Phytochemical analysis of commercially available Spirulina, their activities and Biosynthesis of Nanoparticles

 

Monika M,  K. Krishna Prema*

Department of Microbiology, Ethiraj College for Women, Chennai, Tamil Nadu, India.

*Corresponding Author E-mail: krishnaprema@ethirajcollege.edu.in

 

ABSTRACT:

Spirulina is used as dietary supplement in form of capsules and tablets. It is rich in Bioactive molecules and exhibits various activities. Commercially available Spirulina was collected. Extracts were prepared using various polar and non-polar solvents. The extracts were used for qualitative phytochemical analysis and to determine their Anti-Oxidant, Anti-Inflammatory and Anti-Arthritic activity. Phytochemicals like Tannin, Carbohydrates and Glycosides were present in all the extracts, while others were present only in few extracts. Biosynthesis of Nanoparticles was done by using the extract. Chloroform extract and Nanoparticles showed higher Anti-Oxidant activity, Ethanol extract showed higher Anti-Inflammatory activity and Chloroform extract showed higher Anti-Arthritic activity.

 

KEYWORDS: Nanoparticles, Spirulina, Phytochemical, Extracts, Activity.

 

 


INTRODUCTION:

Spirulina is a filamentous Cyanobacterium, multicellular and it belongs to Phormidiaceae family which appears as blue green filaments under microscope, composed of cylindrical cells arranged in unbranched trichomes. Blue-green algae are used as a source of B-vitamins, iron and dietary protein which is absorbed when taken orally. Blue-green algae has been used in treatment of precancerous growths which are found in the mouth, it also improves memory, boost the immune system, increase energy, lowers cholesterol and helps to improve metabolism, help in healing wounds, preventing heart disease, improves digestion and bowel health. Nanotechnology is the production and manipulation of materials ranging in size from 1 to 100 nanometre scale. They play a top most role in the field of Nanomedicines like Health care and Medicine, Diagnostic and Screening purposes, Antisense and Gene therapy applications, Drug delivery systems, expectations of Nanorobots configuration and Tissue Engineering. Increasing surface area of Nanoparticles increases their biological effectiveness due to the increase in surface energy.

 

Biosynthesis of Nanoparticles using microorganisms, enzymes and plant or plant extracts is eco-friendly alternatives to chemical and physical methods. Different metallic nonmaterial is being produced using Copper, Magnesium, Gold, Zinc, Titanium, Alginate and Silver. Naturally occurring bio molecules play an active role in the formation of Nanoparticles of distinct shapes and sizes thus for the designing of greener, safe and environmental synthesis of Nanoparticles. There is a need for green chemistry synthesis of Nanoparticles using biological systems since it is less toxic, cheap and environmental friendly.

 

Different Phyto-metabolic contents are present in photosynthetic microalgae which includes various biological activities and chemical structures 1. Various lethal toxins are produced by freshwater Cyanobacteria like Microcystins and Anatoxins. In the purification of Microcystins from the bloom samples, a toxic Cyanobacterium produced other peptides types 5. The extraction methods used traditionally have several drawbacks such as laborious, time consuming, have low selectivity and/or low extraction yields; but they make use of large amount of toxic solvents 7.

 

Drug targeting includes the delivery of drugs to specific receptors or organ which may include specific part of the body to deliver the drug exclusively 8. Spirulina is highly nutritious micro salt water plant. The spiral shaped algae were found to be a rich food source 14. Spirulina microalgae is most valuable food which contains macro- and micronutrients including high quality Protein, Gamma-Linolenic Fatty acid, Iron, Carotenoids, Vitamins B1 and B2 16. Spirulina platensis, a blue-green alga is gaining attention due to its nutritional and medicinal value and it also contains Phycobiliproteins like Phycocyanin and Allophycocyanin 20. Compounds like diterpenoid and anthraquinone have been isolated from the cyanobacterium cells of the cultured Nostoc commune (EAWAG 122b) by Bio-guided isolation 11.

 

Nanotechnologies have emerged from Physical, Chemical, Biological and Engineering Sciences as a probe and to manipulate single atoms and molecules 21. Physical and Chemical methods are available to synthesize Nanoparticles. It contains various drawbacks thus various biological agents are used for the synthesis of Nanoparticles. These methods are nontoxic, safe and eco-friendly 22. Various methods available for synthesis of Silver Nanoparticles includes Tollens, Polysaccharide, Irradiation (Microwave, UV, Gamma etc.,), Polyoxometalates and Biological 23. Spirulina platensis (Cyanophyaceae) a one-celled blue green algae that survives in warm alkaline fresh water 24.

 

Nanotechnology is the new trend in the Pharmacy and Biomedical field 2.  Biosynthesis of Nanoparticles has gained attention due to its eco-friendly benign method of production. Nanoparticles synthesis can be done by the use of parts or whole biological component 4. The increasing demand for Nanoparticles includes their wide applicability in areas like Electronics, Energy, Catalysis, Chemistry and Medicine 10. Nanoscience and Nanotechnology includes the study of small nano things and that have applications in all the other Science fields, like Biology, Physics Chemistry, Materials Science and Engineering 19. Nanomaterials can be synthesized using Chemical methods but now synthesized by the use of biological materials 18. Microbial source has been used to produce Silver Nanoparticles and its precipitation due to its metabolic activity 12.

 

Various Modern drugs are isolated from Natural sources which are based on their use in Traditional Medicine. The Traditional Medicinal plants have been used for years to treat disease all over the world 13. Nanoparticles are excellent Research area since they are bridging the gap between the bulk materials and molecular or atomic structural compounds 26. Biological method to synthesis Silver Nanoparticles can be done by Euphorbia hirta 27. Microalgal system provides a simple, cheap alternative source for the biosynthesis of silver nanomaterials which have uses in biomedical applications 28. Characterization of Nanoparticles depends on applications. Characterization can be done by various techniques like those used for determination of parameters like particle size, shape, pore size, crystallinity, fractal dimensions and surface area (29). The advantages are that it increases the absorbance of the Herbal formulation, lowers the dose of formulation and raises its solubility (30). The stabilizing agent that keeps the Silver Nanoparticles stable was identified by FTIR spectrum 31.

 

Branch of Nanotechnology like Nanomedicine which uses nano-sized tools for the Diagnosis, Prevention and Treatment of disease and also helps to gain understanding on the complex underlying pathophysiology 32. Nanotechnology is defined as the technology which allows the manipulation, control, study and to manufacture structure and devices of ‘nanometer’ size range 33.

 

MATERIALS AND METHODS:

Collection of samples:

100g of commercially available Spirullina was purchased from medical shop which was in the form of capsules.

 

Preparation of crude extract 9:

10g of dried algal biomass from Spirullina capsules was weighed and 30ml of solvent (1:3) was added for extraction. Both polar and non polar solvents were used like Chloroform, Aqueous, Acetone and Ethanol. Extraction was done by maceration, in dark at 30C for 24 hours. The extraction was done three times to harvest the maximum of compounds. It was finally filtered by Whatmann filter paper and the filtrate was stored at 4C for further use. These extracts were used for phytochemical analysis and various activity analysis.

 

Qualitative Phytochemical analysis:

Phytochemical analysis was done by standard procedures as stated by Harborne, 1995 6.

 

BIOSYNTHESIS OF NANOPARTICLES 3:

Biosynthesis requires microalgae extract, which is produced by suspending 5g of Spirullina powder from the commercially available capsules in 100ml of double distilled water. It was heated in a water bath at 100C for 15 minutes. It was then cooled and centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected and stored at 4C for further use. This extract was used for Biosynthesis of Nanoparticles.5ml of extract was added drop wise into 200ml of 1 milli molar Silver Nitrate solution. It was kept at 60C for 10 minutes with constant stirring and the colour change was noted. It was finally centrifuged at 15,000 rpm for 20 minutes at 4C.The pellets were washed with distilled water and 90% Ethanol to get pure Silver Nanoparticles.

 

ANTI-OXIDANT ACTIVITY 6:

Test tubes were taken and marked as Blank, Standard, Extracts and Nanoparticles. 40 milli molar solution of Hydrogen Peroxide was prepared in Phosphate buffer.0.1mg/ml of Extract solution was prepared in distilled water. It was mixed with equal amount of Hydrogen Peroxide and left undisturbed for 10 minutes. Absorbance was read using a Colorimeter at 550nm.

 

ANTI-INFLAMMATORY ACTIVITY 15:

Different test tubes were taken for Control, Standard, Extracts and Nanoparticles. 0.5ml of Diclofenac Sodium was added to tube labelled as Standard and 0.5ml of extracts were added to the extract tubes. 2ml of distilled water was added to Control and 1.5ml in other tubes. 3ml of Phosphate buffer was added to Control tube and 1 ml to other tubes. 2ml Hyposaline was added to Standard, Extract and Nanoparticles tubes. 0.5ml of HRBC suspension was added to all the tubes. The HRBC suspension was prepared by mixing 5ml of Human Blood and 5ml of Alsever’s solution. It was centrifuged at 3000 rpm for 15 minutes. The pellets were taken and washed with Hyposaline.10ml of this suspension was mixed with 10ml of Isosaline. The tubes were incubated at 37C for 30 minutes. It was then centrifuged at 3000 rpm for 10 minutes. The supernatant was taken for the further Analysis and Absorbance was noted at 550 nm using a Colorimeter.

 

Anti-Arthritic Activity 17, 25:

Tubes were taken for control, standard, extract and Nanoparticles and labelled respectively.0.5ml of BSA was added to all the tubes. 0.5ml of extract and Nanoparticles were added to the respective tubes.1ml of isosaline was added to all the tubes.0.5ml of Diclofenac Sodium was added to standard tube. Incubate all the tubes at 37C for 20 minutes. Heat all the tubes at 51C for 3 minutes and cool the tubes to room temperature. 2.5ml of Phosphate buffer was added to all the tubes.


 

RESULTS:

Phytochemical Analysis:

Table 1. Phytochemical analysis of various extracts of Spirulina.

S.No.

Test

Aqueous Extract

Acetone Extract

Ethanol Extract

Chloroform Extract

1

Triterpenes

-

-

-

-

2

Terpenoids

+

+

-

+

3

Triterpenoids

-

-

-

-

4

Phenols

+

+

-

+

5

Quinones

+

-

-

-

6

Flavonoids

+

+

+

-

7

Tannin

+

+

+

+

8

Phlobatannins

+

-

-

+

9

Alkaloids

+

+

+

+

10

Saponin

-

-

-

-

11

Carbohydrate

+

+

+

+

12

Glycosides

+

+

-

+

13

Anthraquinone

-

-

-

+

14

Coumarins

+

-

-

+

15

Proteins/amino acids

+

-

+

-

 


Figure 1. Various extracts of Spirulina.

*A-Aqueous Extract, B-Acetone Extract, C-Ethanol Extract and D-Chloroform Extract

 

ANTI-OXIDANT ACTIVITY:

The activity was determined using the formula-

 

                                      Absorbance of Control - Absorbance of Test

Anti-Oxidant Activity =  ----------------------------------------------------

                                                   Absorbance of Control

 

Table 2: Anti-oxidant activity of various extracts

Extract

Od value (550 nm)

% Activity

Control

0.50

-

Ethanol

0.22

56

Acetone

0.31

38

Aqueous

0.23

54

Chloroform

0.05

90

Nanoparticles

0.05

90

 

Chloroform extract and Nanoparticles showed higher anti-oxidant activity followed by Ethanol, Aqueous and Acetone extract respectively.

 

Figure 2. Anti-Oxidant activity

*A-Control, B- Nanoparticles, C-Acetone Extract, D-Ethanol Extract, E- Aqueous Extract and F- Chloroform Extract.

 

Anti-Inflammatory Activity

The activity was determined using the formula –

 

                                      Absorbance of Test- Absorbance of Control

Anti-inflammatory Activity = ----------------------------------------------

                                                         Absorbance of Test

 

Table 3. Anti-Inflammatory activity of various extracts

Extract

OD Value

(550 nm)

% Activity

Control

0.22

-

Standard

0.48

54.1

Ethanol

1.52

85.5

Acetone

1.15

80.8

Aqueous

0.36

38.8

Chloroform

0.38

42.1

Nanoparticles

0.69

68.1

 

The activity was found to be higher than the standard in Ethanol extract followed by Acetone extract and Nanoparticles. Aqueous extract and Chloroform extract showed comparatively lesser activity than the Standard.

 

Figure 3. Anti-Inflammatory Activity

*A-Control, B-Standard, C, Ethanol Extract, D-Acetone Extract, E-Aqueous Extract, F-Chloroform Extract and G-Nanoparticles

 

Anti-Arthritic Activity:

The activity was determined using the formula –

 

                                        Absorbance of Control - Absorbance of Test

Anti-Arthritic Activity = ----------------------------------------------------

                                                     Absorbance of Control

 

 

Table 4. Anti-Arthritic activity of various extracts

Extract

OD Value (660 nm)

% Activity

Control

0.56

-

Standard

0.04

92.8

Ethanol

0.35

37.5

Acetone

0.28

50

Aqueous

0.46

17.8

Chloroform

0.10

82.1

Nanoparticles

76.7

76.7

 

The Chloroform extract had highest activity nearer to standard followed by Nanoparticles, Acetone, Ethanol and Aqueous extracts.

 

Figure 4. Anti-Arthritic Activity

*A-Control, B-Standard, C-Ethanol Extract, D-Acetone Extract, E-Aqueous Extract, F-Chloroform Extract and G-Nanoparticles

 

Biosynthesis of Nanoparticles

Nanoparticles characterization was done by colorimeter. Optical density between 400 to 700 nm was checked for Nanoparticles suspension. Nanoparticles have maximum absorption between 400nm to 480nm. Thus, the production of nanoparticles was confirmed.

                          

Table 5: Absorbance of Nanoparticle suspension at various Nanometers.

Nanometer (nm)

Optical Density(OD)

430

0.69

470

0.68

490

0.60

520

0.53

550

0.48

580

0.38

610

0.32

700

0.26

 

 

Figure 5. Nanoparticles.

 

DISCUSSION:

The extracts were produced by using commercially available Spirulina capsules and were analyzed for qualitative phytochemicals. It was used for the biosynthesis of Nanoparticles. Anti-oxidant activity was found to be higher in Chloroform extract and Nanoparticles. Anti-inflammatory activity was highest in Ethanol and Acetone extracts. Anti-arthritic activity was seen maximum in Chloroform extract and Nanoparticles. The study indicates the presence of phytochemicals in different solvent extracts of commercially available Spirulina tablets. It showed Anti-oxidant, Anti- inflammatory and Anti-arthritic activity higher than the standards. Biosynthesis of Nanoparticles was done and it also showed the activities similar to the extracts.

 

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Received on 07.01.2020            Modified on 19.06.2020

Accepted on 01.09.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(12):6189-6193.

DOI: 10.52711/0974-360X.2021.01071