Antibacterial Effect of Spirulina platensis Extracts on the Viability of Bacterial SpeciesIsolated form Acne Patients in Baghdad
Ghaidaa Alrubaie , Neihaya H. Zaki,Amna Al-Hashimi, Ahmed khuyon
*Biology department, College of Science, Mustansiriyah University Corresponding author E-mail: ghaidaalrubaie @gmail.com
https://orcid.org/0000-0003-0099-583X [email protected],
[email protected]https://orcid.org/0000-0002-9619-6559
[email protected]
Abstract
Considered as Cyanobacteria are very rich source of secondary metabolites that are biologically active metabolites for anticancer activities, antifungal, antibacterial, antiviral. Current study was to determine the antibacterial Isolated from patients with acne.Efficacy of the Hexane and Methanol crude extracts of cyanobacterium Spirulina platensis was determined. Antibacterial activity (In vitro) was estimated by agar disc diffusion assay mod against twelve pathogenic bacteria in which four were Gram-positive and eight of them were Gram-negative bacteria.All bacteria tested with S. platensis extracts found varying degrees of inhibitory activity. The zone of inhibition for different extracts was divers from solvent to solvent. One g of dried S. platensis suspended in 100 μl of distill water to prepare the concentration of (25, 50,100)μl was put in the well.S. platensis was exhibited with maximum inhibition zones (16 and 13) mm in the hexane extract against Aerococcus spp. At 100 and 50 μl respectively, antibacterial activity was found high 13 and 10mm in methanol extract againstAerococcus spp., Enterococcus at 25μl under investigation. Also hexane extractshowed no effect at concentration 25 for all bacterial isolates.GC-MS analysis of hexane and methanol extracts of S. platensis were showed differentactive compounds like, Hexadecene, Heptadecane, Octadecene, 2-Bromopropionic Acid, Methyl-1-Docosene, Benzenedicarboxylic Acid, and Tetradecanol which they are very important as antibacterial agents .
Key wards:Active Compound, Antibacterial Activity, GC-MS Analysis,Spirulina platensis, Solvent Extracts
Introduction
Algae and cyanobacteria have the ability to attract solar energy and convert it into chemical energy that contributes to fixing dioxide gas in the form of an organic compound for the production of food and its metabolites. In addition, they are promising biocatalysts in the field of (green biotechnology) for enhancing production of food, drug, metabolites and green energy source such as biofuel (1).
Spirulina (Arthrospira) is referred to free-floating clusters or fine mats covering substrate with spiral characteristics of its filamentsbelong to class cyanophyta(2). Spirulina is filamentous and multicellular
Spirulina is considering safe for human consuming as obvious by its long history of food use and latest scientific findings. In recent years, Spirulina has gathered great attention from research as well as industries as a prosperous source of pharmaceuticals and nutraceutical. (3)
Spirulina platensis its extract display therapeutic properties, like the reduction blood cholesterol level, decrease toxic metals and nephrotoxicity of pharmaceuticals, ability to curb cancers, Protection from harmful radiation(4,5)
The Spirulina, has been known for its antimicrobial property and nutritionalvalue. cyanobacterial exudates many Antimicrobial compounds such as amino acids, polyphenols, glycolipids, fatty acids,terpenoids,alkaloids, antioxidant pigments,vitamins,minerals. Secondary metabolites that produced from cyanobacteria are related with toxic, antimicrobial effects, antineoplastic effects and hormonal (6, 7).In general, compounds produced naturally by cyanobacteria are considered environmentally friendly because they can be easily biodegradable (8).The aim of the study is to: Detectivethe effect of various Spirulina platensis extractsin vitro against different bacterial isolates from Acne patients and using GC- MS analysis for characterize the structure of active compound.
Material and Methods
Collection of specimens and bacterial identification:
The Acne swabs (20)obtained from Al- Habeebia out patients Hospital, andthe age of patients ranged from 15 to 25 years.collected from patients suffering from acne infections by Sterile swabs,Then transfer the samples immediately to the laboratory .Bacterial isolates were isolated from acne Specimens on Nutrient agar, Bacterial isolates were isolated from acne Specimens on Nutrient agar, Mannitol salt agar,MacConkey agar and Blood agar.According to the standard methods which recommended by, Isolates were identified by microscopic, biochemical tests and cultural (9).
Inoculums' preparation
Few colonies from overnight cultures of tested bacterial isolateswere transferred to 1ml of normal saline to prepare the bacterial suspension, and adjusted to 0.5 McFarland turbidity tube that is equal to 1.5×107 CFU/ml according to(10).
Sample collection of microalgae and culture characterization
Spirulina platensis isolated from the River Diyala in Nhrawan Baghdad city.The isolates according to (11) were identified using taxonomical approaches and morphological variation.cultivation of S. platensis was uses BG-11 medium.The sample was grown under condition (16h light \ 8h dark) at 25±2°C and 268μE/m²/s light intensity, the cultures were harvested until the end exponential phase of the growth ,then collected biomass and dried in oven for 1 h at 60°C h (12).
Preparation of various extracts
According to method (13) with some modification,take one gram of spirulina powder was extracted with 250 ml of 95% methanol solvent using a Soxhlet extraction apparatus at 60 ºC for 3-4 h hours until the solvent becomes colorless, Then, a rotary evaporator is uses at 40 ºC to get the dried crude extract.
Then the extract was weighted and stored at -20°C in hermetic bottle until antibacterial assay. Repeat the same step using a hexane solvent.
Antibacterial activity of S. platensisextracts
The agar well diffusion method was performed to investigate the antibacterial activity of S. platensis extracts.The bacterial suspension was inoculated into Nutrient agar plates using a sterile cotton swab, andthen wells (8 mm) has been done on Nutrient agar medium. Hundred micrograms of dried S.
platensisextracts suspended in 100 μl of distill water to prepare concentration of (25, 50, 100)μland put in the wells. The diameter of inhibition zone around the wells was measured in millimeter. The same procedure has been done to investigate the antimicrobial(14).
Chemical composition of S. platensisextracts
The samples from the methanol and hexane extracts of S. platensis were analyzed by GC-MS Ministry of Industry/ Industrial Research and Development Authority/ Ibn al-Bitar Research Center.At 100 ° C the temperature was placed in the initial column and at 280 ° C the temperatures of the detector and injector were set.In the column, inject 5 ml of the sample using the split method (10.1) after one minute, then 225
° C, raise the oven temperature,at a ramp rate of 7.5 °C/min (hold time 5 min).Through the NIST library and original standards their mass was compared and compounds were identified(15).
Results and discussion
Isolation of acne bacteriaSamples were collected from patients with acne about of 20 swabs.Only twelve samples (60%) were positive for bacterial isolates, including 8 Gram-negative and 4 Gram-positive (Fig 1).
Figure 1: Acne bacteria isolated on Nutrient agar
Characterization of acne bacteria
Table 1 summarizes numbers and percentage of acne bacteria. S.aureus had the highest number of isolates (4), followed by the rest of the species.
Table 1: Numbers and Percentage of acne bacterial isolates
Isolates No. Isolates Percentage%
S. aureus 4 33.33
Ps. aeruginosa 1 8.33
Ps. stutzeri 1 8.33
S.epidermides 3 25
Aerococcus spp. 1 8.33
E.coli 1 8.33
Enterococcus 1 8.33
Total 12 100
Ref (16) showed that varide species of anaerobic and aerobic bacteria were contributory in rising acne infection.The results appear that the elevated percent of the isolates belonged to the Staphylococcus epidermidis, followed by Propionibacterium acnes, Micrococcus and Staphylococcus aureus.And these bacteria play an effective role in the pathogenesis of acnecausingapparent skin infection as an important opportunistic pathogen,the optimal state for growth of Propionibacterium acnes,due to the microenvironment Appropriate of the skin,these bacteria have the ability to produce extracellular products which and are responsible for initiating the inflammatory response for example proteases, lipases and chemotactic factors (17).
Antibacterial activity of S. platensis extracts
Determined S platensis activity extracts against bacteria, were exhibited in the Table - 2.
Table 2: the antibacterial effect of some bacterial isolates treated with Spirulina platensis
Bacterial isolate
Inhibition zone (mm)
Hexane extract Methanol extract
100% 50% 25% 100% 50% 25%
Aerococcus spp. 16 13 - - 15 13
Enterococcus 13 12 - 10 - 10
P. aeroginesa 14 10 - 13 12 -
Ps. stuteri 12 -- - 10 - -
E.coli 15 13 - - - -
Staph. aureus 13 11 - 10 - -
Staph. epidermidis 13 12 - 11 - -
The zone of inhibition of extracts against bacteria was ranged between (16-12mm) at concentration 100μl and ranged between (13-10mm ) at 5μl. Also no effect at 25 μl by hexane extract, while The zone of inhibition of test against bacteria by methanol extract was ranged between (13-10mm) at 100μl , P.
aeroginesa and Aerococcus spp. were the best inhibition at 50μl . In addition no effect at 25μl except Aerococcus spp. and Enterococcus(Fig. 2).
Figure 2: Antibacterial activity of S. platensis on acne bacteria.
(A) Pseudomonas aeruginosa, (B) Aerococcus spp.
Spirulina and its components have been shown to have positive advantage for human health significance from antioxidant properties to malnutrition (18).Algae have been documented to possess and are very rich in antimicrobial compounds in biological systems.In this study, it was found that the active compounds found in the S. platensis extracts inhibit the bacteria in a varied and noticeable manner. The inhibitory effectiveness of S. platensis extract is different from that ofGram negative bacteriaand Gram positive bacteria, also showed in this studies hexane extract better than methanol extracts.
The antimicrobial activity of the extract could be due to the presence of different chemicals that may affect growth and metabolism of bacteria include phenolic, triterpenoids,flavonoids. As well as, they could have an inhibitory or activating effect on microbial growth according to their concentration and constitution such as alkaloids, free hydroxyl group, amides (19).
Has been suggest the S.platensis extracts higher sensitivity to Gram-positive bacteria were more
A B
positive bacteria consists of a one layer, while in Gram negative bacterial consists multilayered structure (21,22) lipids and fatty acids cause rupture the cell wall then peptidoclucan penetrate leading to its disintegration, The variation in the results is due to the reason for producing bioactive compounds correlating to time and location ,seasons, , cultivation medium ,type of organic solvents (23,24).
GC-MS analysis
During GC-MS analysis of hexane extract of S. platensis, itwas observed that haxane showed 19 compounds1-Hexadecene, Eicosane, 10-Trimethyl, 4-Pentadiene,Hexadecanoic Acid Methyl Ester , Methyl 9-Octadecenoate,2-Bromopropionic Acid, E-15-Heptadecenal,1-Octadecene, Methyl Ester, 13- Octadecadienoic Acid, Methyl, Pentadecyl Ester,Alpha,Phytol, Pentadecyl Ester,Alpha,-D- Glucopyranoside,2-BenzenedicarboxylicAcid, 10-Trimethyl, Heptadecane,Glucopyranoside,14- Ethylene1,Allyl N-Octyl Ether (Fig 3).
Figure 3: GC-MS Spectrum of hexane extract of S. platensis
While GC-MS analysis of hexane extract of S. platensis, it was observed 7compounds 1-Tridecene, 9- Octadecenoic acid, 2-ethylhexyl isohexyl ester, 1-Pentadecene, Heptadecene, Octane, Sulfurous acid (Fig.4).
Figure 4: GC-MS Spectrum of methanol extract of S. platensis
According to the outcome of GC-MS Spectrum datum, it was infer that the compound was n- hexadecanoic acid. The results gained This agree with (9) who tilled main fatty acids extracted from the S. platensis as oleic acid,palmitic acid, linoleic acid and stearic acid etc .(5)Identified of 15 compound for volatile components at S. platensis which constituted of the total compounds 96.45%,of the total compounds the major components it is tetradecane and heptadecane.(25) observed that GC-MS analysis, 13 bioactive component were identified of the cyanobacterium S. platensis of the partially purified, the major compound was n-Hexadecanoic acid.
Conclusions
From the currently results it can be concluded that the extract of Spirulina platensis, contains possibility bioactive compound with an efficient antibacterial activity. This compound can be used for the development of natural antibiotics against resistant bacteria.
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داذغت يف باثشلا ةح ىضره نه حلوزعولا حيريركثلا عاىنلأا حيىيح ىلع سيسنذلات انيلوريثس خاصلخرسول ايريركثلل داضولا ريثأرلا يوشاهلا حنهأ ، دوكححياهن ، يعيترلا ءاذيغ
حيرصنرسولا حعهاجلا ، مىلعلا حيلك ، جايحلا مىلع نسق E-mail: ghaidaalrubaie @gmail.com
E-mail :[email protected] E-mail:[email protected] .com
حصلاخلا حقسضًنا ءاشضخنا ةناحطنا ذعذ ايشركتىَايس)
ايشيركثهن جداضي حناعف خاثكشي ىهع اهئاىرحلأ حيجىنىياثنا خايناعفهن اًيُغ ا ًسذصي )
ٌاطشسهن جداضيو خاسوشيفهن جداضيو خايشطفهن جداضيو .
خاصهخرسًنا ىييقذ حيناحنا حساسذنا Spirulina platensis
ايشركثنا ذض
حنوضعًنا باثشنا ةح ىضشي ٍي .
سيسُذلات اُينوشيثس وىيشيركاتىَايسنا ايشيركت ٍي لىَاثيًناو ٌاسكهنا واخ خاصهخرسي حيناعف ذيذحذ ىذ .
حثناس ىهُي حيَاًث حضشًي ايشيركت ششع ىُثأ ذض ساكلأا ظسو يف ساشرَلأا حقيشط داًرعأت شثرخًنا يف ايشيركثهن داضًنا طاشُنا ىييقذ ىذ واشك حثجىي ايشركت حعتسأو واشك حغثصن .
ءاقسضنا ايشيركثنا خشهظأ S. platensis
عيًج ذض ظثثًنا طاشُنا ٍي حذوافري خاجسد
جشثرخًنا ايشيركثنا .
ةيزي ىنإ ةيزي ٍي ٍيفهرخي ٍيصهخرسًن ظيثثرنا حقطُي دفهرخا .
يف قهعًنا ففجًنا سيسُذلات ٍي ذحاو واشغ عضو ىذ
ضيكشذ شيضحرن شيطقرنا ءاي ٍي شرنوشكيي 100 25 (
50,100 , ) جشفح مكن شرنوشكيي .
ذض ظيثثذ حيناعف ىهعا مجس رأ Aerococcus
( spp و 16 )13 ضيكشرنا ذُعًهي و 100
ظيثثذ حيناعف ىهعا ٌأ ذجو اًك، ٌاسكهنا صهخرسي يف يناىرنا ىهع شرنوشكيي 50 و 13
ىهي 10
ذض لىَاثيًنا صهخرسي يف Aerococcus spp.
، Enterococcus ذُع
شرنوشكيي 25 .
ذُع ٌاسكهنا صهخرسي شيثأذ شهظي ىن اًك
ضيكشرنا حيشيركثنا خلاضعنا عيًجن 25
. ميهحذ شهظأ GC-MS
يف لىَاثيًناو ٌاسكهنا خاصهخرسًن S. platensis
حفهرخي حطشَ خاثكشي
Hexadecene مثي Heptadecane و
Octadecene و 2 و
-Bromopropionic Acid Methyl-1-Docosene و
و
Benzenedicarboxylic Acid Tetradecanol و
ىيثاشجهن جداضي مياىعك اًذج حًهي يهو .
حيحارفه خاولك :
،حناعفناذاثكشًنا ايشيركثهن داضي
، ميهحذ صاغناايفاشغىذايوشك ،
، ، سيسُذلات اُينوشيثس ةيزًنا خاصهخرسي
