Akhlesh Singhai^1* , Govind Nayak¹, Ashok Budhwani ² and Ashish Singhai³

¹Lakshmi Narain College of Pharmacy , Bhopal (India).

²Daksh Institute of Pharmacy, Chhatarpur (India).

³VNS Institute of Pharmacy, Bhopal (India).

Abstract

In the present investigation we have attempted the isolation and separation of proteins from the seeds of Sysmbrium irio. Its seeds are used as an expectorant, restorative and externally as stimulating poultice. The protein content was found to be 18.25%. The amino acids were identified by Thin layer and Paper Chromatography. These studies revealed the presence of amino acids, namely- Serine, Glycine, Threonine, and Methionine, Leucine, Lysine, Proline, and Glutamic acid and two remained Unidentified.

Keywords

Sysmbrium irio; Proteins; amino acids

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Copy the following to cite this article: Singhai A, Nayak G, Budhwani A, Singhai A. Isolation and Separation of Proteins from the Seeds of Sysmbrium Irio. Biomed Pharmacol J 2009;2(1).

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Introduction

Sysmbrium irio (Synonym- London Rocket) belongs¹ to the Family Cruciferae and commonly known as “Khubkalan”. 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

The seeds have a hot sharp taste. An extensive survey of literature has revealed that the fixed oil of S.irio has been reported partly.⁴The 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.

Materials and Methods

The seeds of S. irio were dried in a hot air oven at 40-50⁰C 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.

Chemical

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.

Isolation of Proteins

The isolation of the proteins from seeds of S. irio was carried out by salt solution method. About 150 g defatted seeds of S. irio 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 (P^H 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}

Determination of nitrogen content of proteins in seed⁸

Estimation method consisted of two parts:-

(i) Conversion of nitrogen to ammonium Sulfate by Kjehldal method,

(ii) Estimation of ammonia by Nesslerization.

The seed protein of S. irio contains 10.80% nitrogen.

Hydrolysis of Proteins

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}

Thin Layer Chromatographic Studies of Hydrolyzed Protein

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) }.

The plates were air dried & sprayed with ninhydrin reagent and heated at 110 ⁰C for about 10 minutes.^{11, 12}

Results and Discussion

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

The Thin Layer Chromatographic studies (Table -1) revealed that protein hydrolysate of seed proteins of S.irio 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.

Table 1: R_f Values of Amino acids in Protein Hydrolysate.

Spots Detected	Amino acids taken                (Authentic)	RfValues of Authentic Amino acids	Spots from Sample
1.	Serine	0.13	0.13
2.	Glycine	0.23	0.25
3.	Glutamic acid	0.31	0.31
4.	Threonine	0.34	0.35
5.	Methionine	0.41	0.41
6.	Leucine	0.44	0.45
7.	Lycine	0.55	0.54
8.	Proline	0.64	0.64
9.	X	——	0.76
10.	Y	——	0.85

 

Solvent System

n-Butanol : Glacial Acetic acid : Water (8:2:2)

Adsorbent

Silica- Gel G

Detectron Reagent

Ninhydrin- Acetic acid Solution.

References

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2. Kirtikar, K.R. and Basu B.D., India Medicinal Plants, II ^nd, Vol. I, Shri Sat guru Publications, New Delhi, 152 (1975).
3. Bamber J.C., Plants of Panjab, Fellow of Linneam Society, London.247 (1916).
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).
5. Vickery H.B., Physiological Review, 25, 347 (1945).
6. Vickery H.B., Physiological Review, 26, 217(1951).
7. Pirie N.W., Biological Review, 15, 377(1940).
8. Mishra K., Manual of Echology, Banaras Hindu University, Varanasi,146 (1961).
9. Faster F., Introduction to Protein Chemistry, Vol. I., John Wiley, New York, 75 (1957).
10. Felix H.,The Chemistry & Functions of Proteins, 2nd ed. Academic Press, New York, 20(1963).
11. Block R.J.and Boiling D.,The amino acids Composition of Proteins and Food, 2^nd,411 (1951).
12. Stahl E.,Thin Layer Chromatography – A Laboratory Hand book, Academic Press, Inc, London,339 (1965).

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