Science Blog #2 - Vitamin E – Nature’s perfect antioxidant

Vitamin E (Vit-E) is a powerful, fat-soluble antioxidant, which protects cell membranes against damage caused by free radicals. It was one of the first two antioxidant compounds to be sold as dietary supplements, the second being vitamin C. Vitamin E is necessary for structural and functional maintenance of skeletal, cardiac, and smooth muscle. It also assists in the formation of red blood cells and helps to maintain stores of vitamins A and K, iron, and selenium. The term Vit- E encompasses a group of eight compounds, called tocopherols and tocotrienols, with various subsets of each, that comprise the vitamin complex as it is found in nature. Each of these different compounds has distinct chemistries and biological effects.

Chemistry of Vitamin-E

Vitamin-E is made of two compounds viz., tocopherols and tocotrienols together are also called as tocochromanol. Vitamin E exists in eight chemical forms; α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol. The tocopherols are characterized by the 6-chromanol ring structure methylated to varying degrees at the 5, 7, and 8 positions and the C16 saturated phytyl side chain is attached to chromanol ring at position 2. The tocotrienols are characterized by the presence of unsaturated double bonds at the 3', 7', and 11' positions of the phytyl side chain. The different forms of tocopherols and tocotrienols are differing by the number and positions of the methyl groups on the 6-chromanol ring; α-tocopherol/ tocotrienol are tri-methylated; β –tocopherol/ tocotrienol, γ-tocopherol/tocotrienol are di-methylated; and δ-tocopherol/tocotrienol are mono-methylated.

The general structure of tocopherol (A) and tocotrienol (B)

Of the different tocopherol and tocotrienol species present in foods, α-tocopherol has the highest Vit-E activity. Due to the occurrence of α-tocopherols on the surface of the plasma membrane, it has highest antioxidant potential (KamalEldin and Appelqvist, 1996) where in, other forms of Vit-E are buried within the membrane and physically inaccessible to scavenge Reactive Oxygen Species (ROS). Vitamin E is believed to act as membrane stabilizer. The stabilization process is caused by the interaction between the chromanol hydroxyl group of α-tocopherol with the carboxyl group of the ester carbonyl bond of the phospholipid molecules which increases the rigidity of the membrane (Munne-Bosch, 2002).

Biosynthesis of Vitamin-E

The tocochromanols (tocopherols and tocotrienols) are made by same biosynthetic pathway, which takes place in plastids of plant cells (Soll and Schultz, 1979). Tocochromanols result from the condensation of a polar phenolic moiety, p-hydroxy phenyl pyruvic acid (HPP) from the cytosolic shikimate pathway and a polyprenyl side chain derived from iso-pentenyl diphosphate (IPP) produced by the plastidic Methyl erythritol 4-phosphate (MEP) pathway (Lichtenthaler et al. 1999). The side chains of tocopherols and tocotrienols derived from IPP derived phytyl di-phosphate (PDP) and geranyl geranyl di-phosphate (GGPP) respectively. The synthesis of all tocochromanols is initiated by the conversion of HPP into homogentisic acid (HGA) catalyzed by HPP dioxygenase (HPPD). Condensation of HGA and PDP forms 2-methyl-6-phytyl benzoquinol (MPBQ), the committed intermediate of all tocopherols. This reaction is catalyzed by homogentisic acid phytyl-transferase (HPT) and the two subsequent steps catalyzed by tocopherol cyclase (TC) and γ-tocopherol methyl transferase (γ-TMT) are in common for tocopherol and tocotrienol biosynthesis. Tocotrienol formation results from the condensation of HGA and GGPP into 2-methyl 6-geranyl geranyl-benzoquinol (MGGBQ) that is catalyzed by homogentisic acid geranylgeranyl transferase (HGGT). Once the committed intermediates MPBQ and MGGBQ are formed, the pathway branches according to the substrate involved in the next reaction. Direct cyclization of MPBQ forms δ-tocopherol (see below) whereas its methylation at ring position C-3 yields 2,3-dimethyl-6-phytyl-1,4-benzoquinone (DMPBQ), the precursor of γ-tocopherol catalyzed by MPBQ methyl transferase (MPBQ-MT).

The schematic representation of tocochromanol synthesis in plant cell

Sources of vitamin E

In plants, tocopherol composition differs between different species and between different tissues within one species. Usually, leaves commonly accumulate α-tocopherol whereas seeds are rich in γ-tocopherol, β- and δ tocopherol are not very abundant in most plant species. The variations in tocopherol isoforms are detected in several plant species such as soybean, rapeseed and Arabidopsis with most of the tocopherols are present in the form of γ or δ-tocopherol in seeds. In case of sunflower and safflower seeds, α-tocopherol comprises more than 95% of the total tocopherol content. Wide variations in tocopherol content and concentration have been reported in different fats and oils.

A.Tocopherols and tocotrienols content in various fats and oils; Slover, H T. (1971), B. α-tocopherol and total tocopherol content in various crops

Extraction and Estimation of Vit-E

Depending upon the plant species and type of tissues - seeds, leaves and flowers, several methods to extract Vit-E are available. However during sample preparation, it is crucial to consider conditions that support the stabilization of Vit-E, since Vit-E is highly sensitive to light and degrades by photo-oxidation. All the sample manipulations should be carried out under subdued light conditions.

(A) Various methods of Vit-E Extraction

I. Direct Solvent extraction method

Direct solvent extraction method is the most popular method used for Vit-E extraction from various plant species (Lee et al., 2012; Lim et al., 2007). It is a quick and simple method. Solvent extraction processes include basically three steps: preparation, extraction, and solvent solubilization and this method are standardized to extract Vit-E from soybean seeds (Vinutha et al. 2015). The detailed method of extraction and analysis of Vit-E from plant sample is given below.

Direct solvent extraction method is the most popular method used for Vit-E extraction from various plant species (Lee et al., 2012; Lim et al., 2007). It is a quick and simple method. Solvent extraction processes include basically three steps: preparation, extraction, and solvent solubilization and this method are standardized to extract Vit-E from soybean seeds (Vinutha et al. 2015). The detailed method of extraction and analysis of Vit-E from plant sample is given below.

Schematic diagram to show direct solvent extraction method of Vit-E from plant sample

II. Saponification method

Saponification has been widely used to extract Vit-E from many fortified food products, including nuts, meats, fruits, vegetables and infant foods (Kramer et al., 1997; Lim et al., 2007). This process depends upon alkaline hydrolysis to release Vit-E from their conjugates or fatty acids. Fortified foods and feeds are subjected to saponification, because Vit-E is added as their ester form. The disadvantage of this method are formation of problematic emulsion when samples with high fat content are subjected to saponification, pronounced losses of Vit-E even in protective conditions such as darkness, high nitrogen conditions and significant decrease in α-tocopherol occurs when exposed to alkaline conditions for a long time of 2-4 hrs.

III. Soxhlet extraction

The quantification of Vit-E can also be performed by high performance liquid chromatography (HPLC) combined with the Soxhlet extraction method (Peterson et al., 2007). The plant samples will be placed into a Soxhlet apparatus and extracted using hexane solvent with 0.01% BHT for six-hour extraction, extraction; the samples are placed in ultrasound for 3 hours. The extracts will be then filtered through qualitative filter paper and then again filtered through 22µm size filter and analyzed by HPLC as mentioned in the direct solvent method. The advantages and disadvantages of each method of extraction of Vit-E are summarized.

Schematic diagram showing comparison of extraction methods for quantifying Vit-E

(B) Vit-E estimation by HPLC:

Separation of Vit-E was achieved by using HPLC unit with C-30 reverse phase column. 20µl of the syringe filtered sample was manually injected through in the port into the HPLC; column temp. 400 C. The isocratic mobile phase composed of methanol and acetonitrile (75: 25) was used with a flow rate of 1.0 ml/min and wavelength was set at 295nm. Separated Vit-E peaks were identified by comparing the retention time with those of standards peaks.

Vitamin E in human health

Vitamin E functions primarily as a chain-breaking antioxidant that prevents the propagation of lipid peroxidation but its dietary signature affects a cascade of reactions. The absorption of Vit E from the intestinal lumen is dependent upon biliary and pancreatic secretions, micelle formation, uptake into enterocytes, and chylomicron secretion. The liver takes up the chylomicron remnants, containing newly absorbed vitamin E, which are then converted to tocopheroxy radicals or quinone derivatives. During catabolism the chromanol ring and isoprenoid side chain is oxidized and excreted in bile after conjugation with glucuronic acid. Being major antioxidant Vit-E play major role in protecting the PUFAs with in the membrane phospholipids and in plasma lipoproteins and thus maintain membrane integrity, in turn prevent haemolysis in RBC. The ROS scavenging attribute regulates the cell proliferation and play major role in various types of cancer tackling the oxidative stress. Vit-E deficiency has been involved with poor bone calcification and remodeling which justifies its role in play along with vitamin D as well as various hormones (Packer., 1991). The hypothesis that oxidized low-density lipoprotein (oxLDL) is a causative agent in the development of cardiovascular disease continues to dominate experimental protocols aimed at understanding the cause, and potentially the prevention, of cardiovascular disease. Vitamin E does inhibit LDL oxidation whether induced by cells in culture or by copper ion in vitro (Clarke et al., 2008). Vitamin E inhibits smooth muscle cell proliferation through the inhibition of protein kinase C; it inhibits platelet adhesion, aggregation, and platelet release reactions; inhibits plasma generation of thrombin, a potent endogenous hormone that binds to platelet receptors and induces aggregation; mediates up-regulation of the expression of cytosolic phospholipase A2 and cyclo-oxygenase and so on potentiating the risk for cardiovascular diseases. Vitamin E content in the body influences the incidence or rate of progression of age related macular degeneration or cataract. Impact of Vit E supplementation on acute responses and adaptations to strength training is known and mainly due to the positive effects imparted on muscles. Hence its deficiency resulting poor muscle development leads to Muscle hypertrophy or skeletal myopathy. The primary human Vit-E deficiency symptom is a peripheral neuropathy characterized by the degeneration of the large-caliber axons in the sensory neurons. Other Vit-E deficiency symptoms observed in humans include spino cerebellar ataxia, skeletal myopathy, and pigmented retinopathy. Vitamin E, well known as fertility hormone improves sperm health, promotes ovulation, support embryo implantation and reduce risk for miscarriages (Rizvi et al., 2014). The multifaceted dietary role of Vit-E is compiled.

Dietary role of Vitamin E in humans

Potential applications of Vitamin E

Increasing consumer concern regarding toxic chemical additives used in dietary supplements, food, beverage and personal care products are propelling the global market for natural vitamin E. Moreover, economic reforms coupled with growth in the overall health care sector and increasing private equity investments in natural vitamin industry is set to bolster the growth of market globally. The market and the practical utility of Vit-E is segmented into three product types – tocopherols, tocotrienols and others. By application, market is further sub-divided to dietary supplements, food & beverages, cosmetics and others.

Vitamin E as animal feed:

Vit-E supplements in animal feed and diet is increasing at an alarming rate and is due to its antioxidant activity, membrane integrity, immunity and better muscle function. Moreover, it enhances the shelf life of feed by imparting oxidative stability.

Vitamin E as dietary supplements:

Due to high incidence of lifestyle disorders contributed by diet as well as sedentary life style, there is a growing awareness on health concerns and hence great market for wellness products and supplements. As a health supplement, it improves the immune function and maintains vitamin A levels etc. Natural and synthetic forms of Vit-E are well known as anti-cancerous, especially RRR-α-tocopherol ether-linked acetic acid analog (α-TEA).

Vitamin E as sports blend:

As known for muscle developing attributes, Vit-E is used in sports blend for the tired muscles.

Vitamin E in cosmetic industry:

As an antioxidant natural Vit-E is widely used in cosmetic industry in cosmetics and skin care; including lotions, creams, lipsticks, sunscreens etc. It protects skin from harmful radiations like UV and prevents deposition of melanin. It delays the signs of aging, heal scars and acnes, improves moisture content in skin and its texture. It also improves the stability of lipid-based cosmetics.

Other industrial uses:

Mixed tocopherols from soybean oil processing are generally used for stabilizing oxidatively-sensitive lipid supplements such as those from marine oils and other products. In addition, tocopherol-rich oils, such as germ oils from oat, barley and wheat may be mixed with other oils to stabilize them. Among the tocotrienol-rich oils, palm oil, annatto, and rice bran oil are important, and these are often used in product enrichment. Considerable amounts of tocopherols are removed during the refining process of edible oils. Commercially gamma-tocopherol is being used as an anti-oxidative protectant to preserve food emulsions like fish oil-enriched salad emulsions.

Market value of Vitamin E

According to Transparency Market Research (TMR), the global Vit-E market will expand at a12.8% CAGR from 2016 to 2024. With sustainable demand from the elite or well-to-do high profile customer base, the market is estimated to be worth US$2,251.7 mn by 2024. Among the variants of Vit-E, tocopherol is expected to lead in terms of demand as well as in terms of being the highest revenue contributor. The tocopherol segment accounted for 65% of the market back in 2015. High use in dietary supplements is a key reason for the growing demand for tocopherol. As tocotrienols are difficult to absorb during digestion and are also poorly distributed to blood cells, their demand will be low. Moreover, they are rapidly metabolized and eliminated from the body. Thus, its application is limited majorly to cosmetics, while fortified products in the markets mainly favoring the producers are fooling the consumers highlighting its anti-oxidant activity. Therefore, tocotrienols occupies a significantly lower share as compared to tocopherol in the natural source Vit-E market. North America accounts for about 45% of Vit-E market in 2015, followed by Europe with 26%. The increasing awareness as well as affordability by the developing nations of Asia Pacific is escalating the market to grow natural source Vit-E as well as its market. It is estimated that during the forecast period of 2016 to 2024, the Asia Pacific natural source Vit-E market will expand at a 13.3% CAGR. China will be a lucrative market in Asia Pacific region for natural source vitamin E.

Global Natural Source Vitamin E Market Revenue Share in US $ Mn. (Geography, 2015)

The applications for natural source Vit-E include dietary supplements, cosmetics, food and beverage, and animal feed. Of these, the dietary supplements segment not only led in the past but will also witness growth in the coming years on account of the promise supplements hold in preventing chronic diseases or of the promise of longevity. The expansion of distribution chains will also aid the demand for Vit-E-based dietary supplements such as energy drinks, tablets, and capsules. The market for natural source Vit-E is flourishing in Europe and North America. The market for natural source Vit-E market in North America was accounted for over 40% in 2015 and 26% in Europe. The growing awareness about having a healthy lifestyle and the increasing realization of the importance of nutrients by making use of supplements are both driving the natural source Vit-E market in these two regions. The presence of excellent distribution channels will also ensure the growth of the market in Europe and North America. The increasing affordability of natural source Vit-E on account of increasing disposable income in developing nations of Asia Pacific is helping the market in APAC to grow. It is estimated that during the forecast period of 2016 to 2024, the Asia Pacific natural source Vit-E market will expand at a 13.3% CAGR. China will be a lucrative market in Asia Pacific region for natural source Vit-E.

Fortification in Food

Vit-E fortification can act as a double edged sword in improving shelf life of the fortified products like oil as well as the antioxidant property in food and feed to tackling the oxidative stress induced chronic life style syndromes. Fortification with Vit-E shall be critically important where the diet is high in polyunsaturated fatty acids (PUFA). PUFAs are susceptible to oxidation due to the presence of multiple double bonds causing rancid property to the oil as well as at cellular levels where oxidation result in free radicals, which is associated with lifestyle disorders like chronic vascular diseases (CVD), cancer, obesity etc. As a nutrient, it’s been appropriate to fortify products with Vit-E at a level of 65 to 190 mg/Kg. The recommended daily intake of Vit-E increases with intake of PUFA at a ratio of 0.4:1 (mg α-tocopherol: 1gm PUFA). As stated earlier, the Dietary Reference Intakes (DRIs) for Vit-E are based on α-tocopherol only and do not include amounts obtained from the other seven naturally occurring forms historically called Vit-E (β-, γ-, δ-tocopherol and the four tocotrienols). Because the different forms of Vit-E cannot be interconverted in the human, the Estimated Average Requirements (EARs), Recommended Dietary Allowances (RDAs), and Adequate Intakes (AIs) apply only to the intake of RRR-α-tocopherol from food and the 2R-stereoisomeric forms of α-tocopherol (RRR-, RSR-, RRS-, and RSS-α-tocopherol) that occur in fortified foods and supplements (Schneider et al., 2012). As a food additive, tocopherol is labeled with these E numbers: E306 (tocopherol), E307 (α-tocopherol), E308 (γ-tocopherol), and E309 (δ-tocopherol). These are all approved in the USA, EU and Australia and New Zealand for use as antioxidants.

Natural α-tocopherols are not preferred as good fortificant while the synthetic counterparts are preferably used in fortified foods and supplements. Synthetic Vit-E, all rac-α-tocopherol (labeled as dl-α-tocopherol), is produced by coupling trimethylhydroquinone with isophytol; it contains all eight stereoisomers in equal amounts. Esterification of the labile hydroxyl (OH) group on the chromanol ring of Vit-E prevents its oxidation and extends its shelf life. This is why esters of α-tocopherol are often used in Vit-E supplements and in fortified foods. Thus to achieve the RDA recommended in this report of 15 mg/day of α-tocopherol, a person can consume 15 mg/day of RRR-α-tocopherol or 15 mg/day of the 2R-stereoisomeric forms of α-tocopherol (e.g., 30 mg/day of all rac-α-tocopherol) or a combination of the two. It is now known that Vit-E forms are not interconvertible in the human and that their plasma concentrations are dependent on the affinity of hepatic α-tocopherol transfer protein (α-TTP) for them (see section on “Hepatic α-Tocopherol Transfer Protein”). In light of these new findings in humans, it becomes necessary to re-evaluate the relative biological potencies of different forms of Vit-E. Therefore, it is best to measure and report the actual concentrations of each of the various Vit-E forms in food and biological samples.

Supplement forms

RRR-α-tocopherol

RRR-α-tocopherol is the only stereoisomeric form of α-tocopherol found in unfortified foods. The same is not always true for nutritional supplements . Vitamin E supplements generally contain 100 IU to 1,000 IU of α-tocopherol. Supplements made from entirely natural sources contain only RRR-α-tocopherol. RRR-α-tocopherol is the most bioavailable form of α-tocopherol in the body. Synthetic α-tocopherol, which is often found in fortified food and nutritional supplements and usually labeled all-rac-α-tocopherol or dl-α-tocopherol, include all eight possible stereoisomers of α-tocopherol Because half of the isomers present as a mixture in synthetic α-tocopherol are not usable by the body, synthetic α-tocopherol is less bioavailable than natural α-tocopherol. In addition, Vit-E-fortified foods often contain synthetic α-tocopherol, and amounts of Vit-E are provided as a percentage of the daily value of 30 IU (approximately 20 mg of RRR-α-tocopherol).

To calculate the amount (in milligrams) of α-tocopherol bioavailable in a supplement, the conversion factors are as follows:

The main esterified forms of Vit-E in nutritional supplements are α-Tocopheryl succinate and α-tocopheryl acetate, which impart resistance to oxidation during storage than un-esterified tocopherols. When taken orally, the succinate or acetate moieties are removed from α-tocopherol in the human intestine and the bioavailability of α-tocopherol from these synthetic forms gets equivalent to that of natural α-tocopherol. Hence, the conversion factors used to determine the amount of bioavailable α-tocopherol provided by α-tocopheryl succinate and α-tocopheryl acetate are the same as those used for α-tocopherol. In vitro studies indicated that the Vit-E ester, α-tocopheryl succinate, could inhibit proliferation and induce apoptosis in a number of cancer cell lines. Limited data from in vivo onco-animal models found that α-tocopheryl succinate administered by injection inhibited tumor growth. There is currently no evidence in humans that taking oral α-tocopheryl succinate supplements delivers α-tocopheryl succinate to tissues. Of note, current research investigates nanomedicines to increase α-tocopheryl succinate bioavailability before exploring putative benefits in clinical settings. α-Tocopheryl nicotinate is another α-tocopherol ester formed from synthetic α-tocopherol and nicotinic acid (niacin). While α-tocopheryl nicotinate is prescribed as a lipid-lowering agent in Europe and Japan, it is marketed as a supplement only in the US. α-Tocopheryl phosphates (Ester-E®), is another form but currently no published evidence exist on supporting its supplementation affecting the efficiency nor bioavailability. Other minor supplements containing γ-tocopherol, mixed tocopherols, or tocotrienols are also commercially available. In synthetic supplements, the racemic mixture is a combination of various forms which might act antagonistically being structural analogues or reduce the bioavailability, but no report yet exist.

References

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