)catechin. The Cmax values ranged from 25 to 126 nM and Tmax values from 1.6 to 2.3 h. These values suggest uptake of catechins in the small intestine. The appearance of unmetabolised flavonoids in plasma is unusual. The passage of (?)-epicatechin-3-O-gallate and (?)-epigallocatechin-3-O-gallate and uptake through the wall of the small intestine into the circulatory system without metabolic modification could be a consequence of the presence of the 3-O-galloyl moiety. Gallic acid itself is easy absorbed with reported urinary excretions of 37% of intake (Crozier et al. 2009).

Urine collected 0-24 h after green tea ingestion contained an array of flavanol metabolites similar to that detected in plasma. This indicates that the flavanols do not undergo extensive phase II metabolism, in contrast to quercetin metabolites. In total, 8.1% of the ingested green tea flavanols were excreted in urine. Most part of the metabolites in urine were derivatives of (?)-epicatechin and (+)-catechin, which means that these flavonoids are highly bioavailable, being absorbed and excreted to a much greater extent that other flavonoids (Crozier et al. 2009).

3.1.2 Absorption, metabolism, bioavailability and excretion of phenolic acids

Not much research has been performed to the absorption, metabolism, bioavailability and excretion of phenolic acids. Research however has shown that all phenolic acids are absorbed, to some extent. Gallic acid for example is very bioavailable (Manach 2005). As with flavonoids, there are large differences in the bioavailability of phenolic acids. Also for phenolic acids, the nature of the side groups, the presence of other phytochemicals and the foodmatrix are very important for absorbance into the bloodstream.

Two studies are presented in which most polyphenols occurring in LoGiCane are discussed. For the other phenolic acids, no reliable data exist.

Coffee hydroxycinnamic acids

Coffee is a rich source of hydroxycinnamic acids, especially chlorogenic acid. A study was performed in which human volunteers drank coffee as an acute supplement. Plasma and urine were monitored for 4 hours. Caffeic acid, an immediate metabolite of chlorogenic acid, was present in all plasma samples after coffee consumption, contributing on average to 14% of total plasma hydroxycinnamates. Two plasma concentration peaks were observed, the first at 0.5-1.0 h and the second at 1.5-4.0 h after coffee consumption (Monteiro et al. 2007).

Cmax of total chlorogenic acids varied from 4.7 to 11.8 µmol/L. Although Tmax for the different chlorogenic acids was, on average, close to 2 h, there were large interindividual differences (1-4 h). Cmax of caffeic acid (mean = 1.6 µmol/L) occurred at 1.4 h after coffee consumption (Monteiro et al. 2007).

The 2 plasma concentration peaks of the different chlorogenic compounds indicate that absorption and metabolism after coffee ingestion is complex. Absorption of chlorogenic acid and other phenolic compounds, such as ferulic, p-coumaric, gallic, and caffeic acids in the stomach has been reported in rats. In addition, it has been shown that chlorogenic and caffeic acid are also absorbed in the small intestine of rats, mostly in the jejunum. If the polyphenols are first partly absorbed in the stomach, and partly in the jejunum at a later timepoint, the absorption would indeed cause two distinctive peeks. However, it has been shown that phenolic compounds can be returned to the intestine by enterohepatic circulation up to 48 h after intake of the polyphenols. Therefore, the second peak could be caused by enterohepatic recycling (Monteiro et al. 2007).

Scottish strawberries benzoic and cinnamic acids

In another study Scottish strawberries were fed to human volunteers. Scottish strawberries are a rich source of phenolic acids, namely benzoic (1287.95 ± 279.98 mg/kg) and cinnamic (1159.40 ± 233.96 mg/kg) acids. Within 5 h of strawberry consumption, the only phenolic acids detected in the plasma were benzoic acids and their derivatives. Tmax for benzoic acids was reached after 1 hour. Vanillic acid was also detected as free phenolic acid 1 h after consumption. Vanillic acid appears to be rapidly deconjugated, because vanillic acid is not available as a free acid in strawberries. Syringic acid was found to be the dominant conjugated acid in plasma 1 h after consumption. Since syringic acid is both free and esterified in strawberries, it must be conjugated on absorption.

Excretion of phenolic acids in urine was also monitored after ingestion of the strawberries. Also in urine, the major detected phenolic acids were benzoic acids derivatives. Gallic acid was not detected in the plasma and only 1% of the total amount consumed was recovered in the urine, this is not in keeping with other research, which found gallic acid to be readily absorbed (Crozier 2009). Vanillic acid, which was found in the plasma at a low concentration, was also found at a low concentration in the urine (8%). Syringic acid had a relatively high recovery in urine, namely 26%. The majority of phenolic acids detected in the urine were unconjugated. Trace amounts of the cinnamic acids; namely caffeic and p-coumaric were also recovered in the urine (4 and 5%, respectively). Because these acids were not found in the plasma, it is well possible that the cinnamic acids were consumed prior to strawberry intake and were recycled via enterohepatic circulation.

It appears that most cinnamic acid are not absorbed in the small intestine but are metabolized in the colon. A possible explanation for this low absorption is that there was too much competition in the small intestine (Russell et al. 2009). When a food is administered that is particularly high in one cinnamic acid (like chlorogenic acid in coffee) more absorption in the small intestine will occur, as observed in other research (Monteiro et al. 2007).

3.2 Tissue or organ targets of phenolics (distribution)

Research on tissue distribution is very scarce, even in experimental animals. Results found for tissue distribution are summarized below.

Phenolic compounds have been detected in the brain, heart, kidney, spleen, pancreas and reproductive organs of mice and rats. After acute ingestion of the labelled flavonol [2-14C]quercetin-4-glucoside by rats, a number of glucuronide and methylated quercetin metabolites formed in the small intestine and small amounts (4%) were excreted in urine. Flavonols that reached the large intestine were degraded to phenolic acids which were again excreted in urine over a 72 h period. No build-up in the circulatory system took place. There was no marked accumulation of radioactivity in any of the body tissues, including the brain (Crozier et al. 2009).

Research in humans has shown that tea catechins only build up in damaged and inflammatory tissue, where they are associated with positive health outcomes, but not in normal tissue (Crozier et al. 2009).

Library

Chakravartula, S. V. and Guttarla, N. (2007). "Identification and characterization of phenolic compounds in castor seed." Natural Product Research 21(12): 1073-7.

Crozier, A., Jaganath, I. B., et al. (2009). "Dietary phenolics: chemistry, bioavailability and effects on health." Natural Products Reports 26(8): 1001-43.

INRA-France (2009). Phenol-Explorer, A Database on Polyphenol Content in Foods, version 1.0 Beta, www.phenol-explorer.eu.

Monteiro, M., Farah, A., et al. (2007). "Chlorogenic acid compounds from coffee are differentially absorbed and metabolized in humans." The Journal of Nutrition 137(10): 2196-201.

Rice-Evans, C. A., Miller, N. J., et al. (1996). "Structure-antioxidant activity relationships of flavonoids and phenolic acids." Free Radical Biology and Medicine 20(7): 933-56.

Russell, W. R., Scobbie, L., et al. (2009). "Selective bio-availability of phenolic acids from Scottish strawberries." Molecular Nutrition and Food Research 53 Suppl 1: S85-91.

4 Risks and safety of polyphenols

Most studies of polyphenols are aimed to determine the protective effects of polyphenols against diseases, and relatively few investigators have examined their possible toxicity. However, for a number of polyphenols toxic effects have been found(Mennen et al. 2005). Hazards related to polyphenols intake are carcinogenicity/genotoxicity and antinutritional effects (Mennen et al. 2005). In this chapter, the toxic effects found for polyphenols are discussed. The toxicity test used to determine toxicity are described in another document, toxicity tests.

4.1 Flavonoids

Although many positive effects of flavonoids on health have been found, like protection in various types of cancer and cardiovascular disease (Crozier et al. 2009), some toxic effects have been found on the in vitro and in vivo animal level (table 1). Negative effects of polyphenols on humans have not been reported in literature. Studies performed in humans, with for example grape and green tea extracts, have shown that these extracts are safe for human consumption. The toxic effects in vitro and in animals could be caused by high doses often applied in toxicity tests, but this is not always the case. A hypothesis often encountered in the scientific literature is the possibility that flavonoids can become prooxidants under certain circumstances (for example high concentrations) (Mennen et al. 2005; Crozier et al. 2009). As prooxidants they test positive in mutagenicity assays, but these effects are not thought to be relevant in humans. The concentrations in plasma and tissues are not sufficiently high for the polyphenols to become prooxidants.

Below an overview of the toxic effects of flavonoids is shown.

Table 1 Toxic effects of flavonoids

Compound

Toxic effects

Apigenin

Chromosome damage in vitro at high concentration

ECGC

Teavigo, a preparation containing >90% EGCG, is considered safe for human consumption. In vitro genotoxic effects were not confirmed in vivo.

Some dermal irritating en sensitizing effects were found, but these are not relevant for human consumption

Other catechins

Addition to diet (1% or 0.1%) has been found to enhance tumor development in the colon of male rats

Extensive morbidity and mortality after dosing fasted Beagle dogs with a green tea extract at 200, 500 and 1000 mg/kg/day

The same green tea extract is considered safe for human consumption at high concentrations

Extracts of apple and grapes are safe in in vitro and in vivo tests

Diosmin

No toxic effects reported in literature

Kaempferol

Mutagenic and genotoxic potency in vitro

Luteolin

No toxic effects reported in literature

Quercetin

Mutagenic and genotoxic potency in vitro

Formation of toxic oxidation products in vitro

Stimulation of cell proliferation at low doses in vitro

Chronic nephropathy in rats when high doses (2% or 4%) were added to the diet

Addition (0.1%) to the diet of mice significantly reduced life expectancy

Rutin

No toxic effects reported in literature

Apigenin

Apigenin did not increase cell proliferation and was shown to be non cytotoxic (Noel et al. 2006).

The in vitro micronucleus assay, a test for assessing the chromosome damaging (clastogenic) potency of xenobiotics, was used for evaluating the genotoxicity of apigenin in normal human peripheral lymphocytes. This test assesses the number of micronuclei (MN) induced by a xenobiotic. The formation of MN is indicative of chromosome damage.

The number of MN scored per 1000 cells increased in a dose dependent manner and was comparable to Mitomycin C (positive control) at the highest concentration (100?M/5ml culture), indicating potential clastogenicity. No definite conclusions can be drawn from this test since it was performed in vitro, and the results of the in vitro micronucleus assay are not always compatible with human data.

ECGC (Teavigo)

Teavigo is a preparation from C. sinesis tea leaves comprising greater than 90% ECGC. It is developed by DSM Nutritional Products Ltd. and intended for human consumption. The safety of Teavigo is assessed in a great number of toxicology tests, including in vitro, in vivo and clinical tests (Isbrucker et al. 2006).

Genotoxicity

EGCG was found to be non mutagenic in the Ames test, a test that evaluates mutagenic activity using Salmonella typhimurium strains, with and without S9 mixture (a liver extract that mimics the effects of metabolism). Toxic effects were indicated by a reduction of the background growth in the absence of S9 mixture with the highest concentrations ECGC. The toxic effect (antibacterial effect) is most likely accountable to the production of hydrogen peroxide (H2O2), which has strong oxidizing properties. There was no sign of toxicity in the Ames test with S9 mix. S9 mix contains catalase, an enzyme that converts H2O2 to oxygen and water, explaining the absence of toxicity after adding S9.

Mutagenic potential of EGCG was found in mouse lymphoma tk+/- cells at high concentrations (above 100 mm) with addition of S9. This was however considered to be an effect of H2O2 formation. S9 mix contains inherent catalase activity and this is most likely the reason that EGCG showed no clastogenic potency with addition of S9.

The hypothesis that the toxic and mutagenic potential of EGCG in the Ames test and in the mouse lymphoma cells was caused by hydrogen peroxide formation was confirmed by measuring hydrogen peroxide formation in cell culture medium. A concentration and time dependent formation of hydrogen peroxide by EGCG was measured in RPMI culture medium, a medium often used for in vitro mutagenicity testing. The formation of H2O2 is most likely responsible for the toxic and mutagenic activity of EGCG in the performed tests.

No adverse outcomes were shown in an in vivo mouse micronucleus test (an assay that evaluates clastogenic potential) even at the highest dose (2000 mg EGCG/kg bodyweight) in mice. EGCG was also shown to be safe in a study in which mice were fed EGCG in the diet in concentrations up to 1200 mg/kg/day for 10 days. No adverse outcomes were shown at these doses. Because the plasma concentration reached in mice may be too low to assess toxic effects, rats were injected intravenous (i.v.) with 50 mg/kg/day for 2 days. No adverse effects of i.v. injection were observed.

From these studies it can be concluded that the observed adverse effects in in vitro studies were most likely due to H2O2 formation. The in vitro genotoxic effects are not verified in in vivo tests and thus are not relevant for the in vivo situation. It is concluded that EGCG is non-genotoxic, even when given at high doses to animals (Isbrucker et al. 2006).

Dermal, acute and short-term toxicity studies

No acute dermal toxic effect of EGCG was found in a study on rats. EGCG (1860 mg/kg bodyweight) was applied to the exposed skin of rats and left on the skin for 24 hours. Rats were assessed for 15 days for signs of toxicity. No systemic effects were found, but a slight to moderate erythema was noted in all dosed rats after removal of the dressing at 24 h and this lasted for up to 5 days. No abnormal macroscopic findings were observed after necropsy. The acute dermal LD50 to rats was found to be greater than 1860 mg EGCG/kg.

No skin irritation was found in a primary skin irritation study with rabbits. The rabbits were treated with 0.5 g of EGCG preparations (containing 0.47 g EGCG) on the exposed skin for 4 hours. Skin reaction and irritation reactions were assessed until 72 hours after removal of the dressings. No irritation was noted following the dermal exposure.

An open epicutaneous test for skin sensitization by ECGC was conducted in guinea pigs. This assay tests whether a compound is a sensitizer, i.e. whether it is able to induce an allergic inflammatory reaction. The test compound, in this case EGCG, is first applied in a 4 week induction phase, to allow an immune response to develop. An EGCG preparation (80%) was applied to the shaved right flanks of animals at concentrations up to 30% in ethanol at a dose of 100 ml/8 cm2. Immediately after this induction period animals were challenged with up to 10% ECGC on the previously unexposed left flank at a dose of 25 ml/ 2 cm2. After two weeks a second challenge was performed. Challenge reactions were assessed at 24 and 48 h after application. If a skin reaction develops after challenge, the test compound could be a sensitizer. The animals showed skin reactions (erythema) indicative of sensitization to EGCG.

The guinea pig maximization test is based on the same principle, with an induction and a challenge period. Again, erythema developed after induction and challenge periods. No mortalities or symptoms of systemic toxicity were observed and body weights of treatment and control animals were in the same range. Based on these two tests, it is concluded that EGCG is a potential sensitizer.

ECGC was shown to be irritating to the eye in a primary eye irritation test in a rabbit. 0.1 g of EGCG preparation (93%) was applied to the eye of one rabbit. ECGC was not rinsed from the eye. The rabbit was monitored for 17 days. Moderate to severe signs of irritation of the eye was observed. This was reversible and was no longer apparent 17 days following treatment. Due to the severity of the ocular irritation, the test was not repeated in other animals (Isbrucker et al. 2006).

An acute oral toxicity study was performed in rats. Rats were given a single dose by oral gavage of 2000 or 200 mg/kg body weigh EGCG preparation (90% EGCG). All animals were observed daily for 15 days. Almost all animals in the high dose group died after treatment. No treatment related adverse effects were noted in the low dose group. Based on these results, the oral LD50 of the EGCG preparation in rats was determined to be between 200 mg/kg and 2000 mg/kg.

Subchronic oral toxicity was tested in rats. Rats were fed EGCG preparations (77% EGCG) in the feed to deliver doses up to 500 mg EGCG/kg/day for 13 weeks. After the 13 week treatment study, some of the rats were allowed to recover from EGCG treatment for 4 weeks on basal diet without EGCG. There were no treatment related deaths or signs of systemic toxicity during the treatment and recovery periods.

A 13-week toxicity study was performed in fasted Beagle dogs. EGCG was delivered orally up to 400 mg EGCG/kg/day for 13 weeks. The administrations occurred after a minimum 15 h fasting and 3-4 h prior to feeding. Several dogs in the high dose groups died and most dogs showed signs of toxicity.

The study was repeated for 28 days with fed dogs, at concentrations up to 500 mg EGCG/kg/day, to determine the effect of feeding status. None of the dogs died during the study and there were no signs of toxicity observed in any of the groups. Vomiting and diarrhea was observed frequently, and was observed more often in fasted dogs compared to the pre-fed dogs.

A 13-week toxicity study of EGCG (91%) was performed in Beagle dogs after feeding. The dogs were dosed up to 500 mg/kg/day. The total daily doses were divided into twice-daily administrations approximately 1 h after feeding, which also occurred twice daily, for 13 weeks. Several dogs died during the study, but it examination of the dogs showed a lethal bacterial infection. These deaths were not related to EGCG treatment, since there was no dose-effect relationship. There were no signs of toxicity in all the remaining dogs (Isbrucker et al. 2006).

The dermal and sensitizing effects of EGCG are thought to be not relevant for human consumption, because concentrations are too high to be reached in humans, and because tea has a history of safety. There is no indication of sensitization after oral intake. Based on the acute and subchronic toxicity studies, an NOAEL for dogs and rats has been established at 500 mg/kg/day. For fasted dogs the the NOAEL is lower, 40 mg/kg/day, but the systemic concentrations in fed dogs are 10 times lower than those observed in fasted dogs after ingesting comparable amounts of EGCG. An ADI of 5 mg/kg/day for humans is established from this data (a safety factor of 10 is taken into account). This ADI is quite conservative, since 800 mg/day Teavigo for 10 days has been shown safe in humans (Isbrucker et al. 2006).

Teratogenicity and reproductive toxicity studies

To test the safety of EGCG for the fetus, EGCG preparations of >91% purity were administered to pregnant rats during organogenesis and development. In a teratogenicity study, pregnant rats were fed diets supplemented with EGCG at concentrations up to 14,000 parts per million (ppm) during organogenesis. EGCG was non-toxic to dams or fetuses. A two-generation study in rats fed up to 12,000ppm EGCG preparation showed that EGCG has no adverse effects on reproduction or fertility. The highest dose of EGCG used however did reduce the growth rate of offspring, and there was a small increase in pup loss. A NOAEL for rats at all life stages was established at 200mg/kg/day EGCG preparation (Isbrucker et al. 2006).

Overall, the results indicate that EGCG is safe for human consumption at all life stages.

Other catechins

Green tea catechins (1% or 0.1% of the diet) have been found to enhance tumor development in the colon of male rats (Mennen et al. 2005).

A recent study performed on Beagle dogs has found severe toxic effects of Polyphenon E, a formula containing EGCG (200 mg), EGC (37 mg) and EC (31 mg). A 9-month chronic study (0, 200, 500 and 1000 mg/kg/day) was performed in fasted dogs. The dogs were fasted because research in humans has shown a greater bioavailability of catechins with fasting. The study had to be ended at 6.5 months because of extensive morbidity, mortality, and pathology of many major organs of the dogs. A follow-up 13-week study examined the exposure to and toxicity of Polyphenon E. The doses were 176 and 200 mg/kg/day, and there was an additional group in which the dogs were fed prior to administration of Polyphenon E, the dogs were fasted. Toxic effects were less severe than in the chronic study and took place mainly in the gastrointestinal tract and the liver. Feeding Polyphenon E in a fed state led to a lesser bioavailability then in the fasted state. In response to this research, the FDA has recommended that Polyphenon E is to be taken with food and that subjects in future clinical studies have liver function test performed at baseline and repeated every four weeks while on treatment (Kapetanovic et al. 2009).

In contrary to the animal safety data, Polyphenon E has been shown to be safe in humans in a clinical study, even when 4 capsules per day were administered for 4 weeks. The Polyphenon E capsule contained 200 mg EGCG, 37 mg EGC, 31 mg EC and other green tea polyphenols. The reported adverse events were rated as mild events. The more common events include headache, stomach ache, abdominal pain, and nausea, which have been reported in subjects receiving green tea polyphenol treatment as well as in subjects receiving placebo. (Chow et al. 2003). It was therefore concluded that these effects are not treatment related and it is safe to take 4 Polyphenon E capsules a day for 4 weeks.

Grape seed extract (GSE) is a catechins rich extract of grape seeds. It tested negative in the Ames test with and without S9 mix even at high concentration of 5000 mg GSE/plate. GSE was also shown to be safe in an in vitro chromosomal aberration test (a tests that determines the potential of a compound to damage chromosomes) with Chinese hamster lung (CHL)) cells (Wren et al. 2002). Furthermore, GSE at 1000 mg/kg was shown to be safe in the in vivo mouse micronucleus assay (Erexson 2003). Grape skin seed extract (GSKE), which is also rich in catechins, was shown to be safe in the mouse micronucleus test even at 2000 mg/kg (Erexson 2003). GSE en GSKE were also shown to be safe in a subchronic 3-month oral toxicity study at 2.5% w/w of the feed of rats. The NOAEL (no observed adverse effect level) established in this study was approximately 2150 mg/kg body weight/day for female rats and 1780 mg/kg body weight/day for male rats (Bentivegna and Whitney 2002).

In an acute oral toxicity study in rats GSE was shown safe at doses of 2 and 4 g/kg. GSE was also tested in a subchronic 90-days oral toxicity test and was shown safe at a 2% level in the diet of rats. The NOAEL for GSE was established as 1410 mg/kg body weight in male rats and 1501 mg/kg body weight in female rats (Yamakoshi et al. 2002). GSE was shown safe in a 90-day toxicity study in rats when GSE was fed at 2.0% level in the diet (Wren et al. 2002).

These results indicate that GSE and GSKE are safe for human consumption. Several brands of GSE and GSKE are indeed on the market.

Applephenon, an extract containing apple polyphenols (mainly flavonoids like chlorogenic acid, (+)-catechin and (-)-epicatechin), showed mutagenicity at a high concentration of 2500 mg/plate in the Ames test without S9 mixture. However, the Ames test with S9 mixture, the chromosomal aberration test with Chinese hamster lung cells and the micronucleus test found no significant mutagenicity. An acute oral toxicity test in rats and a 90-day subchronic toxicity test in rats also showed applephenon to be safe at a dose of 2000 mg/kg (Shoji et al. 2004). These results indicate that the consumption of Applephenon is safe.

From these data, there is no implication that catechins are not safe in human consumption.

Kaempferol

Kaempferol was mutagenic in the Ames test after addition of S9 mix at a low concentration of 20 mg. Kaempferol also exhibited genotoxic actions against V79 (Hamster) cells in the absence of S9 mix at low concentration of 52 mM in the in vitro chromosomal aberration assay. In the presence of S9 mix kaempferol became even more genotoxic (Silva et al. 2000). From these results it cannot be concluded that kaempferol is genotoxic. The tests performed are all in vitro tests and there is no other research confirming these results. Foods high in kaempferol (for example broccoli and red onions) are considered as safe and even healthy foods.

Quercetin

Quercetin was shown to be mutagenic in the Ames test, both in the presence and absence of S9. Quercetin also exhibited genotoxic actions against V79 (Chinese hamster) cells in the absence of S9 mix in the chromosomal aberration test. In the presence of S9 mix, quercetin loses its genotoxic potency (Silva et al. 2000).

Furthermore, quercetin has been shown to decrease cancer cell proliferation at high doses, but it has also been found to stimulate cell proliferation at low doses (1-5 µmol/L) (Mennen et al. 2005).

During its antioxidative activities, quercetin can become oxidized into various oxidation products. One of these products is quercetin-quinone (QQ), which is toxic because of its ability of modulating proteins. However, the in vivo formation and possible toxicity of QQ has not been demonstrated yet (Boots et al. 2008).

Chronic nephropathy was observed in rats when high doses of quercetin (2% or 4%) were added to their diet. This did not reduce the life span of the rats, while in another study the addition of quercetin (0.1%) to the diet of mice reduced their life expectancy (Mennen et al. 2005).

These results could be indicative of genotoxic effects of quercetin. Foods high in quercetin, like onions, are however considered safe and healthy foods. Human trials in which volunteers were given high amounts of onions and thus quercetin showed no adverse effects (Crozier et al. 2009).

Rutin

Rutin was shown to be safe in both the Ames test as well as the chromosomal aberration test (Silva et al. 2000). The capacity of rutin to cause damage to the DNA was evaluated using the alkaline single-cell gel electrophoresis (also called comet assay) and the micronucleus test in the bone marrow of mice. The mice received i.p. injections, of 2500, 1250 and 625 mg/kg of rutin. All doses were repeated after 24 h. They were observed during a 72-h period, for each drug. The micronucleus test showed that rutin caused no damage to the DNA of the mice bone marrow cells, and the comet assay demonstrated an increase of damage only at the dose of 2 × 1250 mg/kg. Considering the results in this study were obtained with very high doses, it is unlikely that the consumption of rutin produces any clastogenic effects (da Silva et al. 2002).

Furthermore, human trials with food high in rutin have shown no adverse effects (Crozier et al. 2009).

Based on the toxicology data above and the history of use of foods containing flavonoids, there is no reason to expect toxic effects of the (amounts of) flavonoids contained in LoGiCane.

4.2 Phenolic acids

Research on polyphenols often focuses on perceived beneficial effects, not on safety. Little research has been performed to the safety of phenolic acids, probably because they are not so widely used in supplements as for example the flavonoids, which are often found in high concentrations in grape and tea extracts.

The scientific literature shows many beneficial effects of phenolic acids in the development of for example diabetes (Greenberg et al. 2006) and cancer (Crozier et al. 2009), but controversially also shows that some phenolics may have mutagenic effects, which could result in the development of cancer.

Some toxic effects of phenolic acids have been found (see table 2). Most of these effects have been shown in in vitro or animal studies, and it has not been shown that these effects also occur in humans. The toxic effects of phenolic acids could be caused by high doses often applied in toxicity tests, but this is not always the case. Phenolic acids are contained in food in considerable concentrations (like fruits and vegetables), and these foods are recognized as safe or even considered healthy. Intakes from habitual diets are however usually lower than the doses used in the studies investigating toxicity (Crozier et al. 2009). Below an overview of the possible toxic effects of phenolic acids is provided.

Table 2 Toxic effects of phenolic acids

Compound

Toxic effects

Caffeic acid

Clastogenic activity at low concentration (0.2 mg/ml) in in vitro test, which was mitigated after S9 addition

At 2% level in the diet for 4 weeks, induced forestomach and kidney tumors in rats and mice

Chlorogenic acid

No adverse effects after supplementation at 2% level in the diet of rats for 4 weeks

p-Coumaric acid

Minor clastogenic activity in at high concentration (6 mg/ml) in vitro test after S9 addition

Ferulic acid

Minor clastogenic acitivity at very high concentration (25 mg/ml) in in vitro test, which was mitigated after S9 addition

No adverse effects after supplementation at 2% level in the diet of rats for 4 weeks

Gallic acid

Clastogenic activity at low concentration (0.05 mg/ml) in in vitro test, which was mitigated after S9 addition

No adverse effects after supplementation at 2% level in the diet of rats for 4 weeks

Syringic acid

Non cytotoxic against various cell lines

Clastogenic activity at high concentration (3.0 mg/ml) in in vitro test, which was mitigated after S9 addition

No adverse effects after supplementation at 2% level in the diet of rats for 4 weeks

Vanillic acid

Non cytotoxic against various cell lines

Clastogenic activity at very high concentration (3.0 mg/ml) in in vitro test, which was mitigated after S9 addition

Caffeic acid

Caffeic acid showed considerable clastogenic (chromosome damaging) potential in the chromosome aberration test in Chinese Hamster Ovary cells at 0.2 mg/ml. After addition of S9 (metabolism mimicking) mixture, there was no clastogenic potential anymore (Stich et al. 1981).

Caffeic acid, when present at a 2% level in the diet of mice and rats for 4 weeks, induced forestomach and kidney tumors in rats and mice (Hirose et al. 1987), indicating that caffeic acid could be involved in carcinogenesis. However, caffeic acid is present in high concentrations in coffee, and the intake of coffee is considered safe and had even been shown to have beneficial effects for health, it is beneficial in for example the development of diabetes (Greenberg et al. 2006).

Chlorogenic acid

Chlorogenic acid, when present at a 2% level in the diet of male rats for 4 weeks was considered safe (Hirose et al. 1987).

p-Coumaric acid

p-Coumaric acid was tested safe in the in vitro chromosomal aberration test without addition of S9 mix, even at high concentration of 6 mg/ml (The maximum test concentration that needs no be tested according to OECD guideline 473 is 5 mg/ml). After addition of S9 mixture however, p-coumaric acid did show slight clastogenic activity, but only at high concentration of 6 mg/ml. Because of the high concentration used, the perceived clastogenicity is considered not relevant.

Ferulic acid

Ferulic acid was slightly clastogenic at a very high concentration of 25 mg/ml in the chromosomal aberration test. Addition of S9 mix terminated the clastogenic activity (Stich et al. 1981). Ferulic acid, when present at a 2% level in the diet of male rats for 4 weeks was considered safe (Hirose et al. 1987).

Gallic acid

Gallic acid showed profound clastogenic activity in the chromosomal aberration test at a low concentration of 0.05 mg/ml. Addition of S9 mixture terminated the clastogenic activity (Stich et al. 1981). Gallic acid, when present at a 2% level in the diet of male rats for 4 weeks was considered safe (Hirose et al. 1987).

Syringic acid

Syringic acid was considered non cytotoxic against various cell lines (Chen et al. 2006). Syringic acid showed slight clastogenic potential at a relatively high concentration of 3.0 mg/ml. Addition of S9 mixture terminated the clastogenic activity of syringic acid (Stich et al. 1981). Syringic acid, when present at a 2% level in the diet of male rats for 4 weeks was considered safe (Hirose et al. 1987).

Vanillic acid

Vanillic acid was considered safe in a study testing cytotoxicity against various cell lines (Chen et al. 2006). Vanillic acid was tested safe in the in vitro chromosomal aberration test without addition of S9 mix, even at very high concentration (25 mg/ml). After addition of S9 mix, vanillic acid was slightly clastogenic (Stich et al. 1981). The concentration however was 5 times as high as the maximum concentration that needs to be tested according to OECD guidelines (5 mg/ml), so this slight clastogenic activity is considered not to be relevant.

Based on the toxicology data above and the history of use of foods containing phenolic acids, there is no reason to expect toxic effects of the (amounts of) phenolic acids contained in LoGiCane.

4.3 Antinutritional effects

Consumption of polyphenols may also have antinutritional effects. When polyphenol rich foods/beverages are simultaneously taken with foods rich in nonheme iron, the absorption of nonheme iron is strongly inhibited. The high intake of polyphenols may increase the risk of iron depletion, especially in people that already have a low iron status (Mennen et al. 2005).

Polyphenols are also known to bind proteins, which could delay the absorption of protein or even decrease the rate of absorption.

5 Food containing polyphenols

Many foodstuffs contain appreciable amounts of polyphenols. Some foodstuffs that are either high in phenolic acids or in flavonoids have been selected. Because these foodstuffs are considered safe it is likely that the polyphenols that are contained in the food are also safe, if the concentrations are similar. This comparison is not completely valid, because in food there is always a ‘matrix' effect: the influence of components in the food (protein, micronutrients etc.) on the absorption and metabolism of the polyphenols, but it still gives an indication of the safety of polyphenols.

5.1 Coffee

In general, moderate coffee consumption is considered safe, and even has beneficial effects on the body (for example the decreasing risk of diabetes with increased coffee consumption) (Greenberg et al. 2006). The negative effects associated with coffee (i.e. increased risk of cancer, high blood pressure), are usually associated with the caffeine content of coffee (van Dam 2008). According to the Dutch ‘Voedingscentrum' (an organisation that informs and advises Dutch consumers about food and food related subjects), an average Dutch male consumes 7 cups (1 cup equals 125 ml) of coffee a day, and an average female 5 cups, which is considered to be safe (www.voedingscentrum.nl). Coffee contains high amount of phenolic acids, especially hydroxycinnamic acids (like caffeic and chlorogenic acid). Regular consumers of coffee may have a daily intake of hydroxycinnamic in excess of 1 g, and these for many people will be the major dietary phenols (Crozier et al. 2009). No adverse effects are associated to the high phenolic acid intake with coffee consumption.

5.2 Tea

Tea is one of the most widely consumed beverages in the world. Approximately 3.2 million metric tons of dried leaf is produced annually, of which 78% is black tea, 20% is green tea and 2% is oolong. For all three teas the raw material is young leaves, which has high flavanol content and high levels of active enzymes. Although the raw material is the same, the teas differ markedly in their polyphenol nature and content (table 3), due to differences in the manufacturing process.

When harvested, the fresh tea leaf is very rich in polyphenols (ca. 30% dry weight), but during processing the polyphenol content is changed and its concentration is reduced. Green tea is richer in polyphenols then black tea. The consumption of tea is considered to be safe, and also various tea extracts have been shown to be safe for human consumption. According to Pickwick, the average Dutch person drinks 277 ml tea/day (www.pickwicktea.com). No adverse effects are associated with the intake of polyphenols from high amounts of tea.

Table 3 Concentration of the major phenolics in infusions of green and black tea manufactured from the same batch of Camellia sinensis leaves.

Compound

Green tea (mg/l)

Black tea (mg/l)

Black tea content as a percentage of green tea content

Gallic acid

6.0 ± 0.1

125 ± 7.5

2083

5-O-Galloylquinic acid

122 ± 1.4

148 ± 0.8

121

Total gallic acid derivatives

128

273

213

(+)-Gallocatechin B

383 ± 3.1

n.d.

0

(?)-Epigallocatechin

1565 ± 18

33 ± 0.8

2

(+)-Catechin

270 ± 9.5

12 ± 0.1

4

(?)-Epicatechin

738 ± 17

11 ± 0.2

2

(?)-Epigallocatechin-3-O-gallate

1255 ± 63

19 ± 0.0

2

(?)-Epicatechin-3-O-gallate

361 ± 12

26 ± 0.1

7

Total flavanols

4572

101

2

3-O-Caffeoylquinic acid

60 ± 0.2

10 ± 0.2

17

5-O-Caffeoylquinic acid

231 ± 1.0

62 ± 0.2

27

4-O-p-Coumaroylquinic acid

160 ± 3.4

143 ± 0.2

89

Total hydroxycinammate quinic esters

451

215

48

Quercetin-O-rhamnosylgalactoside

15 ± 0.6

12 ± 0.2

80

Quercetin-3-O-rutinoside

131 ± 1.9

98 ± 1.4

75

Quercetin-3-O-galactoside

119 ± 0.9

75 ± 1.1

63

Quercetin-O-rhamnose-hexose-rhamnose

30 ± 0.4

25 ± 0.1

83

Quercetin-3-O-glucoside

185 ± 1.6

119 ± 0.1

64

Kaempferol-rhamnose-hexose-rhamnose

32 ± 0.2

30 ± 0.3

94

Kaempferol-galactoside

42 ± 0.6

29 ± 0.1

69

Kaempferol-rutinoside

69 ± 1.4

60 ± 0.4

87

Kaempferol-O-glucoside

102 ± 0.4

69 ± 0.9

68

Kaempferol-arabinoside

4.4 ± 0.3

n.d.

0

Unknown quercetin conjugate

4 ± 0.1

4.3 ± 0.5

108

Unknown quercetin conjugate

33 ± 0.1

24 ± 0.9

73

Unknown kaempferol conjugate

9.5 ± 0.2

n.d.

0

Unknown kaempferol conjugate

1.9 ± 0.0

1.4 ± 0.0

74

Total flavonols

778

570

73

Total Theaflavins

n.d.

224

Adapted from (Crozier et al. 2009)

5.3 Other sugar products

The most common types of sugars that are widely consumed are white, refined, brown and raw sugar. White sugar, also called blanco directo, is made by precipitating many impurities out of cane juice. Refined sugar is the most consumed form of sugar in North America and Europe and is made by dissolving and purifying brown sugar, in a method similar to the production of white sugar. It is further decolorized by a filtration through a bed of activated carbon or bone char. Raw sugar is far less processed. Raw sugars comprise a range of yellow to brown sugars made from clarified and boiled cane juice, giving rise to a crystalline solid with more mineral salts and phytochemicals then refined or white sugars. Manufacturers sometimes prepare raw sugar as loaves called jaggery in India rather than as a crystalline powder. Brown sugar is either produced in a late stage of sugar refining, when still a significant molasses concentration is present in the sugar; or it is produced by spraying cane molasses syrup on refined sugar. Refined sugar is more pure concerning the sucrose content than white sugar, followed by brown sugar and jaggery sugars (Harish Nayaka et al. 2009). Brown sugars and Jaggery sugar are quite high in their polyphenol content, and their consumption is considered safe throughout the world.

Table 4 Total phenol content and phenolic acid composition of sugar varieties, values expressed as mean±SD (n=3).

Phenolic acids (mg/kg)

Refined sugar

White sugar

Brown sugar

Jaggery

Gallic acid

Trace

Trace

14.6±0.29

122±6.07

Protocatechuic acid

-

-

1.98±0.07

60.0±3.47

Gentisic acid

-

Trace

35.2±0.13

130±5.49

4-Hydroxyphenylacetic acid

-

-

1.75±0.07

29.5±2.08

Vanillic acid

-

-

5.08±0.20

25.6±1.82

Caffeic acid

-

-

-

-

Syringic acid

-

-

11.2±0.26

0.75±0.05

p-Coumaric acid

-

-

6.25±0.17

13.0±0.51

Ferulic acid

-

-

1.2±0.08

34±1.26

t-Cinnamic acid

-

-

-

-

Adapted from: (Harish Nayaka et al. 2009)

5.4 Molasses

Cane molasses is thick syrup, obtained as a byproduct of the manufacture or refining of sucrose from sugar cane, which is high in polyphenols (Guimaraes et al. 2007). Molasses are sold for human consumption and as a supplement in animal feed.

In the production of raw sugar, the raw cane juices are boiled to purify sugar. The remaining syrup is called first molasses, a thick brown sugar. These first molasses are then thinned with water and boiled again, in order to extract more raw sugar. The syrup remaining after this boiling is called second molasses. More boiling steps can be performed, giving yield to third molasses and so on. The more boiling steps are performed, the less sweet the molasses becomes. At some point it is not economically feasible to continue boiling and extraction. The remaining molasses is known as ‘blackstrap molasses'. It has almost no sweetness, but is very rich in polyphenols (www.bgfoods.com). All these byproducts are used for human consumption or as supplement in animal feed.

5.4.1 Human consumption

In the US, molasses products are GRAS (generally recognized as safe), and several molasses brands are available on the market. Molasses has been sold in North America over 200 years (www.grandmamolasses.com).

Treacle is also a byproduct of sugar production, but it is obtained at a different stage of refining. It contains less polyphenols than most kinds of molasses products, because syrups are used in the production instead of molasses. This kind of syrup is very popular in England and is sold worldwide. The oldest brand selling treacle is Tate and Lyle, who started selling Lyle's Golden Syrup (a pale kind of treacle) ( 1) in 1885 (www.lylesgoldensyrup.com). Not all treacle products are molasses products, because treacle can also be produced by treating a sugar solution with acid, in which case no molasses will be used.

Payet et al examined the polyphenolic content of several types of syrups, massecuite (sugar-molasses mixture prior to the removal of the molasses) and molasses (table 5). The compounds are shown in order from low degree of processing to a high degree. Syrups are the least processed product, C molasses the most processed. The polyphenolic content increases with the degree of processing. All these products are suitable for human consumption.

Table 5Contents of polyphenolic constituents of cane sugar products

Compound (mg/kg)

syrups

massecuite

Amolasses

Bmolasses

Cmolasses

protocatechuicacid

8.5

19.4

42.9

71.2

102.3

p-hydroxybenzoicacid

16.4

27.6

45.1

68.3

107.9

chlorogenicacid

8.1

13.1

14.3

vanillicacid

2.2

7.5

7.1

26.1

41.8

caffeicacid

57

25.2

82.1

147.7

88.3

syringicacid

11.1

13.5

37.7

54.8

70.8

vanillin

10.3

14.5

33.1

44.2

46.3

p-coumaricacid

140.9

144.7

289.1

424.6

423.1

ferulicacid

114.4

71.3

168.4

255.1

169.5

benzoicacid

2.1

47.8

128.4

71.1

total

360.9

325.6

761.4

1233.4

1135.5

Contents of polyphenolic constituents by LC-MS of cane sugar products expressed in milligrams per kilogram of dry content of sample, means (n = 3). Standard deviation was always <10% Adapted from (Payet et al. 2006).

5.4.2 Animal feed

Molasses have been used as supplement in animal feed for a long time. One of the early studies evaluating molasses based supplements was performed in 1959 (Bowman et al. 1995), but according to a molasses producing company, R and H Hall (www.rhhall.ie) the use of molasses in livestock and poultry feeds dates back to the nineteenth century. Molasses thus have a long history of use in animals. The concentration of molasses used in animal feed is relatively high: up to 50% of the feed can consist of molasses, but more usual lower concentrations are used (±20%) (FAO, https://www.fao.org/docrep/003/s8850e/S8850E19.htm). Molasses may be fed to livestock in several ways such as molasses meal, molasses blocks, and liquid form to provide energy directly, or be used as a carrier for non-protein nitrogen, vitamins and minerals as well as medicinal compounds (R and H Hall, www.rhhall.ie). A review by Bowman showed that the results of molasses based supplements in ruminants (cattle and sheep) are contradictory. Some studies find no effect of molasses supplementation on feed intake, animal performance and body weight. Other studies do find a positive association between molasses supplementation and feed intake, animal performance and bodyweight. Some studies found a decrease in food intake and body weight after supplementing molasses, but this was most likely due to not including a nitrogen source (for example urea) in the molasses supplement, which is necessary to prevent weight loss. Most authors concluded that feeding molasses-urea supplements to grazing ruminants was not as profitable as feeding dry supplements, but still more profitable then not supplementing the feed (Bowman et al. 1995). This shows that the use of molasses, generally in high concentration (up to 50% of the feed), has no negative effect on ruminants, but likely has positive effects on animal performance and bodyweight.

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Mennen, L. I., Walker, R., et al. (2005). "Risks and safety of polyphenol consumption." American Journal of Clinical Nutrition 81(1 Suppl): 326S-329S.

Noel, S., Kasinathan, M., et al. (2006). "Evaluation of apigenin using in vitro cytochalasin blocked micronucleus assay." Toxicology In Vitro 20(7): 1168-72.

Payet, B., Shum Cheong Sing, A., et al. (2006). "Comparison of the concentrations of phenolic constituents in cane sugar manufacturing products with their antioxidant activities." Journal of Agricultural and Food Chemistry 54(19): 7270-6.

Shoji, T., Akazome, Y., et al. (2004). "The toxicology and safety of apple polyphenol extract." Food and Chemical Toxicology 42(6): 959-67.

Silva, I. D., Gaspar, J., et al. (2000). "Chemical features of flavonols affecting their genotoxicity. Potential implications in their use as therapeutical agents." Chemico-Biological Interactions 124(1): 29-51.

Stich, H. F., Rosin, M. P., et al. (1981). "The action of transition metals on the genotoxicity of simple phenols, phenolic acids and cinnamic acids." Cancer Letters 14(3): 251-60.

van Dam, R. M. (2008). "Coffee consumption and risk of type 2 diabetes, cardiovascular diseases, and cancer." Applied Physiology, Nutrition and Metabolism 33(6): 1269-83.

Wren, A. F., Cleary, M., et al. (2002). "90-day oral toxicity study of a grape seed extract (IH636) in rats." Journal of Agricultural and Food Chemistry 50(7): 2180-92.

Yamakoshi, J., Saito, M., et al. (2002). "Safety evaluation of proanthocyanidin-rich extract from grape seeds." Food and Chemical Toxicology 40(5): 599-607.

6 Efficacy of polyphenols

Inleidend stuk.

6.1 Effects on metabolism

In general, bioavailability of polyphenols is quite low (see section …) (Manach, 2005; Williamson 2005; Scheepens 2009). Although this is a big drawback to the many effects of polyphenols found on cell lines in vitro (e.g. regulation of genes involved in cancer and inflammation, interference with intracellular pathways and nuclear factors) it also means that high concentrations of polyphenols reach the gastrointestinal (G.I.) tract without undergoing extensive metabolism (Williamson, 2005; Scheepens, 2009). Concentrations of polyphenols reached in the small intestine and the colon are sufficiently high to have effects on enzymes and transporters of the G.I. tract in vivo. Furthermore, polyphenols that reach the colon are metabolized by bacteria, but in turn also affect the colonic microbiota. The effects on the enzymes of the G.I. tract also influence the uptake of nutrients and thus influence the energy balance. Below the effects of polyphenols on the G.I. tract are described in detail.

6.1.1 Overview of metabolism
6.1.2 Inhibition of carbohydrate metabolism

Several polyphenols have shown the potency to inhibit certain enzymes involved in carbohydrate metabolism. An inhibition of the metabolism leads to a slower or even declined uptake of glucose, leading to a reduced glucose peak, which correlates with a lower insulin secretion. All this might lead to a better glucose and insulin status, which is beneficial in metabolic syndrome, (pre)diabetes and obesity (Payan, 2003; McDougall, 2008).

6.1.3 Inhibition of protein metabolism

Proteases are the enzymes that break down proteins. They are necessary for many physiological functions in the body, like digestion, blood clotting and dissolution, blood vessel maintenance and bone remodeling. When proteases are overactive however, pathologies like cancer, coagulation diseases and pancreatitis can arise. Therefore, the inhibition of these proteases is a possible way of inhibiting the development of these diseases (Jedinak, 2006). Many effects of polyphenols on proteases have been found, but unfortunately no in vivo effects have been described in the literature yet (to my knowledge). The in vitro effects of polyphenols on proteases are described below.

6.1.4 Inhibition of fat metabolism
6.1.5 Modulation of energy balance
6.2 Effects on intestinal microflora
6.3 Effects on inflammation

Arachidonic acid pathway.

6.4 Antiviral effects

7 Appendix
7.1 Toxicity tests

Below some assays used for toxicity testing will be described.

7.1.1 Ames Test

The bacterial reverse mutation test (Ames test) is a test which is able to detect whether a compound is able to induce point mutations in strains of Salmonella typhimurium or Escheria coli. The ability to induce mutations, called mutagenicity, is a strong indicator of carcinogenicity (the ability to induce or aid the progression of cancer). The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. Its major drawback is that its results are not always relevant, since it is an in vitro test in non-mammalian cells. However, for many compounds it has been shown that Ames test results are compatible with results in mammalian systems and humans.

Test principle

The bacterial test strains are mutated in a way that renders them unable to produce the essential amino acid histidine. In order for the test strains to survive, histidine has to be added to the medium. Certain compounds are able to induce mutations. Such a mutation could lead to a reversal of the original mutation (hence reverse mutation test), leading to the ability of the strain to produce histidine, and thus grow on a histidine lacking medium. In the presence of a mutagen these reversed mutations occur at higher frequency than if no mutagen is present (negative control). Even without adding a suspected mutagenic compound, some revertant colonies will originate by spontaneous mutations (table 1). Only revertant bacteria are able to grow on the histidine lacking medium. Based on this principle, the Ames test detects whether a compound is a mutagen (see 1). The recommended maximum test concentration for the test substances is 5 mg/plate or 5 ml/plate.

At least five strains of bacteria should be used. These should include four strains of S. typhimurium (TA1535; TA1537 or TA97a or TA97; TA98; and TA100) that have been shown to be reliable in laboratories. It is known that these strains may not detect certain oxidizing mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102, which is why at least one of these should be included.

Many of the test strains have several features that make them more sensitive for the detection of mutations, e.g. the elimination of DNA repair systems.

Problems

The bacterial reverse mutation test is not suitable for the evaluation of compounds that are highly cytotoxic or bactericidal, because these will cause a decrease in the number of (revertant) colonies and might thus confound the test results concerning mutagenicity. Cytotoxicity might also result in a clearing or diminution of the background lawn, or the degree of survival of treated cultures. Test substances that are cytotoxic already below 5 mg/plate or 5 ml/plate should be tested up to a cytotoxic concentration. In the case of severe cytotoxic/bactericidal actions it is better to use another mutagenicity test.

Because the test strains are no mammalian cells the effect of metabolism is not taken into account. By adding S9 mixture (a rodent liver extract with metabolic activity) the mammalian metabolism can be mimicked, which increases the relevancy to mammalian situations.

Results

Criteria for determining a positive result (mutagenicity) are a concentration related increase of revertant colonies and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. A compound is considered non-mutagenic when none of the above criteria is met. Generally, when no significant increase in revertant colonies is detected at 5 mg/plate or 5 ml/plate compared to the negative control, a compound is considered non-mutagenic.

https://www.oecd.org/dataoecd/18/31/1948418.pdf

Historical range for the spontaneous revertant control values for some salmonella strains. Adapted from: Mortelmans 2000.

7.1.2 The mammalian in vivo micronucleus test

The mammalian in vivo micronucleus test is used for the detection of damage induced by the test compound to the chromosomes or the mitotic apparatus of erythroblasts (precursors of erythrocytes). When a red blood cell is in the erythroblast stage, it still contains a nucleus, whereas in the erythrocyte stage it lacks a nucleus. If a compound induces damage to the chromosomes or the mitotic apparatus, micronuclei can be formed. Micronuclei are small nuclei, which can be present in the cell, next to the main nuclei of a cell, produced during telophase of mitosis (see 2). The micronucleus test analyses erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents.

The mammalian in vivo micronucleus test is especially useful for assessing mutagenic potential of compounds because the test situation is very relevant to the human situation. Because it is performed in vivo, metabolism and pharmacokinetics are similar to the situation in humans, although the situation may vary among species, among tissues and among genetic endpoints. An in vivo assay is also useful for further investigation of a mutagenic effect detected by an in vitro system, like the Ames test.

If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.

Test principle

Test animals, usually rodents, are treated with a test compound for a predetermined period of time. After the treatment period, the animals are sacrificed and bone marrow (or peripheral blood) cells are harvested.

When a bone marrow erythroblast develops, the main nucleus is extruded. In this phase the cell is called a polychromatic erythrocyte (PCE), an immature erythrocyte which still contains ribosomes and thus can be distinguished from the mature, normochromatic erythrocyte (NCE) which lacks ribosomes. If a compound induces chromosome damage, a micronucleus may be formed which may remain behind in the cytoplasm of the cell while the main nucleus is extruded. Because the main nucleus is extruded, it is easy to detect micronuclei. An increase in the frequency of micronucleated PCEs in animals treated with a test compound is an indication of induced chromosome damage.

The bone marrow of rodents is usually used in this test because PCEs are formed in the bone marrow. Micronuclei can be distinguished by a number of criteria. Most often used is the frequency of micronucleated immature PCEs. The number of mature NCEs in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can also be used as the endpoint of the assay when animals are treated continuously for 4 weeks or more (this time is needed to ensure that a sufficient amount of mature erythrocytes have developed).

Results

There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group. Statistical significance should not be the only determining factor for a positive response. A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test. For an example of results, see table 2.

https://www.oecd.org/dataoecd/18/34/1948442.pdf

www.vub.ac.be

Table 2 Mean of polychromatic erythrocytes with micronuclei (MNPCE) observed in bone marrow cells of female (F) and male (M) Wistar rats treated with a Hypericum brasiliense extract, and respective controls.

These results show that Hypericum extract is not genotoxic in the in vivo micronucleus test, since no dose-effect response could be found and there is no significant increase in micronuclei at any dose. 2000 cells were analyzed per animal.

* Significantly different from negative control (p<0.05).

Adapted from (Esposito, 2005) Evaluation of the genotoxic potentioal of the Hypericum brasiliense (Guttiferae) extract in mammalian cell systems in vivo. Anderson Victorino Espósito; Danielli Maria Vieira Pereira; Leandro Machado Rocha; José Carlos Tavares Carvalho; Edson Luis Maistro

7.1.3 In vitro micronucleus test

The in vitro micronucleus (MNvit) assay is a genotoxicity test for the detection of micronuclei in the cytoplasm of interphase cells. The assay detects the activity of clastogenic and aneugenic chemicals in cells that have undergone cell division during or after exposure to the test substance. Cytochalasin B is the often used in this test to block cytokinesis, the process in which the cytoplasm of a single eukaryotic cell is divided to form two daughter cells. Cytochalasin B prevents citokinesis and thus prevents separation of daughter cells after mitosis, leading to binucleated cells. The evaluation can thus be limited to proliferating cells and a reduction of cell proliferation can be measured simultaneously. The principle of the in vitro micronucleus test is very similar to the principle of the in vivo micronucleus test. S9 has to be added to mimic metabolism. Advantages of the in vitro over the in vivo test are that the in vitro test is easier and faster to perform. The major disadvantage is that the results are less compatible with the situations in humans and test animals.

https://www.oecd.org/dataoecd/45/51/43996258.pdf

7.1.4 In vitro chromosome aberration test

The purpose of the in vitro chromosome aberration test is to test if a compound is able to cause structural chromosome aberrations in cultured mammalian cells. There are two types of aberrations, chromosome aberrations and chromatid aberrations (a chromatid is one copy of the DNA of a replicated chromosome). Most mutagens induce chromatid aberrations, but chromosome aberrations also occur. Chromosome mutations can lead to the development of cancer in humans and experimental animals. Therefore, a positive result in the chromosome aberration test indicates that a substance might induce cancer in humans after exposure to the test compound. Many compounds that are positive in this test are indeed mammalian carcinogens; however, false positives and false negatives have been detected. Compounds that cause cancer by other mechanisms than direct DNA damage are not detected by this test.

Test principle

A variety of cell lines, strains, or primary cell cultures, including human cells, may be used (e.g., Chinese hamster fibroblasts, human, or other mammalian peripheral blood lymphocytes). Cells should be exposed to the test substance both in the presence and absence of a metabolic activation system, usually S9. After a predetermined time of exposure to the test substance, the cells are treated with a substance which arrests the metaphase (e.g. Colcemid or colchicines). After this the cells are harvested, stained and metaphase cells are analyzed with a microscope for the presence of chromosome aberrations.

For relatively non-cytotoxic compounds the maximum concentration should be 5 ml/ml, 5 mg/ml, or 0.01M, whichever is the lowest.

Problems

The in vitro chromosome aberration test is not suitable for the evaluation of compounds that are highly cytotoxic or compounds that cause changes in pH and osmolality. These kinds of compounds could lead to false positive results concerning chromosome damage. This test is used to screen for possible mammalian mutagens and carcinogens.

Results

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations (see table 3). Biological relevance of the results should be considered first. Statistical significance should not be the only determining factor for a positive response. A test substance for which the results do not meet the above criteria is considered nonmutagenic in this system.

Positive results from the in vitro chromosome aberration test indicate that the test substance induces structural chromosome aberrations in cultured mammalian somatic cells. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in cultured mammalian somatic cells.

https://browse.oecdbookshop.org/oecd/pdfs/browseit/9747301E.PDF

Table 3 Possible results of an in vitro chromosome aberration test

Distribution of the different types of chromosomal aberrations in 300 cells analyzed per treatment group and mitotic index (MI) observed in Chinese hamster ovary (CHO) cells after treatment with circumin (9 h) and/or bleomycin (BCM, 10 mg/ml) during S phase. These results indicate that curcumin is able to induce chromosome aberrations in a dose dependent manner, and that it substantially increases the genotoxic damage of the positive control, bleomycin in a dose dependent manner.

https://www.scielo.br/scielo.php?pid=S1415-47571999000300021&script=sci_arttext

7.1.5 The in vitro mammalian cell gene mutation test

The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. Suitable cell lines include L5178Y mouse lymphoma cells, the Chinese hamster ovary (CHO) and the V79 Chinese hamster lung cell line, and TK6 human lymphoblastoid cells.

Test principle

Cells deficient in thymidine kinase (TK), due to a mutation of the TK gene, are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). Cells with TK are sensitive to TFT, which causes the inhibition of cellular metabolism and stops further cell division. Thus, only cells in which the TK gene mutates, rendering it inactive, are able to proliferate in the presence of TFT. If a compound is a mutagen, the chance of the TK gene mutating is higher, which will lead to more cells which are capable of proliferating when TFT is present.

The cells are first incubated with the test compound for a predefined duration. After this incubation time, known numbers of cells are seeded in medium containing the selective agent (TFT) to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.

For relatively non-cytotoxic compounds the maximum concentration should be 5 mg/ml, 5 ml/ml, or 0.01 M, whichever is the lowest.

Results

There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical significance should not be the only determining factor for a positive response. A test substance for which the results do not meet the above criteria is considered nonmutagenic in this system.

Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. Negative results indicate that the test substance does not induce gene mutations in the cultured mammalian cells used.

https://www.oecd.org/dataoecd/18/32/1948426.pdf

7.1.6 In vivo comet assay

The comet assay (also called single cell gel electrophoresis) is a very sensitive assay which is able to detect DNA damage in individual cells. Test animals are treated with the test substance for a predetermined period of time. After this treatment time cells (usually blood cells, but there are many other suitable cell types) are harvested. The cells are captured in agarose and lysed. Each cell forms a cavity in the agarose matrix. After adding a lysing substance, all the cellular constituents dissolve into the agarose matrix except for the DNA, which remains inside the cavity. After an incubation step, the agarose gel is electrophoresed. Damaged DNA is broken into smaller pieces and is able to leave the cavity. Undamaged DNA strands are too large and do not leave the cavity. The smaller the fragments, the farther they are free to move in a given period of time. Therefore, the amount of DNA that leaves the cavity is a measure of the amount of DNA damage in the cell. If a compound is not toxic to the DNA, all the DNA will remain in the cavity, as can be seen in 3. The more genotoxic the compound is, or the higher the concentration of the compound, the longer the tail will be ( 3). According to Collins et al, the best way of measuring comets is by visual scoring. The comets are classified from 0 (no tail) to 4 (almost all DNA in tail). If 100 comets are scored, this leads to a score between 0-400 ‘arbitrary units'. The scores from a concentration range of a test compound are compared with the negative and the positive control. Indications for genotoxicity are a dose-effect relationship or a reproducible increase in ‘arbitrary units'. (Collins, 2004)

Comets are scored visually. A score of 0 means that there is no tail, a score of 4 means that almost all DNA is in the tail.

7.1.7 MTT assay

The MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay is a test which is able to detect whether a compound is cytotoxic to certain cell lines. MTT (yellow) is converted by enzymes to formazan (purple). The change of color can be measured by a plate reader. Only viable cells are able to convert MTT to formazan, thus the developed color is directly related to the viability of cells. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the cytotoxicity of a compound can be deduced. Promega protocol CellTiter 96® Non-Radioactive Cell Proliferation Assay

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