果蔬中维生素C含量测定及其分析

Measure of The Content of Vitamin C in Fruits and An Analysis on The Influence Factor of The Content of Vitamin C Zheqi Li（5111719018） School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China Content of vitamin C, DCPIP titration, Influence factor 1. Background and Introduction Vitamin C, which is also called L-ascorbic acid, is a water soluble vitamin. It is abundant in fruits and vegetables. It is a kind of clear crystal which is not heat stabile. It is bitter in taste and soluble in water and alchol.[5] Figure1 shows the chemical constitution of vitamin C. According to its chemical constitution, following chemical property can be inferred. (1)It is weakly acidic; (2)It is easily led to oxidative decomposition when heated or in solutions; (3)It is easier to be oxidized in alkaline condition; (4)It has a strong reducing property. Vitamin C has many physiological functions. Following are the major functions of it. (1)It helps prevent the scorbutus and improve the anemia. It can promote the conformation of collagen, which is a significant component of connective tissue such as blood capillary and bones, via maintaining the hydroxylase activity. Lack of vitamin C may increase the permeability of vessel wall, which increases the risk of scorbutus. Moreover, it can reduce Fe3+ to Fe2+ in hemoglobin, which may increase the oxygen carrying capacity of red blood cells.[4] (2)It helps prevent cancers. It can prevent nutritious substance in intercellular substance from spreading to cancer cells by blocking up the synthesis of alkyl nitrites in bodies. (3)It helps prevent coronary disease. It can promote cholesterol hydroxylation reaction, which releases bile acids as product. It prevent coronary disease by decreasing the accumulation of cholesterol in vessels. Vitamin C also has the function of detoxification, preventing bronchitis and skin whitening. Based on the abovementioned advantages of vitamin C, it has a significant apply in people’s daily life. Research on the content of vitamin C in different fruits or vegetables and the influence factor of it may guild people to use it properly, which makes a important sense. Vitamin C is abundantly contained in many fruits and vegetables.Table1 and 2 shows the contents of vitamin C in some representive fruits and vegetables. Table1 Content of vitamin C in five fruits Name Content of vitamin C in every 100g fruits/mg Fresh jujube 900 Pomegranate 68 Strawberry 60 Kiwi fruit 58 Lemon 45 Table2 Content of vitamin C in five vegetables Name Content of vitamin C in every 100g vegetables/mg Broccoli 75 Spinacia oleracea 65 Balsam pear 56 Turnip radish 25 Tomato 23 According to the content of vitamin C in several fruits and vegetables, the material of this research should contain a certain amount of vitamin C and not contain too much pigment, which may influence the coloration in titration process. Thus we choose apples and oranges as materials in this research. There are also many factors influencing the content of vitamin C in fruits, such as temperature, illumination, part of fruits and maturity. Further research on these influence factors may obtain the quantitative relationship between the content and these factors, which may guild to reach an access to the accurate extra condition of the maximum content of vitamin C. 2.Principle of Experiment To accurately measure the content of vitamin C, DCPIP[6] titration is chosen to be a correlatively ideal measure method. Vitamin C has a strong reducing property. And it also can be divided into reducing version and dehydrogenation version. The reducing version is able to reduce a dye called DCPIP, which makes the reducing version turn into the dehydrogenation version as a result. In an acidic solution, DCPIP colors red and It turns the solution into colorless after reduced. Thus, when using DCPIP to titrate acidic solutions which contains vitamin C, the dye will turn into colorless immediately with some vitamin C still remained. Once vitamin C in the solution is oxidized up, the dye dropped tures the solution into reddish immediately. Therefore, when the solution turns from colorless into reddish, the vitamin C in the solution is exactly right oxidized, which indicates the end point of titration. Without any disturbances, the amount of used DCPIP is in proportion to the amount of vitamin C in the solution.Figure3 shows the specific process of this reaction. Figure2 The oxidation reaction of reduced ascorbic acid with DCPIP 3.Materials and Instruments (1)Materials some fresh apples and some rotten apples; some fresh oranges. (2)Instruments Erlenmeyer flask(100ml×2); measuring pipet(10ml×1); measuring flask(100ml×1, 250ml×1); microburette (5ml×1);a mortar; a funnel; some gauze; a thermostat water bath pot, an LED flashlight. (3)Reagent 2% oxalic acid solution: 2g oxalic acid dissolved in 100ml distilled water 1% oxalic acid solution: 1g oxalic acid dissolved in 100ml distilled water Standard ascorbic acid solution(1mg/mL); 0.1% dichlorophenol indophenol solution. 4.Methods (1)Extract (2) Standard solution titration (3)Compare the contents of vitamin C in fresh apple pulps and rotten apple pulps ①Extract 10ml extracts of beaker1 and beaker2 in two 100ml erlenmeyer flasks. ②Use standardized DCPIP solution to titrate the solution into reddish with a microburette. Keep the color maintain 15 seconds, which indicates the end point of titration. Each sample is titrated twice. ③Record the titrated volume of standardized DCPIP. (4)Compare the contents of vitamin C in fresh orange pulps and fresh orange peels ①Extract 10ml extracts of beaker3 and beaker4 in two 100ml erlenmeyer flasks. ②Use standardized DCPIP solution to titrate the solution into reddish with a microburette. Keep the color maintain 15 seconds, which indicates the end point of titration. Each sample is titrated twice. ③Record the titrated volume of standardized DCPIP. (5)Research the relationship between temperature and content of vitamin C in fruits ①Extract 10ml extracts from beaker1 to a 100ml erlenmeyer flask. Do it for four times to make 4 same samples. ②Put three of the four samples in the thermostat water bath pot which is set to 80°C. Separately keep them in the pot for 5min, 3min and 1min.Then take them off from the pot. ③Use standardized DCPIP solution to titrate the four samples into reddish with a microburette. Keep the color maintain 15 seconds, which indicates the end point of titration. Each sample is titrated twice. ④Record the titrated volume of standardized DCPIP and the temperature of each samples after measuring. (5)Research the relationship between illumination and content of vitamin C in fruits ①Extract 10ml extracts from beaker3 to a 100ml erlenmeyer flask. Do it for three times to make 3 same samples. ②Use the LED flashlight illuminate the two of the three samples . Separately keep them exposured for 5min and 10 min. Then take them in dark. The other sample is kept in dark from the beginning,. ③Use standardized DCPIP solution to titrate the three samples into reddish with a microburette. Keep the color maintain 15 seconds, which indicates the end point of titration. Each sample is titrated twice. ④Record the titrated volume of standardized DCPIP and the time of exposure in strong lights of each sample. (6)Calculating the content of vitamin C Computational formula: Content of vitamin C(mg/10g sample)= In the formula: VA: the average volume(ml) of the dye which is consumed in titrating the samples; VB: the average volume(ml) of the dye which is consumed in titrating the blank samples; C: the total volume of the sample extract; D: the volume of sample extract for titration; T: the weight(mg) of ascorbic acid that 1ml dye can oxidize; W: the weight of the sample to measure. 5. Data Processing and Analysis Above all, the result of standard solution titration is 9.92ml, which means every 9.92ml dye is equal to 1mg ascorbic acid. We can conduct the following matrixing: 1mg/9.92ml=xmg/1ml x=0.1mg Thus, 1ml dye is equal to 0.1mg ascorbic acid in the titration system. (1)Compare the contents of vitamin C in fresh apple pulps and rotten apple pulps Times Name Fresh apple pulps Rotten apple pulps 1 0.40ml 0.21ml 2 0.46ml 0.18ml Average value 0.43ml 0.20ml Equivalent weight of ascorbic acid 0.043mg 0.020mg Content of vitamin C(mg/100g) 1.04 0.49 Content of vitamin C(in fresh apple pulps): c1=(0.43×50×0.1×100)/(10×20.772)=1.04mg/100g Content of vitamin C(in rotten apple pulps): c2=(0.20×50×0.1×100)/(10×20.147)=0.49mg/100g Brief analysis on the difference of the content of vitamin C: The maturity of botany including fruits results from a phytohormone called ethylene, which is the simplest phytohormone. Moreover, the decomposition of fruit is also relevant to some microbe like bacteria and fungus. To research the reason why the content of vitamin C in fresh apples is higher than that in rotten apples, the relationship between the metabolism of vitamin C and ethylene and microbe should be found out. The physiological mechanism of acceleration of maturation by ethylene is showed following[7]: Some absorbed ethylene increase the permeability of membrane, especially that of tonoplast, which may cause the exosmosis of a great deal of hydrolase. The surpass of hydrolase in cells directly cause the enhancing of respiratory metabolism of plant cells, which causes some conversions of organic compounds in fruits, such as the decomposition of chlorophyll, the accumulating of aromatic substance, the oxidization coagulate of soluble tannic acids. Those conversions cause the softening of fruits, even decomposition. Furthermore, the decomposed part may lead to the growth of microbes, such as bacillus subtilis and saccharomycetes, some of which may accelerate the decomposition of fruits. Figure3 shows a brief process that fruits become decomposed. Figure3 The process of the decomposition of fruit From this figure, we can analyze that the decrease of vitamin C in rotten fruits mainly lies in the following three reasons: (1)The increase of the permeability of tonoplast causes the exosmosis of many nutritive substance including vitamin C. (2)The conversion of organic compounds includes the decomposition of vitamin C. (3)The development of microbes may consume vitamin C as nutritive substance which maintain their growth. (2)Compare the contents of vitamin C in fresh orange pulps and fresh orange peels Times Name Fresh orange pulps Fresh orange peels 1 3.18ml 2.90ml 2 3.14ml 2.86ml Average value 3.16ml 2.88ml Equivalent weight of ascorbic acid 0.316mg 0.288mg Content of vitamin C(mg/100g) 7.22 7.20 Content of vitamin C(in fresh orange pulps): c1=(3.16×50×0.1×100)/(10×21.869)=7.22mg/100g Content of vitamin C(in fresh orange peels): c2=(2.88×50×0.1×100)/(10×20.003)=7.20mg/100g Brief analysis on the difference of the content of vitamin C: The orange peels and pulps belong to different plant tissues. The orange peels actually include two parts: the actual peels and the white tissues called reeling which adhere on the endothecium peels. The reeling just occupied a small part of the whole sample, for which we can omit them. The peels belong to protective tissue. These tissues are usually present in the outermost layer of the plant body such as leaves, stem and roots. It is one cell thick and covered with cutin and protects the underlying tissues present in the plant body. One of the feature of these tissues is lack of vacuole in cells, which cause less nutritive substance including vitamin C.[8] The orange pulps belong to parenchyma. Parenchyma cells are thin-walled cells of the ground tissue that make up the bulk of most nonwoody structures, although sometimes they have lignified cell walls. Parenchyma cells in between the epidermis and pericycle in a root or shoot constitute the cortex, and tissue specialised for food storage commonly is parenchyma. Thus, the pulps of fruit commonly contain the most nutritive substance including vitamin C. In conclusion, the difference of content of vitamin C between orange peels and pulps mainly depends on different plant tissues and the structure and function of them. (3)Research the relationship between temperature and content of vitamin C in fruits Data: Heating time/min 0 1 3 5 Temperature/℃ 25 30 42 60 Volume of dye/ml 0.43 0.34 0.24 0.16 Equivalent weight of ascorbic acid/mg 0.043 0.034 0.024 0.016 Content of vitamin C(mg/100g) 1.04 0.82 0.58 0.37 Content of vitamin C: c1=(0.43×50×0.1×100)/(10×20.772)= 1.04mg/100g c2=(0.34×50×0.1×100)/(10×20.772)= 0.82mg/100g c3=(0.24×50×0.1×100)/(10×20.772)= 0.58mg/100g c4=(0.16×50×0.1×100)/(10×20.772)= 0.37mg/100g Figure4 The relationship between temperature and content of vitamin C Brief analysis on the difference of the content of vitamin C: Vitamin C has a chemical property that it is easy to be oxidized, which becomes easier when heated. Because heating may accelerate the speed of the oxidation reaction. As an irreversible oxidation-reduction reaction, it accords with the Arrhenius equation.[9] (1) or (2) According to this equation, we can clearly infer that the increase of temperature will cause the decomposition of vitamin C, no matter whether the activation energy is positive or negative. (5)Research the relationship between illumination and content of vitamin C in fruits Illumination time/min 0 5 10 Volume of dye/ml 1.42 1.32 1.26 Equivalent weight of ascorbic acid/mg 0.142 0.132 0.126 Content of vitamin C(mg/100g) 3.25 3.02 2.88 Content of vitamin C(in fresh orange pulps): c1=(1.42×50×0.1×100)/(10×21.869)=3.25mg/100g c2=(1.32×50×0.1×100)/(10×21.869)=3.02mg/100g c3=(1.26×50×0.1×100)/(10×21.869)=2.88mg/100g Figure5 The relationship between illumination and content of vitamin C Brief analysis on the difference of the content of vitamin C: In fruits, the kinetic equation of content of vitamin C accords to the following principle[10]: (3) In this formula, f(c)means the potency of vitamin C, k means the degradation rate constant of vitamin C in the condition of illumination and m means the reaction order. According to figure5, it is easy to find out that vitamin C will decompose in illumination and it accords to the primary reaction. To find out how intensity of illumination influences the reaction rate, we conduct a further experiment. We measure the content of vitamin C of different periods in different intensity of illumination .Following is the data calculated: Time 0min 1min 3min 5min 7min 9min 11min 500Lux 3.25 3.17 3.08 3.03 2.99 2.97 2.95 1000Lux 3.25 3.12 3.01 2.92 2.86 2.82 2.80 2000Lux 3.25 3.08 2.92 2.80 2.70 2.64 2.61 Figure6 The relationship between intensity of illumination and the reaction rate It is easy to find out that the intensity is also a significant factor that influences the reaction rate. The stronger the intensity is, the high the rate will be. To find out the relationship between the intensity of illumination and the rate of reaction kL, such equation is characterized: (4) Thus, the equation of the intensity of illumination and the reaction rate may be showed as following: （5） 6. Summary There are many factors which could influence the content of vitamin C in fruits and vegetables. According to this experiment, four conclusions can be inferred. (1)Decomposition can decrease the content of vitamin C. (2)The peels of fruit always have less vitamin C than the pulps do. (3)The higher the temperature is, the less vitamin C is in fruits and vegetable. (4)Illumination can decompose vitamin C and the stronger the intensity of illumination is, the high the rate will be. Those results can guide people how to ingest vitamin C in an efficient way, which makes a significant sense to society. Reference [1] Mark Levine,* Sebastian J. Padayatty, and Michael Graham Espey, Vitamin C: A Concentration-Function Approach Yields Pharmacology and Therapeutic Discoveries, doi: 10.3945/​an.110.000109 Adv Nutr March 2011 Adv Nutr vol. 2: 78-88, 2011 [2] Sebastian J. Padayatty, MRCP, PhD, Arie Katz, MD, Yaohui Wang, MD, Peter Eck, PhD, Oran Kwon, PhD, Je-Hyuk Lee, PhD, Shenglin Chen, PhD, Christopher Corpe, PhD, Anand Dutta, BS, Sudhir K Dutta, MD, FACN and Mark Levine, MD, FACN, Vitamin C as an Antioxidant: Evaluation of Its Role in Disease Prevention, J Am Coll Nutr February 2003 vol. 22 no. 1 18-35 [3] David C. Nieman HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-1" 1, Dru A. Henson HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-1" 1, Steve R. McAnulty HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-1" 1, Lisa McAnulty HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-1" 1, Nathaniel S. Swick HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-1" 1, Alan C. Utter HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-1" 1, Debra M. Vinci HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-1" 1, Shannon J. Opiela HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-1" 1, and Jason D. Morrow HYPERLINK "http://jap.physiology.org/content/92/5/1970.short" \l "aff-2" 2, Influence of vitamin C supplementation on oxidative and immune changes after an ultramarathon, Published online before print February 1, 2002, doi: 10.​1152/​japplphysiol.​00961.​2001 Journal of Applied Physiology May 1, 2002 vol. 92 no. 5 1970-1977 [4] Graham Noctor, Christine H. Foyer, ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under Control, Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998. 49:249–79 [5] The Vitamins Vol. 5, 2nd edn (eds Sebrell, W. H. & Harris, R. S.) 293 (Academic, New York, 1972). [6] M Levine, C Conry-Cantilena, Y Wang, R W Welch, P W Washko, K R Dhariwal, J B Park, A Lazarev, J F Graumlich, J King, and L R Cantilena, Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance, PNAS April 16, 1996 vol. 93 no. 8 3704-3709 [7] Selective Ascorbate Toxicity in Malignant Mesothelioma: A Redox Trojan Mechanism Am. J. Respir. Cell Mol. Bio. 2011 44 (1) 108-117 [8] Vitamin C: Overview and Update Journal of Evidence-Based Complementary & Alternative Medicine 2011 16 (1) 49-57 [9] Adherence to a Mediterranean diet and plasma concentrations of lipid peroxidation in premenopausal women Am J Clin Nutr 2010 92 (6) 1461-1467 [10] Effects of Ascorbic Acid Intake on the Intima-Media Thickness and Blood Flow Velocities of the Carotid Artery in Patients With Sickle Cell Anemia Journal of Diagnostic Medical Sonography 2011 27 (5) 214-219 [10] Dietary ascorbate intake affects steady state tissue concentrations in vitamin C-deficient mice: tissue deficiency after suboptimal intake and superior bioavailability from a food source (kiwifruit) Am J Clin Nutr 2011 93 (2) 292-301 ABSTRACT KEYWORDS Vitamin C, which is also named ascorbic acid, is one of the most important vitamins of human nutrition. Human may suffer from � HYPERLINK "http://www.iciba.com/scorbutus" �scorbutus� without ingestions of vitamin C. Now it has yielded new physiology and pharmacology discoveries.[1]Vitamin C mainly exists in fruits and vegetables.[2]Apples and oranges has a certain amount of contents of vitamin C, which is favored to measure the content of it. There are also many influence factors influencing the content of vitamin C[3], such as temperature, illumination, maturity, part of fruit and so on. This research mainly conducts different measures for the content of vitamin C in a