{"id":700,"date":"2015-02-15T06:15:52","date_gmt":"2015-02-15T06:15:52","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=700"},"modified":"2017-01-04T12:19:25","modified_gmt":"2017-01-04T12:19:25","slug":"isolation-and-separation-of-proteins-from-the-seeds-of-sysmbrium-irio","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol2no1\/isolation-and-separation-of-proteins-from-the-seeds-of-sysmbrium-irio\/","title":{"rendered":"Isolation and Separation of Proteins from the Seeds of Sysmbrium Irio"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Sysmbrium irio<\/em> (Synonym- London Rocket) belongs1<\/sup> to the Family Cruciferae and commonly known as \u201cKhubkalan\u201d. It is used as an expectorant, restorative and used externally as a stimulating poultice. The plant is indigenous to northern India and its distribution extends through Afghanistan to Europe and the Canary Islands.2,3 <\/sup><\/p>\n

The seeds have a hot sharp taste. An extensive survey of literature has revealed that the fixed oil of S.irio<\/em> has been reported partly.4<\/sup>The unsaponifiable part of the oil, however still remains to be studied for its components and its pharmacological activity. Protein fraction of the seed also needs its isolation and separation.<\/p>\n

Materials and Methods<\/strong><\/p>\n

The seeds of S. irio<\/em> were dried in a hot air oven at 40-500<\/sup>C for 48 hrs. The dried seeds were crushed to a coarse powder in an iron pestle & mortar. The powder was sieved & stored in air light container.<\/p>\n

Chemical<\/strong><\/p>\n

Various chemicals used were potassium Iodide, Iodine, Metallic Mercury from CDH chemical. Sodium Hydroxide, Sodium Silicate, Conc. Sulfuric acid, Selenium, Copper Sulfate & Potassium Sulfate of analytical Grade were used.<\/p>\n

Isolation of Proteins<\/strong><\/p>\n

The isolation of the proteins from seeds of S. irio<\/em> was carried out by salt solution method. About 150 g defatted seeds of S. irio<\/em> were crushed and soaked in 10%w\/v Sodium Chloride solution for 10 hrs with occasional shaking. After 10 hrs maceration, the mixture was filtered & marc discarded. To the filtrate 0.5 N HCl was added slowly to get proteins precipitation (PH<\/sup> 3.0- 4.5).The proteins obtained were separated by filtration, washed, dried and weighed. The presence of organic nitrogen was found positive.5, 6, 7<\/sup><\/p>\n

Determination of nitrogen content<\/strong> of proteins in seed8<\/sup><\/strong><\/p>\n

Estimation method consisted of two parts:-<\/p>\n

(i) Conversion of nitrogen to ammonium Sulfate by Kjehldal method,<\/p>\n

(ii) Estimation of ammonia by Nesslerization.<\/p>\n

The seed protein of S. irio<\/em> contains 10.80% nitrogen.<\/p>\n

Hydrolysis of Proteins<\/strong><\/p>\n

About 2 g protein was mixed with 100ml HCl (6N) in a long flask, refluxed for 72 hrs. The hydrolysate was dissolved in 25ml of hot distilled water & filtered. The filterate was evaporated; residue obtained was dissolved in 25ml of 10% aqueous iso-propanol.9, 10<\/sup><\/p>\n

Thin Layer Chromatographic Studies of Hydrolyzed Protein<\/strong><\/p>\n

Approximately 0.10 ml of protein hydrolysate and authentic samples of amino acids were applied at the base line of TLC plates (Silica Gel- G), kept in TLC Chambers saturated with solvent system {n-butanol : Glacial Acetic acid : Water (8:2:2) }.<\/p>\n

The plates were air dried & sprayed with ninhydrin reagent and heated at 110 0<\/sup>C for about 10 minutes.11, 12<\/sup><\/p>\n

Results and Discussion<\/strong><\/p>\n

10% w\/v Sodium Chloride Solution extracted 15.96% of the proteins from defatted seeds of S. irio<\/em>. The proteins gave positive test for nitrogen. Other characteristic tests for\u00a0\u00a0 proteins were also positive. The nitrogen content of proteins was found to be 10.80%.<\/p>\n

The Thin Layer Chromatographic studies (Table -1) revealed that protein hydrolysate of seed proteins of S.irio<\/em> contained ten amino acids. Eight out of these were identified, of which four belongs to category of essential amino acids- threonine, methionine, lysine and leucine. Other four amino acids were serine, glycine, proline and glutamic acids.<\/p>\n

Table 1:\u00a0<\/strong>Rf<\/sub> <\/strong>Values of Amino acids in Protein Hydrolysate.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Spots Detected<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Amino acids taken\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 (Authentic)<\/strong><\/td>\nRf<\/sub>Values of Authentic Amino acids<\/strong><\/td>\nSpots from Sample<\/strong><\/td>\n<\/tr>\n
1.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Serine<\/strong><\/td>\n0.13<\/td>\n0.13<\/td>\n<\/tr>\n
2.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Glycine<\/strong><\/td>\n0.23<\/td>\n0.25<\/td>\n<\/tr>\n
3.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Glutamic acid<\/strong><\/td>\n0.31<\/td>\n0.31<\/td>\n<\/tr>\n
4.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Threonine<\/strong><\/td>\n0.34<\/td>\n0.35<\/td>\n<\/tr>\n
5.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Methionine<\/strong><\/td>\n0.41<\/td>\n0.41<\/td>\n<\/tr>\n
6.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Leucine<\/strong><\/td>\n0.44<\/td>\n0.45<\/td>\n<\/tr>\n
7.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Lycine<\/strong><\/td>\n0.55<\/td>\n0.54<\/td>\n<\/tr>\n
8.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Proline<\/strong><\/td>\n0.64<\/td>\n0.64<\/td>\n<\/tr>\n
9.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

X<\/strong><\/td>\n——<\/td>\n0.76<\/td>\n<\/tr>\n
10.<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

Y<\/strong><\/td>\n——<\/td>\n0.85<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/strong><\/p>\n

Solvent System<\/strong><\/p>\n

n-Butanol : Glacial Acetic acid : Water (8:2:2)<\/p>\n

Adsorbent<\/strong><\/p>\n

Silica- Gel G<\/p>\n

Detectron Reagent<\/strong><\/p>\n

Ninhydrin- Acetic acid Solution.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Hooker J.D., Flora of British India, Vol.-I, John Wiley, New York, 732 (1875).<\/li>\n
  2. Kirtikar, K.R. and Basu B.D., India Medicinal Plants, II nd<\/sup>, Vol. I, Shri Sat guru Publications, New Delhi, 152 (1975).<\/li>\n
  3. Bamber J.C., Plants of Panjab, Fellow of Linneam Society, London.247 (1916).<\/li>\n
  4. Handa K.L. and Chopra I.C., Review of Research on Indian Medicinal and Allied Plants, Forensic Science Industrial Research, New Delhi,16B,45 (1957).<\/li>\n
  5. Vickery H.B., Physiological Review, 25, 347 (1945).<\/li>\n
  6. Vickery H.B., Physiological Review, 26, 217(1951).<\/li>\n
  7. Pirie N.W., Biological Review, 15, 377(1940).<\/li>\n
  8. Mishra K., Manual of Echology, Banaras Hindu University, Varanasi,146 (1961).<\/li>\n
  9. Faster F., Introduction to Protein Chemistry, Vol. I., John Wiley, New York, 75 (1957).<\/li>\n
  10. Felix H.,The Chemistry & Functions of Proteins, 2nd ed. Academic Press, New York, 20(1963).<\/li>\n
  11. Block R.J.and Boiling D.,The amino acids Composition of Proteins and Food, 2nd<\/sup>,411 (1951).<\/li>\n
  12. Stahl E.,Thin Layer Chromatography \u2013 A Laboratory Hand book, Academic Press, Inc, London,339 (1965).<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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