Determination of Bioactive Compounds and Antibacterial of Spirulina sp.
Extract
Malik Muhammad Jabbar and Abdul Wahab Raisan Ayal
Department of Biology, College of Education for Pure Science, University of Thi-Qar, Iraq Corresponding author : [email protected]
Abstract
Objective of this study was to determine of bioactive compounds and antibacterial activity of Spirulina sp extract. Pathogenic bacteria, including Streptococcus mutans, Staphyloylococcus aureus, Morganella morganii, Salmonella Sp.,Pseudomonas aerogenosa and Escherichia coli, as the active chemical compounds were extracted from this algae using solvents (cold water and methanol alcohol) with qualitative detection of secondary metabolites of the aqueous extract of this algae, which indicated the presence of phytochemicals analysis such as Alkaloids, Phenols, Flavonoids, Glycosides, Terpenoids, Taninns, Saponins and Carotenoids . The chemical compounds were determined using the gas chromatography mass spectroscopy (Gc-mass) technique of the methanolic extract of spirulina These compounds were represented Muramic acid, Isopropyl Alcohol, Diglycerol, Neophytadiene, n-Hexadecanoic acid, Heptadecanoic acid, Phytol, 9,12- Octadecadienoic acid (Z,Z)-, 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-, Linoleic acid ethyl ester and Floxuridine . The aqueous extract of this algae did not show any bioactivity against all bacterial species under study. It was noticed that the methanolic extract was more efficient in its effect on the bacterial species, as the rates of inhibition zones for this extract were (23.67, 21.33, 20.67, 18.00, 19.56 and 17.00) mm for each of S. mutans, Staph. aureus, M. morganii, Salmonella Sp. , P.
aerogenosa and E. coli, respectively.
Key words: Spirulina, Antibacterial activity, GC mass, Bioactive compounds Introduction
Infectious diseases are considered one of the main causes of high rates of disease and death among humans all over the world, especially developing countries, as the risk of disease has increased dramatically in recent years due to severe infection and disease-causing bacteria have become resistant to common drugs due to the wrong use of antibiotics and bacteria resistance. The fungi of antibiotics are among the main health problems in the world, and the efficacy has decreased and the resistance of pathogens to antibiotics has necessitated the development of new alternatives, thus algae is a rich source of bioactive compounds (Chowdhury et al., 2015). The last century witnessed a scientific development to study many of the medicinal properties of secondary ovaries present in all organisms, especially algae, and to benefit from natural products in the treatment of many diseases, as the last focus was on algae because they are available and diverse in many places (Amr, 2009). Primary or secondary algae produce potential biologically active compounds of therapeutic, industrial and agricultural importance (Mugilan and Sivakami, 2016). The main reasons for using algae extracts as antibacterial agents are their natural origin and a low chance of developing
resistance to pathogens, as they have minimal adverse side effects on humans and animals and lower environmental risks compared to their synthetic alternatives (Kolanjinathan et al., 2014).
Materials and Methods
Sample collection and preparation of extraction
The algae samples (Spirulina sp) were obtained from the American company Amazon in the form of Powder. The different types of solvent used were absolute methanol 80% and water 100%. All tests were performed at room temperature.
Gas Chromatography mass device.
Some chemical compounds were identified in the methanolic extract of spirulina algae using the Gas Chromatography mass device in the Basra Oil Company / Research and Quality Control Department / Nahran Omar Laboratories .
Antibacterial Assay
Streptococcus mietus, Staphlloccus aureas. Morganella morganii. Salmonella Sp., Pseudomonas aerogenosa and Escherichia coli were used in experiment. Mueller Hinton agar was used in antibacterial assay. spirulina algae extracts were dissolved in methanol to obtain a concentration of 50, 100 and 200 mg/mL. Antibacterial assays were conducted using the disc diffusion method as previously described by (Kumar2014). Zones of inhibition around the discs were measured in mm.
The experiment was repeated in triplicate and the mean of diameter of the inhibition zones was calculated.
Results and discussion
The results shown in Table (1) showed that the aqueous extract of Spirulina sp. on Alkaloids, Phenols, Flavonoids, Glycosides, Terpenoids, Tannins, Saponins and Carotenoids.
Table (1) Phytochemicals analysis of Spirulina sp. extract
Spirulina sp. extract Phytochemicals
+ Alkaloids
Phenols + Flavonoids + Glycosides + Terpenoids + Tannins + Saponins +
Carotenoids +
+ = Presence of constituent; - = Absence of constituent
This is in agreement with Al-Okayli (2019) study on the bluish green alga Spirulina platensis. The results shown in Table (2) showed the chemical compounds diagnosed by the technique of gas chromatography (GC - mass) for methanolian extract of spirulina, as a number of active chemical compounds were found in the methanolate extract of this algae, and among these compounds that occupied the largest area of the total total area of the diagnosed compounds represented by Diglycer The one that occupied an area of (29926205) which is a tertiary alcohol and has an anti-bacterial activity and may be attributed to it because it occupied the largest part of the total area (Wu et al., 2017), followed by the compound n-Hexadecanoic acid, which occupied an area of (18132086) which is one of the solids. Carboxylic activity has antioxidant and antibacterial activities (Saravanakumar et al., 2018). As for the compound 9,12-Octadecadienoic acid (Z, Z) - which is called Linoleic acid, which is one of the basic fatty acids that the body cannot manufacture and which occupies an area of (11757585) as studies indicated that it has anti-bacterial activity (Abd- Elnaby et al., 2016).
Table (2): Chemical compounds diagnosed with GC - mass for methanolic extract of spirulina.
Area Retention
time Molecular
weight Gram / mol Molecular
formula The name of the chemical compound
10916002 9.517
251 C9H17NO7
Muramic acid
1123882 10.565
60 C3H8O
Isopropyl Alcohol
29926205 11.983
166 C6H14O5
Diglycerol
861434 20.564
278 C20H38
Neophytadiene
18132086 21.923
256 C16H32O2
n-Hexadecanoic acid
593591 22.45
270 C17H34O2
Heptadecanoic acid
2191516 23.186
296 C20H40O
Phytol
11757585 23.483
280 C18H32O2
9,12-Octadecadienoic acid (Z,Z)-
8638884 23.535
278 C18H30O2
9,12,15-Octadecatrienoic acid, (Z,Z,Z)-
3261871 23.609
306 C20H34O2
Linoleic acid ethyl ester
349734 26.402
246 C9H11FN2O5
Floxuridine
The results showed that the aqueous extract of this algae at all concentrations did not show any biological activity against all the bacterial species under study, and that result is consistent with the findings of Al-Ghanayem, (2017). On the blue-green algae Spirulina platensis, this finding can be traced back to the fact that most of the antibacterial active ingredients identified are not soluble in water (Stirk et al., 2007). The results shown in Table (3) showed the effect of the different
concentrations of methanolic extract of spirulina algae against the bacterial species under study, as three concentrations (50, 100 and 200) mg / ml were used, and it is clear from the table that there is a clear inhibition against the bacterial species under study in general. There is a direct relationship between the concentrations of algal extract and the average area of inhibition of the bacterial species under study, as when the concentration of algae extract is high, the area of inhibition of the bacterial species is larger, and the results of the study also showed that the higher inhibitory effect is more pronounced against the bacteria positive for the stain of Kram compared to the bacteria negative for the stain of Kram stain.
Table (3) The effect of different concentrations of methanolic extract of spirulina algae against the bacterial species under study.
Mean Concentration mg/ml
Bacterial species
200 100
50
23.67 31.00±2.00
24.00±2.00 16.00±1.00
Streptococcus mietus
21.33 27.00±1.00
23.00±2.00 14.00±2.00
Staphlloccus aureas
20.67 25.67±2.08
22.33±2.08 14.00±1.00
Morganella morganii
18.00 21.67±0.57
19.00±2.00 13.33±1.15
Salmonella Sp.
19.56 25.33±1.53
20.33±1.53 13.00±1.00
Pseudomonas aerogenosa
17.00 22.33±1.15
16.67±1.53 12.00±1.00
Escherichia coli
25.50 20.89
13.72 Mean
L.S.D = 2.14 (P ≤ 0.05)
This is in agreement with what many researchers have stated. The high sensitivity of Gram positive bacteria to algae extracts may be due to differences in the bacterial wall structure. The Gram- negative bacteria wall contains more lipids than the Gram-positive bacteria, and these fats prevent the penetration of the active compounds into the bacteria and thus affect their inhibition (Salem et al., 2011). There are many factors that affect the nature of the results obtained by the researcher in the tests related to the effectiveness of algae extracts against the activity of bacterial growth, there may be differences in the results reached by researchers for the same type of algae, and this difference may be attributed to the regions, time of collection and methods Preserving the samples used in the test before extraction, the environmental factors and the stage of algae growth about the farm harvest, the type of solvent in the extraction and the extraction method.
References
1. Chowdhury, M. M. H., Kubra, K.; Hossain, M. B.; Mustafa, M. G.; Jainab, T.; Karim, M.
R., and Mehedy, M. E. (2015). Screening of Antibacterial and Anti-fungal Activity of
Freshwater and Marine Algae as a Prominent Natural Antibiotic Available in Bangladesh. International Journal of Pharmacology., 11(7): 828-833.
2. Amr , A . (2009) . Protective effect of green algae against Dimethylbenzanthracene (DMBA)-induced Breast Cancer in Rats.International J.Cancer Research , 5(1):12-24.
3. Mugilan, V. and Sivakami, R. (2016). Antimicrobial Activity of Microalgae Isolated from Fresh Water Pond, Tamil Nadu, India. Int. J. Curr. Microbiol. App. Sci., 5(6): 588-595.
4. Ubaoji, K., Nwosu, O., Agu, K., Nwozor, K., Ifedilichukwu, N., & Okaka, A. (2020). Gas Chromatographic Analysis of the Phyto-Constituents and the Assessment of the Anti- Microbial Properties of the Leave Extracts of Nigeria-Grown Gingko biloba. Journal of Scientific Research in Medical and Biological Sciences, 1(2), 45-56.
https://doi.org/10.47631/jsrmbs.v1i2.57
5. Kolanjinathan, K.; Ganesh, P. and Saranraj, P. (2014). Pharmacological importance of seaweeds: a review. World Journal of Fish and Marine Sciences., 6(1): 1-15pp.
6. Harborne , J. B. (1984). Methods of plant analysis. In Phytochemical methods (1-36pp).
Springer, Dordrecht.
7. Foroughi, A.; Pournaghi, P.; Zhaleh, M.; Zangeneh, A.;Zangeneh , M.M. and Moradi ,R.
(2016). Antibacterial Activity and Phytochemical Screening of Essential Oil of Foeniculum vulgare. International Journal of Pharmaceutical and Clinical Research, 8(11):1505-1509.
8.
Kumar V.N, Murthy P.S, Manjunatha J.R. andBettadaiah B.K, Synthesis and quorum sensing inhibitory activity of key phenolic compounds of ginger and their derivatives,Food Chem.,2014, In Press, http://dx.doi.org/10.1016/j.foodchem.2014.03.039.9. Al-Okayli , N. M .(2019) . Study the Effect of the Biological Activities of the Chemical Compounds Isolated from Algae Against the Pathogenic Fungi .M.Sc . Thesis . College of Science . Dhi Qar University. Iraq , p: 23.
10. Sajadi, F. S., Farrokhi, S., Sharifi, M., Saffari, F., and Sepehri, G. (2021). Antibacterial Effect of Two Herbal Extracts on the Level of Salivary Streptococcus mutans in Children.
Journal of Evolution of Medical and Dental Sciences, 10(5), 299-305.
11. Saravanakumar, K., Chelliah, R., Ramakrishnan, S. R., Kathiresan, K., Oh, D. H., and Wang, M. H. (2018). Antibacterial, and antioxidant potentials of non-cytotoxic extract of Trichoderma atroviride. Microbial pathogenesis, 115, 338-342.
12. Abd-Elnaby, H., Abo-Elala, G., Abdel-Raouf, U., Abd-elwahab, A., and Hamed, M. (2016).
Antibacterial and anticancer activity of marine Streptomyces parvus: optimization and application. Biotechnology & Biotechnological Equipment, 30(1), 180-191.
13. Al-Ghanayem, A. A. (2017). Antimicrobial activity of Spirulina platensis extracts against certain pathogenic bacteria and fungi. Advances in Bioresearch, 8(6).
14. Stirk, W.A.; Reinecke, D.L. and van Staden, J. (2007). Seasonal Variation in Antifungal, Antibacterial and Acetyl cholinesterase Activity in Seven South African seaweeds. Journal of Applied Phycology., 19:271-276.
15. Salem, W. M.; Galal, H. and Nasr El-deen, F. (2011). Screening for Antibacterial Activities in Some Marine Algae from the Red Sea (Hurghada, Egypt). Afr. J. Microbiol. Res., 5(15):
2160-2167.
