Identification of the Different Constituents of Spirulina platensis Using HPTLC Fingerprinting.

 

V.R. Ravikumar1*, T. Sudha2 and P.V. Hemalatha2

1Dept. of Pharmacognosy, The Erode College of Pharmacy and Research Institute, Erode-638112, Tamilnadu.

2Dept. of Pharma. Analysis, The Erode College of Pharmacy and Research Institute, Erode-638112, Tamilnadu.

 

ABSTRACT:

Spirulina platensis (fam: Cyanophyaceae) is a blue green fresh water algae. It is a simple, single – celled alga that thrives in warm alkaline fresh water. It is helical in shape and hence the name Spirulina. Spirulina is being developed as the food of the future due to its very high nutritional value. The present work deals with identification of the different constituents of Spirulina using HPTLC finger printing. The dried form of algae was used to prepare extracts of Spirulina in different polar and non polar solvents like petroleum ether, chloroform, ethyl acetate, ethanol and water. The extracts were then subjected to different chemical tests. The ethanol extract presented a good result for the presence of alkaloids, flavonoids and steroids. The ethanol extracts was then subjected to HPTLC analysis in various solvents, suitable for alkaloids, flavonoids and steroids. The HPTLC fingerprinting results confirmed the presence of alkaloids, flavonoids and steroids.

 

KEYWORDS: Spirulina platensis, HPTLC, alkaloids, flavonoids, steroids, immunomodulatory

 

INTRODUCTION:

Spirulina platensis (fam: cyanophyaceae) is a simple one-celled form of blue green algae that thrives in warm alkaline fresh water. Spirulina is helical or spiral in shape forming swirling macroscopic strands. Owing to its high nutritional value, spirulina is being developed as food for the future. Spirulina is one of the few plant sources which is rich in Vitamin B121. The ability of Spirulina to grow in hot alkaline water ensures its hygienic status. Unlike the stereotypical association of microorganism with “germs” and “scum”, Spirulina is in fact one of the cleanest, most naturally sterile food available in nature. Spirulina produces a large number of valuable compounds such as phycocyanine, carotenoids, poly unsaturated fatty acid such as linoleic and linolenic acids and thus have important role in human metabolic pathways2. The present study deals with the development of HPTLC finger printing of the various constituents present in Spirulina platensis. Extensive literature survey reveals that studies on antiviral3, immuno modulatory4, anticancer5, lipid lowering activity6 and anti allergic activity7 on Spirulina have been performed. GC-MS analysis of volatile components of Spirulina platensis resulted in the identification of 15 compounds which constitute about 96.45% of the total compounds8. But there is no evidence of any HPTLC analysis of the algal constituents. Thus an effort has been made to develop HPTLC finger prints of the various constituents present in the algae.

 

MATERIALS AND METHODS:

Collection and extraction:

Spirulina was collected from Ezha Edhlier Maruvazhvu Kazhagam, Erode during the afternoon in high denser condition using muslin cloth (600 holes /


inch). The collected algae were then washed in tap water, dried and milled to a coarse powder suitable for solvent extraction. The dried Spirulina was extracted with different solvents from non polar to polar solvents9 like pet ether, chloroform, ethyl acetate, alcohol and water by continuous hot percolation using Soxhlet apparatus and concentrated by vacuum distillation. The percentage of extractive value of ethanol was good. The qualitative phytochemical analysis of the ethanol extract showed the presence of alkaloids, steroids, flavonoids and saponins.

 

Preparation of test sample10:

The dried powdered form of 100mg of Spirulina platensis was extracted with ethanol and centrifuged at 300rpm for 5 minutes. The supernatant liquid was transferred to a 50 ml volumetric flask and made up to mark with ethanol. The resulting solution was used for the HPTLC analysis

 

Loading of sample:

About 5 µl of the test solution was loaded as 8 mm band length in four 5 X 10 silica gel 60F254 TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.

 

Development of chromatogram:

The TLC plates loaded with the sample were kept in the twin trough developing chamber, after being saturated with the respective mobile phase and the plates were developed up to 90 mm. The different mobile phases for the development of chromatograms of different constituents of Spirulina platensis are given in (Table-1)

 

Photo documentation:

The developed plates were dried in hot air and then kept in the photo documentation chamber (CAMAG REPROSTAR 3) and the images recorded at white light, UV- 254 nm, UV- 366 nm.

 

Spirulina platensis - Alkaloid Profile

 

Spirulina platensis - Flavonoid  Profile

 

Table no.1 Data showing the mobile phase profiles for HPTLC of Spirulina platensis

S no.

Constituents

Mobile phase

Ratio of mobile phase

1.

Alkaloids

n-butanol : acetic acid : water

4 : 4 : 1

2.

Flavonoids

Ethyl acetate : methanol : water

10 : 1.35 : 1

3.

Steroids

Ethyl acetate : chloroform : water

9 : 1: 0.1

4.

Saponins

Chloroform : acetic acid : Methanol : water

6.4 :  3.2 : 1.2 : 0.8

 

Table no.2- Data showing detection reagents used for HPTLC analysis of Spirulina platensis

S. No

Constituents

Spraying reagents

After Derivatization

1.

Alkaloids

Dragedroff’s  reagent and 10% sulphuric acid

Bright orange colored zones in white light

2.

Flavonoids

1% Ethanolic aluminium chloride reagent

Yellow Flourescence at UV 366 nm

3.

Steroids

Anisaldehyde – sulfuric acid reagent

Green colored Zones in White light

4.

Saponins

Anisaldehyde – sulfuric acid reagent

Yellowish green and violet zones in White light

 

 

Derivatization:

The developed plates were sprayed with the respective spraying reagents and dried at 110şC in hot air oven. Again the plates were Photo documented in white light. The spraying reagents for different constituents and the result after derivatization are given in (Table-2)

 

Scanning:

Finally the plates were scanned at 500 nm. The peak table, Peak display and peak densitogram were noted.

 

RESULTS AND DISCUSSION:

The ethanolic extract of Spirulina platensis was used as the sample for the HPTLC analysis for alkaloids, flavonoids, steroids and saponins. 5 X 10 Silica gel 60F254 used. 5 µL of the sample was spotted on the plate as 8 mm band length using Hamilton Syringe and CAMAG LINOMAT 5 instrument. The chromatogram was allowed to develop up to 90mm, in the respective mobile phase combinations. The plates were then dried, Photo documented in white light, UV 254 nm, UV 366nm. Then sprayed with respective spraying reagents, again dried in hot air oven and photo documented. (Fig. 1, 2, 3 and 4). Finally after derivatization, the plates were scanned at 500 nm. The peak table, peak display and peak densitogram were noted as shown in (Table. 3 and fig. 4, 5, 6 and 8).

 

Fig. 7- HPTLC Analysis of Ethanol Extract of Algae

 

Spirulina platensis -  Saponin Profile

 

Track A4- Baseline display (scanned at 500nm

 

Fig.8- Track A4 – Peak densitogram display Saponins in Spirulina platensis

 

 

CONCLUSION:

The different constituents present in the ethanol extracts of Spirulina platensis were analysed by HPTLC. The presence of alkaloids flavonoids, steroids and saponins were confirmed by the HPTLC finger prints.

 

References:

1.        www.naturalways.com

2.        Ratana Chailahan, Nattayapor and Chirasu Wanipid, Fatty acids extraction from the cyanobacterium spirulina, Science Asia, 2008, 34, 299-305.

3.        Toshimitsu H. and Kyoko H. (1996):  Calcium spirulan an inhibitor of Envelope virus replication from a  blue green alga Spirulina  platensis.  Journal of Natural Products. 59, 83-87.

4.        Qureshi M.A, Kidd M.T and Ali R.A. (1996):  Spirulina platensis extract- Enhances Chicken macrophage functions after in vitro exposure. Journal of Nutritional Immunology, 18 (3), 456-476.

5.        Vinod .D. Rangari Pharmacognosy and Phytochemistry I st edition Career publication, Nashik, 2003, Part II, 48.

6.        Colin .J. Barrow and Fereidoon shahidi,   Marine Nutraceuticals and functional foods, Edition, illustrated, published by CRC Press 2007, 346

7.        Karkos P.D. and Leong S.C. (2008): Spirulina in Clinical Practice Evidence-Based Human Applications. Oxford journals. 1-11.

8.        Guven Ozdemir and Ulku Karabay.N, Antibacterial activity of volatile components and various extracts of Spirulina platensis, Phytotherapy Research, 18(9), 754-757

9.        Harbone.J.B, Photochemical method, second Edition, Published by- Chapman and Hall, London, 1984, 4-22.

10.     Beckett. A.H and Stenlake. J.B. (2007): Practical Pharmaceutical Chemistry, 4th edition, Published by-CBS, New Delhi, Part II, 275-337,379-405,115-122.

 

Received on 30.06.2010

Accepted on 07.07.2010        

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(5): Sept.-Oct. 2010, 391-394