Anthocyanins are water soluble pigments that give colours to flower fruits and vegetables. Anthocyanin has ability to act as powerful antioxidant. In this study anthocyanin was extracted from pomegranate and black grapes using TCA and methanol and its stability was increased by nano-emulsifying the anthocyanin present in the samples. The extracts went through stability tests with variation in pH and temperature before and after the nano-emulsion to observe the difference. The amount of anthocyanin was estimated to be more in pomegranate.
Key words: anthocyanin, nano-emulsion, stability.
Anthocyanin are considered to be the largest group of water soluble compounds found in flowers, fruits and vegetables . The class of molecules to which anthocyanin belongs is flavonoids which are synthesised by phenylpropanoid pathway. Anthocyanins are responsible for providing red, blue, or purple colours to fruits, vegetables, and flowers, depending on the pH. The colour attracts insects, birds and animals which help them pollinating . More than 400 naturally occurring anthocyanins have been identified . The difference in anthocyanins can be determined by the number of sugars, hydroxyl groups, aromatic acids and aliphatic groups attached to the structure of anthocyanin . Cyanidin is the most commonly occurring anthocyanin in the nature. The different types of colour anthocyanin provides can be determined by the pattern in which B-ring of aglycone are substituted, pH, temperature, presence of pigments, solvents etc. [5, 6]. Anthocyanin not only provides colours but they are also beneficial in decreasing the risk of heart disease, diabetes, cancer, it also improves the vision.
Black grapes (Vitis vinifera) are berries that grow in clusters of 15 to 300, they could be black, crimson, dark blue, purple in colour. These colours are due to the presence of anthocyanin and other polyphenolic pigments. A compound resveratrol is present in grapes that have antifungal and other defensive properties. Anthocyanin has anti-oxidant strength. Pomegranate (Punica granatum) is a fruit between grapes and lemon. Its diameter is 5-12 cm and number of seed can vary from about 200 to 1400. Pomegranate seeds contain polyphenols and flavonoids. Pomegranate has anti-oxidant values and has shows effective response in reducing factors responsible for heart disease .
Nano-emulsions are non-toxic droplets of lipid having diameter ranges between 100-600 nm. [8, 9]. Nano-emulsion are only kinetically stable. The physical stability of nano-emulsion can be for a very long time, making them unique [10, 11].
2.1. Extraction of anthocyanin:
Pomegranate and black grapes were purchased from the local market of Vellore. They were purchased on the bases of their colour, darker the colour, more is the amount of anthocyanin. The samples were washed and peeled. The 5g of sample was weight and added with the solution of 100ml of trichloroacetic acid in methanol. Ground the sample and solution to make a homogenate. This homogenate was refrigerated for 24 hours. Centrifuged the homogenate, and the supernatant was decanted. A mixture of methanol with a solution of residue: acetic acid: water (10:1:9) was made, and kept on a constant stirrer at the room temperature for 24 hours. These steps were repeated 3 times, and each time the decanted supernatant's volume was measured and filtered by Whatman paper. This extract of anthocyanin was stored at 4??C until its use. The same procedure was followed for both the samples .
2.2. Quantification of anthocyanins:
The quantity of anthocyanin was determined by spectrophotometer. The reading of each sample was observed at a wavelength of 520nm. The total concentration of anthocyanin per 100 g'1 of solid sample, was calculated with the help of volume, weight of sample, absorbance, extinction coefficient 29,600 1/mol'1 . cm'1 and molecular weight 445.2 g/mol .
2.3. Purification and partial identification of anthocyanins by HPLC:
Anthocyanin purification was done with the first extract obtained with 1% of trichloro acidic acid (TCA). The extract with TCA and methanol was run through a LC-18 cartidge. The cartridge was conditioned with acidified water (4 ml), acidified methanol (4 ml), and again with acidified water (2 ml) to remove left out methanol. 1 ml of first extract was added in the cartridge and the anthocyanin was separated by using acidified methanol with TCA at pH 1. HPLC Perkin Elmer c200 was used for analysing the samples with the help of Brown- Iee Analytical C-18 column of 150*4.6 mm. Acidified water with 0.1% TCA was used as mobile phase A, and HPLC grade acetonitrile and 0.035% formic acid was used as mobile phase B, the separation took place with the gradient elusion at 1 ml/min flow rate. The sample was filtered before injecting it into the column. 20??l was injection volume. The gradient used were as follows 10-11% of B in 12 min, 11-12% in 8 min, 12-13% in 5 min, 13- 18% in 10 min, and for 25 min 18% of B was maintained. The anthocyanins were determined at 520nm with a visible detector. And the identification was done using the commercial standards of known retention time.
3. Results and discussion:
The extraction of anthocyanin with TCA and methanol, the anthocyanin content in black grape was found to be 28.7??10.9 and in pomegranate it was found to be 140??28.5 which was obtained and calculated by spectrophotometer. This result was similar to the study performed by Xianli et.al. 2006 . After the extraction and quantification, the purification and partial identification of the extracts was to be done by HPLC. The result of HPLC for both the samples i.e. pomegranate and black grapes were 24.59 and 4.60 respectively. The study till now shows that the amount of anthocyanin in much higher in pomegranate as compared to black grapes.
The extraction of anthocyanin is done from the flesh part of Grapes and Pomegranate with the help of acidified methenol and 1% (TCA) trichloroacetic acid, acidic water is also used in the extraction. It is best to store anthocyanin at 4??C the pH4 should be maintained. It should be stored at dark, so the anthocyanin remains stable. The study shows that the amount of anthocyanin is more in pomegranate than in black grapes. This result contradicts the theory that says that darker the colour more is the amount of anthocyanin. Anthocyanin is quantified by HPLC and to provide stability to it we need to perform nano-emulsion. Anthocyanin fortification is quite a problem in food products, so we need to increase the stability of anthocyanin. The work of is still going on to provide stability to anthocyanin extracted from black grapes and pomegranate.
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