Vitamin C is aWater-soluble vitamin, chemically named L - (+) - Suaraose 2,3,4,5,6-pentahydroxy-2-hexenoacid-4-lactone, also known as L-ascorbic acid, molecular formula CsixHeightOsix, the molecular weight is 176.12.[1-2]
Vitamin C is usually in the form of flakes and sometimes needlesMonoclinic crystal, odorless, sour, soluble in water, with strongReducibility。Participating organism complexmetabolizeThe process can promote growth and enhance resistance to disease, and can be used as a nutritional supplement, antioxidant, and wheat flour improver.However, excessive supplementation of vitamin C is not beneficial to health, but harmful, so it needs to be used reasonably.Vitamin C is used in the laboratoryAnalytical reagent, such asreducing agent、Masking agentEtc.[1-2]
Over 1500 BC, the medical books of Ebes in ancient Egypt first recorded the relationship betweenScurvyA very similar disease.According to textual research, ancient Greek philosophersHippocratesA disease recorded in his works is also scurvy.Because people's understanding of disease was limited at that time, people classified it asplague。[3]
At the end of the 16th century and the beginning of the 17th century, when imperialism was pioneering colonies, the Netherlands and Spain sent a large number of fleets overseas. Tens of thousands of sailors died every year due to lack of vitamin C.Once, a Spanish sailing boat was floating on the sea, and people went to find 25 people on board, all of whom died of scurvy.Later, when the crew of another fleet was on the verge of death, they floated to an island inhabited by Indians, who saved themselves from death by using leaf juice. Only then did they begin to realize the close relationship between vitamins and the human body, a fact that has aroused widespread concern.[3]
At the beginning of the 19th century, the British Navy began to explicitly stipulate that every seafarer must be rationed with lemon juice every day.In 1911, Fengke of Poland put forward his "vitamin hypothesis" in a series of studies on anti beriberi substances, and named the substance he extracted "Vitam ine".In 1920 AD, Drummon unified the name of vitamin and designated it "Vitamin" (the tail letter e was removed).[3]
In 1922, Albert Szent Gy ö rgyi went to the Netherlands to work and began to study the oxidative discoloration of fruits (for example, the surface of an apple will become yellow brown after being cut).He found that cabbage contains a substance that can prevent this yellowing, and there is a similar substance in the adrenal glands of animals, so he studied how to extract this substance from fruits and the adrenal glands of animals.In 1927, St. Germain was invited to work in Frederick Gowland Hopkins Laboratory, a chemist in London, England.There, he was busy extracting this substance from animal and plant tissues.Because the content of oranges, lemons and cabbage is too low, and the cow adrenal gland with a slightly higher content is not easy to get, he was not able to successfully extract a very small amount of this substance until 1928, and obtained the chemical empirical formula C through experimentssixHeightOsix。At first, he didn't know that this substance was vitamin C, which was namedHexuronic acid（L-Ascorbinsäurc）。
In 1929, St. Germain went to Mayo Hospital in the United States to do research. The nearby slaughterhouse provided him with a large number of cow accessory kidneys for free. He separated more vitamin C from them, but only 25 grams.He sent half of them to Walter H. Haworth, a British carbohydrate chemist, for analysis. Unfortunately, the technology was not mature at that time, and Haworth could not determine its structure.
In 1930, when St. Germain returned to Hungary, he found that a local pepper contained a large amount ofHexuronic acid。He finally succeeded in separating 1kg pure hexuronic acid from peppers, and sent another batch to Haworth for analysis.Haworth finally determined the correct chemical structure of vitamin C.Later, Tillmans, Vedder, Harris, etc. also extracted vitamin C from various foods.In 1933, vitamin C began to be artificially synthesized.[3-4]
Vitamin C is a white powder with a molecular weight of 176.12. It is usually a lamellar, sometimes needle shaped monoclinic crystal.Odorless, sour, soluble in water, slightly soluble inethanol, insoluble inEther，chloroform、Petroleum etherAnd other organic solvents.In structure, vitamin C is very similar to sugars, and there are two asymmetric carbon atoms (Cfour，Cfive）, can form four optical isomers.[1][11-12]
Physical properties of vitamin C
Density (25 ° C)
1.65 g/cm³
Henry's law constant (25 ° C)
4.07X10-8Atm cu m/mol at 25 ° C (estimated)
melting point
190-192 ° C (partial decomposition)
Specific rotation (25 ° C)
+20.5 ° to+21.5 °
Solubility (water, 40 ° C)
400 mg/mL
Autoignition point
380 °C
Vapor pressure (25 ° C)
9.28X10-11MmHg (estimated value)
LogP
-1.85
Heat of vaporization (465.15 K)
1.487×10eightJ/kmol
surface tension
4.039×10-2N/m
Dissociation constant
pKone=4.17；pKtwo=11.57
Collision section
127.9 Ų-146.11 Ų
Standard combustion heat of crystalline phase (enthalpy)
The molecular structure of vitamin C has the structure of dienol, and the aqueous solution is acidic.Dip the vitamin C solution with a glass rod and drop it onto theblue litmus paper It can be observed that the test paper turns red;Then dip a small amount of vitamin C into a glass rodPH test paperThe measured pH is about 3;Finally, add 2 drops of vitamin C solutionMethyl orange test solution, the solution turns red.[15]
Since the hydroxyl group on C-2 can form an intramolecular hydrogen bond with the carbonyl group of C-1, the acidity of the hydroxyl group on C-2 is weaker than that on C-3.The acidity of hydroxyl groups on C-3 is strong, which can besodium bicarbonateOr thinsodium hydroxideSolution reaction to produce sodium salt of C-3 enol.However, in a strong base such as concentrated sodium hydroxide solution, the lactone ring is destroyed to form sodium ketoate.[13]
Reducibility
The structure of dienol in vitamin C molecule is easy to release H atom and has strong reducibility.In aqueous solution, it is easily oxidized by oxygen in air to generate dehydroascorbic acid.The two can be transformed into each other, so vitamin C has two forms of oxidation and reduction, both of which have the same biological activity.Dehydrovitamin C is treated by silver nitrateFerric trichloride, alkaline copper tartrate, iodineIodateAnd 2,6-Dichloroindophenol to generate dehydroascorbic acid.[33]Dehydroascorbic acid can be reversed to vitamin C under the action of hydrogen sulfide, hydroiodic acid and other reducing agents.Due to the destruction of the common molecular system of dehydroascorbic acid, dehydroascorbic acid is easier to be hydrolyzed than vitamin C to generate 2,3-dionegulonic acid, which can be further oxidized to generate suanoic acid and oxalic acid and lose its activity.The oxidation rate in aqueous solution is determined by pH and oxygen concentration, and heavy metal ions can catalyze the above reaction.[13]
Instability
Vitamin C is unstable in aqueous solution and quickly oxidized toDehydroascorbic acid, especially in neutral or alkaline solutions, it will be oxidized faster when exposed to light, heat, iron, copper and other metal ions, which can form stableMetal salt；Vitamin C is a relatively strong reducing agent. After being stored for a long time, it turns dark and light yellow to varying degrees.[15]
Differential reaction
Add vitamin C into the aqueous solutionsilver nitrateTest solution, producing black silver precipitation;If 2,6-Dichloroindophenol test solution is added (the test solution itself is cyan and red in acid solution), it can fade.These two reactions can be used to identify vitamin C.[13]
The production methods of vitamin C can be roughly divided into three categories, namelyChemical synthesisChemical synthesis combined with biosynthesis (fermentation) method (sometimes called semi synthesis method) and biosynthesis (fermentation) method.Recently discoveredyeastFermentation synthesis of vitamin C is the only biosynthesis method that does not require intermediate compounds. Although it has great attraction, it is far from reaching the level of industrial production.[16-17]
Chemical synthesis
In 1933, Reichstein and Ault respectively published the chemical synthesis method of vitamin C. However, due to the long synthesis route and low yield, industrial production was not realized.[16-17]
Chemical synthesis combined with biosynthesis
Since 1937, based on the invention of Reichstein and GrussnerglucoseThe "Reichstein procedure" for the production of vitamin C by chemical method combined with fermentation was started.Since then, vitamin C has entered the stage of large-scale industrial production.After continuous improvement and perfection, this method has been widely used in the world.The total yield of industrial production by the Reichsler process is more than 60%.Due to the low cost and easy availability of glucose raw materials, the stable chemical properties of intermediate compounds, especially diacetone-L-sorbose, and the continuous improvement of the process flow, as well as the good product quality, the "Reichsler method" is still the main method for the production of vitamin C abroad.In this method, D-sorbitol is used as raw material, and vitamin C product can be obtained only after four steps of reaction.[16]
one
D-sorbitol was fermented by Gluconobacter melanogenes or Acetobacter cubox ydans to obtain L-sorbitose.
two
Diacetone L-sorbose was prepared by the reaction of L-sorbose and acetone.
three
Diacetone L-sorbose is oxidized to give sodium diacetone-2-keto-L-gulonic acid, and then acidified to give diacetone-2-keto-L-gulonic acid.
four
Vitamin C was obtained by conversion of diacetone-2-keto-L-gulonic acid.
Due to the multiple reaction steps of the "Lai's method" and the serious "three wastes", since the early 1960s, many researchers have made improvements and shortened the reaction steps of the "Lai's method", but no substantial progress has been made.[16-17]
Biosynthesis (fermentation) method
2-keto-L-gulonic acid (2-KGA) is the direct precursor for the synthesis of vitamin C. In the production of vitamin C, the last step from 2-keto-L-gulonic acid to vitamin C is chemical conversion.From glucose, there are at least 6 known pathways to produce 2-keto-L-gulonic acid: ① D-sorbitol pathway; ②L-sorbose pathway (i.e. two-step fermentation); ③L-iduronic acid (or L-gulonic acid) pathway; ④2-keto-D-gluconic acid pathway; ⑤2,5-diketo-D-gluconic acid pathway; ⑥The pathway of producing 2-keto-L-gulonic acid directly from glucose fermentation.Among the six synthetic routes, only the second route has achieved industrial production, that is, the "two-step fermentation method" developed by China itself. The process is as follows.[17]
Chemical Synthesis of D Sorbitol
Sorbitol and glucose are both 6-carbon carbohydrate compounds, and the difference between them lies in the C-1 group.Therefore, the aldehyde group on D-glucose C-1 is reduced to alcohol group to obtain D-sorbitol.In industrial production, D-sorbitol is prepared by catalytic hydrogenation of D-glucose, pressure control, reduction of aldehyde group to alcohol hydroxyl under the condition of hydrogen as reducing agent and nickel as catalyst.Add 50% glucose solution to activated carbon at 75 ℃ to remove impurities.useLime milkLiquid regulating pH8.4, pressing into the hydrogenation reactor, addingNickel catalyst, inject hydrogen, and react at 3.43 × 103kPa and 140 ℃. The reaction end point is when hydrogen is not absorbed.The reaction process shall strictly control pH8.0-8.5, otherwise mannose, the isomerization product of the C-2 differential position of glucose, will be reduced to formmannitol。After the reaction, the catalyst is removed by static sedimentation. After the reaction solution is treated with ion exchange resin and activated carbon, it is concentrated under reduced pressure to obtain D-sorbitol with a content of 60% - 70%, which is a colorless transparent or yellowish transparent viscous liquid with a yield of about 97%.[16]
Microbial fermentation of 2-keto-L-gulonic acid
Two microorganisms were used for two-step biotransformation to prepare 2-keto-L-gulonic acid.First, D-sorbitol was converted to L-sorbose, and then to 2-keto-L-gulonic acid.
The first step of fermentation: from D-sorbitol to L-sorbose, only the hydroxyl at C-2 position is oxidized to the meryl group, keeping other groups unchanged. The specificity of this reaction can be achieved through microbial transformation.Although many kinds of Acetobacter can be used as transformants, black acetate rod is better.Acetobacter was expanded through seed culture, and then put into fermentation tank. The seed and fermentation medium mainly included sorbitol, corn syrup, sudami, yeast extract, calcium carbonate and other components, with pH of 5.0-5.2. The concentration of sorbitol was controlled at 24% - 27%, the culture temperature was 29-30 ℃, and the ventilation ratio was 1 ∶ 1-0.7VVM.Determine the sorbose in the fermentation liquid. When the concentration no longer increases, end the fermentation, about 10h.The biotransformation rate of D-sorbitol to L-sorbose was over 98%.The fermentation liquid is sterilized at 60 ℃ for 20min and cooled to 30 ℃ as the raw material for the second step of fermentation.
The second step of fermentation: from L-sorbose to 2-keto-L-gulonic acid, the C-1 hydroxyl group needs to be oxidized to the carboxyl group, keeping other groups unchanged.Many microorganisms, including Pseudomonas, GluconobacterAcetobacter, AerobacteriumBacillusCan convert L-sorbose into 2-keto-L-gulonic acid.Among them, Acetobacter, Gluconobacter and Bacillus had the strongest transformation activity, and the yield of the product reached the highest level when the two mixed strains were used together.Generally, the yield of a single strain is very low.Gluconobacter, with long or short rod like cells, flagellate or non flagellate, can grow at pH4.5, oxidize glucose to produce gluconic acid and have the function of polyol ketogenesis.The optimum growth temperature is 30-35 ℃ (Gluconobacter oxydans) or 18-21 ℃ (Gluconobacter oxydans).In industrial production, Gluconobacter orydans and Bacillus megaterium are used for mixed culture.The former is acidogenic bacteria, and the latter is a companion bacteria.The possible mechanism is that the former converts L-sorbose to L-idulose (oxidizes the C-1 hydroxyl group to aldehyde group), and the latter further converts 2-keto-L-gulonic acid (oxidizes the C-1 aldehyde group to carboxyl group).
The fermentation tanks for vitamin C production are all above 100m3, and they are slender airlift reactors without mechanical agitation.The components of the seed and fermentation medium are similar, mainly including L-sorbose, corn syrup, urea, calcium carbonate, potassium dihydrogen phosphate, etc., and the pH value is 7.0.The large and small bacteria are expanded through secondary seed culture, and then transferred into the fermentation tank containing the fermentation liquid in the first step. A large amount of sterile air is introduced at 29-30 ℃, and the fermentation is ended after about 72h of culture. The residual sugar is less than 0.5%.The conversion rate of 2-keto-L-gulonic acid from L-sorbose can reach 70%~85%.
The early stage of fermentation is the growth stage of bacteria, which should be in a high dissolved oxygen state with sufficient oxygen supply to promote growth and shorten the early stage.The middle stage of fermentation is the main acid production stage. The acid production rate and sugar consumption rate are constant. The concentration of dissolved oxygen should be controlled within an appropriate range, generally 20%.At the later stage of fermentation, the vitality of the bacteria decreased and the production capacity slowed down. The fermentation was ended in time according to the acid production concentration and residual sugar.
During the whole fermentation period, maintaining a certain number of Gluconobacter oxydans is the key to fermentation.Can be based onSporeTo control fermentation.When the associated bacillus starts to form spores, the acid producing strain starts to produce 2-keto-L-gulonic acid, until the complete formation of spores and the emergence of free spores, the acid production reaches the peak.Add lye drops to adjust the pH value to keep it at about 7.0.When the temperature is slightly higher (31-33 ℃), the pH is about 7.2, and the residual sugar content is below 0.8mg/mL, it is the fermentation end point.At this time, the free spores and residual Bacillus have gradually dissolved into fragments, and it is impossible to distinguish the difference between the two bacterial cells by microscopic observation, so the whole acid production reaction is ended.[16]
Separation and purification of 2-keto-L-gulonic acid
After two steps of fermentation, the fermentation broth only contains about 8% 2-keto-L-gulonic acid, and there are impurities such as mycelium, protein and suspended solid particles left, which are often purified by heating precipitation, chemical coagulation and ultrafiltration.The traditional process is heating and sedimentation. After static sedimentation, the fermentation broth is adjusted to the isoelectric point of protein with hydrochloric acid, and the protein is heated to coagulate, then the mycelium, protein and particles are separated by high-speed centrifuge.
The acidified supernatant passes through the 732 hydrogen type ion exchange resin column to control the pH value of the effluent.When the effluent reaches a certain pH value, replace the resin for exchange.Collect the effluent and eluate, adjust the pH in the heating tank to the isoelectric point, heat it at 70 ℃, add active carbon, heat it at 90-95 ℃ for 10-15min, rapidly cool it, and filter it.
The filtrate passes through the cation exchange column again, controls the effluent pH1.5-1.7, and acidifies into an aqueous solution of 2-keto-L-gulonic acid.The 2-keto-L-gulonic acid was obtained by decompressing concentration at 45 ℃, cooling crystallization, centrifugation separation, ice ethanol washing, and the extraction rate was more than 80%.[16]
Preparation of vitamin C
Vitamin C can be obtained by esterification of 2-keto-L-gulonic acid at the C-4 position and alcoholization of 2-keto-L-gulonic acid at the C-2 position under the action of acid or base catalysts.In industrial production, the alkali conversion method is often used to catalyze the formation of vitamin C from 2. - keto-L-gulonic acid.[16]
one
Acid conversion:The ingredient ratio is 2-keto-L-gulonic acid: 38% hydrochloric acid: acetone=1:0.4 (mass/volume): 0.3 (mass/volume).First add acetone and half of the gulonic acid into the conversion tank for mixing, then add hydrochloric acid and the rest of the gulonic acid.Open the steam valve, slowly raise the temperature to 30-38 ℃, and close the steam valve.The reaction reached its climax and crystallized after natural heating to 52-54 ℃ and holding for about 5h.The temperature in the tank rises slightly, up to 59 ℃, and the strictly controlled temperature cannot exceed 60 ℃.After the high tide period, maintain the temperature at 50-52 ℃ until the total holding time is 20h.Cool down for 1h, add proper amount of ethanol, cool down to - 2 ℃, and discharge.After being filtered for 0.5h, it is washed with ice ethanol, dried, washed again, dried for about 3h, and dried to obtain crude vitamin C.The equipment of acid conversion process is simple and the process is short, but vitamin C is seriously damaged and the quality is poor.The equipment was severely corroded and the problem of "three wastes" was not solved well, so it was gradually eliminated.
two
Alkali conversion:2-keto-L-gulonic acid is catalyzed by concentrated sulfuric acid in methanol to produce 2-keto-L-gulonic acid methyl ester, and NaHCO is addedthree。It is converted into sodium salt of vitamin C, acidified by hydrogen type ion exchange resin, and dried under reduced pressure at 50-55 ℃ to obtain crude vitamin C.The alkali conversion process has a long process and a large investment, but the equipment is less corroded, the intermediate is easy to separate, and the product quality is good.However, due to the use of sodium bicarbonate, a large number of sodium ions are brought in;A large amount of sodium sulfate is produced in the mother liquor after conversion, which seriously affects the application of mother liquor and the quality of finished products.[16]
Laboratory preparation
extract
Use a torsion balance to accurately weigh 10g of fresh samples (fruits and vegetables rich in vitamin C, etc.), and put a small amount of 2% HCl (5-10ml) into the milk bowl.Fully grind and extract (do not poke out the solution), grind and extract for 3-4 times in this way, and filter the n-time extract solution into a 50ml volumetric flask through a funnel through two layers of gauze (do not drop the extract solution from the gauze outside the bottle).Finally, dilute to the scale with 2% HCl and mix well to obtain crude solution of vitamin C.The stability of vitamin C is the best under the acidic condition of pH 3-4.The single acid extractant 2% oxalic acid is most widely used, 5% trichloroacetic acid and 10% hydrochloric acid have better extraction effect, and the effect of metaphosphoric acid is better than oxalic acid, but metaphosphoric acid is expensive and highly toxic, so it is not recommended.The extraction effect of mixed acid solvent is better than that of single acid extractant. The reported literature mostly uses 2% oxalic acid mixed acid, and the extraction effect is more stable and efficient.[18-19]
physiological function
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antioxidant
Most of the biological functions of vitamin C are determined by its unique structure.The molecular formula of vitamin C is CsixHeightOsixThere are two active hydroxyl groups in the structure of lactone ring, which is very easy to occurionization（pKa11.6 and 4.2 respectively), generate ASC andDHADHA can be reduced to ASC in a series of enzymatic reactions.Therefore, ASC has become a natural antioxidant, and the reduced glutathione (GSH) - dependent DHA reductase mediated reaction is the most representative of the above reactions.[5]
A large number of reactive oxygen species (ROS) can be produced during oxidative phosphorylation of mitochondria and inflammatory reaction induced by bacteria and viruses.Many proteins and enzymes need sulfhydryl (- SH) groups to perform their physiological functions, but sulfhydryl groups are vulnerable to oxidation by oxidative free radicals. If active oxygen free radicals cannot be eliminated as soon as possible, DNA oxidation and lipid peroxidation can occuroxidation reaction , amino acid oxidation, etc.Protect the sulfhydryl group on the erythrocyte membrane to prevent hemolysis and protecthemoglobinPrevent oxidationMethemoglobinEtc.In addition, vitamin C can interact withvitamin ECooperate with each other to timely remove the oxidation free radicals in the hydrophobic region;Electron transfer mediated by reduction reaction can also effectively maintain the balance of redox environment, which is very important for maintaining cell integrity and normal physiological activities of cells.[5]
collagen synthesis
collagen protein(collagen) mainly exists in skin, bone, viscera and other parts, which can maintain the shape of skin and other organs.Collagen-4-hydroxylase (C-P4H) is required to promote the cross-linking of collagen molecules and ensure the integrity of tissue structure. C-P4H is a ferrous ion and 2-oxoglutarate dependent dioxygenase that catalyzes the hydroxylation of collagen proline residues to produce (2S, 4S) - 4-hydroxyproline.Vitamin C is a cofactor for C-P4H to exert enzyme activity.[5]
In addition to C-P4H, dopamine beta hydroxylase (D - β H) in such family can catalyzedopaminegenerateNorepinephrine, γ - butyroletaine dioxygenase (GBBH) can catalyze the synthesis of carnitine, and peptidylglycine α - hydroxylation monooxygenase (PHM) can catalyze theAmidation reactionEtc.In case of lack of vitamin C, large amount of Fe2+Oxidized to Fe3+C-P4H quickly lost its enzyme activity, which seriously affected the hydroxylation of proline residues on collagen, leading to vitamin C deficiency.The damage of capillaries in specific parts can not be repaired in time, resulting in blood stasis, delayed wound healing, arthralgia, purpura, etc., which can cause death in serious cases.If vitamin C is added again, a large amount of Fe3+Is reduced to Fe2+C-P4H enzyme activity can be completely recovered.In addition, vitamin C can also promote Fe3+To Fe2+Transformation can also increase the body's absorption of iron and promote the expression of ferritin in cells.[5]
Delay cell aging and apoptosis
Vitamin C can participate in the remodeling of chromatin structure and the regulation of effector gene expression through the regulation of 5-methylpyrimidine modifying enzyme 1, histone demethylase of Jumonji C domain.Vitamin C can enhance the activity of HIF PHs, induce the degradation of HIF-2 α, promote the maturation of precursor i PSCs, and inhibit the expression of p53 gene during reprogramming.Vitamin C can also regulate protein ALKBHs. ALKBHs can not only remove the methylation modification on histone H2A, but also interact with core pluripotent factors to jointly regulate the expression of embryonic stem cell specific miRNA.These reactions can not only delay cell senescence and apoptosis, but also promote the repair of some tissues.[5]
Anticancer and anti-tumor
Since the 1970s, people began to study the anti-cancer and anti-tumor effects of vitamin C.After many timesClinical trialsAnd animal experiments, gradually revealing the secrets of vitamin C in anti-cancer and anti-tumor.[5]
Abnormal hypermethylation of the genome is an important cause of cell carcinogenesis. Hypermethylation in the CpG island of the DNA gene promoter region can inhibit the expression of tumor suppressor genes.Most of the causes are hyperfunction of DNA methyltransferases (DNMTs) and loss of function of TET family proteins. Vitamin C can increase the activity of most of the enzymes in the TET family, correspondingly reduce or even avoid DNA hypermethylation, promote the inhibition of tumor gene expression, promote stem cell differentiation, and enhance DNA methyltransferases inhibitor,DNMTi) induced immune information.[5]
HIF-1 α is a transcription factor. HIF-1 α in cancer cells will show a higher expression level, which can regulate the transcription of hundreds of malignant tumor related genes. By up regulating cell glycolysis, erythropoiesis, cell survival pathway, angiogenesis and tissue remodeling, cells can adapt to hypoxia and metabolic stress caused by rapid growth,These include vascular endothelial growth factors that determine tumor angiogenesis.Vitamin C can not only regulate the hydroxylation of HIF-1 α, but also act as Fe2+/The cofactors of α - KGDDs enhance the function of HIF hydroxylase, inhibit the transcription of HIF-1 α, block its downstream pathway, and promote its protein degradation.[5]
In the process of mutual transformation of different active forms of vitamin C, whenDehydroascorbic acidWhen the concentration reaches above 100nmol/L, H can be generatedtwoOtwo；HtwoOtwoEntering the cell interior can first cause DNA damage, which will consume a lot of energy (ATP) to repair the damage.Tumor cells can show unique Warburg effect, that is, active glycolysis, high glucose consumption, high lactic acid content, and low energy generation efficiency.Vitamin C can be rapidly reduced to vitamin C prototype after entering tumor cells, further increasing ROS level and producing similar HtwoOtwoThe effect of TNF can lead to cell energy depletion, mitochondrial damage and apoptosis.Some studies have shown that vitamin C can kill normal cells (ECfifty）More than 20mmol/L, but the EC of cancer cellsfiftyLess than 4 mmol/L.[5]
Regulate cell signaling pathway
The traditional research field of vitamin C focuses on its oxidation-reduction property and cofactor coenzyme, and the related research as a factor regulating cell signal pathway is a new and active field in the past two decades.[5]
It has been found that vitamin C can inhibit P38MAPK activity and P53 induced cell senescence by reducing the level of ROS, and can also promote the regeneration and maturation of endothelial cells in damaged tissues by activating ERK signaling pathway.Vitamin C can inhibit NFkThe signal activation of β is involved in biological processes such as inflammation, tumor formation and apoptosis.Some studies have shown that vitamin C can promote the proliferation of cardiomyocytes by activating MEK-ERK1/2 pathway, reduce the accumulation of cAMP in cells by inhibiting part of adenylate cyclase, and inhibit the maturation and differentiation of precursor adipocytes.[5]
Cholesterol metabolism
cholesterolIt is not only an important component of cell membrane and plasma lipoproteins, but also a precursor of many important substances, such asbile acid、Adrenocortical hormone、vitamin D、sex hormoneEtc.In the process of cholesterol metabolism, vitamin C is a coenzyme for the hydroxylation of cholesterol ring, and the side chain is decomposed into bile acid.Therefore, vitamin C can regulate the metabolism and synthesis of cholesterol in both directions.[5]
application area
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food industry
one
Fresh keeping of fruits and vegetables:The rapid senescence of fruits after picking is closely related to the production of a large number of reactive oxygen species (ROS) in the pulp metabolism after picking. If the ROS in the cells cannot be cleared by the antioxidant system in time, it will lead to oxidative stress.Vitamin C is an important antioxidant in plants. Some studies have shown that if the vitamin C content of fruits and vegetables is increased during postharvest storage, the postharvest fresh-keeping effect of fruits and vegetables can be improved.[23]
two
Anti browning:Fruits and vegetables are easily discoloured after being damaged during transportation, packaging or processing, which is called enzymatic browning.The basic step of enzymatic browning reaction is to oxidize phenolic compounds to o-quinones under the catalysis of phenolase, and then quinones will undergo a series of oxidative condensation polymerization to form brown products, which will lead to browning.Vitamin C is a common anti browning agent, which can prevent browning through two different mechanisms: in the absence of polyphenol oxidation (PPO) substrate, it makes PPO irreversibly inactivated, possibly by combining with its active site;In the presence of PPO substrate, vitamin C will reduce the reaction products of PPO oxidation, that is, the quinone formed by oxidation will be coupled with oxidation, and the quinone will be reduced, leading to the regeneration of phenol, thus inhibiting browning.[23]
three
Improve protein properties:Because vitamin C is unstable in aqueous solution, there is an oxidation-reduction ascorbic acid system.Studies have shown that the active carbonyl group of DHA, the first stable oxidation product of vitamin C, and other further oxidation products (threose, oxalic acid, etc.) can be crosslinked with amino acid residues. Some scholars also believe that DHA participates in the exchange reaction between SH group and disulfide bond, through which molecular expansion can be increased,The protein protein interaction is enhanced by the formation of intermolecular disulfide bonds, which may affect the properties of proteins.[23]
four
Inhibition of fat oxidation:Vitamin C can not only inhibit lipid oxidation, but also improve the antioxidant property of emulsion.Oil oxidation is a serious problem of oil food, which has a negative impact on the shelf life and nutrition of food.Because vitamin C has good reducibility, it can remove oxygen from food and protect oil from oxidation.[23]
five
Reduce nitrite content:nitriteGenerated by reaction with amine in meat productsNitrosamine, potentially carcinogenic after consumption.As an antioxidant, vitamin C can reduce nitrite to NO and consume NO2 under acidic conditions-So as to reduce the nitrite concentration.[23]
six
As SOtwoSupplement:SOtwoIt is usually added to wine as a protective agent. It is not only a bactericide, which can effectively kill the miscellaneous bacteria in grapes, but also an antioxidant. It can protect the natural fruit characteristics of wine liquor while preventing wine liquor from aging.In wine, for SOtwoThe residue is strictly limited, so look for SOtwoThe ideal supplement or substitute has become a research topic.In 1956, the United States approved the use of vitamin C in the production of wine and fruit juice.[23]
seven
Protect the stability of tea polyphenols and improve the quality of black tea:Vitamin C is beneficial to increase the content of theaflavin and thearubigin and reduce the occurrence of polymerization.The content of theaflavins and thearubigins is directly related to the quality of black tea, and the polymerization products have adverse effects on the quality of black tea.[23]
Chicken breeding:Appropriate use of vitamin C can promote the survival of chickens, promote the growth of broilers, increase the egg production rate of layers, improve the quality of egg shells, enhance the immunity of chickens, and improve the ability of chickens to resist stress.[24]
two
Aquaculture:Vitamin C mainly improves the taste and meat quality of aquatic animals by promoting the synthesis of collagen.Research has proved that collagen plays an important role in maintaining the muscle structure, flexibility and swimming ability of fish.Adding vitamin C to feed can improve the immunity and survival rate of aquatic animals.Vitamin C can reduce the secretion of glucocorticoid, avoid the immunosuppression caused by it, and thus improve the anti stress ability of aquatic animals.[25]
Pharmaceutical use
indication
oneScurvy, infectious diseases and purpuraKeshan diseaseWhen the patient has cardiogenic shock.2. Chronic iron poisoning.3. Idiopathic methemoglobinemia.4. Increased demand for vitamin C: patients receive chronicHemodialysis、Gastrointestinal Diseases、AIDS、tuberculosis、cancer、ulcerHyperthyroidism, fever, infection, trauma, burn, post operation, etc;Patients receiving parenteral nutrition due to strict control or choice of diet, malnutrition, sudden weight loss, and pregnant and lactating women;When barbiturates, tetracyclines, salicylic acids, or vitamin C are used as acidifiers of the urinary system.[26]
Pharmaceutical preparations
Vitamin C is used in several pharmaceutical preparations, including vitamin C tablets, vitamin C injection, vitamin C granules, vitamin C effervescent tablets, vitamin C effervescent granules, and compound sodium vitamin C chewable tablets.[27]
Calculate chemical data
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1. Number of hydrogen bond donors
four
2. Number of hydrogen bond receptors
six
3. Number of rotatable chemical bonds
two
4. Number of tautomers
eight
5. Topological molecular polar surface area
one hundred and seven
6. Number of heavy atoms
twelve
7. Surface charge
zero
8. Complexity
two hundred and thirty-two
9. Number of isotope atoms
zero
10. Determine the number of atomic structure centers
two
11. Number of uncertain atomic structure centers
zero
12. Determine the number of chemical bond stereocenters
zero
13. Uncertain number of chemical bond structural centers
zero
14. Number of covalent bond units
one[1]
Safety measures
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Environmental hazards
Unless specifically excluded, residues produced by the use of vitamin C as an inert or active ingredient in pesticide chemicals (including anti microbial pesticide chemicals) may be exempt from the tolerance requirements in Section 408 of FFDCA if their use complies with good agricultural or manufacturing practices.[11]
Vitamin C is flammable, but there is no open flame.Dust forms explosive mixture in the air, and the explosion severity is moderate.[11]
Health hazards
Excessive use of vitamin C has the following health hazards:
Children in growth stage are more than 3 g/day, which will affect the absorption of calcium and phosphorus by bone after several months, and they are prone to osteopathy in adulthood.
If the adult is more than 3 g/day, the urine output will increase. Because vitamin C and glucose have the same reaction in reduction, the reduction of vitamin C can produce a positive reaction of urine sugar, which will hinder the treatment of diabetic patients and the exact urine will become weakly acidic;About 10 days later, urinary calcium oxalate stones and kidney stones may occur. In severe cases, frequent urination, urgency and hematuria may occur.
Serious hemolytic reaction may occur when intravenous injection of 4-5g/day.Because vitamin C increases the sensitivity of erythrolysis.5g/day can increase the absorption of iron, and vitamin C can make the intestinal absorption of iron difficult3+Reduction to easily absorbed Fe2+And then form ferruginous erythrocytic anemia, and can reduce the intestinal impact on BtwelveIt can accelerate the deterioration of megaloblastic anemia.
If the dosage is more than 3-4g/day during treatment, the anticoagulant effect of anti heparin and dicoumarin may occur, leading to thrombosis.
Female fertility can be reduced if it is greater than 5g/day.Because massive use of vitamin C can cause the change of glycoprotein disulfide bond in cervical mucus, prevent sperm penetration, and cause infertility.[32]After several months of continuous use of 3g/day by pregnant women, if their fetus is not given the same large dose of vitamin C after birth, the fetus can develop scurvy.After induced abortion, women given 2g/day can cause menstrual bleeding.
When vitamin C is used in large quantities for more than 60 days, the content of vitamin C in the body decreases because it changes the regulatory mechanism of vitamin C in the body and accelerates the decomposition and excretion.Therefore, once the drug is stopped, the body still maintains accelerated decomposition and excretion of vitamin C, resulting inVitamin C deficiency, early bad blood symptoms, such as gum swelling and bleeding, gum loosening, etc.
Medium contentVitamin B12Add 0.1g vitamin C and vitamin B to the diet oftwelve40% is destroyed. If 0.5g is added, 95% is destroyed.Because vitamin BtwelveIts chemical structure is easily destroyed and fails under acidic conditions.vitamin BtwelveAfter a large amount of damage, the human body is prone to anemia.
When there is inflammation in the body, a large amount of vitamin C is used every day, which can reduce the body's resistance and is not conducive to the absorption of inflammation.Because there is too much vitamin C around white blood cells, it not only prevents white blood cells from destroying bacteria, but also protects bacteria and cancer cells.Therefore, cancer patients undergoing radiotherapy and chemotherapy should not use vitamin C in large quantities.
Abuse of vitamin C may accelerate arteriosclerosis.[20]
Safety signs
Safety signs
S26 S36 S24/25
Danger signs
R20/21/22 R36/37/38[1]
Toxicological data
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Reproductive impact
Routes/Organisms
dose
influence
Intraperitoneal/mouse
6680 mg/kg (11 days of pregnancy)
Reproduction: effects on embryos or fetuses: fetal death
Intravenous injection/mice
800 mg/kg (8 days of pregnancy)
Reproduction: specific developmental abnormalities: central nervous system
Reproduction: specific developmental abnormalities: musculoskeletal system
Oral cavity/guinea pig
19500 mg/kg
(30-58 days pregnant/10 days after birth)
Reproduction: effects on newborns: biochemistry and metabolism
Oral cavity/guinea pig
5800 mg/kg (1-58 days of pregnancy)
Reproduction: impact on newborns: stillbirth
Reproduction: impact on newborns: vitality index (e.g., survival on day 4)
Oral cavity/guinea pig
2471 mg/kg (multi generation)
Reproduction: impact on newborns: development (e.g. weight gain and loss)
Oral/rat
2500 mg/kg (1-22 days of pregnancy)
Reproduction: impact on fertility: implantation mortality
reference material:[21]
Carcinogenic data
Routes/Organisms
dose
influence
Oral/rat
Published minimum toxic dose: 1802500 mg/kg;103 weeks - continuous
Carcinogenicity: according to RTECS standard, leukemia
reference material:[21]
Storage and transportation
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Storage method
Vitamin C oxidizes rapidly in air and alkaline medium, so it should be sealed in brown glass bottles and stored in a cool, dry place away from light. It should be stored separately from strong oxidants and strong alkalis.[1][22]
Transport method
matters needing attention
Dust dispersion shall be prevented during transportation of vitamin C. Local exhaust or respiratory protection devices, protective gloves and safety glasses can be used.Avoid direct contact with light and air during transportation.[11]
Disposal of expired drugs
Expired or abandoned vitamin C should not be disposed of by flushing the drug into the toilet or throwing it into the garbage can.If possible, return the drug product to the manufacturer for proper disposal, carefully mark it correctly and safely pack the material.Alternatively, waste vitamin C shall be labeled, safely packaged and transported by the medical waste contractor and disposed of in a licensed hazardous or toxic waste landfill or incinerator by burial.[11]
test method
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Capacity analysis
Iodometry
Vitamin C can be directly titrated with iodine titrant in acidic solution with starch as indicator.
Determination method: Take about 0.2g of this product, precisely weigh it, add 100ml of newly boiled cold water and 10ml of dilute acetic acid to dissolve it, add 1ml of starch indicator solution, immediately titrate it with iodine titrant (0.05mol/L) until the solution turns blue, and it will not fade within 30s.Every 1ml of iodine titrant (0.05mol/L) is equivalent to 8.806mg of vitamin C.
Attention shall be paid during operation: titration reaction is carried out in acidic solution (acetic acid, sulfuric acid or metaphosphoric acid), which can slow down the oxidation rate of vitamin C by oxygen in air;Add new boiled cold water to dissolve, so as to reduce the influence of dissolved oxygen in water.Even so, when the test article is dissolved in dilute acid, it still needs to be titrated immediately to reduce the interference of oxygen in the air.
This method is applicable to the determination of vitamin C raw materials.Almost all national pharmacopoeias use this method to determine the content of API.Since there are often reducing substances in the preparation, which interfere with this method, this method is generally not used for the determination of vitamin C in the preparation. However, considering that this method is extremely simple and fast, the Chinese Pharmacopoeia (2020 Edition) still uses this method for the determination of tablets and injections.However, in order to eliminate interference, some necessary processing should be done before measurement.If the tablet is dissolved, it should be filtered, and the primary filtrate should be discarded, and the subsequent filtrate should be taken for determination;The injection often contains sodium bisulfite as an antioxidant. Before titration, acetone (or formaldehyde) should be added to make it react with sodium bisulfite to form an adduct and mask it to eliminate interference.[2]
2,6-Dichloroindophenol method
2,6-Dichloroindophenol is an oxidation-reduction indicator dye. With the strong reducibility of vitamin C, it can be reduced from oxidized red (in acid solution) to reduced colorless phenol imine, which can significantly change the color and be used for volumetric analysis or colorimetric determination of vitamin C.
When vitamin C is dropped with 2,6-dichloroinophenol in acid solution, titration is performed until the solution shows rose red, which is the end point, and no additional indicator is required.
Precisely measure an appropriate amount of this product (about 50mg of vitamin C), place it in a 100ml volumetric flask, add 20ml of metaphosphoric acid acetic acid test solution, dilute it with water to the scale, and shake it evenly;Precisely measure an appropriate amount of diluent (about 2mg of vitamin C), place it in a 50ml conical flask, add 5ml of metaphosphoric acid acetic acid test solution, titrate with dichlorindophenol titrant until the solution shows rose red, and continue for 5s;Take 5.5ml of metaphosphoric acid acetic acid test solution, add 15ml of water, titrate with dichloroinophenol titrant, and use it as blank test for correction.Calculate the titre of vitamin C with dichloro indophenol titrant.
The specificity of this method is higher than that of iodometry, and it is mainly used for the analysis of vitamin C preparations and food.However, this method is not a specific reaction of vitamin C. Other reducing substances, such as vitamin B, ferrous compounds, nicotinic acid reducing derivatives, and compounds containing hydroxyl groups, have interference, but because the oxidation rate of vitamin C is far faster than that of interfering substances, rapid titration can reduce the impact of interfering substances.Therefore, it should be titrated quickly. According to the International Pharmacopoeia, the titration should be completed within 2 minutes.
For the content determination of tablets, firstly, extract repeatedly with metaphosphate acetic acid test solution (also with metaphosphate test solution), combine the extract solution, filter, wash the residue with metaphosphate acetic acid test solution, combine the lotion with the filtrate, and then carry out the content determination. This is to keep vitamin C stable during the extraction process, and extract all vitamin C,Ensure that the determination results are accurate and reliable.
As quasi 2,6-dichloroinophenol solution is not stable enough and easy to decompose slowly during storage, it needs to be prepared and calibrated before use.[12]
N-bromosuccinimide (NBS) titration
N-bromosuccinimide is weakly oxidizing, while vitamin C is a strong reducing agent. When other reducing agents interfere, N-bromosuccinimide is selective, and vitamin C is oxidized quantitatively first.
Add potassium iodide and starch in the titration solution as indicators. When vitamin C is completely oxidized, a slight excess of N-bromosuccinimide oxidizes potassium iodide to release free iodine, which will appear blue with starch to indicate the end point.
This method can be used for the determination of vitamin C in preparations, biological fluids, vegetables and fruits. It has the advantages of rapidity, accuracy and specificity.However, when there are sulfites, thiosulfates and thiourea in the test solution, they can be oxidized before the oxidation of potassium iodide, so they interfere with the determination.[12]
colorimetry
It is colored with 1,10-phenanthroline iron (Ⅲ) reagent [1,10 phenanthroline iron (Ⅲ)].In acid solution, iron (Ⅲ) oxidizes vitamin C to produce dehydrovitamin C and is reduced to ferrous (Ⅱ) ion, which can complexe with 1,10-phenanthroline to generate red ferrous phenanthroline ion, with maximum absorption at the wavelength of 510nm.
This method can be used for the determination of vitamin C and its preparations.This method has a fast color development speed, and the darkest color can be obtained in 1-2min at room temperature.The optimal pH range is 1.5-6.5, and the pH value of the final colorimetric solution is about 5.5, so it is unnecessary to adjust the pH value of the test solution.After color development, the color can be stable for 24 hours without change, and the color development test solution can also be stable for several weeks without change.
The test shows that 10 times of the following compounds have no interference on the determination: nicotinic acid, nicotinamide, urea, thiourea, methionine, starch, glucose, fructose, mannose, sucrose, maltose, aspartic acid, tartaric acid, glutamic acid, citric acid, calcium, magnesium and copper salts.Contains vitamin A and vitamin DoneVitamin Bone, Vitamin BtwoVitamin B6，The compound vitamin preparations such as nicotinamide and calcium pantothenate did not interfere with the determination of vitamin C.The reason why many of the above compounds, especially reducing compounds, do not interfere with the determination is that the determination is carried out at room temperature, the conditions are mild, and the reaction time is extremely short.[12]
Ultraviolet spectrophotometry
Vitamin C has the maximum absorption at 267nm wavelength between pH5-10, and its molar absorption coefficient is 1.5 × 10 '. Assume that the absorbance is A (I) at this time. If vitamin C is oxidized to dehydrovitamin C by Cu * catalysis, and further hydrolyzed to diketogulonic acid, then the absorbance A (II) of the oxidation product is measured under the same conditions, and its molar absorption coefficient is very small. This property is applied,The content of vitamin C can be determined by measuring the difference of absorbance of vitamin C before and after Cu catalytic oxidation.[12]
High performance liquid chromatography
The concentration of vitamin C in body fluid is usually determined by 2,4-dinitrophenylhydrazine colorimetry and high performance liquid chromatography.[12]
Relevant standards
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The national standard "Food Additive Vitamin C (Ascorbic Acid)" (GB 14754-2010) was implemented on March 21, 2010, and is applicable to the food additive vitamin C, which is chemically synthesized from D-glucose or sorbitol after fermentation.[7]
The national standard Chemical Reagent L (+) - Ascorbic Acid was implemented on December 1, 2015, which stipulates the properties, specifications, tests, inspection rules, packaging, storage, transportation and marks of the chemical reagent L (+) - Ascorbic Acid (Vitamin C).This standard is applicable to the test of chemical reagent L (+) - ascorbic acid (vitamin C).[9]
The national standard Feed Additive L-ascorbic acid (vitamin C) (GB 7303-2018) was implemented on January 1, 2020, which stipulates the requirements, test methods, inspection rules and labels of feed additive L-ascorbic acid (vitamin C) products, and is applicable to vitamin C produced by chemical synthesis after fermentation with D-sorbitol as raw material.[8]
derivative
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Sodium Ascorbate
Sodium AscorbateAlso known as sodium L-ascorbate, it has two types: crystal and particle.Sodium ascorbate is mainly used as a nutritional supplement and antioxidant, and its function is the same as that of vitamin C.Because it has no sour taste and is easily soluble in water, it is widely used for the fresh-keeping and color fixation of ham, sausage, cake and mildew prevention of moon cakes.There are two synthetic routes: one is to add sodium bisulfate in the aqueous solution of vitamin C, place it for a while, and then add ethanol to precipitate, filter and dry it;The other route is to dissolve vitamin C in organic solvent, add sodium bicarbonate, and then heat, react, cool, precipitate and filter.[6]
Vitamin C calcium
Vitamin C calciumAlso called L-ascorbic acid calcium, the price of vitamin C calcium in the international market is 15% - 30% higher than that of vitamin C.Vitamin C calcium is mainly used as a food antioxidant. Its addition to food does not change the taste of the original food, can supplement calcium easily absorbed in food, and does not lose the physiological activity of vitamin C. It is a dual functional auxiliary for the prevention and treatment of rickets and scurvy.The synthesis method is to dissolve vitamin C in water, add calcium carbonate under intense agitation, and the resultant reactant will crystallize at room temperature by itself to obtain the crystal product calcium vitamin C dihydrate with a purity of 98%.The synthesis process is simple and easy to produce.[6]
magnesium ascorbyl phosphate
magnesium ascorbyl phosphate It is a kind of odorless and tasteless powder, which is quickly enzymatically hydrolyzed into vitamin C after entering the human body. Its advantages are that it is not easy to be oxidized, is not afraid of alkali, is not affected by iron and other metal ions, and can be stored for a long time.Its production process is mainly vitamin C or sodium salt of vitamin C and POCl under certain conditionsthreeThe reaction generates vitamin C phosphate or sodium vitamin C phosphate, and then reacts with magnesium oxide to generate magnesium vitamin C phosphate.[6]
Vitamin C polyphosphate
Phosphatase exists in animal digestive organs, which can hydrolyze vitamin C phosphate and free vitamin C, so it can be used by animals.Its advantage is that it is stable in water for a long time, and it still has antioxidant effect after certain pressure treatment. This feature enables it to maintain stable performance in the process of processing into required pellets under pressure.The experiment proves that compared with ordinary vitamin C, vitamin C polyphosphate has stable quality and excellent performance, and its antioxidant capacity, immersion stability, thermal stability, heat resistance and ultraviolet resistance are greatly improved. It has obvious application effect in shrimp and freshwater fish, and is worth popularizing in the feed industry.The raw materials used for its synthesis mainly include vitamin C, sodium trimetaphosphate and related reaction aids.[6]
Vitamin C palmitate
Vitamin C palmitateThe molecular formula is Ctwenty-twoHthirty-eightOsevenIt is an efficient, safe, non-toxic, fat soluble antioxidant, which is mainly used in baby food, canned food, cream, etc. It can be added to drug ointment and capsule preparation to increase drug stability.Its price is almost four times that of vitamin C, and its profit is very high.In recent two years, this variety has attracted people's attention in China. The main raw materials for its synthesis are palmitic acid, vitamin C, and dichloro sulfoxide.[6]
Vitamin C Stearate
Vitamin C StearateThis product is also called L-ascorbyl stearate, and its molecular formula is Ctwenty-fourHforty-twoOseven。This product is a slightly shiny white crystalline powder, which is basically stable in the dry state, extremely easy to oxidize in the moisture absorption state, insoluble in water, and soluble in peanut oil and cottonseed oil.This product is a lipophilic substance with the effect of vitamin C and can be used as a food nutrition supplement.The production process of this product is to dissolve vitamin C and stearic acid in 95% HtwoSOfourPlace it for 24h, add crushed ice to precipitate it, and then extract it with ether and other organic solvents, wash it with water to neutral, recover the organic solvent to precipitate the product, recrystallize it with petroleum ether, ether and other organic solvents, and then vacuum dry it.[6]
Vitamin C-glucose compound
Vitamin C-glucose compound is a new product called "stable vitamin C".In addition to the traditional use of vitamin C, it is also expected to develop new uses such as injections, drops, eye drops, etc.The production method is to utilize the sugar transfer reaction of cyclic maltose paste glucose transferase from microorganisms.The product is very stable, soluble in water, sour, powdery, and can form salts with many metals. Compared with the original vitamin C, its performance has not changed.[6]
