- What is Carrageenan
- What is degraded carrageenan?
- What is poligeenan?
- Regulatory status of carrageenan
- Potential health benefits of carrageenan
- Carrageenan Foods and Other Uses
- Clinical studies in infants
- Carrageenan side effects
What is Carrageenan
Carrageenans or carrageenins are a family of linear is a high molecular weight sulfated galactose polymer (linear sulfated polysaccharides or sulfated polygalactans) that are derived from certain members of red seaweeds (class Rhodophyceae) 1. Carrageenan is very commonly used as food additives (food‐grade carrageenan also known as E 407), primarily used as a stabilizer and thickener in processed foods to improve the texture of processed foods by increasing the solubility of a variety of ingredients, particularly milk proteins 2. Carrageenan is a vegetarian and vegan alternative to gelatin in some applications or may be used to replace gelatin in confectionery. Carrageenan is used in a variety of commercial applications as gelling, thickening, and stabilizing agents, especially in food products such as frozen desserts, chocolate milk, cottage cheese, whipped cream, instant products, yogurt, jellies, pet foods, and sauces. Carrageenan is found in hundreds of food products, including ice cream, chocolate milk, yogurt, soymilk, beer, deli meats, infant formula, salad dressings, nutritional supplements, and many other processed foods 3. In addition to its use in food products, carrageenan is added to a variety of other products, including toothpaste, room air deodorizers, cosmetics and industrial applications such as mining 4. Although carrageenan is well known to cause inflammation and has been widely used in the laboratory to produce inflammation in animal and cell-based models for decades 4, its inclusion in processed foods, pharmaceuticals, cosmetics, and other commercial products has continued to increase 3. Previous studies showed that carrageenan initiates inflammation in intestinal epithelial cells by activating a pathway of innate immunity mediated by TLR4-BCL10 and by production of reactive oxygen species (ROS) 5. The Scientific Committee for Food 6, endorsed the acceptable daily intake (ADI) of 0–75 mg/kg body weight per day for carrageenan (E 407) previously established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) 7. The Scientific Committee for Food maintained the original group acceptable daily intake (ADI) for all types of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) of 0–75 mg/kg body weight because of some uncertainty ‘about the general immuno-reactive potential of the various carrageenans now in use as food additives’ 8. In 2003, the Scientific Committee for Food had no objection to the use of carrageenan in follow-on formulae up to a maximum level of 0.3 g/L 9. In its latest evaluation, JECFA in 2015, concluded that ‘the use of carrageenan in infant formula or formula for special medical purposes at concentrations up to 1,000 mg/L is not of concern’ 10.
Based on experimental studies in animals, ingested carrageenan is excreted quantitatively in the feces 11. Studies have shown that carrageenan is not significantly degraded by low gastric pH or microflora in the gastrointestinal tract. Due to its molecular weight, structure and its stability when bound to protein, carrageenan is not significantly absorbed or metabolized 12. Carrageenan also does not significantly affect the absorption of nutrients. Subchronic and chronic feeding studies in rodents indicate that carrageenan at doses up to 5% in the diet does not induce any toxicological effects other than soft stools or diarrhea, which are a common effect for non-digestible high molecular weight compounds 12. Review of several studies from numerous species indicates that food grade carrageenan does not produce intestinal ulceration at doses up to 5% in the diet 12. Effects of carrageenan on the immune system following parenteral administration are well known, but not relevant to food additive uses. The majority of the studies evaluating the immunotoxicity potential were conducted with carrageenan administered in drinking water or by oral gavage where carrageenan exists in a random, open structured molecular conformation, particularly the lambda form; hence, it has more exposure to the intestinal mucosa than when bound to protein in food 12. Based on the many animal subchronic and chronic toxicity studies, carrageenan has not been found to affect the immune system, as judged by lack of effects on organ histopathology, clinical chemistry, hematology, normal health, and the lack of target organ toxicities 12. In these studies, animals consumed carrageenan at orders of magnitude above levels of carrageenan in the human diet: ≥1000 mg/kg/day in animals compared to 18-40 mg/kg/day estimated in the human diet 12. Dietary carrageenan has been shown to lack carcinogenic, tumor promoter, genotoxic, developmental, and reproductive effects in animal studies 12. Carrageenan in infant formula has been shown to be safe in infant baboons and in an epidemiology study on human infants at current use levels 11.
Carrageenan is extracted from seaweeds in two ways. In native extraction, the seaweed is made into an aqueous solution, and the residue is filtered, leaving nearly pure carrageenan. The alkaline-modified method is less expensive and easier. The seaweed is mixed in an alkali solution, leaving a mixture of carrageenan and cellulose that can be sold as semirefined carrageenan. Carrageenan is yellowish or tan to white, coarse to fine powder that is practically odorless.
Indonesia, the Philippines, and Chile are three main sources of raw material and extracted carrageenan.
The use of dried carrageenan-containing seaweeds to produce puddings from milk reportedly goes back at least two centuries, and the Irish are reported to have used a seaweed in foods and medicines for about six hundred years 13. Carrageenan was first extracted only in 1837; the structure was not investigated until the 1840s and was not clarified until the mid-1950s. Commercial production of carrageenan started in 1937; it remained an almost exclusively US industry until the 1950s when it began to be carried out on a large scale in European countries 14.
Carrageenan is recovered commercially from Chondrus seaweed in the US and from Chondrus and Gigartina seaweed in Europe 15. Processing steps used to recover carrageenan vary considerably and are closely-guarded trade secrets. Cottrell and Baird 14 state that patent literature indicates that the process generally includes the following steps: The seaweed is washed to remove soluble salts and debris before being extracted with slightly alkaline hot water. The extract is concentrated to about 3% carrageenan before alcohol is added to precipitate it; alternatively, the extract can be drum dried to produce a less pure product.
There are three main varieties of carrageenan [lambda-(λ), kappa-(κ) and iota-(ι)], which differ in their degree of sulphation 16.
- Kappa-carrageenan has one sulphate group per disaccharide. Kappa (κ-carrageenan) forms strong, rigid gels in the presence of potassium ions; it reacts with dairy proteins. It is sourced mainly from Kappaphycus alvarezii (the elkhorn sea moss, which is a species of red algae) 17.
- Iota-carrageenan has two sulphate group per disaccharide. Iota (ɩ-carrageenan) forms soft gels in the presence of calcium ions. It is produced mainly from Eucheuma denticulatum (a species of red algae) 18.
- Lambda-carrageenan has three sulphate group per disaccharide. Lambda (λ-carrageenan) does not gel, and is used to thicken dairy products.
The solubility of carrageenan is influenced by various factors, such as temperature, pH, the presence of other solutes, the type of carrageenan (sulphate groups), and their associated cations (K+, Ca++). All carrageenans are soluble in hot water. Lambda carrageenans (λ-carrageenans) are soluble in cold water, but kappa-carrageenan and iota-carrageenan are soluble in cold water only as their sodium salts, although potassium salts are not. Carrageenans are also insoluble in organic solvents, such as alcohol, ether, and oil 19.
Commercial carrageenans are generally presented as sodium, potassium, and calcium salts or a mixture of these 20. These salts provide stability and the average molecular mass of commercial carrageenan varies between 100 and 1000 kDa 20. Sodium forms of carrageenan are more readily soluble, while potassium forms are less so 21. The minor differences in their chemistry impart different functional properties that are very useful to the food industry. For example, κappa-carrageenan is known to provide a strong and brittle gel in the presence of potassium salts. Meanwhile, iota-carrageenan requires calcium to form an elastic gel with thixotropic behavior at low concentrations, while lambda-carrageenan does not form gels, but can be used as a thickener 2. In the pharmaceutical industry, carrageenan is used as an excipient, and is included in the US FDA Database of Inactive Ingredients approved in drugs 22.
According to the Commission Regulation (EU) No 231/2012, ‘carrageenan (E 407) consists chiefly of the potassium, sodium, magnesium and calcium sulphate esters of galactose and 3,6-anhydrogalactose polysaccharides. The prevalent polysaccharides in carrageenan are designated as κ-, ι-, λ- depending on the number of sulphate by repeating unit (i.e. 1,2,3 sulphate)’ 12. In the processed Eucheuma seaweed (E 407a), the main polysaccharide is kappa-carrageenan (κ-carrageenan).
Gel formation is one of the most important properties for the pharmaceutical and commercial application of carrageenan. It has been observed that kappa-carrageenan and iota-carrageenan form three-dimensional gels through interactions with metal ions (potassium or calcium) that may be either clear or turbid, rigid or elastic, tough or soft, and heat stable or thermally reversible 23.
Potassium ions are the best gelling agent for kappa-carrageenan, forming a rigid and brittle gel, while calcium ions are less effective for gelation. The combination of both ions, however, produces a strong gel. Kappa-carrageenan gels are the strongest and most rigid, and synerise, while iota-carrageenan gels best with calcium ions to produce soft and flexible gels with good freeze-thaw stability that do not synerise 24.
Gelation of carrageenan occurs on cooling the solutions of kappa-carrageenan and iota-carrageenan; the linear molecules originate double or triple helices with a restricted length due to the absence of an α-d-galactopyranosyl unit containing a 3,6-anhydro ring. The linear helicoidal proportions therefore bind and form a firm and stable three-dimensional gel network with the appropriate ions 25. In contrast, rather than forming a three-dimensional gel, lambda-carrageenan improves the viscosity of the medium and is used as a thickener.
Table 1. The carrageenan composition in red seaweeds differs from one species to another
|Chondrus crispus||mixture of kappa and lambda.|
|Kappaphycus alvarezii||mainly kappa.|
|Eucheuma denticulatum||mainly iota.|
|Gigartina skottsbergii||mainly kappa, some lambda.|
|Sarcothalia crispata||mixture of kappa and lambda.|
There are two basic grades of carrageenan:
- Refined carrageenan. Refined carrageenan is the original carrageenan and until the late 1970s-early 1980s was simply called carrageenan. It is now sometimes called filtered carrageenan. It was first made from Chondrus crispus, but now the process is applied to all of the above algae. The seaweed is washed to remove sand, salts and other foreign matter. It is then heated with water containing an alkali, such as sodium hydroxide, for several hours, with the time depending on the seaweeds being extracted and determined by prior small-scale trials, or experience. Alkali is used because it causes a chemical change that leads to increased gel strength in the final product. In chemical terms, it removes some of the sulphate groups from the molecules and increases the formation of 3,6-AG: the more of the latter, the better the gel strength. The seaweed that does not dissolve is removed by centrifugation or a coarse filtration, or a combination. The solution is then filtered again, in a pressure filter using a filter aid that helps to prevent the filter cloth becoming blocked by fine, gelatinous particles. At this stage, the solution contains 1-2 percent carrageenan and this is usually concentrated to 2-3 percent by vacuum distillation and ultrafiltration.
- Semi-refined carrageenan.
In the United States both grades are labeled as carrageenan. In the European Union, refined carrageenan is designated by the E number E-407, and semi-refined carrageenan as E-407a 27. Refined carrageenan has a 2% maximum for acid insoluble material and is produced through an alcohol precipitation process or potassium chloride gel press process. Semi-refined carrageenan contains a much higher level of cellulosic content and is produced in a less complex process.
Figure 1. Red seaweeds (source of Carrageenans)
Carrageenan is a hydrocolloid consisting mainly of the ammonium, calcium, magnesium, potassium and sodium sulfate esters of galactose and 3,6-anhydrogalactose polysaccharides. This gives them the ability to form a variety of different gels at room temperature.
- Carrageenans are widely used in the food and other industries as thickening and stabilizing agents. Their main application of carrageenan is in dairy and meat products, due to their strong binding to food proteins.
- When used in food products, carrageenan has the EU additive E-number E407 or E407 27.
- Daily intake of carrageenan in food was calculated to be about 100 mg/day in adults in the United States in the 1970s 3. More recently, intake has been estimated by food industry publications to be 18-40 mg/kg/day, indicating potential intake of several grams daily 11. Average daily carrageenan consumption in the United States was estimated in one report to be 250 mg/day or ~4.2 mg/kg body weight (250 mg/60 kg) 4, considerably less than the amount reported by the industry 11.
- In its evaluation of carrageenan (E 407) and processed Eucheuma seaweed (E 407a), the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (ANS) noted that the absorption, distribution, metabolism and excretion database was sufficient to conclude that carrageenan was not absorbed intact; in a subchronic toxicity study performed with carrageenan almost complying with the European Union specification for E 407 in rats, the no-observed-adverse-effect level (NOAEL) was 3,400–3,900 mg/kg body weight per day, the highest dose tested; no adverse effects have been detected in chronic toxicity studies with carrageenan in rats up to 7,500 mg/kg body weight per day, the highest dose tested; there was no concern with respect to the carcinogenicity of carrageenan; carrageenan and processed Eucheuma seaweed did not raise a concern with respect to genotoxicity; the no-observed-adverse-effect level (NOAEL) of sodium and calcium carrageenan for prenatal developmental dietary toxicity studies were the highest dose tested; data were adequate for a refined exposure assessment for 41 out of 79 food categories 12. However, the Panel noted uncertainties as regards the chemistry, the exposure assessment and biological and toxicological data. Overall, taking into account the lack of adequate data to address these uncertainties, the European Food Safety Authority Panel concluded that the existing group acceptable daily intake (ADI) for carrageenan (E 407) and processed Eucheuma seaweed (E 407a) of 75 mg/kg body weight per day should be considered temporary, while the database should be improved within 5 years after publication of this opinion 12.
- This pilot study suggests that in individuals with prediabetes the removal from the diet of the food additive carrageenan is a novel intervention that may help prevent type 2 diabetes 4. The study diet completely eliminated the commonly used food additive carrageenan from the diet. Contents of the study diets were determined by study dieticians and study nutritionist-coinvestigator. Energy from dietary fat (~35-40% of energy), carbohydrate (~40-50% of energy), and protein (~15% of energy) at baseline and postintervention were similar for the no-carrageenan and the carrageenan-containing type of diet 4. Similar foods with or without carrageenan were selected for the majority of the dietary items. In twelve weeks, HbA1c declined by an average of 0.12% in study participants on the no-carrageenan diet. It is uncertain whether or not the HbA1c would continue to decline with ongoing removal of carrageenan from the diet. Further studies are needed to clarify the precise mechanisms by which the specific elimination of carrageenan-containing foods can affect glucose tolerance in the long term 4. Although food industry scientists have published several studies in support of the safety of carrageenan 2, other study indicate that carrageenan exposure could cause inflammation and leads to significant physiological consequences 28. The impact of removing carrageenan from the diet in patients with established diabetes and impaired beta cell function needs further investigation. Effects of carrageenan on the fecal microbiome, lymphocyte subsets, lipid parameters, and other inflammatory parameters may also contribute to the impact of carrageenan on glucose metabolism and human health.
There has been significant confusion in the scientific literature and the public realm between the high molecular weight (MW) food additive carrageenan (200 kDA to 800 kDa) and the products of acid hydrolysis of carrageenan, which are “degraded-carrageenan” (20 kDA to 40 kDa) and poligeenan (10 kDA to 20 kDa). In early works poligeenan was often incorrectly called “degraded carrageenan” 2. In some instances, early studies referred to, “degraded-carrageenan” as carrageenan, furthering the confusion. However, in 1988, the United States Adopted Names Council (USAN 1988) assigned the name ‘‘poligeenan’’ to pharmaceutical aids and dispersing agents in the 10 kDA to 20 kDa range 2. Degraded carrageenan and poligeenan are not food additives and have a completely different physical/chemical and toxicological properties from carrageenan 29. Poligeenan is not approved for use in food products, though it does have use in medical imaging, primarily in barium sulfate slurries used in X-ray studies of the mouth, throat, and esophagus during swallowing. poligeenan provides lubrication to aid in swallowing and the viscosity aids in maintenance of the suspension as well as prevention of barium sulfate aggregation and “caking” 30. Unfortunately, the terms ‘‘poligeenan’’ and “degraded-carrageenan” have not been used in the literature on a consistent basis. In fact, many studies published today still use the term carrageenan when describing the use of “degraded-carrageenan”, which continues to confuse both the scientific community and consumers 2. Over the past several years several groups have attempted to set the record straight regarding issues of size and nomenclature 29. This confusion has resulted not only in some innocent, but incorrect conclusions in research papers and reviews 29, but has also fueled misinterpretations of toxicological data by research groups 31 and by consumer groups 32. The basic research reported by these groups using cell models have since been proven to be non-reproducible 33 and their assertions that carrageenan used as a food additive is harmful have been rejected by regulatory authorities 34.
Some groups have postulated that poligeenan can be formed by hydrolysis of carrageenan in the acidic conditions found in animal and human stomachs. Poligeenan is not produced biologically, it is produced in the laboratory or commercially by subjecting carrageenan to very low pH (0.9–1.3) and non-physiological temperatures (>80°C) for several hours 2. These harsh conditions do not exist in the stomach or intestinal tract of animals or humans 2. In rodents, the pH of the stomach is 3–5 and, as such, acid hydrolysis of carrageenan cannot occur 35. In humans, gastric pH averages 2–3, but as food is taken in the pH rises rapidly to nearly 5 and then gradually goes back down to 2–3 as food leaves the stomach 36. Even with an average gastric emptying time in humans of 3–4 hours, there is insufficient time, temperature, and acid to cause carrageenan hydrolysis 2. Some studies have reported the formation of degraded carrageenan using shorter times (1 hour) and lower temperatures (35°C) 37. However, in the Watt, McLean, and Marcus study 37, concentrated hydrochloric acid (HCl) (12 molar) was added to dry carrageenan powder. This concentration of acid is far more concentrated than the in the human stomach. At a pH of 1–2 the concentration of HCl in the human stomach is approximately 0.1 to 0.01 molar 2. Thus, the strong acidic concentration used by Watt, McLean, and Marcus 37 would not be found under normal physiologic conditions in the human stomach. In a study by Chen et al. 38, lambda-carrageenan was purified, freeze-dried and resuspended with distilled water then adjusted to pH 1.9 with concentrated HCl. The incubation was incubated for 4 hours at 37°C. After neutralizing the mixture with sodium hydroxide (NaOH) it was subjected to ultracentrifugation through a membrane with a molecular weight cut off of 50 kDa. Then it was subjected to a second filtration through a membrane with a molecular weight cutoff of 10 kDa. This non-physiological preparation of degraded carrageenan yielded some degraded carrageenan, but the efficiency of the reaction model was not described 38. To date, the formation of poligeenan from carrageenan in the gastrointestinal tract has not been demonstrated 2. This is an important point because the toxicological effects in the gastrointestinal tract of animals have only been observed when using poligeenan (molecular weight 10 kDA to 20 kDa.) or degraded carrageenan (molecular weight 20 kDA to 40 kDa.). Both of these weight average molecular weight ranges are encompassed in the polydispersity molecular weight profile of poligeenan 2. In dietary studies using food grade carrageenan (molecular weight 200 kDA to 800 kDA) and conducted under Good Laboratory Practices (GLP) there were no intestinal lesions 39.
Table 2. Comparison of chemical/physical and toxicological properties of carrageenan and poligeenan
|Manufacture||Alkaline extraction at room temperature.||Acid hydrolysis (pH 1) at high temperature, 95ºC for up to 6 h.|
|Uses||Food additive, pharmaceutical excipient at <2.0% in food||Medical Imaging in Barium Sulfate slurries at 10% w/w|
|Molecular Weight||200–800 kDa.||10–20 kDa.|
|% below 50,000||<5%||>90%|
|% below 20,000||<0.5%||About 70%|
|% below 10,000||<0.1% (not detected)||About 50%|
|Strongly bound to protein by 3 cross-linking mechanisms, including carrageenan helical formation for complex 3-D carrageenan-protein structures.||Bound to protein strongly with no helical formation and complex 3-D structures.|
|Gel formation||No gel formation|
|Functional Properties in Food||Stabilizer (proteins and emulsions), gelation, thickener||None|
|Toxicological Properties||Not absorbed by gastrointestinal tract.||Absorbed to some extent by gastrointestinal tract.|
|Safe via oral exposure in laboratory animals and man.||Causes gastrointestinal ulceration and tumors in laboratory animals via food, water.|
|Not carcinogenic, tumorigenic, genotoxic.||Carcinogen by oral route in animals.|
|No immune system effects.||Immune system toxicity, suppress immune response.|
|Not a tumor initiator or promoter.|
|Not a developmental or reproductive toxicant.|
Carrageenan production methods
The manufacturing of carrageenan consists of extraction, purification, concentration, precipitation, filtration, and drying, although the process may vary according to the family of red algae used to extract the sulphated polysaccharide. Specific extraction methods are considered trade secrets by their manufacturers but generally follow a similar process 40. There are several methods to extract the carrageenan. The original method for manufacturing carrageenan is to extract the polysaccharides by means of aqueous solutions, filtering to eliminate the remaining residues, recovering the solution by precipitation using alcohol, and finally separating, drying, and milling the precipitate to produce refined carrageenan. This method is time-consuming and energy-intensive and does not have a high extraction efficiency, which is why more precise methods have been developed. Another method described in the literature is to extract the carrageenans as insoluble residue after washing out any residual minerals, soluble proteins, and lipids. This insoluble residue is sold as semi-refined low-purity carrageenan 41.
There are two different methods of producing carrageenan, based on different principles.
- In the original method – the only one used until the late 1970s-early 1980s – the carrageenan is extracted from the seaweed into an aqueous solution, the seaweed residue is removed by filtration and then the carrageenan is recovered from the solution, eventually as a dry solid containing little else than carrageenan. This recovery process is difficult and expensive relative to the costs of the second method.
- In the second method, the carrageenan is never actually extracted from the seaweed. Rather the principle is to wash everything out of the seaweed that will dissolve in alkali and water, leaving the carrageenan and other insoluble matter behind. This insoluble residue, consisting largely of carrageenan and cellulose, is then dried and sold as semi-refined carrageean. Because the carrageenan does not need to be recovered from solution, the process is much shorter and cheaper.
Other more complex methods include extraction using enzyme 42 or fungal 43, extraction with deep eutectic solvents 44. Microwave-assisted extraction, ultrasound-assisted extraction, reactive extrusion, and photobleaching processes have also been reported in recent years 45. Each extraction method may offer certain advantages over traditional methods, enabling the improvement of the physico-chemical, gelling, and bioactive conditions of the carrageenan. Finally, the variables used in the extraction process, such as temperature, pH, time, and alkaline pre-treatment, must always be taken into account, since these will condition the expected results 46.
Carrageenans were originally extracted from some types of seaweed of the phylum Rhodophyta, until growing demand and the expansion of their use led to the cultivation of new species to ensure their year-round availability.
Most carrageenans are therefore extracted from Kappaphycus alvarezii and Eucheuma denticulatum 20. Kappa-carrageenan is mostly extracted from Kappaphycus alvarezii, commercially known as Eucheuma cottonii, while iota-carrageenan is predominantly produced from Eucheuma denticulatum, also known as Eucheuma spinosum 20. The advantage of these two species over the others that are traditionally used is the types of carrageenan that are extracted, since species, such as Chondrus crispus, contain a mixture of kappa- and lambda-carrageenan that cannot be separated during commercial extraction. Lambda-carrageenan is obtained from seaweed in the Gigartina and Chondrus genera 47.
Figure 2. Carrageenan production method of refined Carrageenan
What is degraded carrageenan?
The term “degraded carrageenan” refers exclusively to the test material products used in the feeding study papers published in the literature from the mid-1950s through the mid-1970s 2.
By the mid-1950s and early 1960s, there were two sources of degraded carrageenan available. The degraded carrageenan product “Ebimar” was produced by Evans Medical Ltd., Liverpool, England from Irish Moss (species Chondrus crispus and Gigartina stellata) and could be used at concentrations above 5% without gelation 2. The molecular structure of Ebimar is comprised primarily of ideal kappa-carrageenan and ideal lambda-carrageenan. The degraded carrageenan product “C16” was produced by Laboratoires Glaxo, Paris, France from species Eucheuma spinosum, and comprised primarily ι-carrageenan. Both “Ebimar” and “C16” are referenced multiple times in scientific journals and patents of that time period 30.
There is confusion in this early literature between Mw (weight-average molecular weight) and Mn (number-average molecular weight), and often neither “Mw” nor “Mn” are specified 2. However, these specific degraded carrageenan test materials had a “weight average molecular weight” range of about 20 kDA to 40 kDa., slightly higher than ideal poligeenan (10 kDA to 20 kDa.), but still part of the poligeenan molecular weight profile (see Figure 3 below). Blakemore and Dewar 48 measured Mn (number-average molecular weight) of C16 at 16 kDa. and 19 kDa. by two difference chemical methods. The Polydispersity Index (PDI, a value calculated by dividing Mw/Mn) of single degraded carrageenan extracts is typically about 1.75, which means that the Mw of C16 calculates to 28 kDa. and 33 kDa. respectively for the two methods used, both of these falling within the 20 kDA – 40 kDa. range quoted earlier 2.
The term “degraded carrageenan” would also include poligeenan as this is can be made in the laboratory by the acid hydrolysis of carrageenan. On the other hand, it has been suggested that degraded carrageenan products (i.e. C16 or Ebimar) are simply poligeenan 30, while the European Commission’s Scientific Committee on Food stated “degraded carrageenan, also called poligeenan, has a weight average molecular weight of 20–30 kDa.” 49, hence adding to the confusion. Although the Mw (weight-average molecular weight) ranges of degraded carrageenan and poligeenan as described above are clearly different, they are both part of the poligeenan molecular weight profile and thus can be considered poligeenan (see Figure 3). Based on the acid hydrolysis conditions published in these papers, the Mw (weight-average molecular weight) profile of “degraded carrageenan” would be similar to those shown in Figure 3 for poligeenan.
From the discussion above and below, the meaning of Mw (weight-average molecular weight) should be clear with regard to describing poligeenan or carrageenan. Moreover, it should be apparent that poligeenan and degraded carrageenan are both part of the poligeenan molecular weight profile and both are made artificially in the laboratory and not by seaweed plants 2. Poligeenan and degraded carrageenan are separate and distinct molecules, and are not part of the carrageenan molecular weight profile (Figure 3) 2.
What is poligeenan?
Poligeenan is manufactured by subjecting carrageenan to acid hydrolysis at a pH of 0.9–1.3 at non-physiological temperatures (>80°C) for several hours 2. The resulting liquor is neutralized to about pH 7.5 and the poligeenan isolated by roll-drying or spray drying. This degradation converts carrageenan with molecular weight (MW) = 200 kDA to 800 kDa to poligeenan with molecular weight (MW) = 10 kDA to 20 kDa 50. The biological and toxicological activity of poligeenan is completely different from carrageenan. This is due to the lower molecular weight of poligeenan (Mw = 10 kDA to 20 kDa.), which reduces the strength of protein binding and which may allow absorption from the gut and interaction with cell systems. In comparison, the high molecular weight of carrageenan, combined with its strong affinity for proteins means that it is not absorbed from the gut and does not interact with cellular processes of the intestinal mucosal surface. Figure 3 compares the average molecular weight (Mw) profiles of food grade carrageenan and poligeenan. Molecular cleavage of the carrageenan during acid hydrolysis is random and the resulting molecular weight profile is shown in Figure 3. The carrageenan shown in Figure 3 is the same typical commercial product and is used in infant formulations at about 300 µg/mL. The Mw is 707 kDa. and the profile comprises individual molecules from approximately 30 kDa. to approximately 5,000 kDa. This MW profile is typical of all carrageenan extracts and represents the range of carrageenan molecule sizes in the live growing seaweed at the time of harvesting. The percentage below 50 kDa. of this carrageenan was calculated to be about 2.2% 51.
Figure 3. Molecular weight profiles of poligeenan and carrageenan
Footnotes: Carrageenan is synthesized by red seaweed and displays the common characteristic of polydispersity, which is seen with many hydrocolloid molecules. Poligeenan can only be formed when carrageenan is subjected to harsh acid hydrolysis under laboratory conditions. In order to understand the molecular weight of a polymer it is subjected to size exclusion chromatography (SEC). The instrument compares the weight fraction per change in Log Mw (Y-axis) to the Log of the molecular weight of each fraction. Each molecular weight fraction produces a signal proportional to its concentration. The graph depicts the molecular weight (MW) profiles of both carrageenan and poligeenan. Both carrageenan and poligeenan profiles are made up of molecules of various sizes (polydispersity). Polydisperse molecules are often described by their weight average molecular weight (Mw). The vertical arrows show the accepted Mw range for poligeenan (PGN) (10,000 – 20,000 Da.), degraded carrageenan (d-CGN) (20,000 – 40,000 Da.), and carrageenan (200,000 – 800,000 Da.). Note that the Mw for poligeenan and degraded-carrageenan are both clearly part of the poligeenan molecular weight profile and both are formed by harsh acid hydrolysis in the laboratory, also known as the “poligeenan process.” In the example sample above the Mw for poligeenan is 19,000 Da. and the Mw for the carrageenan 707,000 Da. Mw requires that you know the fraction of the total weight represented by each individual molecular size. The total mass of each molecule in a sample is NiMi. To get the weight contribution as a fraction of the whole sample, each NiMi is divided by the SNiMi (the sum of all the NiMi values). This fraction is then multiplied by NiMi to yield WiMi. The weight average molecular weight (Mw) for the sample is then the sum of WiMi, where Ni is the number of molecules at any given weight, Mi is the molecular weight of each of those molecules, and Wi is the weight fraction of each type of molecule.[Source 2 ]
The poligeenan Mw (weight-average molecular weight) profile shown in Figure 3 is a typical commercial product used in barium suspensions at about 10% for enhanced X-ray procedures and diagnostics. As expected from random acid hydrolysis, the poligeenan molecular weight profile has a similar shape to that of carrageenan, just moved to the left of carrageenan, because it contains fractions of lower molecular weight (Figure 3) (note that the x-axis is logarithmic scale). The molecular weight of this poligeenan is 19 kDa (19,000 Da), and there is a skew towards the lower molecular weight region at about 3 kDa (3,000 Da), which is typical of poligeenan products. The range of molecular weight is from about 2 kDa (2,000 Da) to about 300 kDa (300,000 Da). The percentage below 50 kDa. of this poligeenan was calculated to be about 96%, with about 48% below 10 kDa. 52. Figure 3 shows a profile overlap between these typical and individual carrageenan and poligeenan products, the molecular weight profiles crossing at about 70 kDa. This does not mean that the lower molecular weight section of the carrageenan profile is poligeenan. These lower molecular weight carrageenan components have not been produced by deliberate acid hydrolysis (poligeenan process); so they are not poligeenan. There is no poligeenan in carrageenan. Similarly, the higher molecular weight components of the poligeenan profile are not carrageenan. These higher molecular weight components of the poligeenan Mw profile are poligeenan. These higher molecular weight fractions have been produced by deliberate acid hydrolysis (poligeenan process); they are poligeenan. There is no carrageenan in poligeenan, though both are polydisperse, containing a range of molecular weight molecules or fractions. Again, this is a key point with respect to many misinterpretations of molecular weight data and toxicological conclusions 2.
Poligeenan cannot form gels because the molecules are not long enough to form meaningful helical structures due to their lower weight-average molecular weight 50. However, poligeenan has high protein reactivity, but only through direct cross-linkages with the protein. This means that poligeenan has no functionality in foods, whether stabilization, gelation, or thickening. Carrageenan and poligeenan are two totally different commercial products, manufactured by two different processes to two different specifications, with two completely different regulatory positions, and with no overlapping applications 50. Carrageenan is used in food, pharmaceutical, and personal care applications. Poligeenan is used solely as a clinical diagnostic tool for the suspension of barium sulfate. When used at about 10% (w/w) in this application, poligeenan has three primary clinical functionalities; short-term suspension of barium sulfate fine solids (thickening), ease of swallowing (lubricity), and prevention of barium sulfate particles “caking” on storage of liquid products (high charge density).
Poligeenan has been shown to induce gastrointestinal lesions, and tumors, and to induce a severe inflammatory response in the gastrointestinal tract of most laboratory animals 2. In addition, poligeenan has been shown to be absorbed through the gastrointestinal wall, reaching cells in the liver and being excreted in the urine of animals after prolonged administration. Carrageenan, on the other hand, is not absorbed orally and does not cause gastrointestinal lesions. carrageenan has never been shown to be converted to poligeenan in the gastrointestinal tract (or anywhere) in vivo. Carrageenan is not carcinogenic, tumorigenic, genotoxic, or a tumor promoter or initiator, or a reproductive toxicant, and has no adverse effects on the immune system. In fact, very preliminary research suggests that there may be some health benefits to consuming carrageenan. These studies suggest that there may be a link between consumption of carrageenan and decreased cholesterol and low-density lipoproteins (LDL, the “bad” cholesterol), as well as, increased immune status parameters and a decrease in inflammation biomarkers in human volunteers 53. Collectively, these data support the regulatory agencies’ decision that carrageenan is safe for human consumption.
Regulatory status of carrageenan
In the U.S., carrageenan is allowed under the U.S. Food and Drug Administration (FDA) regulations “Carrageenan” (21 CFR 172.620) as a direct food additive and is considered safe 54 when used in the amount necessary as an emulsifier, stabilizer, or thickener in foods, except those standardized foods that do not provide for such use. FDA also reviewed carrageenan safety for infant formula. The European Food Safety Authority concluded “there is no evidence of any adverse effects in humans from exposure to food-grade carrageenan, or that exposure to degraded carrageenan from use of food-grade carrageenan is occurring” 55, Furthermore, the Joint FAO/WHO expert committee on food additives stated in a July 2014 review of carrageenan “that the use of carrageenan in infant formula or formula for special medical purposes at concentrations up to 1000 mg/L is not of concern” 56.
Regarding the food additive carrageenan (E 407), the International Agency for Research on Cancer (IARC), in 1983 classified carrageenan in group 3 considering that the available data did not provide evidence that carrageenan is carcinogenic to experimental animals, in the absence of human data 12. IARC also evaluated ‘degraded carrageenan’ and concluded that ‘experiments in rats with doses of degraded carrageenan comparable to those used to test carrageenan provide sufficient evidence for the carcinogenicity of degraded carrageenan in rats. No data on humans were available’ (category 2B) 12.
A review of carrageenan (E 407) was published from the Nordic Council of Ministers. In the report, it was concluded for carrageenan (E 407) that: ‘Carrageenan has been extensively studied and in most studies seems to pose no toxicological problem. However the question about the potential promotion of colon carcinogenesis by carrageenan should be clarified. The Scientific Committee on Food has expressed the wish to review carrageenan in the light of this aspect. JECFA, however, at its 57th meeting June 2001, withdrew the temporary status and allocated a full acceptable daily intake (ADI) “not specified”. It may be necessary to clarify whether all types of carrageenan are sufficiently covered by the toxicological evaluation’ 57.
In the most recent review by an independent panel, the Joint Expert Committee of the Food and Agriculture Organization of the United Nations and World Health Organization on Food Additives (JECFA) released a technical report in 2015 on the use of carrageenan in infant formula and found that the additive was ‘not of concern’ in infant formula as food for special medical purposes at concentrations up to 1000 milligrams per liter 58. The use of carrageenan in infant formula, organic or otherwise, is prohibited in the EU for precautionary reasons, but is permitted in other food items.
In its evaluation of carrageenan (E 407) and processed Eucheuma seaweed (E 407a), the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) noted that 12:
- According to industry carrageenan (E 407) is defined as having a weight-average molecular weight of 200–800 kDa;
- Uno et al. 59 determined in a survey on samples of food-grade carrageenan, representing κappa-, iota- and lambda-carrageenan, a weight-average molecular weight of 453–652 kDa and detected no obvious peak of poligeenan with a detection limit for poligeenan of about 5%;
- The absorption, distribution, metabolism, and excretion (ADME) database was sufficient to conclude that high molecular weight carrageenan (e.g. κappa/lambda-carrageenan with a number-average molecular weight of approx. 800 kDa, study in rhesus monkeys) was not absorbed intact, while low molecular weight carrageenan (number-average molecular weight: 88 kDa or less) was found in tissues of rats or guinea pigs after administration of this material by gavage or diet;
- In one subchronic toxicity study in rats performed with κappa-carrageenan with an average molecular weight range of 196–257 kDa, almost complying with the European Union specification, the No Observed Adverse Effect Level (NOAEL) was equal to 3,394–3,867 mg/kg body weight per day for males and females, respectively, the highest dose tested; No Observed Adverse Effect Level (NOAEL) is the highest exposure level at which there are no biologically significant increases in the frequency or severity of adverse effect between the exposed population and its appropriate control; some effects may be produced at this level, but they are not considered adverse effects.
- No adverse effects have been detected in several chronic toxicity studies in rats with carrageenan (mostly κappa/lambda-type, no adequate indication of molecular weight distribution); from the available rat studies, NOAELs up to 7,500 mg/kg body weight per day, the highest dose tested, were identified 60. However, in another study in rats given carrageenan preparations with a molecular weight of 244 and 252 kDa, a Lowest Observed Adverse Effect Level (LOAEL) of 1% in the diet (equivalent to 500 mg/kg bw per day) was identified by the Panel (Documentation provided to EFSA n. 43). The Panel noted that the characterisation of the test material in all the chronic studies was limited; Lowest Observed Adverse Effect Level (LOAEL) is the lowest exposure level at which there are biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control group.
- Carrageenan (different types) and processed Eucheuma seaweed did not raise a concern with respect to genotoxicity;
- There was no concern with respect to carcinogenicity for carrageenan (mostly κappa/lambda-type);
- The No Observed Adverse Effect Level (NOAEL) of sodium and calcium κappa/lambda-carrageenan for developmental effects found in dietary prenatal developmental toxicity studies was 3,000 and 5,000 mg/kg body weight per day for rats and hamsters (Documentation provided to EFSA n. 46), and 3,060 mg calcium carrageenan/kg bw per day for rats 61, the highest doses tested.
In addition, the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) observed that 12:
- From all the data received, data were adequate for a refined exposure assessment for 41 out of 79 food categories;
- In the general population, based on the reported use levels, a refined exposure, in the brand-loyal scenario of up to 758.6 mg/kg body weight per day for toddlers (from 12 months up to and including 35 months of age) was estimated;
- For populations consuming foods for special medical purposes and special formulae, the 95th percentile of maximum exposure assessments calculated based on the maximum reported data from food industry (equal to the maximum permissible level) were up to 49.4 mg/kg body weight per day for infants (from 12 weeks up to and including 11 months of age).
- For the food additives carrageenan (E 407) and processed Eucheuma seaweed (E 407a), the fraction of low molecular weight carrageenan is limited in the European Commission specifications by the purity criteria. The Panel was informed by one interested party that the material on the market does not necessarily comply with the European Union specifications regarding the limit of the low molecular weight fraction. This fraction has been associated with potential health hazards similar to those reported for preparations of degraded carrageenan, such as poligeenan or C16, to which this fraction shows similarity in molecular structure and in weight-average molecular weight. Although, full identity of degraded carrageenan such as poligeenan or C16 with the low molecular weight fraction of carrageenan has not been specifically demonstrated.
Concerning degraded carrageenan (e.g. poligeenan, C16) the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) therefore noted the following 12:
- Degraded carrageenan has been described to be absorbed and to be present in various tissues, namely the liver, and the urine of animals when administered in drinking water or via the diet;
- Degraded carrageenan did not raise concern with respect to genotoxicity;
- Rats exposed to degraded iota-carrageenan (weight-average-molecular weight of 20–40 kDa) via drinking water, diet or by gavage for up to 24 months developed in first instance colitis, secondary metaplasia and finally tumours (squamous cell carcinomas, adenocarcinomas, adenomas);
- Monkeys given degraded iota-carrageenan (C16) via drinking water showed histopathological lesions in the colon which varied from slight mucosal erosions at the low dose (750 mg/kg body weight per day) to ulceration associated with inflammatory infiltration of the lamina propria at the high dose (2,900 mg/kg body weight per day). In this study, all monkeys on degraded iota-carrageenan (C16) lost blood frequently from the intestinal tract in a dose-related degree and developed some degree of anaemia. A Lowest Observed Adverse Effect Level (LOAEL) of 750 mg degraded iota-carrageenan (C16)/kg body weight per day was noted;
- Toxicity studies have been mainly conducted with degraded iota-carrageenan, thus, degraded κappa- and lambda-carrageenan being sparsely toxicologically characterized.
The following uncertainties were noted by the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) as regards the chemistry and fate of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) 12:
- No data are available showing the molecular weight distribution for different food-grade carrageenan preparations within the defined weight-average molecular weight range of 200–800 kDa;
- No data are available showing the molecular weight distribution for individual food-grade processed Eucheuma seaweed preparations;
- The weight-average molecular weight range of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) is not defined in the European Union specifications allowing for the presence of a low weight-average molecular weight fraction of carrageenan. Based on the information provided on weight-average molecular weights of commercially available carrageenans, the low molecular weight material needs to be accurately quantified;
- In most of the toxicological studies the carrageenan used is not well specified and its weight-average molecular weight and its content of low molecular weight fragments are not given;
- Although it has been claimed that there is no adequate analytical method available to measure the low molecular weight fraction, the Panel noted that there is indication of the percentage of the low molecular weight fraction in the food additive carrageenan (E 407) tested in a few toxicological studies;
- Only limited information on the stability of carrageenan in food was available. No data on stability of carrageenan and/or processed Eucheuma seaweed addressing the usual variation of parameters (temperature, pH) relevant for the authorised food uses were available. Information on possible degradation products under acidic conditions in relevant food products is missing;
- Studies investigating the hydrolysis of the κappa-, iota- and lambda- carrageenan showed contradictory results.
The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) further noted the following uncertainty as regards the exposure assessment scenario 12:
- The refined estimates were based on 41 out of 79 food categories in which carrageenan (E 407) and processed Eucheuma seaweed (E 407a) are authorised and result in an overestimation of the real exposure to carrageenan (E 407) and processed Eucheuma seaweed (E 407a) as food additives in European countries considering only food additives uses for which data have been provided were considered.
Among the uncertainties from the biological and toxicological data, the Panel considered the following 12:
- The lack of reliable comparative toxicokinetic and toxicological studies between the different types of carrageenan and their corresponding low molecular weight fractions;
- The theoretical possibility that limited degradation could occur under conditions representative of the in vivo situation;
- No firm conclusion on the other types of carrageenan could be drawn on the observation of occult blood in feces of rhesus monkeys dosed with a commercial carrageenan;
- The characterisation of the test material in most of the toxicological studies was limited;
- There were no adequate toxicological studies performed with low weight-average molecular weight carrageenan (around 200 kDa), apart from one 90-day study (with an average molecular weight carrageenan in the range of 196–257 kDa, not specified if it was a number-average or a weight-average);
- Testing for chronic toxicity and reproductive and developmental toxicity was performed almost exclusively with κappa/lambda-carrageenan; almost no data on iota-carrageenan was available;
- Inadequate data on the possible relevance of carrageenan exposure for existing inflammatory bowel diseases (IBD) in humans;
- The relevance for humans of the observations in animal studies pointing to the induction of glucose intolerance and glucosuria by carrageenan is unclear;
- The possible role of sulfate and the interactions of the various forms of carrageenans with the gut microflora in some of the reported inflammatory effects of carrageenans.
In most toxicological studies, the molecular weight distribution of the carrageenan tested is not or only inadequately described 2. The low molecular weight fraction of carrageenan is associated with potential adverse effects 2. This is due to its similarity in molecular structure and weight-average molecular weight with those of degraded carrageenan, such as poligeenan or C16, known to cause inflammatory intestinal effects 2. Moreover, results suggesting that carrageenan might enhance inflammatory bowel disease (IBD) in humans need clarification. The test compounds used in a large number of the toxicity tests are thus not considered to reflect adequately the diversity of preparations of the food additive on the market, particularly with respect to the broad range of weight-average molecular weights reported. The Panel noted that most of these issues were also raised and discussed in a recent review 62.
The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) also considered other pending uncertainties regarding the relevance of the studies available to assess the safety of the authorized food additive carrageenan (E 407). The physicochemical properties of carrageenan depend on the chemical conformation (helical or random coil) in which it exists in the preparations and in foods (solid or liquids foods) and are influenced by the presence of cations, proteins and the pH and temperature. These conditions could affect the toxicity of carrageenan and are thus relevant for the safety assessment of the food additive in the authorized uses. Furthermore, findings in some studies suggest that carrageenan exposure could be related to the induction of glucose intolerance and glucosuria. In the view of the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS), all this information still needs clarification.
Overall, taking into account the lack of adequate data to address the above mentioned uncertainties, the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) concluded that the existing group acceptable daily intake (ADI) for carrageenan (E 407) and processed Eucheuma seaweed (E 407a) of 75 mg/kg body weight per day should be considered temporary, whilst the database should be improved within 5 years after publication of this opinion 2. Within the given time frame, high importance should be ascribed to the establishment of an adequate interlaboratory validated analytical method to quantify, at the existing 5% limit, the low weight-average molecular weight fraction of carrageenan, and to establish whether or not this fraction is associated with health risks.
The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) further concluded that in the refined brand-loyal exposure assessment scenario the exposure estimates exceeded, in some cases by up to approximately 10-fold, the temporary existing acceptable daily intake (ADI) at the high level (95th percentile) for all population groups and at the mean for all population groups except for infants and the elderly. Although the exposure may be overestimated the extent of the exceedance of the acceptable daily intake (ADI) (10-fold) may be a safety concern 2.
Potential health benefits of carrageenan
Carrageenans have shown potential bioactive qualities, including antiviral, antibacterial, antihyperlipidemic, anticoagulant, antioxidant, antitumor, and immunomodulatory properties 63. Figure 4 shows a summary of these properties and the carrageenan types that display them.
In Table 2, the bioactive properties of carrageenan and its various applications in the biomedical field are summarized.
Table 2. Bioactive properties of carrageenan and their biomedical applications
|Bioactive Properties||Type of carrageenan||Applications||Reference|
|Inhibits Herpes Simplex Virus (HSV), Human Papillomavirus (HPV), Varicella Zoster Virus (VZV) and Human Rhinoviruses||64|
|Lambda-carrageenan/kappa-carrageenan||Bioactivity against HPV and HSV-2||65|
|carrageenan||Genital HPV infection||66|
|Iota-carrageenan||Reduction in cold symptoms and reduces the growth of Human Rhinoviruses (HRV)||67|
|Iota-carrageenan||Potential inhibitor of the Influenza A Virus infection||68|
|Kappa-carrageenan||H1N1/2009 and other similar viruses||69|
|Influenza A Virus strains (pandemic H1N1/09, H3N2, H5N1, H7N7)||70|
|Iota-carrageenan||Human Rhinovirus (HRV) 1a, hRV8 and Human Coronavirus OC43||71|
|Kappa-carrageenan||Enterovirus 71 (EV 71)||72|
|Lambda-carrageenan||Rabies Virus (RABV)||73|
|Varicella Zoster Virus (VZV)||74|
|Iota-carrageenan||Severe acute respiratory syndrome coronavirus 2|
|Antibacterial effects||Iota-carrageenan||Inhibits the growth of the bacterial strains||76|
|Iota-carrageenan||Ocular Chlamydia trachomatis infection||77|
|Kappa-carrageenan||Activity against Saccharomyces cerevisiae||78|
|Kappa-carrageenan||Activity against Gram-positive and Gram-negative bacteria||79|
|Kappa-carrageenan||Reduced the production of interleukin-6 in cells treated with kappa-carrageenan||80|
|Kappa-carrageenan||Activity against S. aureus and E. coli||81|
|Kappa-carrageenan||Activity against S. aureus, Bacillus cereus, E. coli and Pseudomonas aeruginosa||82|
|Antihyperlipidemic effects||carrageenan||Hypocholesterolemic effect||83|
|Kappa-carrageenan||Reduces serum levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol (LDL-C), and increasing high-density lipoprotein cholesterol (HDL-C)||84|
|Modulate prostaglandin E2 synthesis and stimulate IL-1β and IL-6 synthesis||85|
|Kappa-carrageenan/iota-carrageenan||Reduces in serum levels of total cholesterol||86|
|Anticoagulant and antithrombotic activity||Lambda-carrageenan||Highest anticoagulant activity in the rabbit whole blood test||89|
|Antitumor and immunomodulatory activity||Lambda-carrageenan||Anticancer effects, immunomodulation||91|
|Lambda-carrageenan||Improve the antitumor activity of 5-Fluorouracil||92|
|Lambda-carrageenan||Inhibits tumour growth in mice with murine melanoma cell lines||93|
|Kappa-carrageenan delays the cell cycle in the G2/M phase|
Lambda-carrageenan stalled the cell cycle in both the G1 and G2/M phase
|Iota-carrageenan||Suppressed tumour growth, induced apoptosis, and halted the G1 phase||95|
|Cytotoxic effect on LM2 tumour cells||96|
|Lambda-carrageenan/epsilon-carrageenan||Inhibits colorectal cancer stem-like cells||97|
|Antitumour and immunotropic effects||98|
|Antioxidant activity||Kappa-carrageenan||Antioxidant activity in the multilayer coating||99|
Figure 4. Bioactive properties of carrageenan20 ]
The antiviral effect of carrageenan is due to the specific screening of the cellular structures involved in binding the virus to its receptor 100. For example, the antiviral activity of lambda-carrageenan may be due to the irreversible formation of stable virion-carrageenan complexes, and therefore, the viral envelope sites essential for the binding of the virus to the host cells are occupied by carrageenan, preventing the virus from completing the infectious process 101.
Carrageenan is a selective inhibitor of several viruses, including the herpes simplex virus (HSV), the human papillomavirus (HPV), the varicella zoster virus (VZV), human rhinoviruses, and others 102.
Several in vitro (test tube studies)/in vivo (animal) studies have reported that carrageenans act as potent and selective inhibitors of the herpes simplex virus-1 (HSV-1) and the herpes simplex virus-2 (HSV-2) 103. The combination of carrageenan with lectin has also shown strong activity against HSV-2 and human papillomavirus (HPV) in in vitro and in vivo studies 104. Carrageenan has demonstrated activity against HPV, and is 1000 times more effective against HPV (human papillomavirus) in cell culture tests than against HSV (herpes simplex virus) and HIV (human immunodeficiency virus). Carrageenan acts primarily by preventing the binding of HPV virions to cells 105.
Carrageenans have been applied with other active ingredients to enhance or direct their bioactivity against HPV and HSV-2. An intravaginal ring was reported containing four active ingredients: lambda-/kappa-carrageenan, zinc acetate, levonorgestrel, and MIV-150 (microbicide); this formulation had a prolonged action and allowed protection against HIV-1, HSV-2, HPV, and unwanted pregnancies 106.
More recently, Perino et al. 66 evaluated the safety, satisfaction, and antiviral effect of a new carrageenan-based vaginal microbicide in a population of fertile patients with genital human papillomavirus. The gel was formulated with 0.02% from different types of carrageenan and Propionibacterium extract, and it was administered in two treatment phases. During the first phase, gel therapy was applied once daily for 30 days continuously; the second phase began with an application on alternate days for 45 applications. In both phases, the patients could also have sexual intercourse without a condom. The results showed that 60% of the patients presented negative HPV; the gel was safe and well tolerated by women. This research also supports the hypothesis that carrageenans play a role in accelerating the normal clearance of genital HPV infection in women with a positive HPV-DNA test 66.
Iota-carrageenan was active against respiratory viruses in vitro and was effective as a nasal spray in clinical trials. Eccles et al. 107 investigated iota-carrageenan in patients with early common cold symptoms. Their results indicated a significant reduction in cold symptoms in the iota-carrageenan group compared to placebo during the first four days when symptoms were most severe and also corroborated that iota-carrageenan reduces the growth of human rhinoviruses (HRVs) and inhibits the virus-induced cytopathic effect of infected HeLa cells 107. Consequently, it is observed that iota-carrageenan, like the other types of carrageenan, acts by preventing the binding or entry of virions into host cells 108.
Iota-carrageenan was tested as a potential inhibitor of the influenza A virus infection. Half-maximal inhibitory concentration (IC50) is the most widely used and informative measure of a drug’s efficacy. The half maximal inhibitory concentration is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of potency of an antagonist drug in pharmacological research. The inhibitory potential of iota-carrageenan with IC50 values of around 0.2 µg/mL in H1N1 and 0.04 µg/mL in H3N2 infections was up to 10 times higher than with kappa-carrageenan 68. The authors thus confirmed that iota-carrageenan reduced the spread of the influenza virus in the surface epithelia of infected animals, providing sufficient benefit for animals to promote survival. This research determined that iota-carrageenan was safe and effective in the treatment of influenza infection, even above other types of carrageenan, making it a suitable antiviral candidate for the prophylaxis and treatment of this infection 68.
Shao and co-workers 69 investigated the ability of kappa-carrageenan to inhibit the swine flu pandemic 2009 H1N1 influenza virus. The results demonstrated that kappa-carrageenan could significantly inhibit A/Swine/Shandong/731/2009 H1N1 (SW731) replication by interfering with a few replication steps in the SW731 life cycles, including adsorption, transcription, and viral protein expression. Kappa-carrageenan inhibited SW731 mRNA and protein expression after internalization into cells. This indicates that kappa-carrageenan may be suitable for use against H1N1/2009 and other similar viruses 69, but this is not always the case, since in the study previously described, kappa-carrageenan was less effective against influenza A virus infection. This disadvantage related to antiviral efficacy could be related to various factors, such as the molecular weights, sulfate content, and the origin of carrageenans.
Many authors have proposed improving the antiviral effectiveness of carrageenan by combining it with other drugs. The combination of an anti-influenza drug Zanamivir with carrageenan in a nasal formulation was evaluated in vitro and in vivo, and the results indicated that carrageenan and Zanamivir act synergistically against several influenza A virus strains (pandemic H1N1/09, H3N2, H5N1, H7N7). In this study, it can be observed that the combination of two types of carrageenan (iota-carrageenan and kappa-carrageenan) and an antiviral drug increase the efficacy of the formulation, since it was observed that the combined use of the compounds significantly increases the survival of infected animals in comparison with mono-therapies or placebo 70.
A recent study reported the evaluation of a nasal spray containing xylometazoline hydrochloride and iota-carrageenan 71. In vitro experiments revealed that the combination of the vasoconstrictive properties of the drug and the antiviral activity of iota-carrageenan were effective against human rhinovirus (hRV) 1a, hRV8, and human coronavirus OC43. In this study, it was clearly observed that the combination of iota-carrageenan with the drug does not produce a negative effect, but rather the efficacy and safety of both components remain unchanged, producing the desired therapeutic effect, showing that the combination of carrageenan with drugs fails to produce harmful interactions. The formulation was well tolerated at the application site, with no occurrence of erythema or edema in the nostrils of any of the rabbits or any signs of toxicity in any of the organs and tissues examined 71. These studies generally mentioned that carrageenan created a physical barrier in the nasal cavity against respiratory viruses, preventing their binding to host cells 109.
Chiu et al. 72 reported that kappa-carrageenan has a strong and effective anti-enterovirus 71 (EV 71) activity able to reduce plaque formation, prevent viral replication before or during viral adsorption, and inhibit EV 71-induced apoptosis. In the virus binding assay, they demonstrated that kappa-carrageenan can bind firmly to the EV 71 to form carrageenan-virus complexes, so the virus–receptor interaction is likely to be disrupted 72.
The potential role of lambda-carrageenan P32 on the inhibition of the rabies virus (RABV) has also been studied, and the results show it to be a promising anti-rabies virus agent that can effectively inhibit RABV infection in vitro by affecting viral internalization and cell fusion mediated by viral G protein 73. A comparison between lambda-carrageenan P32 and the P32 structural analogues (heparan sulphate and heparin) revealed that the effect of P32 inhibition on rabies virus infection was stronger than heparan sulphate and heparin, suggesting that the notable anti-rabies virus activity of P32 can be attributed to more than its structural similarity to heparan sulphate. The authors also investigated the inhibitory effect of lambda-carrageenan on the vesicular stomatitis virus (VSV), and observed a null inhibition 73.
A more recent study by Abu-Galiyun et al. 74 reported the inhibitory effects of kappa-carrageenan, iota-carrageenan, lambda-carrageenan, and other natural polysaccharides on varicella zoster virus (VZV) infection in vitro. Almost all the polysaccharides tested were very active against varicella zoster virus compared to acyclovir as a reference drug and exhibited dose-dependent behavior. The results suggested that iota-carrageenan may inhibit the early step/s of the virus infection, such as virus attachment or penetration to the host cells, and the late step/s after the penetration of the virus into the host cells, showing that iota-carrageenan has strong antiviral activity on various types of viruses 74.
Finally, a recent study, developed during the Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), determined that marine sulfated polysaccharides, such as iota-carrageenan, can inhibit viral binding and entry into host cells. Iota-carrageenan could prevent the infection at concentrations ≥125 μg/mL 75. Morokutti-Kurz and colleagues show that iota-carrageenan can inhibit the cell entry of the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike pseudotyped lentivirus in a dose dependent manner 110. SARS-CoV-2 spike pseudotyped lentivirus particles were efficiently neutralized with an IC50 value of 2.6 μg/ml iota-carrageenan. Experiments with patient isolated wild type SARS-CoV-2 virus showed an inhibition of replication in a similar range. In vitro data on iota-carrageenan against various Rhino- and endemic Coronaviruses showed similar IC50 values and translated readily into clinical effectiveness when a nasal spray containing iota-carrageenan demonstrated a reduction of severity and duration of symptoms of common cold caused by various respiratory viruses 110. Therefore, it was observed that marine polysaccharides have adequate antiviral activity in various viruses, inhibiting viral binding, which allows their use in the treatment and prevention of Coronavirus disease 2019 (COVID-19) 111, 112.
In general, studies have shown that the antiviral efficacy of carrageenan is very broad, which would make it possible to suppress the replication of various viruses with or without envelopes. The antiviral effect of carrageenan occurs alone or in combination with other compounds, such as drugs, and this combination allows substantial improvement of the therapeutic efficacy and generally does not produce interactions. The antiviral activity of carrageenan continues to be promising since various viral strains, such as SARS-CoV-2, continue to be analyzed, and these polysaccharides fulfill this proposed antiviral efficacy, but there is still a long way to go in order to fully evaluate the efficacy and safety of these polysaccharides as highly effective antiviral components.
Carrageenan antibacterial activity
Carrageenan can inhibit infection caused by a variety of bacteria. Yamashita et al. 76 evaluated the antimicrobial action of dietary polysaccharides on foodborne pathogenic bacteria and showed that carrageenan had the most pronounced inhibitory effect of all polysaccharides, significantly inhibiting the growth of almost all the bacterial strains studied. A growth-inhibition experiment using Salmonella enteritidis showed that the inhibitory effect of the carrageenan was bacteriostatic. The investigation also indicated that the removal of sulphate residues eliminates the bacteriostatic effect of iota-carrageenan, suggesting that the sulphate residue (s) in carrageenan play an essential role in this effect 76, being very similar to that observed in the antiviral effect; thus, the sulfate content and the molecular weight are characteristics that determine the bioactive capacity of carrageenan.
In other research, Inic-Kanada and co-workers 77 tested the effects of iota-carrageenan on ocular Chlamydia trachomatis infection. Iota-carrageenan tends to reduce the infectivity of Chlamydia trachomatis in vitro and when it was evaluated in vivo, the results showed slightly reduced ocular pathology and significantly less shedding of infectious elementary bodies, which suggested that iota-carrageenan could be a promising agent for reduction of the transmission of ocular chlamydial infection, with more in-depth studies to support these first results 77.
Another way to evaluate the antibacterial efficacy of carrageenans was through the use of kappa-carrageenan oligosaccharides against Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae, Penicillium citrinum, and Mucor spp., determining the antibacterial efficacy by measuring the diameter of the inhibitory zone. Consequently, it was observed that all kappa-carrageenan oligosaccharides had inhibitory activity against the bacteria studied, presenting a greater inhibitory activity against Saccharomyces cerevisiae 78.
Another work evaluated a modification of carrageenan as an antibacterial agent (oxidized kappa-carrageenan). The results mentioned that the oxidized kappa-carrageenan could damage the bacterial cell wall and cytoplasmic membrane and suppress the growth of both Gram-positive and Gram-negative bacteria. Oxidized kappa-carrageenan possessed broad-spectrum antibacterial activity and could be a suitable candidate for the development of a new antibacterial agent, requiring further studies 79.
Kappa-carrageenan was added to a sinus rinse that is commercially available on the Australian market (Flo CRS® and Flo Sinus Care®) and applied directly to cultured human primary nasal epithelial cells at the air–liquid interface, and to human bronchial epithelial cells in the presence of different Staphylococcus aureus strains 80. The addition of kappa-carrageenan to commercially available sinus rinses reduced the production of interleukin-6 in cells treated with kappa-carrageenan and Flo Sinus Care®. The addition of kappa-carrageenan to both Flo CRS® and Flo Sinus Care® rinses also reduced the intracellular infection rate by an average of 2% 80. These results suggest that carrageenan can be used in inhalation drug delivery systems, inhibiting infection caused by a facultative Gram-positive anaerobic bacterium.
PVA, a hydrogel based on carrageenan crosslinked with silane, showed strong antibacterial activity against Staphylococcus aureus and slight activity against E. coli. The antibacterial activity may be due to the interaction of carrageenan molecules with the cell membrane of the bacterial strains, and more specifically with the structure of the carrageenan, as carrageenan comprises negatively charged SO42− suspended groups, and Staphylococcus aureus (Gram-positive) has an outer covering of mucopeptide and peptidoglycan lipids; whereas E. coli consists of phospholipids and lipopolysaccharides, which gives its surface a strongly negative charge. These interactive sites on the Gram-positive bacteria favor the alteration of the bacterial cell membrane that controls bacterial growth. The authors also indicate a second factor that could control bacterial growth, namely the bonding of carrageenan and PVA with the DNA of the bacterial strain, thereby limiting transcription and translation by DNA 81.
A recent study described the carboxymethylation of kappa-carrageenan with monochloroacetic acid to achieve different degrees of substitution of carboxymethyl-kappa-carrageenan, in order to improve the properties of the polysaccharide. In antibacterial assays, carboxymethyl-kappa-carrageenan with degrees of substitution of 0.8, 1.0, and 1.2 exhibited growth inhibition against S. aureus, Bacillus cereus, E. coli, and Pseudomonas aeruginosa 82. The antibacterial activity could be due to the presence of sulphate, and carboxylate groups may create an acidic pH environment; it is also possible that the carboxylate groups could increase the nucleophilicity of the polymer. Although the authors recommended further studies on this subject, they noted that antioxidant, antibacterial, and biocompatibility tests could confirm potential applications of this polymer, such as for wound dressings and scaffolds 82
Studies that describe antibacterial effects tend to be scarcer compared to antiviral effects, and perhaps this is because in most cases, the antibacterial effect of carrageenan occurs when they are modified by various processes, such as oxidation or carboxymethylation, thus allowing fulfilment of the antimicrobial effect in few bacteria.
Carrageenan antihyperlipidemic effects
Carrageenan also presents biological activity in the gastrointestinal tract, generally associated with oral administration. When ingested it increases the viscosity of the intestinal content and decreases the rate of digestion and absorption, which in turn reduces the diffusion of enzymes, substrates, and nutrients in the intestinal absorption phase, resulting in a lower absorption of nutrients, including cholesterol (the hypocholesterolemic effect) 83. In other words, the bioactive potential of carrageenan derives from its ability to decrease the cholesterol absorption rate and increase the rate of synthesis of endogenous cholesterol 113.
Several research works have demonstrated the bioactive role of carrageenan, mainly kappa-carrageenan, in reducing serum levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-C also known as “bad” cholesterol), and increasing high-density lipoprotein cholesterol (HDL-C also known as “good” cholesterol) in the peripheral blood, due to the interaction of their chemical structure in digestion 84.
Sokolova et al. 85 reported the effect of kappa-carrageenan, kappa/β-carrageenan and iota/kappa-carrageenan individually and in combination with lipopolysaccharide on the synthesis of prostaglandin E2 and cytokines (interleukin [IL]-1β and IL-6) in a whole blood model in vitro. At high concentrations, carrageenans have a substantial ability to modulate prostaglandin E2 synthesis and stimulate IL-1β and IL-6 synthesis, confirming the possible mechanism of the cholesterol-reducing properties of carrageenan 85.
In a recent study, the authors evaluated the bioactive potential of carrageenan in the lipid profile in individuals with total cholesterol levels equal to or higher than 200 mg/dL after the ingestion of a jelly composed of a hybrid kappa-carrageenan/iota-carrageenan polysaccharide 86. In total, 100 mL of jelly per day were ingested for 30 and 60 days. The results showed a statistically significant decrease in total cholesterol and high-density lipoprotein cholesterol (HDL-C) in both periods (30 and/or 60 days). Daily intake for 60 days also showed a reduction in serum levels of total cholesterol and low-density lipoprotein cholesterol (LDL-C) in women 86.
Kappaphycus alvarezii (red seaweed) was used as a whole-food supplement to attenuate the development of obesity in rats fed a high-carbohydrate high-fat diet that mimics symptoms of human metabolic syndrome, including central obesity, hypertension, dyslipidemia, and impaired glucose tolerance, coupled with the cardiovascular and liver complications of metabolic syndrome 87. The study highlighted the potential of Kappaphycus alvarezii (red seaweed) as a functional food with possible application for the prevention of metabolic syndrome. The researchers also demonstrated that Kappaphycus alvarezii (red seaweed) may reverse metabolic syndrome through the selective inhibition of obesogenic gut bacteria and the promotion of health-promoting gut bacteria 87.
A new in vivo study investigated the potential of red seaweed (Sarconema filiforme) as a functional food for the reversal of metabolic syndrome and its possible mechanisms in male Wistar rats 88. Rats fed a high-carbohydrate high-fat diet supplemented with red seaweed (Sarconema filiforme) as a source of iota-carrageenan decreased their body weight, systolic blood pressure, abdominal and liver fat, and plasma total cholesterol concentrations compared to controls. Iota-carrageenan attenuates symptoms of diet-induced metabolic syndrome in rats. The correlations between changes in the gut microbiota and physiological changes following administration of red seaweed (Sarconema filiforme) suggest that the likely mechanism is that carrageenans act as prebiotics, and through systemic anti-inflammatory responses in organs, such as the heart and liver 88.
The antihyperlipidemic effect of carrageenan is little studied. It should be mentioned that the main advantages at the metabolic level produced by carrageenan are due to the fact that they are not degraded or absorbed in the gastrointestinal tract, allowing a decrease in the rate of digestion and absorption of all nutrients included in the diet 20.
Carrageenan antioxidant activity
Studies carried out in recent years have shown that carrageenan also has significant antioxidant activity, a property associated with sulphate group content 82. Gomez-Ordoñez et al. 114 tested carrageenan sequentially extracted from Mastocarpus stellatus with water, acid, and alkali. Extraction with water produced polysaccharides with the highest degree of sulfation and the highest molecular weight. The analysis of these polysaccharides showed that they had the best results in terms of in vitro antioxidant and anticoagulant capacity, confirming that the number of sulphate groups influences antioxidant activity, and that a high molecular weight plays a role in anticoagulant capacity. These authors found that the different extraction methods influence the bioactive capacity of the carrageenan 114. Another study demonstrated the antioxidant capacity of a multilayer coating based on kappa-carrageenan and lecithin/chitosan loaded with quercetin using the layer-by-layer technique, and determined that the chemical structure of the layers is an important factor in obtaining antioxidant activity in the multilayer coating 99.
Carrageenan anticoagulant and antithrombotic activity
Anticoagulant activity could be influenced by the glycosidic bond (either (1 → 3) or (1 → 4)) and the neighboring sulphate groups 115. Liang et al. 89 studied the anticoagulant potential of different types of carrageenan and their derivatives: kappa-carrageenan oligosaccharide, sulphated kappa-carrageenan, and desulphated kappa-carrageenan. Lambda-carrageenan demonstrates the highest anticoagulant activity in the rabbit whole blood test, while kappa-carrageenan oligosaccharide and desulphated kappa-carrageenan show no anticoagulant activities. In the case of oligosaccharide, this is probably due to the lack of a secondary structure caused by the decrease in molecular weight, and in desulphated kappa-carrageenan to the absence of favorable sulphate groups. The substitution position of the sulphate groups has a greater impact than the degree of substitution on both anticoagulant activity and cell proliferation. C-2 of 3,6-anhydro-α-d-Galp is the most favorable position for substitution, whereas C-6 of β-d-Galp is the most disadvantageous. Secondary structures of glycans also play a key role in biological activities 89.
Among carrageenan types, lambda-carrageenan has approximately twice the activity of unfractionated carrageenan and four times the activity of kappa-carrageenan 40. Lambda-carrageenan showed greater antithrombotic activity than kappa-carrageenan, probably because anticoagulant or antithrombotic activity is influenced by the number of sulphate groups present in the chemical structure. The main basis for carrageenan’s anticoagulant activity appears to be an antithrombotic property 64.
In a recent study, selective oxidation was performed on five carrageenan-kappa-carrageenan, iota-carrageenan, iota/nu-carrageenan, Theta-carrageenan, and lambda-carrageenan at C-6 of the β-d-Galp in order to evaluate the in vitro anticoagulant activity of oxidized derivatives 90. The results showed a synergic effect of the carboxyl groups on the anticoagulant activity, which was dependent on the regiochemistry of the sulphate groups in the polysaccharide backbone. Sulphate groups at C-2 of the β-d-Galp units appeared to positively influence the anticoagulant effect compared to C4-sulphate samples. The partially oxidized kappa-carrageenan derivative also exhibited a better anticoagulant effect than the fully oxidized carrageenan. These results support the synthesis of new carrageenan derivatives to increase anticoagulant properties 90. These results show that carrageenans have anticoagulant and antithrombotic properties but require structural modifications that allow them to develop this bioactive activity. This leads to the development of chemical processes that perhaps hinder studies in this area, with few published works being observed.
Carrageenan anticancer and immunomodulatory activity
Several studies have shown that carrageenan has immunomodulatory and antitumor activity 91. The antitumor activity of carrageenan could be related to the destabilization of the interaction of the glycosaminoglycans (GAGs) portion of the proteoglycans and the extracellular matrix proteins, thus eliminating the adhesion of cancer cells to matrices, which is necessary for the spread of metastasis 116. The bioactive properties of carrageenan depend on their chemical structure, molecular weight, and the quantity and position of sulfation, so lambda-carrageenan can be degraded into several products with different molecular weights, all exhibiting anticancer effects, probably through immunomodulation. The lower molecular weight compounds, 15 and 9.3 kDa, showed higher anticancer and immunomodulation effects 91.
Carrageenan has been considered as an adjuvant in cancer immunotherapy. Tumor-inhibiting activity of low-molecular-weight lambda-carrageenan and its mixture with 5-Fluorouracil (5-Fu) on mice transplanted with S180 tumor was investigated 92. The results suggested that the degraded lambda-carrageenan could enhance the antitumor activity of 5-Fu and improve immunocompetence damaged by 5-Fu 92. Lambda-carrageenan was also reported to inhibit tumor growth in mice with murine melanoma cell lines and murine mammary tumor cell lines through intratumoral injection. Lambda-carrageenan exhibited an efficient adjuvant effect in an ovalbumin-based preventative and therapeutic vaccine for cancer treatment, which significantly enhanced the production of anti-ovalbumin antibody. The toxicity analysis suggested that lambda-carrageenan had a good safety profile. Therefore, this study suggests that lambda-carrageenan could be used as a potent antitumor agent and as an adjuvant in cancer immunotherapy 93.
The effects of carrageenan on the tumor cell cycle were investigated using human cervical carcinoma cells (HeLa) and human umbilical vein endothelial cells (HUVECs) 94. The results indicated that carrageenan interrupted the cell cycle at specific stages and delayed the time necessary for the cell to progress through the cell cycle. Kappa-carrageenan was found to delay the cell cycle in the G2/M phase, while lambda-carrageenan stalled the cell cycle in both the G1 and G2/M phase. Lambda-carrageenan also suppressed the cell’s ability to divide, demonstrating a strong antiproliferative effect 94. Degraded iota-carrageenan suppressed tumor growth, induced apoptosis, and halted the G1 phase, which improved the survival rate of tumor-bearing mice 95.
Calvo et al. 96 tested purified native and degraded carrageenans and their disaccharides, obtained from extracts of the potential cytotoxic and antitumor compounds Hypnea musciformis, Iridaea undulosa and Euchema spinosumas. The results showed that kappa-carrageenan and iota-carrageenan and carrageenan oligosaccharides had a cytotoxic effect on LM2 tumor cells. Some oligosaccharides are also more cytotoxic than their parent compounds, indicating that a lower molecular weight is one of the factors that improves its cell ability. These results point to the potential use of disaccharide units, such as carrabioses coupled to antineoplasics, to improve their cytotoxicity and antimetastatic properties, and the use of iota-carrageenan as an adjuvant or carrier in anticancer treatments 96.
In a recent study, Cotas et al. 97 showed that the carrageenans extracted from the two Gigartina pistillata life cycle phases, particularly the T (tetrasporophyte) carrageenan, have potential against colorectal cancer stem-like cells. This could be explained by the higher content of sulphated ethers in T carrageenan (lambda-carrageenan/epsilon-carrageenan) compared to the female gametophyte carrageenan (kappa-carrageenan/iota-carrageenan) 97.
Antitumor and immunotropic effects were reported in kappa-carrageenan and lambda-carrageenan isolated from Chondrus armatus and their low-molecular-weight degradation products 98. The results showed that low-molecular-weight carrageenan degradation products not only retain the biological activity of their high-molecular-weight precursors but also increase their efficacy in a type-dependent manner. carrageenan degradation is a viable solution to increase their biomedical applicability by overcoming the limitations of their chemical and physical properties 98.
Within the antitumor activity, it is completely clear that the effect is fulfilled when carrageenans are modified or degraded, since these processes have been a viable solution to increase the biomedical applicability of carrageenans, overcoming certain limitations of their chemical and physical properties. Furthermore, many studies agree that low molecular weight influences the therapeutic efficacy of carrageenan.
Carrageenan in Drug Delivery Systems
Initially, excipients were used almost exclusively as components that contributed to the manufacturing processes of the pharmaceutical forms, but, thanks to the boom in the obtaining, purification, and use of biodegradable, biocompatible, and non-toxic compounds in pharmaceutical formulations, it led to their transformation into multifunctional components, which can provide bioactive and functional properties in drug development 20. Hence, the biomedical importance of carrageenan as bioactive components that make it possible to improve pharmaceutical formulations and in certain cases be adjuncts to other active principles. The biological and chemical properties of carrageenan are the main reasons they are used in drug delivery systems 117.
The chemical structure of carrageenan is a factor that explains the increase in its applications in drug delivery systems, since it has three important characteristics 118: (a) its glycosidic bonds allow it to be cleaved by hydrolase enzymes, producing biodegradability; (b) the sulphate groups in the carrageenan are anionic and enhance the behavior of polyelectrolytes; and (c) the presence of hydroxyl groups provides the necessary interactions to produce chemical modifications.
Carrageenans are applied in various pharmaceutical formulations, including tablets 119, suppositories 120, films 121, fast-dissolving inserts 122, beads 123, pellets 124, microparticles 125, nanoparticles 126, inhalable systems, injectables 127 and hydrogels 128. In addition, recent studies have shown that carrageenans are promising candidates in tissue engineering, thanks to their similarity to native glycosaminoglycans 129.
Carrageenans perform various functions in these pharmaceutical formulations, ranging from the formation of matrices, stabilizers, binders, disintegrators, solubilizers, thickeners, and coatings, to more complex processes, such as drug release control 20. Often, they do not fulfil a single function but are cumulative. Carrageenan readily form a gel, so they are commonly used in drug delivery systems, generally formed through heat-reversible gelation, ionic crosslinking, and the modification of the carrageenan main chain 20. The chemical structure determines their functional properties, stability, biodegradability, and biocompatibility.
Carrageenan Foods and Other Uses
- Desserts, ice cream, cream, milkshakes, yogurts, salad dressings, sweetened condensed milks
- Sauces: to increase viscosity
- Beer: clarifier to remove haze-causing proteins
- Pâtés and processed meats (e.g., ham): substitute for fat, increase water retention, increase volume, or improve slicing
- Toothpaste: stabilizer to prevent constituents separating
- Fruit Gushers: ingredient in the encapsulated gel
- Fire fighting foam: thickener to cause foam to become sticky
- Shampoo and cosmetic creams: thickener
- Air freshener gels
- Marbling: the ancient art of paper and fabric marbling uses a carrageenan mixture on which to float paints or inks; the paper or fabric is then laid on it, absorbing the colours
- Shoe polish: to increase viscosity
- Biotechnology: to immobilize cells and enzymes
- Pharmaceuticals: used as an inactive excipient in pills and tablets
- Soy milk and other plant milks: to thicken
- Diet sodas: to enhance texture and suspend flavours
- Pet food
- Personal lubricants
- Vegetarian hot dogs
Clinical studies in infants
Carrageenan is currently used in some cow milk– and soy-based formulas. These include both powders and liquids. The predominant type of carrageenan used in infant formula is kappa-carrageenan (κ-carrageenan). The typical level of carrageenan used in reconstituted powdered and liquid cow milk– and soy-based formulas is 0.009–0.1 g/100 mL (90–1000 mg/L), with the higher levels being used in formulas containing hydrolysed proteins.
There are brief reports of two studies in human infants given formula containing carrageenan.
In one study, 1269 full-term infants given liquid formula containing carrageenan at 300 mg/L (0.03%) for the first 6 months of life were compared with 149 infants given powder-based formula not containing carrageenan for frequency of symptomatic upper respiratory tract infection. There were no statistically significant differences between the two groups; a slightly higher proportion of infants given formula containing carrageenan were illness-free during the first 6 months of life. The authors concluded that carrageenan-containing liquid infant formula is not immunosuppressive 130, 131.
In a masked, randomized study on healthy newborn infants aged 0–9 days at enrolment in the study, 95 infants were fed powdered casein hydrolysate–based formula that did not contain carrageenan, and 100 infants were fed liquid ready-to-feed casein hydrolysate–based formula containing carrageenan at 1000 mg/L (0.1%). This is at the high end of carrageenan concentrations used in formulas for special medical purposes. One hundred and thirty-seven infants completed the study; no information was given on the numbers in each of the two groups at the end of the study, but intolerance to the formulas accounted for dropout for 21 infants in the group fed formula containing carrageenan and 16 in the group fed formula not containing carrageenan. Intake, stool patterns and anthropometric measurements were monitored at entry and on days 14, 28, 56, 84 and 112 of the study. There were no differences between the two groups in weight gain, length, head circumference or tolerance to the formulas. Infants on powdered formula had significantly lower intakes of formula and passed significantly fewer stools per day. Stool consistencies were similar except for liquid formula infants, who had firmer stools on entry 132.
In 2003, the Scientific Committee for Food re-evaluated carrageenan in the revision of the essential requirements of infant formulae and follow-on formulae intended for the feeding of infants and young children 9. The Scientific Committee for Food reconfirmed its view that carrageenan should not be used in infant formulae. However, the Committee had no objection to the use of carrageenan in follow-on formulae up to a maximum level of 0.3 g/L. ‘The Scientific Committee for Food further recommended maintaining the concept that if more than one of the three substances locust bean gum, guar gum or carrageenan are added to a follow-on formula, the maximum level established for each of those substances is lowered with that relative part as is present of the other substances together 9.
In its 2015 evaluation, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) reviewed data published since 2007 taking into account new studies relevant to the evaluation of the use of carrageenan in infant formula and formula for special medical purposes, including studies on absorption and toxicity in both the neonatal minipig and neonatal pig 10. JECFA also considered other data available in the literature related to carrageenan and to the signalling pathways involved in inflammation. The Committee noted that although the margins of exposure were small, these were derived from a neonatal pig study in which no adverse effects on the gut or on immune parameters were observed at the highest dose tested 10. JECFA also took into account the previous toxicological database on carrageenan, which did not indicate other toxicological concerns. Overall, JECFA concluded that ‘the use of carrageenan in infant formula or formula for special medical purposes at concentrations up to 1,000 mg/L is not of concern’ 10.
In conclusion, in the absence of any further information on possible absorption of carrageenan by the immature gut in the very young infant, the Scientific Committee for Food reaffirms its earlier view 133 that it remains inadvisable to use carrageenan in infant formulae that are fed from birth, including those in the category of foods for special medical purposes. The Scientific Committee for Food has no objection to the use of carrageenan in foods for older infants, such as follow-on milks 134 and weaning foods 135.
Carrageenan side effects
Carrageenan has been proven safe for human consumption; however, there has been significant confusion in the literature between carrageenan and the products of intentional acid-hydrolysis of carrageenan, which are “degraded carrageenan” and poligeenan 2. In part, this confusion was due to the nomenclature used in early studies on carrageenan, where poligeenan was referred to as “degraded carrageenan” and “degraded carrageenan” was simply referred to as carrageenan 2. Although this nomenclature has been corrected, confusion still exists resulting in misinterpretation of data and the subsequent dissemination of incorrect information regarding the safe dietary use of carrageenan 2. The lack of understanding of the molecular weight distribution of carrageenan has further exacerbated the issue. Degraded carrageenan and poligeenan are not food additives and have a completely different physical/chemical and toxicological properties from carrageenan 29. Poligeenan can only be formed when carrageenan is subjected to harsh acid hydrolysis under laboratory conditions [non-physiological acid hydrolysis (pH of 0.9–1.3) and non-physiological temperatures >80°C for several hours)] 2. The resulting liquor is neutralized to about pH 7.5 and the poligeenan isolated by roll-drying or spray drying. This degradation converts carrageenan with Mw = 200 kDA to 800 kDa. to poligeenan with Mw = 10 kDA – 20 kDa. 50. The biological and toxicological activity of poligeenan is completely different from carrageenan. This is due to the lower molecular weight of poligeenan (Mw = 10,000 – 20,000 Da.), which reduces the strength of protein binding and which may allow absorption from the gut and interaction with cell systems. In comparison, the high molecular weight of carrageenan (Mw = 200 kDA to 800 kDa.), combined with its strong affinity for proteins means that it is not absorbed from the gut and does not interact with cellular processes of the intestinal mucosal surface 2.
Studies in a variety of species of experimental animals, including rats, guinea pigs and primates, have shown that there is negligible absorption of food grade carrageenan from the gastrointestinal tract 136, 58. A review of carrageenan (E 407) and processed Eucheuma seaweed (E 407a) was published from the Nordic Council of Ministers. In the report, it was concluded for carrageenan (E 407) that: ‘Carrageenan has been extensively studied and in most studies seems to pose no toxicological problem 57.
Carrageen an has been extensively evaluated for genotoxicity (agents that damages the DNA) using a range of in vitro and in vivo assays, including the bacterial reverse mutation assay ± S9 fraction, sister chromatid exchange assays, cytogenetic assays, rec-assay in Bacillus subtilis, mouse micronucleus assay, host-mediated assays and dominant lethal assays in rats. Results of these various assays have been negative 137.
There is no robust evidence of allergic reactions to ingested carrageenan 138.
In the toxicity studies, in addition to a wide range of toxicological parameters, a detailed examination of the histology of all segments of the gastrointestinal tract and quantification of mast cells along the gastrointestinal tract were undertaken in both the minipig and pig. In the pig, an appropriate array of serum and gut cytokines was also assessed, together with blood leukocyte immunophenotyping. From these new investigations, there was no evidence of any inflammation in the gut or any effects on immune parameters. A no-observed-adverse-effect-level (NOAEL) of 430 mg/kg body weight per day, which was the highest dose tested, was derived from the neonatal pig study. The no-observed-adverse-effect-level (NOAEL) of 430 mg/kg body weight per day from the neonatal pig study is almost identical to that from the earlier infant baboon study of 432 mg/kg body weight per day 58.
In the 10-day neonatal minipig study, animals were given infant formula containing carrageenan at concentrations up to 3000 mg/kg (0.3%). Concentrations of carrageenan above approximately 2500 mg/kg (0.25%) become highly viscous, and this appears to have adversely affected palatability and growth in the minipigs. Accordingly, the amount of carrageenan added to the formula fed to piglets in the main study was reduced to 2250 mg/kg (0.225%). As a consequence of this limitation, the margins of exposure (MOEs) between the no-observed-adverse-effect-level (NOAEL) from the pig study and human infant exposures at 2–4 weeks of age range from 2 to 12 on a body weight basis and from 2 to 8 on a concentration basis 58.
The Joint Food and Agriculture Organization of the United Nation (FAO) and the World Health Organization (WHO) Food Additives Committee noted that although the margins of exposure (MOEs) are small in magnitude, they are derived from a neonatal pig study in which the highest dose tested was without adverse effects on the gut or on immune parameters, supported by a neonatal minipig study. The neonatal pig and minipig are appropriate models for the young human infant up to at least 12 weeks of age, for whom infant formula may be the sole source of nutrition. These new studies allay the earlier concerns that carrageenan, which is unlikely to be absorbed, may have a direct effect on the immature gut. The Joint FAO/WHO Expert Committee on Food Additives also took account of the previous toxicological database on carrageenan, which did not indicate other toxicological concerns.
The FAO and WHO Committee concluded that the use of carrageenan in infant formula or formula for special medical purposes at concentrations up to 1000 mg/L is not of concern 58. The Committee recognizes that there is variability in medical conditions among infants requiring formulas for special medical purposes that contain the higher levels of carrageenan, and the Committee notes that these infants would normally be under medical supervision 58.
Carrageenan and degraded carrageenan on the mechanisms of inflammation
A number of studies with samples of carrageenan and “degraded carrageenan”, which were not always adequately characterized analytically, has been performed in order to evaluate if these compounds were able to induce or increase the production of proinflammatory mediators by various cells. These cell models (mostly intestinal or macrophage-like) were selected by the authors on the ground of their relevance to the oral administration of the additive E 407. Only the most recent ones are reported below as described by their authors.
In vitro studies
Borthakur et al. 139 reported that when human (intestinal epithelial cells from colonic surgeries, cell line NCM 460) cells, and normal rat ileal epithelial cells were treated for 1–96 hours with lambda-carrageenan (not analytically characterized) at a concentration of 1 μg/mL, increased BCL10, nuclear and cytoplasmic nuclear factor κB (NF-κB), and IL8 secretion were observed.
In a series of studies with various cell models (RAW 264.7 and 23ScCr mouse macrophage cell lines, NCM 460 human colonic epithelial cell line, primary cultures of human colonic cells), Bhattacharyya et al. 140 studied the mechanisms of carrageenan-induced intestinal inflammation. Toll-like receptor (TLR) and the protein B-cell lymphoma/leukaemia 10 (BCL10) were identified as important components of the cell membrane for binding of carrageenan to the cell membrane. The authors reported that in their studies, binding of carrageenan (purity and composition not reported) to the receptor resulted in stimulation of NF-κB, and activation of interleukin-8 (IL-8) production and of a reactive oxygen species (ROS)-mediated pathway 140.
Choi et al. 141 evaluated the effects of carrageenan on proinflammatory transcription factor NF-κB (subfamily RelA/p65) and early growth response gene 1 product (EGR-1) in relation to human intestinal epithelial barrier integrity. Human colonic cancer cell lines HCT-8, HT-29 and Caco-2 were exposed to 0 or 1 μg/mL of carrageenan (type not specified) for up to 24 hour. NF-κB activation (but not EGR-1 activation) was involved in the induction of proinflammatory cytokine IL-8. According to the authors, the results showed that both NF-κB and EGR-1 play a role in maintaining the epithelial barrier integrity in response to carrageenan 141.
Another study assessed ‘whether binding of carrageenan to TLR4 was specific or due to the mechanical coating of the membrane as a result of the large molecular weight of carrageenan and of the conditions used in vitro (Documentation provided to EFSA n. 47). Human embryonic kidney cells transfected with a TLR4 reporter system were employed to determine the binding, using alkaline phosphatase (SEAP) as the reporter molecule. Cells were exposed for 24 h to various types of carrageenan, all compliant with the JECFA specifications: food-blend carrageenan from a manufacturer, commercial λ, κ and ι-carrageenan, and a commercial mixture of κ- and ι-carrageenan, at concentrations of 0, 0.1, 1, 10, 50, 100, 500, 1,000 or 5,000 ng/mL’. Positive (lipopolysaccharide) and negative (clarified locust bean gum (CLBG) and sodium alginate) controls were included. Cell viability, as measured by the levels of cellular adenosine triphosphate and released lactate dehydrogenase released, was unaffected by any of the carrageenan or by clarified locust bean gum, sodium alginate or lipopolysaccharide at any exposure concentration tested. Overall, no measurable changes in cell viability were observed under these test conditions, and the three types of carrageenan tested were not TLR4 agonists or antagonists.
McKim et al. 142 evaluated the ability of the different types of carrageenan (κappa-, iota- and lambda-, food-grade purity 51–85%) to bind and activate TLR4 signalling by using a TLR4/MD-2/CD14/NF-κB/SEAP reporter construct in a HEK293 cell line. The carrageenan used was characterised with respect to identity and purity, and it was observed that commercial carrageenan samples contained sugars (dextrose or sucrose). Cells were exposed for 24 hour to concentration of 0.1, 1, 10, 50, 100, 500, 1,000, and 5,000 ng/mL of the various compounds. The results showed that carrageenan did not bind to TLR4 and was not cytotoxic to the HEK293 cells at the concentrations and experimental conditions tested. In addition, it was shown that carrageenan binds tightly to serum proteins, in particular proteins from the fetal serum used in the cell cultures. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) noted that the authors claimed that carrageenan was described as having a weight average molecular weight (mw) of 200–800 kDa but that no data presenting the MW profile of the different forms of carrageenan used in the study were reported in this publication.
In another study, McKim et al. 143 evaluated the intestinal permeability, cytotoxicity and carrageenan-mediated induction of proinflammatory cytokines using a standard Caco-2 absorption model and both iota-, κappa- and lambda-carrageenan. They found no carrageenan permeability or cytotoxicity at concentrations of 100, 500 and 1,000 μg/mL. In two human intestinal cell lines (HT-29 and HCT-8) carrageenan (0.1, 1.0 and 10.0 μg/mL) did not induce IL-8, IL-6 or MCP-1 nor produced cellular toxicity at 24 hour. The authors concluded that carrageenan did not cross the intestinal epithelial cells and were not cytotoxic to these cells. Carrageenan did not increase cellular oxidative stress nor did they induce expression of proinflammatory genes. Positive control substances produced the expected results indicating that the test systems were responsive. According to the authors, this study was unable to reproduce any of the previously reported in vitro findings that carrageenan may cause inflammation or disrupt insulin signalling pathways. They also reported that when a commercial batch of ʎ-carrageenan was carefully characterised for identity and purity, only 64% of the material sold was carrageenan, with 34% being comprised of sugars. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) noted that the authors claimed that carrageenan was described as having a weight average molecular weight (mw) of 200–800 kDa but that no data presenting the MW profile of the material used in this study were reported in the publication.
Chan et al. 144 studied the influence of the three major types of carrageenan polysaccharides on monocyte (human THP1) behaviour in vitro, only the λ-type induced monocyte adhesion and only in the presence of serum. Further analyses indicated lambda-carrageenan bound IL-8 in the serum and activated the cultured monocytes through an IL-8-dependent pathway.
Fahoum et al. 145 investigated if ‘carrageenan’ may modify gastric proteolysis and if ‘physiologically (partially) digested carrageenan’ (i.e. carrageenan incubated in a simulated gastric fluid may affect gut epithelial structure and function. Food-grade κappa-, iota- and lambda- types of carrageenan (no indication of molecular weight distribution) were used.
The authors reported that 145:
- native carrageenan bound milk, soya or egg proteins,
- native carrageenan impaired digestibility by pepsin of these proteins,
- in Caco 2 cells, pdcarrageenans induced increased expression of inflammatory markers and affected the epithelial structure,
- Some of these effects were associated with the degree of sulfation of the different carrageenan forms, with kappa being less potent.
The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) noted that:
- the simulated gastric fluid used was close to the ‘physiologic’ conditions and distinctly different from the treatment used to yield ‘polygeenan’ or C 16 from carrageenan,
- there was no information on the changes in MW of carrageenan after partial digestion,
- when looking at the biological effects, it was unclear what the partially digested material used in the experiments with ‘pdcarrageenans’ was: either ‘protein-carrageenan complexes’, or ‘pure carrageenan’ (not in complex with a protein),
- as regards the biological effects (e.g. secretion of mediators of inflammation), there was a difference between the types of partially digested carrageenan i.e. iota and lambda having an effect in contrast to kappa. The results with native iota carrageenan were negative.
Therefore, the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) considered that although the reported effects may have been relevant for this evaluation, it was not possible to use the data from this study 12.
Applying Caco 2, THP-1 and HT-29 cells as models, the same group 146, reported the effects of κappa-carrageenan on the inflammatory reaction in vitro. The results showed that κappa-carrageenan could induce significant increase in secretion of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and to participate in the Bcl10-NF-κ B-mediated pathway to enhance lipopolysaccharide stimulated secretion of IL-8 in HT-29 cells. The Panel noted that the MW distribution of the κappa-carrageenan used was not given by the authors who only indicated that: ‘the average molecular weight of 1 × 106 of kappa carrageenan was used in this experiment’ 146.
Overall, the interaction of carrageenan with inflammatory pathways at the molecular level was investigated in several in vitro cell models. These studies were based on signalling pathways involving the transcription protein nuclear factor κB (NF-kB), which, among others, regulates the expression of genes associated with inflammation. In many studies, the three types of carrageenan (commercial material, data on purity not available) were reported to activate inflammatory cascades that are related to innate immunity and to the generation of reactive oxygen species (ROS), which may lead to inflammation. A recent study conducted with κappa-, iota− and lambda-carrageenan suggested that the difference in their capacity to react with food and to induce the expression of inflammatory markers may be due to the difference in sulfation of the different forms 145. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) noted that the available in vitro studies with ‘carrageenan’ were discussed several times 147. Main pitfalls were that the studies on signalling pathways did not establish neither concentration-related responses, nor direct and specific binding to receptors or entry of carrageenan inside the cells. The author considered that high molecular weight carrageenan might coat the receptors. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) acknowledged that the in vitro studies are useful to investigate the mechanisms of toxicity. However, the European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) also noted that they were often conducted using established cell lines, which are not fully representative of the normal human intestinal epithelium in vivo, and that several confounding factors, including poor characterisation of the material used and limited consideration of the interactions with food were identified in the protocols used 12.
In vivo studies
Bhattacharyya et al. 148 investigated the pathways for induction of inflammation in Bcl10 wild-type, heterozygous and null mice. Groups of 3–8 male mice were given drinking-water containing 0 or 10 μg/mL of carrageenan (commercial lambda- and κappa-types) for 14 weeks, amounting to averaged 50 μg carrageenan/mouse (equivalent to 1.7 mg/kg body weight per day). Markers of intestinal inflammation (fecal calprotectin), blood cytokines (including keratinocyte chemokine, the mouse analogue of human IL-8) and markers of the inflammatory cascade were measured. Body weights were unchanged. Fecal calprotectin and circulating keratinocyte chemokine were significantly increased in both wild-type and heterozygous mice exposed to carrageenan compared with controls. In all three types of mouse, serum IL-6 and monocyte chemotactic protein-1 (MCP-1) were similarly increased by carrageenan treatment. No treatment-related effects were observed on serum levels of cytokines TNF-α, interferon gamma (IFN-γ), IL-1β, IL-10, IL-12 and IL-23. No gross changes or macroscopic lesions were apparent in the intestine of carrageenan-treated mice, except in one wild-type mouse. Microscopically, in carrageenan-exposed mice, the extent of inflammatory infiltrate throughout the intestine was greater in the wild-type than in the Bcl10 null mice, being significantly greater in the small intestine than in the colon and rectum for each of the groups. IL-10-deficient mice exposed to carrageenan showed an increase in activation of NF-kB (RelA) activation, but no increase in RelB or phospho-Bcl10. According to the authors, these findings demonstrated ‘a requirement for Bcl10 to obtain maximum development of the inflammatory pathway by carrageenan and lack of complete suppression by IL-10 of activation of the inflammatory pathway by carrageenan’.
In the study by Shang et al. 149 C57/BL/6J mice (6/groups) were given normal autoclaved water or lambda, κappa or iota-carrageenan (purchased from Sigma Shangai; no further precision) in drinking water (20 mg/L) for 6 weeks. Blood (for the estimation of serum TNF-α, IL-1β, IL-6 and IL-10), cecum and colon were collected at the end of the treatment period. A sample of the colon was fixed in 10% formaldehyde then the degree of inflammation was scored after staining with haematoxylin and eosin. The colonic content was collected aseptically for analysis of the microbiota by DNA amplification and high-throughput sequencing. The authors reported that the treatment with carrageenan induced disruption of the epithelium of the colon, colitis, and increased (about three times) the serum TNF-α levels, whatever the type of carrageenan used; serum levels of IL-1β, IL-6 and IL-10 were unchanged. The treatment with lambda, κappa and iota-carrageenan had different effects on the various components of the gut microbiota but all of them were able to decrease the amount of a bacterium known to have anti-inflammatory properties (Akkermensia muciniphila).
Wu et al. 146 used the Citrobacter freundii DBS100-induced intestinal inflammation model with NHS male and female mice to study if κappa-carrageenan administered by gavage in water for 1 week at daily doses of 1.7 mg/kg body weight, 8.3 mg/kg body weight or 41.7 mg/kg body weight, could aggravate the inflammatory reaction of the colon to pathogen exposure. They reported that κappa-carrageenan modulated cytokine production, down regulated the proportion of T regs and up regulated NF-κB. According to the authors, these results suggested that κappa-carrageenan acted as a potential inflammatory agent that could magnify existing intestinal inflammation. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) noted that the source of kappa carrageenan used in this study was not indicated. The authors stated that: ‘the average molecular weight of 1 × 106 was used in this experiment’, but no indication was given about the molecular weight distribution. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) also noted that in the absence of lipopolysaccharide stimulation, kappa carrageenan alone did not significantly modify the responses of the cells.
Overall, the Panel noted that the material used for most of the in vivo experiments presented was commercial carrageenan, which was however generally not further characterized in particular as regards the amount of low weight-average molecular weight carrageenan, in the region of 50 kDa. In addition, the Panel noted that carrageenan was given in drinking water, which was not representative of the dietary exposure where carrageenan is bound to protein. Therefore, the extent to which these studies might be representative of the situation when carrageenan is used as a food additive, and consequently the possible use of these studies for the safety assessment of the food additive carrageenan (E 407) where, according to the European Union specifications, the amounts of compounds with molecular weight below 50 kDa must be low (< 5%), was deemed limited 12.
The Panel also noted that intake of carrageenan has been reported to influence the composition of the gastro-intestinal microflora 149. Interplays between the bacterial gut microflora and sulfated glycans 150 seem to play a significant role in the development of inflammation in the gut 151. Because sulfate and undigested sulfur compounds have been implicated in the etiology of ulcerative colitis 152, the sulfate moiety of carrageenan might be involved in the proinflammatory effects 145 through the production in the colon of hydrogen sulfide from sulfate, by sulfate-reducing bacteria 153. Finally, from the reported studies, the purity of the test material used appeared to be of primary importance, and the amount of low molecular weight carrageenan it contained likely influenced the capacity of the test material to induce some effects. However, there is still uncertainty about the ‘safe’ level of this low molecular weight material. These considerations raised the issue of how the samples used in the studies could be considered as representative of the food additive E 407 as sold in the market. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) could not conclude on the potential of carrageenan used as a food additive (E 407) to induce immunotoxic and/or inflammatory effects in the absence of relevant studies using well characterized carrageenan 12.
Carrageenan in vivo studies on the gastro-intestinal tract
In two consecutive studies 154, 155, groups of Wistar rats (males and females) were exposed to carrageenan (5% in diet equivalent to 4,050 mg/kg body weight per day) or degraded carrageenan (0.25%, 0.5%, 1% and 5% in drinking water equivalent to 225, 450, 890 and 4,450 mg/kg bw per day) for 21 or 56 days (commercial products for both types of carrageenan were from Glaxo Laboratories, Paris; interpreted as isolated from E. spinosum with an approximate ratio of iota:κappa:lambda type 100:0:0). Carrageenan was a fine cream-coloured powder prepared from E. spinosum (moisture content, 6%; viscosity of a 0.5% aqueous solution at 25°C, 22 cP; total heavy metals, 20 ppm) and degraded carrageenan was a fine cream-coloured powder prepared by controlled hydrolysis of carrageenan extracted from E. spinosum (moisture content, 5%; viscosity of a 0.5% aqueous solution at 25°C 7 cP; sulfate content, 33.2%; total heavy metals, 20 ppm). Rats developed diarrhea with watery stools when given 5% degraded carrageenan. Only a slight diarrhoea, marked chiefly by feces which were semi-solid in consistency, was observed in rats given 1% degraded carrageenan or 5% carrageenan. The whole of the gastrointestinal tract was examined macroscopically for ulceration, but no adverse effects were noted. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) considered that the no-observed-adverse-effect-level (NOAEL) for iota-carrageenan was 5% in the diet equivalent to 4,050 mg/kg body weight per day, the highest dose tested.
Groups of 10 male and 10 female Sprague–Dawley rats were given either a 5% solution of degraded iota-carrageenan (C16; a low weight-average molecular weight commercial product produced via mild acid hydrolysis of an extract of E. spinosum obtained from Laboratories Glaxo, France) as drinking water (daily intake of degraded carrageenan was 6–10 g/kg body weight) or a dose of 5 g degraded carrageenan per kg body weight in aqueous solution by gavage (once daily; 6 days/wk; 0.5–5 g/kg/body weight per day) for 15 months 156. Number-average and weight-average molecular weights for this carrageenan fraction were reported to be in the range of 16–19 kDa and 20–30 kDa, respectively. Groups of 5 rats/sex given distilled water as drinking fluid by gavage served as controls. Rats were necropsied at intervals ranging from 1 to 15 months. At 15 months, treatment of one female and one male which were given degraded carrageenan via drinking water or gavage stopped. These rats were necropsied at 17 and 16.5 months, respectively. After 6 months, squamous metaplasia of the rectal mucosa was reported, together with accumulation of metachromic material (presumed by the authors to be carrageenan) in macrophages. No lesions in the cecum were found, whereas ulcerative lesions in the distal colon could explain the occurrence of occult blood in the stools.
Germfree (n = 9) and conventional (n = 12) female Wistar rats were exposed to degraded carrageenan with a sulfate content of about 30% and an average molecular weight of 20–40 kDa (obtained from Glaxo Labs and produced by mild acid hydrolysis of carrageenan) 157. No information on the type of carrageenan is reported. The degraded carrageenan used in this study was reported as having an average molecular weight of 20–40 kDa. Degraded carrageenan was added at a level of 10% to the basal diet. The germfree rats were necropsied on day 7 (n = 3); day 20, 35 and 63 (n = 2). Three conventional rats (n = 3) were necropsied according the same schedule. Histopathological examination of Swiss-roll preparations of the intestines. Mucosal lesions associated with erosion of the large intestine induced by degraded carrageenan were much more extensive in germfree than in conventional rats.
Six pregnant Wistar rats received drinking water with 5% of degraded carrageenan as 158. The carrageenan was derived from the red seaweed E. spinosum and was degraded by mild acid hydrolysis to retain a 29% sulfate content, interpreted to be mainly iota-carrageenan. No indication about the weight-average molecular weight was given. Six pregnant rats received drinking water without degraded carrageenan. Twelve female offspring from each group were separated and the exposure to 5% degraded carrageenan via drinking water was continued for 6 months. Ulceration of the caecum was observed in 4 of the twelve treated rats. In two of the four rats, the ulceration was accompanied by inflammatory cell infiltration (polymorphonuclear cells and macrophages) and occasionally by glandular hyperplasia at the ulcer margins. In the remaining 2 rats, the ulcers were healed, the mucosa showing atrophy, distortion of glands and fibrosis of the lamina propria.
Degraded carrageenan derived from the red seaweed E. spinosum (obtained by mild acid hydrolysis to retain 30% sulfate content and an average molecular weight of 20–40 kDa; interpreted by the Panel to be mainly ι-carrageenan) was given to Sprague–Dawley rats 159. In experiment 1, 4 groups of 30 males and 30 females was given a diet with 0%, 1%, 5% or 10% degraded carrageenan ad libitum for 24 months (equivalent to 0, 500, 2,500 or 5,000 mg/kg body weight per day). In experiment 2, 2 groups of 20 males and 20 females was given 0% or 5% aqueous solution of degraded carrageenan ad libitum as drinking water for 15 months (equivalent to 0 or 2,500 mg/kg body weight per day). In experiment 3, 3 groups of 15 males and 15 females were given 0, 1,000 or 5,000 mg degraded carrageenan/kg body weight in aqueous solution by gavage for 15 months. Degraded carrageenan induced consecutively: colitis, secondary metaplasia, then tumours (squamous cell carcinomas, adenocarcinomas, adenomas). When degraded carrageenan was given by gavage, the incidence of malignant tumors was lower than when given by diet or drinking water. The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) noted that this study has been taken into consideration in IARC (International Agency for Research on Cancer)’s evaluation of carcinogenicity of low molecular weight degraded carrageenan (category 2B) 160.
Carrageenan derived from red seaweed E. spinosum, and degraded by acid hydrolysis to retain 30% sulfate content, and obtained from Glaxo, France (iota-carrageenan; no indication about the weight-average molecular weight was given) was given to male Fischer 344 rats via the diet (10%) for varying periods of time: group I (n = 39) for 2 months (equivalent to 8,100 mg/kg body weight per day); group II (n = 42) for 6 months and group III (n = 42) for 9 months (equivalent to 5,000 mg/kg body weight per day) 161. The degraded carrageenan was reported as having an average molecular weight of 20–40 kDa. After termination of the administration on degraded carrageenan, all the rats were given the normal basal diet until 18 months. At that time all rats were autopsied. The controls (n = 46) received normal diet for 18 months. Colorectal squamous metaplasia (100%) and squamous cell carcinomas, anaplastic carcinomas and adenomas (incidence 5/39 animals) were seen after exposure to degraded carrageenan for 2 months. The incidence of colorectal tumours increased with exposure time and was 17/42 (40.5%) after exposure to degraded carrageenan for 9 months. The Panel noted that this study was taken into consideration in IARC (International Agency for Research on Cancer)’s evaluation of carcinogenicity of low molecular weight degraded carrageenan (category 2B) 160.
In two consecutive studies, Watt and Marcus 162 exposed groups of 10 male adult guinea pigs (strain not given) exposed to a 1% drinking solution (not more than 1,500 mg/kg body weight per day according to the authors) of iota-carrageenan (commercial product from Cumming & Son Ltd., Salford) via drinking water, or to 5% aqueous drinking solution of degraded carrageenan (commercial product from Laboratoires Glaxo-Evans, Paris; both types interpreted by the Panel as isolated from E. spinosum with an approximate ratio of iota:κappa:lambda type 100:0:0) (not more than 2,000 mg/kg body weight per day according to the authors) during 20 or 30 days 162. No indication about weight-average molecular weight was given. In animals exposed to iota-carrageenan, 2/4 showed ulcerative lesions in the caecum after 20 days and the other 6 animals had lesions after 30 days. No such effects were seen in controls (drinking water). In animals exposed to degraded iota-carrageenan, most of the animals showed looseness of the stools by the end of 10 days; from 20th to the 30th days, all had occult blood in the faeces. Incidence of ulcerative colitis was 100%. In five animals killed between the 20th and 25th days the lesions were mainly in cecum, while in the remaining animal, killed between the 26th and 30th days, ulceration had extended into the lower colon and rectum. The ulcerations involved mainly the mucosa and showed features of both acute and subacute inflammatory infiltration as well as crypt abscesses.
Multiple cecal ulcerations were noted in guinea pigs (strain not given) dosed with 5% (about 2,000 mg/kg body weight per day) carrageenan (mainly iota carrageenan; interpreted by the Panel as isolated from E. spinosum with an approximate ratio of iota:κappa:lambda type 100:0:0) over 2–4 weeks via diet 163. No indication about the weight-average molecular weight was given. Sequential studies showed that the lesion first develops as an accumulation of macrophages in the lamina propria and subsequently in the submucosa leading to the formation of pale raised areas which can easily be seen macroscopically. Ulceration of the mucosa then occurs, particularly in these areas. The ulcers were small and superficial and affected only the mucosa. A mixed cellular infiltrate, consisting predominantly of macrophages accompanied by polymorphonuclear, lymphocytes, and plasma cells, surrounded the ulcerated area.
In two consecutive studies 154, 155, white guinea pigs (males and females) were exposed to carrageenan (5% in diet equivalent to 5,900 mg/kg body weight per day for rats) or degraded (0.25%, 0.5%, and 2% in drinking water equivalent to approximately 300, 600 and 2,500 mg/kg body weight per day) carrageenan for 21–45 days. For both types, commercial products from Laboratoires Glaxo, Paris; interpreted by the Panel as isolated from E. spinosum with an approximate ratio of iota:κappa:lambda type 100:0:0). No indication about the weight-average molecular weight of the tested compound was given. Details of the compounds used are reported in the rat studies described above 154, 155. Groups of four male animals were exposed to 1% degraded carrageenan in drinking water during 3 weeks and sacrificed week 4, 7, 11, 15. Multiple pin-point cecal and colonic ulcerations were developed after 3–5 week of treatment in guinea pigs given 5% carrageenan in the diet or 2% degraded carrageenan in the drinking water. Ulceration was not observed in the animals receiving degraded carrageenan 0.25% in drinking water. Ulcers appeared as extensive macrophage infiltration at the base, cover a thin layer of fibrin; polymorphonuclear cells and lymphocytes occurred in considerable numbers. Macrophages, polymorphonuclear and lymphocytes deeply infiltrated the epithelium around the ulcer. In some animals, microabscesses were formed close to the ulcerated areas due to substantial polymorphonuclear cell infiltration. No cecal or colonic ulceration was observed in animals sacrificed 1–4 weeks after the end of the treatment, but distinct granulomas with evident carrageenan within the macrophages were observed. In animals scarified at a later stage, the histology of the cecum and colon was unchanged compared to the controls, and no carrageenan could be shown in the macrophages within the lamina propria.
Guinea pigs (8 females of the Hartley strain, average weight 360 g) given 2% partially degraded iota-carrageenan (C16) (commercial products from Glaxo); interpreted by the Panel as isolated from E. spinosum with an approximate ratio of iota:κappa:lambda type 100:0:0) in sterile distilled drinking water for 2 weeks developed caecal ulceration 164. No indication about the weight-average molecular weight was given. This effect was not observed when the same level of degraded iota-carrageenan (C16) was given in milk, or when lower levels of C16 (0.02% or 0.2%) were given in drinking water for 12 or 10 months, respectively. The authors reported that the ability of macrophage lysosomes in the lamina propria of the guinea pig to endocytose and store degraded iota-carrageenan (C16), which was not seen in the other species, was apparently closely related to the caecal ulceration observed in guinea pigs species.
Engster and Abraham 165 compared the cecal response of various degraded carrageenan (Marine colloids, 7 iota-fractions of number-average molecular weight between 5 and 145 kDa from E. spinosum, 3 κappa-fractions of number-average molecular weight between 8.5 and 314 kDa and 3 lambda-fractions of number-average molecular weight between 20.8 and 275 kDa from C. crispus) administered to female guinea pigs as a 1% solution in the drinking water for 2 weeks. Six iota-fractions were also given to female guinea pigs in the diet at a 2% level for 10 weeks. When administered in the drinking water, all iota-fractions, with the exception of those of lowest (5 kDa) and highest (145 kDa) number-average molecular weights, were absorbed and inflammation, erosion or ulceration were observed in the caecum. By contrast, κappa- and lambda- carrageenan fractions given in drinking water or iota-fractions given in the diet for 10 weeks produced no caecal damages. According to the authors, these results indicated that caecal ulceration in the guinea pig was caused only by certain molecular weight iota-fractions and when administered in drinking water 165.
Twenty male Californian rabbits (average body weight 2,950 g) received drinking water with 0, 0.1, 1 or 5% degraded carrageenan derived by mild acid hydrolysis of iota-carrageenan from E. spinosum (Glaxo, France, retaining about 29% sulfate) for 6–12 weeks 166. No indication about the weight-average molecular weight was given. Animals fed degraded carrageenan at 5% concentration in their drinking water received an average dose of 1,400 mg/kg body weight). Diarrhea associated with occult blood in the feces developed by the end of 7 days and persisted. All animals of this group showed severe ulceration of the colon. One rabbit fed degraded carrageenan at 1% (average daily dose of 800 mg/kg body weight) over a 7-week period developed diarrhea, occult blood in the faeces was present in all animals after 2 weeks. All animals showed moderate ulceration in the colon. Animals fed 0.1% degraded carrageenan (daily dose of 70 mg/kg body weight) for 12 weeks, developed no diarrhea but occult blood was seen in three rabbits by the end of week 10. Multiple ulcers were found in the colon in three of five rabbits.
Fifteen mature rabbits (not further specified) weighing about 2,200 g and sensitized intramuscularly with 1 mg lambda-degraded carrageenan in 1 mL, were administered lambda-degraded carrageenan (approximately 30 kDa; not further specified) dissolved in drinking water at a concentration of 1%. No indication about weight-average molecular weight of the tested compound was given. Ten rabbits were given the substance continuously for 12 months and the remaining five were treated for 28 months. They were sacrificed at the end of the treatment period. Histopathological examination showed mild inflammatory changes of the colonic mucosa in all animals and a focal high-grade dysplasia involving the mucosal epithelium in 3/5 animals treated for 28 months. Tumors of the colorectum were not observed 167.
Overall, degraded carrageenan administered in water is known to be a model for the development of colitis in various animal species 168. Degraded iota-carrageenan given in drinking water or diet induced ulceration in the caecum and colon of rats, guinea pigs and rabbits. A mixed cellular infiltrate consisting predominantly of macrophages, polymorphonuclear cells, lymphocytes, and plasma cells, surrounded the ulcerated area. Preulcerative changes consisted mainly in accumulation of macrophages in the lamina propria and they were associated with the presence of material that was assumed to be degraded carrageenan in the subepithelial tissue. Rats exposed to degraded iota-carrageenan (weight-average molecular weight reported as 20–30 kDa, or average molecular weight as 20–40 kDa) via drinking water, diet or by gavage developed in first instance colitis, secondary metaplasia and finally tumors (squamous cell carcinomas, adenocarcinomas, adenomas).
Hypersensitivity, allergenicity and food intolerance
The α-1,3-galactosidic linkage, which is present in the three types of carrageenan is specifically recognized by human IgG and IgM anti-galactose natural antibodies. Binding of these antibodies to this saccharide epitope may lead to an immune reaction and, for instance, they were involved in acute rejection by humans of pig xenografts 169. These antibodies developed with age in human; they are not present in the neonates but develop within a few months, as soon as the newborn’s gastrointestinal tract becomes colonized by microorganisms.
The European Food Safety Authority Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS) noted that an ELISA was available for the detection/quantification of carrageenan in various foods 170, which indicated that carrageenan could elicit the production of and react with, specific antibodies thus revealing the presence of immunogenic sites in carrageenan. It was not known whether these antibodies also recognized poligeenan and other degraded carrageenans.
Vojdani and Vojdani 171 reported that 27%, and 17% of the sera from 288 healthy, asymptomatic volunteers contained specific IgG and IgE antibodies to carrageenan, respectively. Fixation of IgE but not of IgG antibodies was largely inhibited by several common food antigens, indicating that these IgE may be formed in response to cross-reacting antigens present in common foods.
The Panel noted that despite the presence in human serum of antibodies capable to recognize saccharidic epitopes on the carrageenan molecules, no case reports of significant allergic and/or anaphylactic reactions after ingestion of foods containing carrageenan were identified in the available literature 12.References
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