anthocyanin – study of extraction from basella rubra l and verifying antioxidant activity, application in identificating di sodium tetra borate in food

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY ANTHOCYANIN – STUDY OF EXTRACTION FROM BASELLA RUBRA L. AND VERIFYING ANTIOXIDANT ACTIVITY, APPLICATION IN IDENTIFICATING DI SODIUM TETRA BORATE IN FOOD SUPERVISOR STUDENT MSc. TRAN THI XUAN MAI NGO TRAN HUU NGHIA Student code: 3102836 Session: 36 (2010 – 2014) Can Tho, 2014 APPROVAL SUPERVISOR STUDENT MSc. TRAN THI XUAN MAI NGO TRAN HUU NGHIA Can Tho, December , 2014 PRESIDENT OF EXAMINATION COMMITTEE i ABSTRACT Study of extracting anthocyanins from Basella rubra L. and antioxidant activity, identifying of Di Sodium Tetra Borate in food. Through the experiment with different solvents, temperatures and compared the total anthocyanin content among products, the most effective method with the highest outcome was recorded for further experiment. The anthocyanin content was calculated by mean of pH differential method at pH 4.5 and 12.0, with light absorbance at maximum wave length and 700nm. Extracted juice from fruits were used for estiamating antioxidant activity and verifying the ability of identifying borax in food due to the specific color change in reaction with borax solution pH from 1 to 8. In this research, the treatment was carried out with distilled water as solvent and temperature of 30oC was recorded as the most effective extraction method, the total anthocyanin content yield was 3.33%.Moreover, the IC50 index from antioxidant activity experiment was 1. 8 µg/mL and lower when compared with ascorbic acid and garlic acid 8.4 µg/mL and 2.6 µg/mL respectively, this result demonstrated the high potential of antioxidant activity from Basella rubra L.. Finally, the identification of Di Sodium Tetra Borate experiment resulted in a positive abilities from anthocyanins: both 20% anthocyanins indicator kits (pH =2) and indicator papers gave clear purple color and high contrast displays with the minimum concentration of borax at 0.05% for kits and 0.1% for papers. The extend experiment of the two indicators also achieved the further ability that could identify Sodium hydroxide with high and low concentration. Key word: anthocyanin, antioxidant, borax, di sodium tetra borate,. ii CONTENT APPROVAL ...................................................................................................... i ABSTRACT ..................................................................................................... ii CONTENT ...................................................................................................... iii CHAPTER I. INTRODUCTION ................................................................... 1 1.Introduction ................................................................................................ 1 2.Objective..................................................................................................... 2 CHAPTER II. MATERIALS AND METHODS .......................................... 3 1.Materials .................................................................................................... 3 1.1.Sample ................................................................................................. 3 1.2.Apparatus ............................................................................................. 3 1.3.Equipment ............................................................................................ 3 1.4.Chemicals ............................................................................................ 3 2. Methods ................................................................................................ 3 2.1.Sample preparation .............................................................................. 3 2.2.Anthocyanin extraction method ........................................................... 3 2.3.Moisture determination........................................................................ 4 2.4.Total anthocyanin content analysis ...................................................... 4 2.5.Verification of antioxidant activity and IC50 index ............................ 5 2.6.Preparation of indicator solution: ........................................................ 5 2.7.Data analysis ........................................................................................ 5 3. Experiment arrangement .................................................................... 5 3.1.Experiment 1: Verification of color change among the pH range ....... 5 3.2.Experiment 2: Verification of maximum absorbance wave length ..... 6 3.3.Experiment 3: The effect of solvent and temperature on the total anthocyanin content ................................................................................... 6 3.3.1.Determination of moisture ............................................................ 6 3.3.2.The effect of solvent and temperature on the extraction process.. 6 iii 3.4.Experiment 4: Antioxidant activity verification .................................. 7 3.5.Experiment 5: Application in Identification of di sodium tetra borate 8 3.5.1.Identification of Borax .................................................................. 8 3.5.2.Producing indicator solution ......................................................... 8 3.5.3.Producing indicator papers ........................................................... 9 3.5.4.Identification of sodium hydroxide in food .................................. 9 CHAPTER III. RESULT AND DISCUSSION ........................................... 10 1.Verification of color change among the pH range .................................... 10 2.Verification of maximum absorbance wave length .................................. 11 3.The effect of temperature and solvent on total Anthocyanin content ....... 12 3.1.Moisture content ............................................................................ 12 3.2.Total Anthocyanin content ............................................................ 12 4.Experiment 4: Antioxidant activity verification ....................................... 14 5.Application in Identification of Di Sodium Tetra Borate ......................... 16 5.1.Identification of Borax....................................................................... 16 5.2.Possibilities of indicator solution....................................................... 17 5.3.Possibilities of indicator papers ......................................................... 19 5.4.Identification of sodium hydroxide ................................................... 20 5.4.1.Identification of sodium hydroxide by indicator solution: ......... 20 5.4.2.Identification of sodium hydroxide by indicator papers: ............ 21 CONCLUSIONS AND SUGGESTIONS ..................................................... 23 1.Conclusions .............................................................................................. 23 2.Suggestions ............................................................................................... 24 REFFERENCES ............................................................................................ 25 iv CHAPTER I. INTRODUCTION 1. Introduction Anthocyanin are water-soluble vacuolar pigments occur in tissues of higher plans, play an important role beside Chlorophyll and Carotenoid. This polyphenol act as main constituent of natural color on flowers, fruits, seeds that can not be replaced by artificial colored products. In fact, those man – made colorant only adapt to coloring function but still existed problems in food safety like cancer, neurological disorder, intestinal or poisoned lead to lethal situations. In contrast, anthocyanins – polar organic components are safe, soluble in polarized solvents with characteristic of antioxidant, anti-cancer, antiinflammatory and prevent cardiovascular diseases (Joseph,1999; Pawlowicz, 2000). Thanks to the natural qualities and valuable functions, anthocyanins have been leading scientists and companies to study and exploit this natural pigment in food consumption to make sure the safety in society. Basella rubra L. are vegetable that evaluated as easy crops because of short time growth, simple cultivate methods, suitable for many kinds of soil; they have been using as high nutrient vegetable in daily dishes due to both delicious and medicinal properties. Farmers usually use only stems and leaves for sale, the left over fruits after harvest are kept as breeding purpose in few amount, the rest are useless. The flesh contain red or purple – red color, these pigments can soluble in water easily, which are high potential for food colorants. Pigment content inside fruit can supply for food color, food preservatives or functional food. By mean of using Basella rubra L. fruits for extraction anthocyanins purpose, the problem with leftover fruits after harvested can be solved among with produce natural colorant safe for health. Problems in food safety are not only cause by chemical colorant but also food preservative. There is a fact that some banned preservatives that still have been using, are threatening consumers health. Di Sodium Tetra Borate or Borax 1 are using as an anti-fungal compound in food preservation, that can help fishes and meat become tough and elongate the storage period. The color of anthocyanins can change along with the pH range, this property can be applied as an indicator to identify the present of borax in food through the color change. In total, this thesis aim to: study of extraction from Basella rubra L. and verifying antioxidant activity, application in identifying disodium tetra borate in food. 2. Objective ‒ Study of extracting anthocyanins from fruits of Basella rubra L. ‒ Verifying antioxidant activity of anthocyanin from Basella rubra L. ‒ Determining the identical possibility of borax in food by anthocyanin from Basella rubra L. fruits. 2 CHAPTER II. MATERIALS AND METHODS 1. Materials 1.1. Sample Fruits of Basella rubra L. were harvested from Biotechnology R&D Institute, Can Tho University and Dong Thap province, Vinh Long province, Tien Giang province. 1.2. Apparatus Beaker, buret, crucible and cover, Erlenmeyer flask, filter paper, funnel, graduated cylinder, micropipette, mortar and pestle, test tube, crack, thermal metter, volumetric flask, volumetric pipet, vacuum bottle, vacuum filter bottle, wash bottle. 1.3. Equipment Analytical balance, balance, centrifuges, dry cabinet, pH parameter, stir plate, ultraviolet–visible spectroscopy, vacuum pump, water bath. 1.4. Chemicals ‒ Anthocyanin extraction: ethanol, H2O (1:1 ; 1%HCl), HCl 0.1N, acetone 0.01% HCl, distilled water, Na2HPO4 0.2M, Citric acid 0.1M, KCl 0.2M, NaOH 0.1N. ‒ Antioxidant activity: DPPH, methanol. ‒ Borax identification: Di Sodium Tetra Borate, pH buffers (1-8). 2. Methods 2.1. Sample preparation ‒ Fresh fruits was collect and separated into 2 part, one were used in moisture experiment and the other part were used in the rest experiment. ‒ Fruits were treat through experiments of extracting anthocyanins, verifying chemical property, antioxidant activity and the application of identifying borax in food safety 2.2. Anthocyanin extraction method 3 2.3. Moisture determination Weigh 1g fresh fruits sample (M1) and put in each of 3 crucibles which are dried to a constant weight. Dry sample in drying oven at 70oC in 36 hours until the weight remains constant, record the final unchanged weight (M2) using analytical scale. Moisture content was determined by using formula: Moisture = (M1-M2) x100/M1 (%) In which: M1: The weight of eggfruit powder sample before drying M2: The final unchanged weight of eggfruit powder sample after drying This experiment was repeated 3 times. 2.4. Total anthocyanin content analysis Total anthocyanins content was calculated by pH differential method. The principle of method: anthocyanins have flavium form at pH 4.5 and carbinol form at pH 12. Method: Measuring light absorbance at maximum wave length and 700nm within pH 4.5 and pH 12.0. Equations: a A.M .K  .l A = (Amax.pH4.5 – A700nm.pH4.5) - (Amax.pH12 – A700nm.pH12) With Amax, A700nm: light absorbance at maximum wave length and 700nm, at pH = 4.5 and pH = 12 a: Anthocyanin concentration, g/L; M: molecular weight of Anthocyanin, g/mole; l: path length of cuvette, cm; K: dilution factor; ɛ = 26900 L/mole Total Anthocyanin content: % Total Anthocyanin content = a.V 100% m100  w). 4 V: volume of extracted liquid, mL. m: mass of sample, g. w: moisture 2.5. Verification of antioxidant activity and IC50 index The Antioxidant activity was calculated by: AA% = Abscontrol - Abssample 100% Abscontrol AA%: antioxidant activity Abssample : Light absorbance of sample at 517nm. Abscontrol: Light absorbance of control sample at 517nm IC50 value (mg/ml) is the effective concentration at which DPPH radicals were scavenged by 50%. IC50 was calculate from trend line that built up by the result of AA% 2.6. Preparation of indicator solution: Indicator solution were produced by diluted centrifuged extracted juice with pH buffer solution and store in 2.2mL tubes within dark cabinet. 2.7. Data analysis Data from experiment were processed by Microsoft word, document software. Statistic was handled by SPSS 20. 3. Experiment arrangement 3.1. Experiment 1: Verification of color change among the pH range Purpose: Extracted juice from fruits were centrifuged 13000p/minute and used for verifying the color change among different pH. The result could predict the main ingredient of anthocyanins from Basella rubra L. fruits and their effect to the result of lateral experiment. Objective: anthocyanins extracted juice. Method: 1mL extracted juice was diluted with 24mL distilled water in each test tube. The adjustment of pH in each tube was carried out by the addition 5 of HCl 1N or NaOH 1N. The color change were observed and recorded at each grade of pH from 1 to 14. 3.2. Experiment 2: Verification of maximum absorbance wave length Purpose: Define the maximum absorbance wave length Object: Extracted juice from fruits Method: Diluted 1mL juice with 24mL pH = 1 buffer solution in a 25mL volumetric flask. Absorbance spectra were recorded by continuous scanning range from 450 nm to 650 nm. 3.3. Experiment 3: The effect of solvent and temperature on the total anthocyanin content 3.3.1. Determination of moisture Aim: determining the moisture content of fruits, then used for calculating total anthocyanins content. Object: fresh fruits of Basella rubra L. Method: dried sample at 70oC for 48 hours, experiment was repeated 3 times. 3.3.2. The effect of solvent and temperature on the extraction process The experiment was carried out for the aim of verifying the effect of solvent and temperature on the same object when extracted anthocyanins from fruits. There were two factors in this experiment, solvent and temperature; solvent with 3 treatments: distilled water added 0.1% HCl, Ethanol: H2O (1:1) added 1% HCl, Acetone: H2O (1:1) added 0.01% HCl; temperature with 2 treatments: 30oC and 35oC. Total 3x2 = 6 treatments and repeat 3 times. 6 Grinded sample Solvent added Ethanol : H2O Acetone : H2O (1:1) (1:1) HCl 1% HCl 0.01% H2O Dark cabinet 30oC 35oC Vacuum filter E1 A1 H1 E2 A2 H2 3.4. Experiment 4: Antioxidant activity verification Aim: Verifying Antioxidant activity Object: extracted juice from Basella rubra L. fruits Method: Extracted juice was centrifuged 13000 spins/minute and diluted into different concentration. Sample was mix with DPPH and measure light absorbance at 517 nm. Repeat 3 times for each dilution. (Brand-Williams et al., 1995; Huang et al., 2005) ‒ Dilution range: 10-1, 10-2, 10-3, 10-4 times. 7 ‒ Tested sample included 1mL DPPH, 1mL sample for each dilution in one 2.2 mL tube. The sample was incubated in dark cabinet for 60 minutes and measured the light absorbance. ‒ Control sample included 1mL DPPH and 1mL Methanol. 3.5. Experiment 5: Application in Identification of di sodium tetra borate Aim: identify borax in different concentration due to the color change of Anthocyanin when reacted with borax. Object: Anthocyanin from Basella rubra L. 3.5.1. Identification of Borax Anthocyanin extract from fruits was diluted into 20%, 10% and 5% in pH buffer solutions from 1 to 8 and reacted with Di Sodium Tetra Borate in 5 concentration 2%, 1%, 0.1%, 0.05% and 0.005%. The results with clear and visible color change were used for the next part of experiment. 3.5.2. Producing indicator solution Aim: producing indicator solution that could quickly identify the present of borax. Object: anthocyanin solution and di sodium tetra borate solution From the color change of Anthocyanin within borax solution at specific pH, the indicator kits were produced. Anthocyanin was diluted into 20% and 10% solution with pH buffer solution and kept in dark cabinet with room temperature. The possibilities were test by the change color of indicator kits with borax in 5 concentration 2%, 1%, 0.1%, 0.05% and 0.005%. Experiment arrangement: ‒ Anthocyanin at specific pH 20% reacted with borax concentration 2%, 1%, 0.1%, 0.05% and 0.005%. ‒ Anthocyanin at specific pH 10% reacted with borax concentration 2%, 1%, 0.1%, 0.05% and 0.005%. 8 3.5.3. Producing indicator papers Aim: producing indicator pappers that could quickly identify the present of borax. Object: di sodium tetra borate solution Filter papers were cut into 0.5x5cm sheets and preserved in extracted juice from Basella rubra L. fruits with different dilutions: 20%, 30%, 40%, 50%, and 60% in 24 hours. Then these papers were dried under room temperature and used for identifying borax solution: 2%, 1%, 0.1%, 0.05% and 0.005%. Experiment arrangement: ‒ Indicator papers preserved in 20% solution with borax at concentrations of 2%, 1%, 0.1%, 0.05% and 0.005%. ‒ Indicator papers preserved in 30% solution with borax at concentrations of 2%, 1%, 0.1%, 0.05% and 0.005%. ‒ Indicator papers preserved in 40% solution with borax at concentrations of 2%, 1%, 0.1%, 0.05% and 0.005%. ‒ Indicator papers preserved in 50% solution with borax at concentrations of 2%, 1%, 0.1%, 0.05% and 0.005%. ‒ Indicator papers preserved in 60% solution with borax at concentrations of 2%, 1%, 0.1%, 0.05% and 0.005%. 3.5.4. Identification of sodium hydroxide in food Base on the most clear and high contrast result at specific pH from identification experiment, the indicator kits and papers were used to identify NaOH with concentration of 1%, 0.5% and 0.05%. Sodium hydroxide is representative of alkaline food preservatives, which are added as anti fungus and antibacterial, these preservatives can threaten consumers‘ health. The result were compared with the other indicators that were used in market to estimate the possibility of indicator products from this research. 9 CHAPTER III. RESULT AND DISCUSSION 1. Verification of color change among the pH range Anthocyanin change color due to the transfer of ion H+ and OH- that produce colored salt contain flavium, quinonoidal, chalcone and colorless salt as carbinol. Figure 1. The colored change of Anthocyanin among pH range The color of Anthocyanin solution changed through the pH range from 1 to 14 as shown on the figure 1. The rising of pH from an initial point of 1 to 7 witnessed the stable color of Anthocyanin with pink and red-purple color. At pH nearly 8, Anthocyanin remained purple color and then changed to violet, blue respectively when pH rose from 9 to 11 due to the present of quinoidal ion. Solution gradually lose blue color and became colorless of carbinol salt at pH = 12.0. The rest pH range gave result of yellowish color due to the present of chalcome salt. In comparison with the color range of Anthocyanin from berry, Anthocyanin from Basella rubra L. had a specific difference that it could maintain pink color among pH range from 1 to 7. Base on the result of colored change, flavium salt could be the main contributions of Anthocyanin from Basella rubra L. Those flaivium salt affect strongly to the result of extraction experiment. 10 2. Verification of maximum absorbance wave length The light absorbance was at 450nm then rose gradually and peaked at high of 539nm as the maximum absorbance wave length. The light absorbance stably reduced when wave length rose from 540nm to 650nm. Absorbance (AU) 2.5 2 1.5 1 0.5 0 0 200 400 600 800 Wave length (nm) Figure 2. The light absorbance of anthocyanin from 450nm – 650nm Base on the research out come of Sullivan in 1998, the maximum absorbance wave length of anthocyanin ranged fluctuated between 510 to 540. For instance, maximum wave length of mulberry is 527 (Nhan, 2011), strawberry is 520 nm (Francisco et al., 1998). Due to the outcome of reported paper, the pigments from extracted juice from Basella rubra L. fruits were anthocyanins. 11 3. The effect of temperature and solvent on total Anthocyanin content 3.1. Moisture content Table 1. Moisture of Basella rubra L. fruits Repeat M2 (g) M1(g) D (%) 1 0.2015 1.1018 81.71 2 0.2072 1.2028 82.77 3 0.1851 1.1283 83.59 Mean 82.69 The moisture of fresh fruit was 82.69%. This data was used for calculated total anthocyanin content. 3.2. Total Anthocyanin content Table 2. The total Anthocyanin content Temperature 30oC 35oC Solvent Total Anthocyanin content (%) CV H2O 3.33a 10.2% Ethanol 1.59b 22.1% Acetone 1.28b 5.8% H2O 1.74a 12.8% Ethanol 0.83b 23.3% Acetone 0.58b 7.6% *Those data within a column with same temperature have the same subset b have a statistically significant effect on Percent at the 5.0% confidence level . Temperature and solvent effected significantly to the total Anthocyanin content of extracted liquid in both single and correlative impact. Treatment with distilled water as solvent resulted in high yield for both temperature 30 oC and 35oC. Since the main contributions of Basella rubra L.’s Anthocyanin are flavium salts with negative charge, this polyphenols become anionic and dissolved well in protic solvent like water through hydrogen bonds. In addition, 12 under condition of 30oC temperature, hydrogen bonds become more stable than those at 35oC, therefore, the total Anthocyanin content increased. There was a significant different when compare with result of Nhan et al. 2011 that acetone gave high outcome while distilled water in this result gave high yield. These factors have a statistically significant effect on Percent at the 95.0% confidence Total Anthocyanin Content (%) level. 4.00 3.33 3.00 2.00 1.74 1.59 0.83 1.00 1.28 0.58 0.00 H2O Ethanol Acetone Solvent 30oC 35oC Figure 3. Chart of total Anthocyanin content Treatment with distilled water as solvent at 30oC temperature ranked first, at 3.33% total Anthocyanin content, followed by treatment with the same solvent but at 35oC temperature. Solvent acetone came last at both 30oC and 35oC temperature with 1.28% and 0.58% respectively. Compared with other Anthocyanin content from reported journal as mulberry 1.88%, red cabbage 0.909%, red tea 0.335% (Cuc et al., 2009); the total amount of Basella rubra L. higher than the other fruits. Extraction method used distilled water as solvent at 30oC was the most effective method for Anthocyanin extraction for both high yield of Anthocyanin and simple condition as room temperature and distilled water. 13 4. Antioxidant activity verification Figure 4. The colored change of diluted sample at 10x after 1 hour with DPPH Figure 4 showed the color of sample from different dilutions after 1 hour had reacted with DPPH. Dilution 10-1 gave nearly red color, dilution 10-2 resulted in yellow color. The other dilutions gave the same color with DPPH. Table 3. Antioxidant of Anthocyanin in different dilutions Concentration (µg/mL) Means (%) CV 2 67.7a 3.51% 0.2 20.93b 3.1% 0.02 4.55c 21.9% Antioxidant activity verification experiment was carried out with extracted juice from Basella rubra L. fruits, the concentrated solution has concentration of 200 µg/mL. Dilution of 10-1 (equal to 20 µg/mL) result in minus data because DPPH was not enough to react with Anthocyanin molecular. The antioxidant activity decreased directly proportional to the decrease of Anthocyanin concentration. The reason of decreasing AA% was the decline of 14 Anthocyanin molecular through dilutions. The result of antioxidant activity was shown in table 6, the data have a statistically significant effect on Percent at the Antioxidant activity (AA%) 95.0% confidence level 80 70 y = 29.498x + 9.2317 R² = 0.9713 60 50 40 30 20 10 0 0 0.5 1 1.5 2 2.5 Concentration of anthocyanin (µg/mL) Figure 5. Trend line of Basella rubra L. anthocyanin antioxidant activity. The IC50 index was determined as 1.8 µg/mL, significant low compared to ascorbic acid and garlic acid with IC50 index as 2.6 µg/mL, 8.4 µg/mL respectively (Brighente et al., 2007). This result proved the fact that Anthocyanin from Basella rubra L. need fewer molecular to scavenge DPPH by 50%, therefore, the antioxidant activity of Anthocyanin is higher. Since the high antioxidant activity, Basella rubra L. can be considered as potential sources of functional foods in prevent cancer, anti-inflammatory and prevent cardiovascular diseases or acting as food preservatives. 15 5. Application in Identification of Di Sodium Tetra Borate 5.1. Identification of Borax Figure 6. The identification of Borax in different pH (a) Anthocyanin 10% solution, (b) Anthocyanin 20% solution Extracted juice displayed specific violet color when reacted with Di Sodium Tetra Borate at pH = 2.0 and 8.0. The principles of this experiment are the colored change of Anthocyanin at specific pH and the pH produced by Borax. This violet color was similar to color of Anthocyanin at pH higher than 8 and the pH result that measured from borax solution was approximately 8.4, therefore, the reason that make anthocyanin change to violet is that borax provide a medium with pH >8 that make Anthocyanin change to specific violet color. 16 Anthocyanin 20% solution at pH = 2.0 was chosen for the indicator kits purpose because of the clear and visible colored change – red to violet display. Result at pH = 8.0 could not be chosen because its color was similar to Anthocyanin color at pH higher than 8, this colored change can make wrong decision because of no difference before and after test. 5.2. Possibilities of indicator solution Figure 7. The indicator kits (a) 20% Anthocyanin (b) 10% Anthocyanin Both of the two indicator kits were sensitive and possible to recognize borax with the minimum concentration of 0.05%. Di Sodium Tetra Borate solution change from colorless to violet when added 1 drop of kit 20% and 3 drop of kit 10%. Figure 8. Possibility of solution 10% in identification of Borax 17 Figure 9. Possibility of solution 20% in identification of Borax Indicator kit 20% Anthocyanin at pH = 2.0 was most suitable for identifying borax in food because it need just fewer than one drop for one identifying time and more visible colored change than kit 10%. 18 5.3. Possibilities of indicator papers Figure 10. Possibility of paper 20% in identification of Borax Figure 11. Possibility of paper 40% in identification of Borax Figure 12. Possibility of paper 60% in identification of Borax Indicator papers could identify Di Sodium Tetra Borate in solution by specific violet color. Most of papers could recognize the present of borax at 19 minimum concentration of 1%, especially indicator papers preserved in Anthocyanin 20% solution could clearly identify borax at minimum concentration of 0.1%. Indicator papers produced from anthocyanin can normally identify borax with the same level as the other indicator from market with the visible display at 1% borax minimum in concentration. However, those market indicator can not display clearly when the concentration of borax under 0.1% as Anthocyanin papers. Figure 13. Indicators from market reacted with Borax solution From the result of indicator papers from identification figure and comparison with market indicators, 20% anthocyanin indicator papers was the most suitable indicator for identification of borax by mean of clear and high contrast color change. 5.4. Identification of sodium hydroxide 5.4.1. Identification of sodium hydroxide by indicator solution: Indicator kits from Anthocyanin could identify Sodium hydroxide through the color change. There was a difference from identification of borax that indicator kits could recognize and distinguish the concentration of NaOH, namely violet color for low concentration and yellow color for high concentration. 20 Figure 14. Identification of NaOH in different dilutions by indicator solution 5.4.2. Identification of sodium hydroxide by indicator papers: Indicator papers gave the similar result to indicator kits. They could both recognize and distinguish Sodium hydroxide at high and low concentration, better than market indicator, which only could identify without distinguish the concentration of NaOH. Figure 15. Identification of NaOH 1% by indicator papers 21 Figure 16. Identification of NaOH 0.1% by indicator papers Figure 17. Identification of NaOH 0.05% by indicator papers In the situation of Vietnamese food safety in recently, the use of food preservatives are widely beside borax and NaOH, therefore, the ability of variable identification of indicators from this research is possible and necessary for pre-test of food preservatives before carrying more further tests in food safety. 22 CONCLUSIONS AND SUGGESTIONS 1. Conclusions ‒ From an initial pH of 1, Anthocyanin from Basella rubra L. had the color ranging from red to pink color at the pH of 7. This polyphenol maintain purple color in solution when pH = 8. Then anthocyanin’s color changed to blue when pH rose from 9 to 11 and remain colorless at pH 12. The last two pH (13, 14) witnessed the changed color to yellow. ‒ Since the high ingredient of flavium salt content in Anthocyanin, the extraction experiment resulted most effectively with distilled water as solvent under the temperature of 30o. From this outcome, Basella rubra L. can be considered as a potential vegetable in Anthocyanin yielded when compared with other fruit that has been report in recent years. This extraction method can be simple carried out with room temperature and distilled water. ‒ The IC50 index of Basella rubra L. was 1.8 µg/mL, profusely lower than garlic acid and ascorbic acid. The lower of IC50 the more effective of antioxidant activity, therefore, the antioxidant of Anthocyanin from Basella rubra L. is more effective than the other two acids. This kind of vegetable is suitable for using as antioxidant food and acts as an effective food preservation. ‒ The result from Anthocyanin’s application in indenting di sodium tetra borate by indicator solution and papers show that 20% anthocyanin content products made the most stable result. These two kind of indicator can recognized Borax with high contrast display at the minimum concentration of 0.05% for kit and 0.1% for indicator paper. Moreover, the possibilities of identifying and classifying Sodium hydroxide at high and low concentration through yellow and purple color in extended experiment strengthen the possibilities of producing Borax ‘s indicator products. 23 2. Suggestions ‒ Specific analyses like photo spectrometry on the constituent of Anthocyanin from Basella rubra L. is necessary for further experiment. ‒ Study on sources of papers and suitable dying methods to make sure that identifiable kit and papers can display in a clear and regular result, prevent the browning process under moist condition. ‒ Further experiment in purification of Anthocyanin after extraction should be carried out because the distilled water solvent can dissolve many other polar substances in the product. 24 REFFERENCES Vietnamese: Huỳnh Thị Kim Cúc, Phạm Châu Huỳnh, Nguyễn Thị Lan và Trần Khôi Uyên. 2009. Xác định hàm lượng Anthocyanin trong một số nguyên liệu rau quả bằng phương pháp pH vi sai. Tạp chí Khoa học và Công nghệ. Trường Cao đẳng Lương thực thực phẩm Đà Nẵng. Huỳnh Thị Thanh Tuyền và Trần Xuân Hồng. 2011 Anthocyanin và những nguyên liệu chứa Anthocyanin. Đồ án Môn học: Công nghệ Thực phẩm. Đại học Kỹ Thuật Công Nghệ TP.HCM. Nguyễn Thị Mỹ Nương.2012. Nghiên cứu tách chiết và khảo sát hoạt tính chống oxy hóa của dịch chiết từ vỏ trái thanh long. Luận văn tốt nghiệp trường Đại Học Nha Trang. Phạm Thị Thanh Nhàn. 2011. Tách chiết anthocyanin từ các mẫu thực vật khác nhau. Tạp chí sinh học, 33: 79-85. Trần Thị Thanh Uyên. 2011. Nghiên cứu tách chiết anthocyanin từ bắp cải tím và khả năng ứng dụng để nhận diện hàn the trong thực phẩm. Luận văn tốt nghiệp Đại học. Đại học Đà Nẵng. English: Bertuglia, S., S. Malandrino and A. Colantuoni. 1995. Effect of Vaccinium myrtillus anthocyanosides on ischemia reperfusion injury in hamster cheek pouch microcirculation. Pharmacol Res, 31(3-4):183-187. Bhojwani S. S. and M.K. Razdan .1996. Plant tissue culture: Theory and practice, a revised edition, Elsevier science B.V., ISBN 0-444-81623-2, pp.39-45. Blois Marsden S. 1958. Antioxidant Determinations by the Use of a Stable Free Radical. Nature 181, 1199-1200. Borissova, P., S. Valcheva and A. Belcheva. 1994. Anti-inflammatory effects of flavonoids in the natural juice from Aronia melanocarpa, rutin, and rutinmagnesium complex on an experimental model of inflammation induced by histamine and serotonin. Acta Physiol Pharmacol Bulg, 20(1):25-30. 25 Brand-Williams W., M.E. Cuvelier and C. Berset. 1995. Use of a free radical method to evaluate antioxidant activity. Lebenson Wiss Technol, 28:2530. Brighente, M.C., M. Dias, L.G. Verdi, and M.G. Pizzolatti. 2007. Antioxidant Activity and Total Phenolic Content of Some Brazilian Species. Pharmaceutical Biology, 45(2): 156–161. Deshmukh, S.R. 2009. Antioxidant activity of Morinda citrifolia cultures: Prevention for major diseases. Awishkar SGB Amravati University Journal. 1: 6-9. Elliot, A.J., S.A. Scheiber, C. Thomas and R.S. Pardini. 1992. Inhibition of glutathione reductase by flavonoids. A structure-activity study. Biochem Pharmacol, 44(8): 1603-1608. Francis, F.J. 1999. Colorants. Egan Press. ISBN 1-891127-00-4. Garcia-Viguera, C., P. Zafrilla and F. A. Tomás-Bárbern. 1998. The use of Acetone as an Extraction Solvent for Anthocyanins from Strawberry Fruit. Phytochemical Analysis, 9:274-277. Giusti, M. M., and Wrolstad, R. E. 2001. Anthocyanins. Characterization and measurement with UV visible spectroscopy. In R. E. Wrolstad (Ed.), Current Protocols in Food Analytical Chemistry. New York: Wiley. Hu, C., and Kitts, D.D. 2000. Antioxidant acticity of Echinacea root extract. J. Agric. Food Chem. 48, 1466-1472. Huang, D, B.X. Ou, Prior R.L. 2005. The chemistry behind antioxidant capacity assays. J agric Food chem, 53:1841-1856. Joseph, J.A. 1999. Reversals of age-related declines in neuronal signal transduction, cognitive, and motor behavioral deficits with blueberry, spina strawberry dietary supplementation. J Neurosci, 19(18):8114-8121. Kadam, V.J, Y.M. Yoshi, H.P. Sawant and T.A. Jahdav. 2010. Free radical scavenging activity of aqueous solution of black salt. International Journal of Pharmacy and Pharmaceutical Sciences, 2:95-96. Kim, J.-K., Noh, J.H., S., Choi, J.S., Suh, H., Chung, H.Y., Song, Y.-O. and Choi, W.C. 2002. The first total synthesis of 2,3,6-tribromo-4,5dihydroxybenzyl methyl ether (TDB) ang its antioxidant activity, 23, 661-662. 26 Ko, F.N., Cheng, Z.J., Lin, C.N., and Teng, C.H. 1998. Scavenger and antioxidant properties of prenylflavones isolated from Artocarpus heterophyllus. Free Radic Biol. Med. 25: 160-168. Lee, J. 2005. Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the pH Differential Method: Collaborative Study. Journal of AOAC International, 88(5):1269-1278. Luis E. Rodriguez-Saona and Ronald E. Wrolstad, 2001. Extraction, Isolation, and Purification of Anthocyanins. Current Protocols in Food Analytical Chemistry Manetas, Y. 2006. Why some leaves are anthocyanic, and why most anthocyanic leaves are red. Flora, 201:163.177. Mensor, L.L., F.S. Menezes, G.G. Leitao, A.S. Reis, T.C. dos Santos, C.S. Coube and S.G. Leitão. 2001. Screening of Brazilian plant extracts for antioxidant activity by the use of dPPH free radical method. Phytother res, 15:127-130. Michaelis, Leonor; M. P. Schubert, C. V. Smythe. 1936. Potentiometric Study of the Flavins. J. Biol. Chem, 116 (2): 587–607. Retrieved 2008-04-25. (coi lại tên) Nurliyana, R., Syed Zahir, I., Mustapha Suleiman, K., Aisyah,M.R. and Kamarul Rahim, K. 2010. Antioxidant study of pulps and peels of dragon fruits: a comparative study. International Food Research Journal 17: 367375. Oliveira, A.M.F., L.S. Pinhero, C. K. S. Pereira, W. N. Matias, R. A. Gomes, O. S. Chaves, M. F. V. Souza, R. N. Almeida and T. S. Assis. 2012. Total Phenolic Content and Antioxidant Activity of Some Malvaceae Family Species. Antioxidants, 1:33-43. Pawlowicz, P. 2000. Administration of natural anthocyanins derived from chokeberry retardation of idiopathic and preeclamptic origin. Influence on metabolism of plasma oxidized lipoproteins: the role of autoantiboides to oxidized low-density lipoproteins. Ginekol Pol, 71(8):848-53. Prakash, A., Rigelhof , F., and Miller, E. 2000. Antioxidant activity. Analytical progress Medallion Laboratories. 1- 4. Sakakibara, H., Y. Honda, S. Nakagawa, H. Ashida and K. Kanazawa. 2003. Simultaneous determination of all polyphenols in vegetables, fruits, and teas. J. Agric. Food Chem., 51(3): 571-581 27 Stafford, H. A. 1994. Anthocyanins and betalains: evolution of the mutually exclusive pathways. Plant Science, 101(2): 91–98. Stintzing, F. C. and R. Carle. 2004: Functional properties of anthocyanins and betalains in plants, food, and in human nutrition. Trends Food Sci. Technol, 15(1): 19-38 Sullivan, J. 1998. Anthocyanin. Carnivorous Plant Newsletter (CPN) September 1998. Archived from the original on 1 November 2009. Retrieved 6 October 2009. Tsuda, T. 1998. Dietary cyanidin 3-0-beta-D-glucoside increases ex vivo oxidative resistance of serum in rats. Lipids, 33(6):583-588. Wang, H. and T.B. Ng. 2001. Novel antifungal peptides from Ceylon spinach seeds. Biochem Biophys Res Commun, 288(4):765-770 Wrolstad, R.E. 1993. Color and Pigment Analyses in Fruit products, Agricultural Experiment Atation. Oregon State University, Station Bulletin 624. Website: http://www.worldcrops.org/crops/Malabar-Spinach.cfm (Acessed 23/10/2013) http://database.prota.org/dbtwwpd/exec/dbtwpub.dll?AC=QBE_QUERY&B= http%3A%2F%2Fdatabase.prota.org%2Fsearch.htm&TN=PROTAB~1 &QB0=AND&QF0=Species+Code&QI0=Basella+alba&RF=Webdispl ay (Acessed 23/10/2013) http://parkseed.com/product.aspx?p=05660-PK-P1 (Acessed 23/10/2013) 28 [...]... Anthocyanin with pink and red-purple color At pH nearly 8, Anthocyanin remained purple color and then changed to violet, blue respectively when pH rose from 9 to 11 due to the present of quinoidal ion Solution gradually lose blue color and became colorless of carbinol salt at pH = 12.0 The rest pH range gave result of yellowish color due to the present of chalcome salt In comparison with the color range of. .. of Anthocyanin from berry, Anthocyanin from Basella rubra L had a specific difference that it could maintain pink color among pH range from 1 to 7 Base on the result of colored change, flavium salt could be the main contributions of Anthocyanin from Basella rubra L Those flaivium salt affect strongly to the result of extraction experiment 10 2 Verification of maximum absorbance wave length The light... Anthocyanin from Basella rubra L need fewer molecular to scavenge DPPH by 50%, therefore, the antioxidant activity of Anthocyanin is higher Since the high antioxidant activity, Basella rubra L can be considered as potential sources of functional foods in prevent cancer, anti-inflammatory and prevent cardiovascular diseases or acting as food preservatives 15 5 Application in Identification of Di Sodium. .. preservatives before carrying more further tests in food safety 22 CONCLUSIONS AND SUGGESTIONS 1 Conclusions ‒ From an initial pH of 1, Anthocyanin from Basella rubra L had the color ranging from red to pink color at the pH of 7 This polyphenol maintain purple color in solution when pH = 8 Then anthocyanin s color changed to blue when pH rose from 9 to 11 and remain colorless at pH 12 The last two pH (13, 14)... Experiment 5: Application in Identification of di sodium tetra borate Aim: identify borax in different concentration due to the color change of Anthocyanin when reacted with borax Object: Anthocyanin from Basella rubra L 3.5.1 Identification of Borax Anthocyanin extract from fruits was diluted into 20%, 10% and 5% in pH buffer solutions from 1 to 8 and reacted with Di Sodium Tetra Borate in 5 concentration... temperature and distilled water ‒ The IC50 index of Basella rubra L was 1.8 µg/mL, profusely lower than garlic acid and ascorbic acid The lower of IC50 the more effective of antioxidant activity, therefore, the antioxidant of Anthocyanin from Basella rubra L is more effective than the other two acids This kind of vegetable is suitable for using as antioxidant food and acts as an effective food preservation... The result from Anthocyanin s application in indenting di sodium tetra borate by indicator solution and papers show that 20% anthocyanin content products made the most stable result These two kind of indicator can recognized Borax with high contrast display at the minimum concentration of 0.05% for kit and 0.1% for indicator paper Moreover, the possibilities of identifying and classifying Sodium hydroxide... Ashida and K Kanazawa 2003 Simultaneous determination of all polyphenols in vegetables, fruits, and teas J Agric Food Chem., 51(3): 571-581 27 Stafford, H A 1994 Anthocyanins and betalains: evolution of the mutually exclusive pathways Plant Science, 101(2): 9 1–9 8 Stintzing, F C and R Carle 2004: Functional properties of anthocyanins and betalains in plants, food, and in human nutrition Trends Food Sci... Valcheva and A Belcheva 1994 Anti-inflammatory effects of flavonoids in the natural juice from Aronia melanocarpa, rutin, and rutinmagnesium complex on an experimental model of inflammation induced by histamine and serotonin Acta Physiol Pharmacol Bulg, 20(1):25-30 25 Brand-Williams W., M.E Cuvelier and C Berset 1995 Use of a free radical method to evaluate antioxidant activity Lebenson Wiss Technol,... range Anthocyanin change color due to the transfer of ion H+ and OH- that produce colored salt contain flavium, quinonoidal, chalcone and colorless salt as carbinol Figure 1 The colored change of Anthocyanin among pH range The color of Anthocyanin solution changed through the pH range from 1 to 14 as shown on the figure 1 The rising of pH from an initial point of 1 to 7 witnessed the stable color of Anthocyanin ... application in identifying disodium tetra borate in food Objective ‒ Study of extracting anthocyanins from fruits of Basella rubra L ‒ Verifying antioxidant activity of anthocyanin from Basella. .. Basella rubra L ‒ Determining the identical possibility of borax in food by anthocyanin from Basella rubra L fruits CHAPTER II MATERIALS AND METHODS Materials 1.1 Sample Fruits of Basella rubra L. .. in food safety 22 CONCLUSIONS AND SUGGESTIONS Conclusions ‒ From an initial pH of 1, Anthocyanin from Basella rubra L had the color ranging from red to pink color at the pH of This polyphenol