What Are L Glutathione Uses?

Glutathione (GSH, glutathione), γ-glutamic acid-L-cysteine-glycine, is a nonprotein thiol tripeptide synthesized by glutamylcysteine synthetase (GCS) and glutathione synthetase (GS) by the cascade of glutamic acid, cysteine, and glycine.

1.Introduction to Glutathione

1.1Structure of Glutathione

In plant, animal and microbial cells, three amino acids, glutamate, cysteine and glycine, are synthesized into a biologically active small tripeptide compound by the sequential action of two enzymes, glutamylcysteine synthetase (GCS) and glutathione synthase (GS), known as glutathione (also known as γ-L-glutamylglutamate, Glutathione)[1] . -L-cysteinylglycine, Glutathione)[1] .

In eukaryotic cells, 80-85 % of glutathione is found in the cytoplasm, while the rest is found in the mitochondria and endoplasmic reticulum[2-4] . However, in the cells of some saline bacteria or parasitic protozoa, glutathione is present in the form of sulfides such as S-nitrosoglutathione, glutamylcysteine (γ-EC), thiosulfate, and N1,N8-bis(glutathione) spermidine [5-6]. Glutathione homologs are also found in legumes, which are distinguished by the fact that the C-terminal residue amino acid is not glycine, but other types of amino acids, such as Homoglutathione (H-Glu , Cys-β-Ala-OH)[7] .

Glutathione exists in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione is the main form of glutathione in cells, accounting for more than 98% of the total glutathione content. The molecular formula of reduced glutathione is C10H17O6N3S. In the cell, glutathione is oxidized and dehydrogenated to form GSSG, which is then reduced to GSH by GSSG reductase (GR) with the participation of NADPH (reduced coenzyme II), thus forming a redox cycle, and the two forms of glutathione are shown in Figure 1.1.

Figure 1.1 Two forms of glutathione

2.Uses of Glutathione

The special structural characteristics of glutathione determine its functional properties, and the γ-mercapto group on its cysteine structure, as a free active group, provides a strong ability to supply electrons or proton hydrogen. In recent years, the attention of domestic and foreign scholars to glutathione has gradually increased, and the use and commercial value of glutathione are rapidly coming into people's attention. Currently, the application markets of glutathione can be categorized into food, cosmetic and medical fields, etc.[8-11] The specific applications are as follows:

2.1 Glutathione in Food Applications

Glutathione is a potential preservative and flavor enhancer in the food industry due to its unique antioxidant ability. In the storage of seafood or livestock meat food, the appropriate amount of glutathione can effectively extend the freshness and shelf life of the food, and at the same time, to a certain extent, it can inhibit the growth and reproduction of microorganisms and inhibit the degradation of nucleic acids in meat food, which can help to prolong the shelf life of the food, and ensure the quality of food [12-14].

During the winemaking process, oxidative browning of wine is mostly caused by the oxidation of phenolics in grape juice catalyzed by polyphenol oxidase (PPO) to form o-benzoquinone, and the formation of dark substances through the polymerization or condensation of o-benzoquinone, and the appropriate addition of glutathione during the winemaking process can enable it to react with the oxidized phenolics to form a thioether (2-S-glutathione adipic acid) or grape reaction product (GRP) [15-17], which can prevent the oxidation of phenolics in the wine. The appropriate addition of glutathione during the winemaking process enables it to react with these oxidized phenolics to form a sulfide (2-S-glutathione adipate) or grape reaction product (GRP), which prevents the oxidation of phenolics in wine [15-17], and prevents the formation of quinones, thus maintaining the good color of wine.

At the same time, the addition of glutathione can effectively reduce the loss of aroma components of wine, protect its fresh and mellow special flavor, and play the function of enhancing the sensory stability of wine [18-21]. In addition, glutathione can be used as a flavoring agent in food processing to add unique flavors to food and achieve the effect of flavor enhancement [22-23], and it can also be added to functional foods to facilitate the metabolism and absorption of the human body [24-25].

2.2 Application of Glutathione in Cosmetics

Glutathione plays a key role in the regulation of cellular activity as an antioxidant and enzyme cofactor capable of eliminating free radicals. In cosmetic applications, glutathione derivatives (S-acyl glutathione derivatives) can be used topically for the treatment of skin aging and for the treatment of hyperpigmentation, to enhance skin permeability and to accelerate the rate of skin metabolism [26]. The formation of skin melanin is due to the formation of dopaquinone (DQ) catalyzed by Tyrosinase (TYR), which in turn forms melatonin and eumelanin in a series of catalytic oxidation reactions[27] .

Glutathione and its derivatives can reduce dopamine to form brown pigment, which is lighter in color than melatonin, and glutathione helps to keep the redox-sensitive active site on the enzyme in the necessary reduced state, which can inhibit tyrosinase activity to a certain extent, thus achieving the whitening effect [28-29].

In addition, it has been shown that the ability of reduced glutathione to inhibit tyrosinase activity is stronger than that of oxidized glutathione, and reduced glutathione and oxidized glutathione can be mixed according to the ratio to achieve the best antioxidant and whitening effect [30].

2.3Glutathione in Medical Applications

As a natural endogenous substance, glutathione is widely distributed and participates in redox processes in the human body, and is also known as an endogenous detoxifier because of its strong ability to supply electrons or proton hydrogens[31-32] . Glutathione mainly plays a role in inhibiting oxidative stress: on the one hand, glutathione can neutralize free radicals or stabilize the activity of immune cells to enhance human immunity; on the other hand, nitrosoglutathione (GSNO) and its reductase, GSNOP, can inhibit excessive inflammation and protect immune cells and related tissues by modulating the nitric oxide signaling pathway in the body. On the other hand, nitrosoglutathione (GSNO) and its reductase GSNOP can inhibit excessive inflammation by regulating the nitric oxide signaling pathway in the body and protect the immune cells and related tissues in the body [33-34].

Reduced glutathione is also valuable in the detoxification of exogenous substances or their metabolites, the regulation of immune function, and the formation of fibrosis. Glutathione can directly quench reactive hydroxyl radicals, other oxygen-centered radicals, and free radical centers on DNA and other biomolecules[35-37] . Glutathione protects the skin, lens, cornea and retina from radiation damage, and is the biochemical basis for detoxification in the liver, kidney, lungs, intestinal epithelium and other organs [38-39].

In the defense metabolic system of animals, the central pillar of antioxidant metabolism is the selenium-dependent glutathione peroxidase (GPX), which, in order to regulate redox-dependent cellular signals, binds to -SH on cysteine residues of proteins to form a glutathionylation, which alters the oxidative state of proteins and protects sensitive protein thiols from irreversible oxidation [40]. In plants, glutathione can improve plant tolerance, including drought resistance, resistance to high or low temperature, and resistance to heavy metal stress. Glutathione can directly or indirectly scavenge free radicals in plants by regulating metabolism-related enzymes, or combine with toxic peroxides in vivo and then metabolize and excrete them, so as to achieve the effect of maintaining the stability of the organism and improving the resistance of plants [41-45].

3. Glutathione Production Methods

With the in-depth research on the functional application of glutathione, it has been emphasized in the fields of food, medicine, beauty care and biomedicine, etc. The commercial production methods of glutathione are constantly being updated, and the main production methods are chemical synthesis, enzyme method and microbial fermentation method[46-47] , as follows:

3.1 Chemical Synthesis

In the 1970s, glutathione was mainly produced by chemical synthesis, in which three precursor amino acids (L-glutamic acid, L-cysteine, and glycine) were condensed in a series of chemical reactions, which consisted of three phases: group protection, condensation, and deprotection.

The chemical synthesis of glutathione has been in a relatively mature stage, but due to the complexity of the process, and the fact that the chemically synthesized glutathione is a racemate, the process of racemization and separation has an impact on the activity of glutathione, and there are also problems such as different purity of the product and uneven bioefficacy. Considering the influence of other active amino groups, carboxyl groups or side-chain groups of the precursor amino acids on the yield, purity or racemization during the reaction, researchers [48-49] proposed to protect the unwanted groups in the synthesis reaction of glutathione, and then eliminate these protective groups after the reaction is completed. S-benzyl cysteine glycine was synthesized by using benzyloxycarbonyl (C6H5-CH2-O-CO , Cbz) as the protecting group for the amino group, and then N-Cbz-L-glutamyl anhydride was synthesized by protecting the glutamic acid amino group with the Cbz protecting group, and then glutathione with protecting group was obtained by the reaction of S-benzyl cysteine glycine and N-benzyloxycarbonyl-L-glutamyl anhydride, and then glutathione was obtained by deleting the protecting group under the corresponding conditions. group was then removed under the corresponding conditions to obtain glutathione.

3.2 Bio-enzymatic Methods

Enzymatic synthesis of glutathione is mainly based on the use of natural glutathione synthetase in living organisms, which is able to catalyze the synthesis of glutathione specifically using three precursor amino acids as substrates, as well as an appropriate amount of ATP, cofactors necessary to maintain glutathione synthetase enzyme activity (Mg+), and an appropriate pH environment [50-51].

Glutathione synthetase systems are mainly derived from microbial cells, such as Escherichia coli, Saccharomyces cerevisiae, etc. However, due to the instability of the free enzyme activity in the reaction environment and the complexity of the isolation and purification process from the reaction system, the enzyme is not reusable in industrial production. However, due to the instability of the free enzyme activity in the reaction environment and the complexity of the separation procedure from the reaction system in the purification process, which cannot be reused, glutathione synthesis in industrial production is mostly carried out by immobilized cells or immobilized enzymes [52-55].

The immobilization method not only simplifies the production process and increases the enzyme recovery, but also improves the thermal stability of the enzyme and increases the product yield. Since ATP is required for the synthesis of glutathione, and the production of ADP has a certain inhibitory effect on the enzyme activity, the construction of ATP regeneration system, in addition to the immobilization method of the enzyme, is the key to the effect of glutathione synthesis. Researchers [56] established a cascade reaction between polyphosphate kinase (PPK) and glutathione bifunctional enzyme GshF, and utilized the catalytic synthesis process of PPK enzyme that can regenerate ATP to construct an energy regeneration system, and then constructed a low-cost and high-efficiency glutathione enzyme synthesis system.

3.3 Biofermentation

Microbial fermentation is a method of converting inexpensive hairspray raw materials into glutathione by microbial metabolism using bacteria or yeast that synthesize glutathione intracellularly. Since the realization of glutathione production from yeast in 1938, the process and method of glutathione production by fermentation have been improved continuously. Since the bacteria or yeast used in fermentation are easy to cultivate, in industry, biofermentation is generally chosen to use Saccharomyces cerevisiae and Candida utilis as the bacterial strains for fermentation. Fermentation is carried out with Saccharomyces cerevisiae and Candida utilis. Fermentation has become the most common method for glutathione production due to its easy availability of raw materials and controlled conditions [57]. In order to increase the yield of fermented glutathione, researchers have improved the study from the stages of fermentation strain selection [58-61], fermentation process optimization, fermentation culture method [62-63] and isolation optimization [64], respectively.

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