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العنوان
Assessment of Nutritional and Bioactive Constituents from some Microalgae Dunaliella salina, Scenedesmus obliquus
and Spirulina platensis /
المؤلف
Yousef, Asmaa Samy Abd-Elsamiae.
هيئة الاعداد
باحث / Asmaa Samy Abd-Elsamiae Yousef
مشرف / Abdel Nasser Badawi Singab
مشرف / Nabaweya Ali Ibrahim
مناقش / Azza Abdelmageed Matloub
تاريخ النشر
2019.
عدد الصفحات
316 P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
العلوم الصيدلية
تاريخ الإجازة
1/1/2019
مكان الإجازة
جامعة عين شمس - كلية الصيدلة - قسم العقاقير
الفهرس
Only 14 pages are availabe for public view

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Abstract

7. General summary
Microalgae, making photosynthesis, produce half of the atmospheric oxygen. Over 15,000 novel compounds like carotenoids, fatty acids, enzymes, polymers, peptides, toxins and sterols , originating from algal biomass, have been chemically determined. Microalgae show promise for remediation of waste waters and producing value adding biproducts for large markets such as biofuels, biofertilizers, and biopolymers.
1- Dunaliella salina, belonging to chlorophyta, is a type of green microalgae especially found in sea salt fields. Known for its antioxidant activity because of its ability to create large amount of carotenoids especially β-carotene. Dunaliella salina contained many active constituents having antiviral, anticancer and antibacterial activities.
2- Scenedesmus obliquus, belonging to chlorophyta, is a type of green microalgae especially found in fresh water. Scenedesmus obliquus contained many active constituents like lipids and carotenoids having antibacterial and anticancer activities.
3- Spirulina platensis, belonging to Cyanobacteria , is a blue-green algae occuring naturally in tropical , subtropical lakes and brackish water. Arthrospira is very rich in proteins used in malnourished patients and contained many active constituents having antibacterial, anticancer and anti-inflammatory effects.
So, our study aimed to production of the three microalgae and studying the photochemical and biological properties (anticancer, antiviral, antioxidant and anticholinesterase) of their extracts as well as carotenoids, protein and polysaccharides.
This study includes the following:
Firstly the microalgae under study were subjected to mass production in the biotechnology department in the national research centre (NRC) then photochemical and biological studies were performed. Some stress conditions (increasing salinity concentration to 2.0% Sodium chloride, 45 mM organic carbon as Sodium acetate and 125 ppm iron as ferrous sulfate) were applied to microalgae (spirulina platensis and scenedesmus obliquus).
Chapter I: Preliminary phytochemical screening and proximate analysis of microalgae (Dunaliella salina, scenedesmus obliquus and Spirulina platensis)
1- Preliminary phytochemical screening
The results of phytochemical screening of air dried microalgae show the presence of (glycosides and/or carbohydrates) and (sterols and/or triterpenes) in the three microalgae.
2- Physical and chemical characters of successive extracts of microalgae (Dunaliella salina, scenedesmus obliquus and Spirulina platensis)
Results showed that (Sterols and/or triterpenes) were present in the extracts prepared with non-polar solvents which are pet.ether, chloroform and carotenoids. Carbohydrates appear only in the cold and hot water extracts. Chemical characters were same for the three microalgae.
3- Determination of certain pharmacopeial constants
The percentages of the total, water soluble and acid insoluble ash were determined. Spirulina had the highest percent of total ash (9.5%) followed by Dunaliella (7.4%) then Scenedemus (6.3%). The total ash increased in the stress forms.
Chapter II: Phytochemical and biological study of the Lipoidal Content of the three microalgae
1. Phytochemical investigation of the Lipoidal Content of the three microalgae
The petroleum ether extracts were subjected to saponification methods then the unsaponifiable matter and fatty acid methyl esters identified by GC/MS analysis. The used stress conditions decreased the lipid percent in Scenedesmus and had little increase effect in Spirulina.
1.1. GC/MS analysis of the unsaponifiable matter (USM) of the microalgae (Dunaliella salina, scenedesmus obliquus and Spirulina platensis)
1- The results of GC/MS analysis of the USM of Dunaliella salina revealed the identification of 23compounds representing (98.41%) of the total identified unsaponifiable matter. The results revealed a high perecentage of phenyl hydrocarbons (94.59%) where the major constituents were 1-butyloctyl benzene (10.94%), 1-pentylheptyl benzene (10.88%) and 1-butylheptyl benzene (9.94%). On the other hand, low percentage of hydrocarbons (0.65%) represented in 2-Methylnonadecane was observed.
2- The results of GC/MS analysis of the USM of S. obliquus revealed the identification of 44 compounds representing (97.25%) of the total unsaponifiable matter. The results revealed a high perecentage of hydrocarbons (63.19%) and the major constituent was n-heptadecane (14.43%). The identified sterols were cholesterol, campesterol and β-sitosterol representing (0.71, 0.29and 0.59%, respectively).
3- The results of GC/MS analysis of the USM of S. platenis revealed the identification of 51 compounds representing (88. 26%) of the total USM. The results revealed a high perecentage of hydrocarbons (59.82%). The major constituents were n-heptacosane (12.98%) and hexacosane (9.68%). The identified sterols were chondrillasterol, (6-Fluoro-4,6-cholestadien-3beta-ol) and (22,23-Dihydroergosterol) representing (1.19%, 1.16% and 1.11%, respectively).
1.2. GC/MS analysis of the fatty acid methyl ester (FAME) of the microalgae (Dunaliella salina, scenedesmus obliquus and Spirulina platensis)
1- The results of GC/MS analysis of FAME of D. salina revealed the identification of 3 major fatty acids. The total relative percentage of unsaturated fatty acids was (66.6%) of the total fatty acids represented in 7-Hexadecenoic acid (35.04%) and 6-Octadecenoic acid (39.92) while the total percentage of saturated fatty acids was (35.57%) represented as palmitic acid.
2- The results of GC/MS analysis of FAME of Scenedesmus revealed the identification of 3 major fatty acids where the total relative percentage of saturated fatty acids was (81.9%) represented as palmitic acid (57.76%) and stearic acid (24.14%).On the other hand, the total percentage of unsaturated fatty acids was (18.1%) represented as oleic acid (omega 9 fatty acid).
3- Results of GC/MS analysis of FAME of S. platensis revealed the identification of 24 fatty acids. The total relative percentage of unsaturated fatty acids was (80.13%) where 9,12-octadecadienoic acid (24.65%), 7,10,13-hexadecatrienoic acid (19.29%) and4,7,10,13-hexadecatetraenoic acid (10.07%) represented the major ones .On the other hand, the total percentage of saturated fatty acids was (19.87%) where stearic acid (8.3%) and myristic acid (3.5%)represented the major. Omega3 fattyacids [5, 8, 11, 14, 17-Eicosapentaenoic acid (2.94%), 7,10,13-hexadecatrienoic acid (19.29%) and stearidonic acid (3.34%)] were present representing 25.57% of the total fatty acids in S. platensis which agree with Sayeda et al.,2015 who reported the presence of omega 3 fatty acids in S. platensis. Also, omega 6 fatty acids [6, 9, 12-octadecatrienoic acid (γ-linolenic acid) (2.49%), arachidonic acid (2.4%) and 8, 11, 14-Eicosatrienoic acid (dihomo γ-linolenic acid) (0.4%)] were present representing 5.29%, so S. platensis is very rich in unsaturated fatty acids, omega3 and omega6 fatty acids.
2. In-vitro bioactivity studies of the lipidoidal matter of the three microalgae
2.1. Cytotoxic activity of lipidoidal matter
With respect to microalgal pet.ether extracts, S. obliquus showed highly significant (100% at 100 µg/ml dose) anticancer activity against HePG2, MCF7 and HCT116 cell lines while D. salina showed highly significant (100% at 100 µg/ml dose) anticancer activity against HePG2 and MCF7 cell lines in comparison with Doxorubicin as a positive control. On the other hand, S. platensis showed weak cytotoxic activity. Furthermore S. obliquus and D. salina showed obvious highly significant cytotoxic activity against MCF7 which was higher than standard doxorubicin at 25 and 50 µg/ml doses. Also, S. obliquus showed promising highly significant cytotoxic activity against HCT116 which was higher than standard doxorubicin at all doses even 6.25µg/ml very low dose.
Concerning microalgal chloroform extracts, D. salina showed promising highly significant cytotoxic activity against MCF7 which was higher than standard doxorubicin at all doses even 6.25µg/ml very low dose while S. platensis showed highly significant (99.6%) activity against MCF7 at 100 µg/ml dose and moderate activity (56.1% at 100 µg/ml dose) against HePG2
2.2. Antiviral study:
The pet.ether and chloroform extracts of the three microalgae had weak activity against the tested HCV genotype 4, rotavirus Wa strain, Herpes simplex virus (HSV1) and Coxsackievirus B4 except the pet.ether extract of S. platensis had good antiviral activities (50% and 40%
2.3. Antioxidant bioassay (DPPH free radical scavenging activity):
The antioxidant activities of all the microalgal pet.ether and chloroform extracts were dose dependant. At 100 μg/ml dose, all the studied microalgal pet.ether and chloroform extracts showed highly significant antioxidant activity (higher than the standard ascorbic acid) except D. Salina chloroform extract showed moderate antioxidant activity. The most promising antioxidant activity was S. obliquus followed by S. platensis then D. Salina.
2.4. Cholinesterase inhibition activity
The anticholinesterase activity of D. salina pet.ether and chloroform extracts were dose dependant while it was inversely proportioned with the dose in Spirulina. Furthermore, pet.ether and chloroform extracts of D. salina and also chloroform extract of S. obliquus exhibit marked cholinesterase inhibition activity at all doses in comparison with galanthamine standard and they could be used in the treatment of Alzhiemer,s disease.
Chapter III: Phytochemical and biological study of the carotenoids Content of the three microalgae
1. Phytochemical investigation of the carotenoid content of the three microalgae
1.1. Quantitative estimation of carotenoids in the three microalgae:
Dunaliella salina had the highest carotenoids content followed by Scenedesmus obliquus then Spirulina platensis. Also, the carotenoids/ chlorophyll ratio of Dunaliella and Scenedesmus were nearly equal and lower than Spirulina. Carotenoids content increased in the stress forms of both Spirulina and Scenedesmus more than twice times.
1.2. ESI+-MS results:
Six compounds (β-carotene, lutein, echinenone, diatoxanthin, violaxanthin and antheraxanthin) were identified by ESI+ mass spectra and MS fragmentation and were present in Scenedesmus obliquus and Spirulina platensis. Four of them (β-carotene, echinenone, diatoxanthin and antheraxanthin) were present in Dunaliella salina. β-carotene, diatoxanthin and echinenone were predominant in Dunaliella salina while echinenone, diatoxanthin and antheraxanthin were predominant in Scenedesmus obliquus. Furthermore, lutein, beta-carotene and diatoxanthin were predominant in spirulina platensis.
2. In-vitro bioactivity studies of the carotenoid content of the three microalgae
2.1. In vitro cytotoxic activity study of the carotenoids
In comparison with Doxorubicin as a positive control, Spirulina and Dunaliella showed highly significant inhibition against liver cancer cell line (HepG2) (100% at 100 µg/ml dose), significant inhibition against breast cancer (70.5 % and 69.7%, respectively at 100 µg/ml dose) and also significant inhibition against colon cancer (91% and 80.7%, respectively at 100 µg/ml dose) while Scenedesmus showed good inhibition against liver cancer (60.2% at 100 µg/ml dose).
2.2. Biological In vitro antiviral study:
The extracted carotenoids from the three microalgae had weak activity against the tested HCV genotype 4, rotavirus Wa strain, Herpes simplex virus (HSV1) and Coxsackievirus B4.
2.3. Investigation of in-vitro antioxidant bioassay (DPPH free radical scavenging activity):
The antioxidant activities of all the microalgal carotenoid extracts were dose dependant. At 100 μg/ml, S. platensis carotenoids showed the highest significant antioxidant activity followed by D. salina then S. obliquus.
2.4. Investigation of in-vitro Cholinesterase inhibition activity
The anticholinesterase activities of Dunaliella and Scenedesmus were dose dependant while it was inversely proportioned with the dose in Spirulina. Also, Dunaliella exhibit the highest significant cholinesterase inhibition activity in comparison with galanthamine standard and it could be used in the treatment of Alzhiemer,s disease.
Chapter IV: Phytochemical and biological study of the isolated proteins from the three microalgae
1. Phytochemical investigation of the isolated proteins
1.1. Estimation of total protein content of the three microalgae (Micro-Kjeldahl method):
Estimation of total protein revealed that the highest protein content exist in Spirulina followed by Dunaliella then Scenedesmus. Also, the protein content decreased in the stress forms of both Spirulina and Scenedesmus.
1.2. Polyacrylamide gel Electrophoresis:
It was noticed that there was one common band identified at Rf 0.95 and Mwt 5.27 KDa. Furthermore, three protein bands were noticed in Dunaliella salina (Mwts 171.77, 20.48 and 5.75 KDa) while in Scenedesmus obliquus vegetative form, it was found that 9 denatured bands (Mwts 214.68, 137.74, 71.54, 42.37, 30.18, 20.40, 17.00, 12.44 and 5.46 KDa) and in Scenedesmus obliquus stress form, the protein pattern was represented electrophoretically by 4 bands (Mwts 176.42, 112.29, 17.27 and 5.75 KDa). The electrophoretic protein pattern in Spirulina platensis was expressed by 10 denatured protein bands (Mwts 174.08, 115.40, 71.80, 48.58, 27.66, 19.92, 15.90, 13.91, 5.27 and 4.27 KDa).
1.3. Amino acids analysis of the separated proteins
Fifteen amino acids were observed in Spirulina and Scenedesmus while 17 amino acids in Dunaliella. The most predominant amino acid in all studied microalgae was glutamic acid having percentages of 4.96%, 3.3% and 7.58% in Dunaliella, Scenedesmus and Spirulina, respectively. With respect to Spirulina, the more predominant amino acids were alanine & isoleusine while the more predominant amino acids in Scenedesmus were alanine & leucine. Also, the more predominant amino acids in Dunaliella were aspartic acid & leucine.
2. In-vitro bioactivity studies of the separated proteins
2.1. Biological In vitro cytotoxic activity of the separated proteins
Non significant cytotoxic activity was observed in the three microalgae on HePG2, MCF7 and HCT116 cell lines in comparison with standard doxorubicin
2.2. Antiviral activities
Non significant antiviral activity (20%) at non-toxic doses (0.7, 0.8 and 0.9 mg/ml) for (D. salina, S. obliquus and S. platensis, respectively) was observed against all tested viruses.
2.3. DPPH free radical scavenging activity
The antioxidant activity of the isolated proteins from the three microalgae was dependant dose. With respect to D. salina, the isolated protein exhibit highly significant antioxidant activity (300.16% and 308.78% at 10 μg/ml and 100 μg/ml doses, respectively) which is higher than standard ascorbic acid (80.00% and 94.82% at 10 μg/ml and 100 μg/ml doses, respectively). Additionally, S. obliquus and S. platensis had significant antioxidant activity (124.49% and 140.81%, respectively) at 100 μg/ml dose in comparison with standard ascorbic acid.
2.4. Acetylcholinesterase inhibition activity
At low dose (1 μg/ml), all the three microalgae exhibit marked anticholinesterase activity while at the highest dose (100 μg/ml), D. salina and S. obliquus exhibit marked anticholinesterase activity in comparison with standard galanthamine.
Chapter V: Phytochemical and biological study of the isolated polysaccharides from the three microalgae
1. Phytochemical investigation of the polysaccharides
Scenedesmus obliquus cold and hot extraction methods (CEM and HEM) polysaccharides constituted highest content of total polysaccharides than other tested microalgae estimated by phenol-sulphuric acid method. The isolated polysaccharides characterized as heterogeneous polysaccharides bounded protein using IR spectra, GLC and microelemental analysis. The polysaccharides constituted 47- 66 % of sugar and 14.88 - 41.06 % of protein contents whereas galactose, mannose, glucose and rhamnose were represented as predominant sugars. The polysaccharide content increased in the stress form of Scenedesmus while decreased in the stress form of Spirulina.
2. In-vitro bioactivity studies of the isolated polysaccharides
2.1. In vitro cytotoxic activity study of isolated polysaccharides
The polysaccharide of D. salina HEM with an IC50 value 64.2 μg/ml exhibited significantly a potent cytotoxic effect on HCT116 human cell line than other tested isolated polysaccharides while S. obliquus CEM inhibited 50.4% of HepG2 cell line at 100 µg/ml dose.
2.2. Antiviral activities
The non-toxic dose of the isolated polysaccharides were ranged from 1.1 to 1.8 mg/ml. The polysaccharides (CEM and HEM) of S. platensis have promising antiviral activity against HCV genotype 4a replicon, coxsackievirus B4, rotavirus and herpes simplex virus 1 which reduced replication of tested virus about 50 – 87.6% at non toxic doses 1.8 and 1.5 mg/ml, respectively.
2.3. DPPH free radical scavenging activity
D. salina HEM and S. obliquus CEM showed the significantly highest percent of inhibition (308.16 and 308.69%, respectively) at 100 μg /ml, followed by S. platensis HEM and S. obliquus HEM polysaccharides as they recorded (206.12 and 200.20%, respectively) inhibition at 100 μg/ml dose.
2.4. Acetylcholinesterase inhibition activity
The cholinesterase inhibition activity was dose dependant relationship of both D. salina and S. obliquus while S. platensis showed inverse relation. S. platensis HEM showed significant inhibition activity compared to S. platensis CEM. On the other hand, S.obliquus CEM and HEM polysaccharides exhibited the significant highest cholinesterase inhibition activity followed by D. salina CEM and HEM polysaccharides compared to galanthamine standard.