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Ascorbic acid also known as vitamin C is an essential water-soluble vitamin and it is the first vitamin being discovered by the Hungarian physician Professor Albert Szent-Györgyi from the adrenal cortex of cattle. (Davies et al., 1991). Nowadays, a tablet of vitamin C has become a part and parcel in daily life for many people. Vitamin C acts as a supplement or an antioxidant, which helps to prevent various kind of disease such as scurvy, cancer aging disease and so on. So it can also be said that "a vitamin c a day, keeps doctors away".
Antioxidant simply means the molecule or a compound capable of prevent oxidation of other compounds. Hence vitamin C can provides protection against harmful free radicals which can help people to reduce the risk of some disease include cardiovascular disease, cancer, diabetes, Alzheimer's disease, cataracts, and age-related functional decline. (Zulueta et al., 2006). L-ascorbic acid has antioxidant ability because it can easily reversibly oxidized to L-dehydroascorbic acid (DHA) and the irreversibly be oxidized to 2,3-diketogulonic acid as shown in figure 1. (Hernández et al., 2005)
Figure : Oxidation of l-ascorbic acid (Hernández et al., 2005)
Besides antioxidant, Vitamin C is also a cofactor in various physiological processes such as post-translational hydroxylation of proline and lysine in collegen and other connective tissue protein, collegen gene expression, activation of many peptide hormone, synthesis of carnitine and synthesis of adrenaline and nor adrenaline. (Phillips et al, 2010). According to Eitenmiller and Landen (1999), the involvement of L-ascorbic acid in collagen synthesis is directly related to the etiology of scurvy. Moreover, it also participates in immune system, biosynthesis and metabolism of certain compounds.
According to Institute of medicine (IOM), the recommended Dietary Reference Intakes (DRIs) for vitamin C for adult men and women is 90 mg/day and 75 mg/day respectively. (Food and Nutrition Board, Institute of Medicine, 2004). Deficiency of vitamin C cause scurvy, which is a disease Characterized by bleeding gums, impaired wound healing, anemia, depression and fatigue. (Phillips et al, 2010).
Ascorbic acid is usually added during the manufacture of commercial juices or soft drinks in order to improve the nutrition value or to avoid the autoxidation of the products. (SalkiÄ‡ and KubiÄek, 2008). Due to the ascorbic acid has been widely used in canned fruits, juices and drugs, various methods had been adapted to determine vitamin in many industry, which include spectrophotometry, titrimetry, voltammetry, fluorometry, potentiometry, kinetic-based chemiluminescence (CL), flow injection analyses and chromatography. (SalkiÄ‡ and KubiÄek, 2008; Arya et al, 2000).
Among all the methods, spectrophotometry is the most widely used method in determination of ascorbic acid. This is because it can provide a fast, simple, and reliable method for the determination of ascorbic acid. (SalkiÄ‡ and KubiÄek, 2008). However, the major problem by using this method is the absorption of UV light by the sample matrix. Various techniques has been developed to solve this problem include, thermal degradation, UV light decomposition, enzymatic or metal catalytic oxidation and photodestruction of L-ascorbic acid. However the thermal degradation and the UV light decomposition is too slow to be used practically. (SalkiÄ‡ and KubiÄek, 2008)
Due to vitamin C can be oxidized easily, so the determination procedure has to be designed to avoid the loss of the vitamin. Factors including the temperature, light, pH and oxygen exposure can affect the vitamin C content. (Phillips et al, 2010).
The objective of this study is to evaluate the feasibility of using ultraviolet spectrophotometry method to determine concentration of vitamin C. Furthermore, it is also to perform a direct and simple ultraviolet spectrophotometric method for the determination of vitamin C concentration in fruits. Lastly is to determine the effect of different temperature to the storage of ascorbic acid solution.
2.0 LITERATURE REVIEW
2.1 L-Ascorbic Acid
L-ascorbic acid (C6H8O6) is the trival name for vitamin C, while its chemical name is 2-oxo-L-threo-hexono-1,4-lactone-2,3-enediol. Vitamin C refers to the compound exhibiting full or partial biological activity of L-ascorbic acid, which are esters of ascorbic acid, synthetic forms such as 6-deoxy-L-ascorbic acid and dehydroascorbic acid. (Eitenmiller and Landen, 1999)
Ascorbic acid is a white, crystalline powder, which is highly soluble in water. (Pauling, L.1970). There are two enantiomers of ascorbic acid, which are L and D-ascorbic acid. (Davies et al., 1991) According to Pauling (1970), only L-ascorbic acid shows the vitamin C activity and if the prefix of the ascorbic acid is not shown, it is referred to L-ascorbic acid or vitamin C itself.
Unfortunately, due to lack of enzyme gulonolactone oxidase, human cannot synthesize ascorbic acid in vivo. (Naidu, 2003). Hence, it is important to make sure an individual to consume sufficient amount of vitamin C to prevent certain disease for example scurvy. As a saying goes, "prevention is better than cure".
2.1.1 History of L-Ascorbic Acid and Scurvy
During the time between 1497 and 1499, a disease which produces haemorrhaging into tissue, bleeding gums, loose teeth, anaemia and general weakness caused a Portuguese navigator, Vasco da Gama lost about 100 of his complement of men when he sailing from Lisbon to Calicut and this disease continued to take its toll of sea travellers for four hundred years after this time. (Davies et al., 1991; Pauling, L.1970.). This disease is called Scurvy.
According to Verrax (2008), Scurvy is a vitamin C deficiency disease which occurs in humans whose diet is deficient in fresh fruits and vegetables. Symptoms of the scurvy include defect in collegen synthesis and failure of wounds to heal, defects in tooth formation, bleeding gums, anaemia, fatigue, and depression. (Verrax et al., 2008; Phillips et al, 2010).
Besides that, Naidu (2003) also mentioned that sailors developed scurvy when they were on sea. This is due to eating non-perishable items and lack of fresh fruits and vegetables in their diet.
In mid-18th century, a British physician, James Lind proposed that some substance in fresh citrus fruits can cure scurvy. Hence, in order to help the sailors to prevent and cure scurvy, Lind was then created a method to concentrate and preserve the citrus fruit juices for the sailors. (Verrax et al., 2008; Naidu, 2003).
The L-ascorbic acid was first isolated by a Hungarian physician Professor Albert Imre Szent-Györgyi von Nagyrapolt. In the year 1928, he was successfully to accumulate a less amount of an off-white crystalline substance from the cortex of cattle. After the discovery of this off-white crystalline compound, he had done a series of test on it. The result showed that, the substance can decolorize iodine and lower the vapour pressure of the water, which gave the relative molecular mass of the crystal about 180 g/mol. (correct value=176.4 g/mol).
Moreover, Szent-Györgyi also did a combustion analysis on the compound, which gave 40.7% carbon, 4.7% hydrogen and 54.6% oxygen. This data enable him to deduce the C6H8O6 as the molecular formula for this compound. Later, he named the compound as 'hexuronic acid' but finally changed to ascorbic acid which means prevention of scurvy. (Davies et al., 1991). In the year 1937, Szent-Györgyi was awarded Nobel prize for Medicine in recognition of his discoveries concerning the biological oxidation processes with special reference to vitamin C. However, ascorbic acid was first synthesized by Haworth and Hirst. (Naidu, 2003)
2.1.2 Chemistry and the Structure of L-Ascorbic Acid
L-ascorbic acid (AA) and dehydroascorbic acid (DHA) are the major form of the vitamin C. The chemical name for the L-ascorbic acid is 2-oxo-L-threo-hexono-1,4-lactone-2,3-enediol. (Naidu, 2003). L-ascorbic acid has the relative molecular mass of 176.14g and the melting point is between 190-192 oC. The density for L-ascorbic acid is 1.65g cm-3 and the specific rotation in water is +23o. (Davies et al., 1991)
L-ascorbic has molecular formula C6H8O6 and it has chiral centers at the forth and the fifth carbon. Besides that, it can exists in four stereoisomeric form which are L- and D-ascorbic acid and L- and D-araboascorbic acid. (Eitenmiller and Landen, 1999). D-araboascorbic acid is also known as D-isoascorbic acid (isoAA) is the epimers that has the orientation of hydrogen group and hydroxyl group at the fifth carbon that differ from the L-ascorbic acid. IsoAA is usually added to food as an antioxidant and because of this, high vitamin C values will be found if improper methodology is used. (Eitenmiller and Landen, 1999)
The structure of L-ascorbic acid makes it a good reducing agent and so it is usually uses as a nutritional food additive, antioxidant, browning inhibitor, reducing agent, flavour stabilizer, modifier and enhancer, colour stabilizer, dough modifier and in many other capacities. (Zümreoglu-karan, 2006; Eitenmiller and Landen, 1999).
2.1.3 Stability of L-Ascorbic Acid
According to Odriozola-Serrano et al (2007), Vitamin C has a labile nature. Ascorbic acid (AA), and dehydroascorbic acid (DHA) are readily oxidize especially when they are exposed to elevated temperatures, divalent cations, oxygen, alkaline pH, light or degradative enzymes. (Odriozola-Serrano et al, 2007). However, the crystalline form of L-ascorbic acid is highly stable in the presence of oxygen when water activity remains low. (Eitenmiller and Landen, 1999).
When the crystalline L-ascorbic acid (AA) dissolved in water or high in water activity, it undergoes rapid and excessive oxidative change to reversibly convert to its reduce form, dehydroascorbic acid (DHA). Reducing agent and enzyme glutathione dehydrogenase can convert AA back to DHA. (Eitenmiller and Landen, 1999). DHA can irreversibly hydrolysed to produce a biologically inactive 2,3-diketogulonic acid. (Hernández et al., 2005).
Eitenmiller and Landen (1999) also mentioned that, L-ascorbic acid can oxidize through one- or two-electron transfers. One-electron reductions utilize the transition through the L-ascorbic acid free radical. While, the two-electron reductions is caused by transition metals which form complex with L-ascorbic acid. (Eitenmiller and Landen, 1999).
Due to the labile nature of the vitamin C, it is important to make sure that the nutrients do not degrade during the preparation and the processing of foods. Preparation procedure needs to be well designed to avoid the loss of vitamin C. Do not overcook the vegetables or food to avoid the degradation of nutrient due to temperature.
In the determination methods, the used of metaphosphoric acid is common to inhibit the L-ascorbic oxidase and metal catalysis. (Odriozola-Serrano et al, 2007). According to Eitenmiller and Landen (1999), fully protonated form of L-ascorbic acid is quite stable in acidic condition and the stability decreases as the as the pH approaches pKa 4.04.
2.1.4 Synthesis, Biosynthesis and Sources of L-ascorbic acid.
Ascorbic acid can be generated de novo by many of the species of plants and animals. According Naidu (2003), L-ascorbic acid presences in fruits such as, citrus, watermelon, barriers, papaya and mango; in leafy greens such as, tomatoes, broccoli, green and red peppers, cauliflower and cabbage. Figure 2 shows the vitamin C content of some common fruits and vegetables.
Figure 2: Vitamin C content of some common fruits and vegetables (Davies et al., 1991)
Most of the plants and animals synthesize ascorbic acid from D-glucose or D-galactose. (Naidu, 2003). Figure 3 shows the biosynthesis pathway in animals. Unfortunately, a small number of species of higher organisms, including Homo sapiens are not able to synthesized ascorbic acid due to lack of the genetic material necessary to synthesize the last enzyme, L-gulonolactone oxidase which is responsible to catalyst the reaction from L-gulonolactone to 2-keto-L-gulonolactone. (Davies et al., 1991). However, this is not a problem these species as vitamin C can easily obtained from various fruits and vegetables.
Besides that, L-ascorbic acid can also be synthesized chemically for commercial purpose. The synthesis of L-ascorbic acid can be started by using different C5 sugar as a precursor, for example xylose, lyxose and arabinose. (Davies et al., 1991). Recently, the synthesis process involved the reduction of D-glucose to D-sorbitol which is then fermentatively oxidised to L-sorbose with Acetobacter suboxydans or Acetobacter xylinum. (Davies et al., 1991) The detail synthetic pathway is shown in Appendix 1.
Figure 3: Biosynthesis of L-Ascorbic acid in animals (Naidu, 2003)
2.2 Vitamin C in prevention of various diseases
2.2.1 Vitamin C and Scurvy
As the meaning of the ascorbic acid is the prevention of scurvy, vitamin C is a very powerful supplement in prevention of scurvy. Linus Pauling mentioned in his publication of Vitamin C and the common cold in year 1970 about that, a person will become sick and eventually die in a few months if he eats no vitamin C even though his diet is adequate in other respect. The dying is caused by the vitamin C deficiency disease called scurvy. Pauling also mentioned that, a small amount of vitamin C range from 5mg to 15mg daily is enough to prevent scurvy.
Davies and co-workers also mentioned some of the factors that cause the scurvy for most of the sailor in the early century. This was because sailors at that time were lack of the knowledge about the scurvy and they consumed foods with low vitamin C content and lack of vegetables and fruits due to long voyaging in the sea. Even they brought along with fruits and vegetables, Vitamin C can degrade along with times.
There was a case studied by Laura Pimentel (2002) in a 79-year-old black woman. She was admitted to the internal medicine hospital service with a provisional diagnosis of scurvy and she was treated with multivitamins (500 mg vitamin C, three times per day, 10 mg vitamin K, subcutaneously for 3 days, and 200 mg vitamin B12 intramuscularly for 1 dose). Finally the symptoms were decreased and she was discharged home on continued vitamin C therapy. This has proven that Vitamin C is essential in the prevention and treatment of scurvy.
2.2.2 Vitamin C and common cold or influenza
Linus Pauling proposed that ingestion of 1-2 g of ascorbic acid effectively prevents or ameliorate common cold. However, ascorbic acid in prevention and ameliorate common cold has been a long-standing debate among the researchers. (Naidu, 2003).
Douglas and his co-workers found out in their review that, vitamin C does not exert any effect to the prevention of common cold, but a very high doses of vitamin C does helps in shorten the length of the illness. Ascorbic acid enhances T-cell proliferation in response to infection to control inflammatory reaction and thus reduce the illness of the common cold. T-cells are the cells that capable to lyses the infected targets and help B-cells to synthesize immunoglobulins. (Naidu, 2003).
The research by Banerjee and Kaul had shown that oral intake of vitamin C unable to provide a protection against influenza. However, there is a possibility that the combination of oral and inhalational routes of vitamin C delivery helps in anti-influenza activities. (Banerjee and Kaul, 2010)
Rahman Khan and co-workers (2006), also mentioned that, vitamin C can effectively reduces the symptoms of cold and prevents secondary viral and bacterial diseases. Besides that, human should keep our body enough with vitamin C to keep a good health and for prevention from common cold. (Rahman Khan et. al., 2006)
2.2.3 Vitamin C and cancer
Vitamin C may affects carcinogenesis by the mechanisms such as antioxidant effects, blocking the formation of nitrosamines and fecal mutagen, enhances the detoxification of liver enzymes and helps in immune response (Hercberg et. al. 1998). Free radical can damage DNA and initiate tumor growth (Naidu, 2003). Vitamin C scavenges free radical formation during cellular metabolisms and thus play an indirect role in protecting cell membranes from free radical induced damage. (Wilis and Wians, 2003). Vitamin C prevents by neutralizing free radicals or acts as a pro-oxidant helping body's own free radicals to kill tumor sells in their early stages (Naidu, 2003).
2. Structural Details by Instrumental Methods
3.0 Experimental Part
3.1 Ascorbic acid determination in Supplement tablet by using Hitachi u-2000 UV-vis spectrophotometer
3.1.1 Materials and Methods
188.8.131.52.1 Stabilizer solution preparation
A solution of 1M hydrochloric acid (HCl) was prepared by dissolving 180 ml of concentrated hydrochloric acid in the distilled water and making up a volume of 2 liters.
184.108.40.206.2 Vitamin C stock solution (1mg/1ml) preparation
0.25g of ascorbic acid was weighted in 100 ml beaker. Then 50ml of 1M hydrochloric acid was added to dissolve the ascorbic acid. The solution was then transfer to a 250 ml volumetric flask and was topped up with 1M hydrochloric acid to the mark. This solution was prepared freshly before each experiment since the ascorbic acid can oxidize easily.
220.127.116.11.3 Standard solution preparation for calibration
The stock solution (1mg/ml) was diluted by using 1M hydrochloric acid to 1, 2, 3, 4, 5 mg/100ml. The calculated volumes that needed to be transferred and diluted are as follows:
Volume needed to be transferred (ml)
Diluted volume (ml)
The concentration of the stock solution (mg/ml) and the standard solution (mg/100ml) was calculated by using the equation below:
After that, a calibration curve, absorbance against ascorbic acid concentration was plotted and the equation was obtained for calculation.
18.104.22.168 Sample Preparation
22.214.171.124.1 Soluble vitamin C tablet sample solution preparation
Sample from the two soluble product (A and B) were prepared in the same manner. First, one tablet was placed in a 100 ml beaker and 50ml of distilled water was added to dissolved the tablet. Then the solution was transfer to a 500 ml volumetric flask and the beaker was washed with distilled water a few times to make sure all the vitamin c had been transferred into the volumetric flask. The solution was then topped up with distilled water to the 500 ml mark and was mixed well. Next, 1 ml of the solution was transferred to a 50 ml volumetric flask and was topped up with 1M hydrochloric acid. The solution was mixed well and the vitamin c content was measured by using the Hitachi u-2000 UV-Vis spectrophotometer and the result was analyzed by MicrosoftÂ® Office Excel 2007.
126.96.36.199.2 Chewable vitamin C tablet sample solution preparation
A tablet of vitamin C sample was minced with mortar and 20ml of distilled water was added. Then the sample solution was transferred into a 500ml volumetric flask and was topped up with distilled water. Next, proper amount of sample solution (1ml for sample c, 2ml for sample d) was transfer to a 50 ml volumetric flask. The solution was then topped up with 1M hydrochloric acid to the mark. Finally, the vitamin c content was measured by using the Hitachi u-2000 UV-Vis spectrophotometer and the result was analyzed and processed by MicrosoftÂ® Office Excel 2007.
3.1.2 Result and Discussion
188.8.131.52 Calibration curve
The Î»max of the highest concentration standard solution was determined by using Hitachi u-2000 UV-vis spectrophotometer and it was found to be at 242.4 nm. Then the absorbances of all the standards were measured to construct a calibration curve in absorbance against concentration. The equation was obtained for calculation and was tabulated in the table in appendix 2. One of the calibration curves was shown in the figure below:
184.108.40.206 Determination of vitamin C in sample