Enhancement of Pharmaceutical and Bioactive Components of Scenedesmus...

[Full title: Enhancement of Pharmaceutical and Bioactive Components of Scenedesmus obliquus Grown Using Different Concentrations of KNO3]

OPEN ACCESS International Journal of Pharmacology ISSN 1811-7775 DOI: 10.3923/ijp.2018.758.765 Research Article Enhancement of Pharmaceutical and Bioactive Components of Scenedesmus obliquus Grown Using Different Concentrations of KNO 3 1 Ragaa A. Hamouda, 2 Noura El-Ahmady El-Naggar and 3 Ghada Wagih Abou-El-Seoud 1 Department of Microbial Biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat, Egypt 2 Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt 3 Department of Botany, Faculty of Science, Menoufia University, Shibin El Kom, Egypt Abstract Background and Objective: The growth rate, primary and secondary metabolites are influenced by nitrogen concentration of media This study aimed to determine the effect of different concentrations of KNO 3 as a source of nitrogen on the growth, phytochemical components, cholesterol reduction, antioxidant and antibacterial activities of the green alga , Scenedesmus obliquus ( S. obliquus ) Materials and Methods: Scenedesmus obliquus was cultured in Kuhlʼs medium and KNO 3 was added with different concentrations in the medium at different concentration (0.12, 0.75, 1.5, 2.25 and 3 g L G 1 ). The effect of different five concentrations of nitrogen on protein and carbohydrates was determined. Antioxidant activity, total phenolic content (TPC) of alga extracts, cholesterol reduction and antibacterial activity were evaluated. The data analyses were carried out using SPSS software version 16. Results: The results revealed that the best KNO 3 concentration for algal growth and carbohydrate content is 1.5 g L G 1 . Meanwhile, the high lipid content was obtained with KNO 3 starvation. Medium containing 0.75 g L G 1 of KNO 3 has the highest effect on the protein production of S. obliquus . The contents of dry alga extracted with chloroform: methanol and the extracted contents were determined by GC/MS chromatogram; the major contents were 5-Hydroxymethylfurfural followed by hexadecanoic acid (palmitic acid), cis-9-octadecenoic acid (oleic acid) and hexadecanoic acid and methyl ester, respectively. Conclusion: Potassium nitrate limitation caused the highest effect on the total phenol content (TPC), increased antioxidant capacity, cholesterol reduction activity and also antibacterial activity against Staphylococcus aureus and Escherichia coli . Key words: Potassium concentrations, green algae, lipids, primary metabolites, secondary metabolites, antibacterial Received: October 12, 2017 Accepted: January 08, 2018 Published: July 15, 2018 Citation: Ragaa A. Hamouda, Noura El-Ahmady El-Naggar and Ghada Wagih Abou-El-Seoud, 2018. Enhancement of pharmaceutical and bioactive components of Scenedesmus obliquus grown using different concentrations of KNO 3 . Int. J. Pharmacol., 14: 758-765 Corresponding Author: Ghada Wagih Abou-El-Souod, Department of Botany, Faculty of Science, Menoufia University, Shibin El Kom, Egypt Tel: 002/048/01100018458 Copyright: © 2018 Ragaa A. Hamouda et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists Data Availability: All relevant data are within the paper and its supporting information files.

Int. J. Pharmacol., 14 (6): 758-765, 2018 INTRODUCTION Microalgae are good producers of a broad variety of valuable compounds like lipids, carbohydrates, proteins, vitamins, amino acids and Beta-carotene that are used as feedstock for energy production, feed additives, food, cosmetics and medicine 1,2 . Numerous applications of microalgae such as aquaculture feeding, manufacture of active ingredients for cosmetics and food formulations due to promising sources of fine chemicals 3 . Scenedesmus sp contains high nutritional and bioactive metabolite contents So, it is used in numerous biotechnological applications 4 Hamouda et al 5 reported that S. obliquus produce high amount of carbohydrates over short periods. Also contains protein, all the essential amino acids, a good amount of minerals and lipid 6 . Antiproliferation and antioxidants agents could be used as ingredients that helps in health promotion and disease prevention had been extracted from green microalgae S. obliquus 7 . The favorable antioxidant compounds have been extracted from S. obliquus and used in food additives and active ingredients for therapeutics 3 . Green alga Scenedesmus showed antibacterial effect against Staphylococcus aureus and Bacillus subtilis 8 . Carotenoids and phenolic compounds are good potential source of natural antioxidants that had been extracted from microalgae Chlorella sp. and S. obliquus 9 . A major variety of microalgae commercialized for human nutrition such as Spirulina , Chlorella , Dunaliella salina and Aphanizomenon flos-aquae 10 The present research aimed to study the influence of KNO 3 concentrations on S. obliquus growth, tannins, flavonoids, phenolic contents, cholesterol reduction effect, antibacterial and antioxidant activities those can be used in many biotechnological applications MATERIALS AND METHODS The studying was carried out in the Microbial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City; Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt and Department of Botany, Faculty of Science, Menoufia University, Egypt (2016) and all chemicals were used from Sigma Aldrich Alga: Scenedesmus obliquus was collected from river Nile and then purified and identified according to the method of Prescott 11 . The axenic culture was maintained on Kuhl agar slants 12 at 4 E C S. obliquus cultivation using different KNO 3 (nitrogen) concentrations: Standard Kuhl's medium was prepared and used for growth of the microalgae 12 . KNO 3 was added with different concentrations (0.12, 0.75, 1.5, 2.25 and 3 g L G 1 ) to the medium Determination of algal growth parameters: Algal growth was followed by measuring optical density of growth using Unico UV-2000 spectrophotometer 13 . Cell numbers were estimated using Neubour Haemocytometer. Growth rate (µ) and doubling time (T 2 ) were calculated by the following Eq:   2 1 2 1 ln N N µ 100 T T     where, N 1 , N 2 cell number at Time T 1 and T 2 while T 2 = 0.6391/µ. Total carbohydrate content, total soluble proteins and lipids were estimated after 18 days of incubation period 14-16 Analysis of extracted alga by GC/MS analysis: Oil content of alga was extracted by chloroform: methanol (1:1) was analyzed by GC/MS analysis 17 Influence of different KNO 3 concentrations on antioxidant activity and total phenolic contents (TPC) of alga extracts: Half gram of each treatment of fine grind dried alga ( S. obliquus ) was soaked in 10 mL methanol for 48 h. The extracts were filtered and used for determination of total phenol content. Cholesterol reduction effect: The cholesterol reduction by algal extracts was determined by using enzymatic colorimetric kit 18 Antibacterial activity of algal methanol extracts that grown under different nitrogen concentrations: The antibacterial activity of methanol extracts of alga in comparison with Vancomycin as positive control was assessed against both Escherichia coli and Staphylococcus aureus using agar well diffusion method according to Perez et al 19 . Algal methanol extract was dissolved in dimethyl sulfoxide (DMSO) which also used as negative control. Exactly 200 µL from algal methanol extract (1 mg mL G 1 ) was used for each well. The inhibition zones diameters were measured in mm after 24 h of incubation 759

Int. J. Pharmacol., 14 (6): 758-765, 2018 Statistical analysis: Results of the study were expressed as ±standard error of the mean. Significant differences between the means of parameters (LSD) were estimated using Duncanʼs multiple range tests (p<0.05). All the above mentioned data analyses were carried out with SPSS software version 16 20 . RESULTS AND DISCUSSION Influence of KNO 3 concentrations on S. obliquus growth: The effect of different concentrations of KNO 3 on S. obliquus growth is shown in Fig. 1. The best growth of alga was at 1.5 g L G 1 KNO 3 after 13 days of cultivation. The lowest biomass was obtained in medium containing 0.75 g L G 1 KNO 3 . The best biomass was obtained when alga cultivated in concentration of 1.5 g L G 1 KNO 3 followed by 2.25, 3, 0.75 and 0.12 g L G 1 KNO 3 , respectively. The minimum amount of S. obliquus biomass was obtained with KNO 3 limitation in Kuhlʼs medium. Table 1 shows the specific growth rate and doubling time of alga that cultivated under various KNO 3 concentrations. The exponential growth phase of alga grown in Kuhlʼs medium was achieved at concentrations of 1.5 g L G 1 of KNO 3 at 10 days with specific growth rate of 0.427 and doubling time of 1.620 The green microalga Parietochloris incisa that grown on (+N) was possessed higher final biomass than the nitrogen-forbid (-N) cultures 21 Effect of different KNO 3 concentrations on primary metabolites of S. obliquus : Table 1 shows the impact of KNO 3 concentrations on total carbohydrates, protein and lipids contents of S. obliquus . The total carbohydrates and protein of alga are significantly reduced at low KNO 3 concentrations, lead to an increase in lipids. The carbohydrate contents were 18.3, 17.49, 17.2, 15.31 and 9.65% of dry weight at 1.5, 2.25, 3.0, 0.75 and 0.12 g L G 1 KNO 3 of medium, respectively. The protein contents of S. obliquus under different concentrations of KNO 3 were increased to 30.56 and 30.06 with 0.75 and 2.25 g L G 1 of medium, respectively compared to control (1.5 g L G 1 KNO 3 ). The lowest concentration of KNO 3 (0.12 g L G 1 ) significantly increased the lipid content to double compared with control, the lipid content was 26% with 0.12 g L G 1 KNO 3 medium while it was 12% with control (1.5 g L G 1 KNO 3 of medium). Lipids content of S. obliquus was increased when KNO 3 contents in media decrease, meanwhile total carbohydrate and protein contents were decreased. The lowest amount of protein was achieved with nitrogen limitation 22 reported that nitrogen starvation is essentially characterized by a large reduce in the protein pool Thompson 23 reported that lipid accumulation of green algae increase of up to 2-3 folds might be expected under conditions of nitrogen deprivation. Nigam et al 24 demonstrated that lipid content rises as nitrogen concentration decrease in the medium. Nitrogen is the highest Table 1: Effect of KNO 3 concentrations on specific growth rate, doubling time carbohydrate, protein and lipids contents of S. obliquus KNO 3 concentrations (g L G 1 of medium) 0.12 0.75 1.5 2.25 3.0 Specific growth (µ/day) 0.391202 0.401638 0.427667 0.422926 0.415888 Doubling time (T 2 days) 1.771718 1.725682 1.620655 1.638819 1.666553 Carbohydrate (%) 9.65 15.31 18.13 17.49 17.2 Protein (%) 14.87 30.56 25.18 30.06 26.62 Lipids (%) 26 11.5 12 13 15.33 Fig. 1(a-b): Influence of KNO 3 concentrations on S. obliquus growth measured by (a) Optical density and (b) Cell numbers Values were taken as SE 0.6 0.5 0.4 0.3 0.2 0.1 0.0 O p tic a l d en sity ( n m ) 0 2 4 6 8 10 12 14 16 18 Days 0.12 0.75 1.50 2.25 3.00 (a) 90 80 70 60 50 40 30 20 10 0 Nu m b e r o f c e ll s 1 0 H 4 0 2 4 6 8 10 12 14 16 18 Days 0.12 0.75 1.50 2.25 3.00 (b) 760

Int. J. Pharmacol., 14 (6): 758-765, 2018 O OH OH OH OH HO HO HO HO HO O O O O O O O O O O O O O O OH OH OH OH OH OH HO HO HO HO HO OH OH O O O O OH HO HO O O Table 2: GC/MS chromatogram of chloroform: methanol extraction from S. obliquus Compounds Formula and structure Area (%) 5-Hydroxymethylfurfural C 6 H 6 O 3 39.92 1-Octadecene C 18 H 36 1.28 Hexadecanoic acid methyl ester C 17 H 34 O 2 10.07 Hexadecanoic acid (palmitic acid) C 16 H 32 O 2 16.32 Octadeconic acid methyl ester C 19 H 38 O 2 10.90 Cis-9-octadecenoic acid (oleic acid) C 18 H 34 O 2 9.54 Octadecanoic acid (stearic acid) C 18 H 32 O 2 2.35 Octadecadienoic acid (linoleic acid) C 19 H 38 O 4 1.16 Acetohydrazide 2-(3-Hydroxy-2-pentyl cyclopentyl) C 24 H 38 O 4 1.27 Hexadecanoic acid 2-hydroxy-1-(hydroxymethyl) C 24 H 38 O 4 1.47 1, 2-1, 2-Benzenedicarboxylic acid, diisooctyl ester C 24 H 38 O 4 0.84 Dioctyl phthalate C 24 H 38 O 4 4.87 Fig. 2: Type of tannins (tannic acid, polyphenol compounds) critical nutrient affecting in algae lipid metabolism. The accumulation of lipids, particularly TAG, related to nitrogen limitation has been noticed in a large number of species or strains of various microalgae 25 . In various microalgae, nitrogen starvation or limitation conditions are shown to enhance the biosynthesis and accumulation of lipids or carbohydrates or both 26 GC/MS chromatogram of chloroform: methanol extraction of S. obliquus : The results obtained by GC/MS chromatogram of methanol: chloroform extraction of S. obliquus indicate the presence of twelve components of extract. These components are 5-Hydroxymethylfurfural, 1-Octadecene,hexadecanoic acid methyl ester, hexadecanoic acid (palmitic acid), octadeconic acid methyl ester, cis-9-octadecenoic acid (oleic acid), octadecanoic acid (stearic acid), octadecadienoic acid (linoleic acid), acetohydrazide 2-(3-Hydroxy-2-pentyl cyclopentyl), hexadecanoic acid 2-Hydroxy-1-(hydroxymethyl), 1,2- Benzenedicarboxylic acid, diisooctyl ester and dioctyl phthalate (Table 2). Chloroform: methanol extract of dry S. obliquus biomass showed many compounds that had many biotechnological applications. 5-Hydroxymethylfurfural (HMF) used for the production of biofuels and plastics 27 . Also it has multi-functional compounds such as intermediate for polymers, pharmaceuticals, fine chemicals and for the synthesis of other organic derivatives 28 . Li et al 29 reported that 5-HMF has new marine natural antioxidant and prospective precursor for practical applications in the food, cosmetic and pharmaceutical fields. Hexadecanoic acid methyl ester has also been observed to cause autolysis of membranous structures, inhibit phagocytic activity, stimulate significant aortic dilation and nitric oxide production of various cells, diminish levels of tumor necrosis factor-alpha (TNF) and prostaglandin E 2 (PGE 2) 30 . Palmitic acid, oleic acid and linoleic acid were the three main compounds in the high-acid oil-biodiesel 31 . 1, 2-Benzenedicarboxylic acid, di-isooctyl ester has antimicrobial and antifouling 32 Linton et al 33 stated that the octadecanoic acid (OA) methyl ester had antiviral activity against measles disease virus. Stearic acid is used in the manufacture of pharmaceutical products 34 . Also used for a cyclosporine-A drug carrier system 35 and used for vanishing the bitter taste of pharmaceutical compounds 36 Influence of different KNO 3 concentrations on tannins, flavonoids and phenolic contents of S. obliquus : Free radicals are controlled by natural products before attack cells and causes many diseases, these natural products are antioxidant. Tannins, phenolic compounds (Fig. 2) and flavonoids are accumulated by plants as secondary metabolites and considered antioxidant substances that advantage in the pharmaceutical industry 761

Int. J. Pharmacol., 14 (6): 758-765, 2018 Fig. 3(a-c): Influence of different KNO 3 concentrations on bioactive components (a) Tannins, (b) Phenolic and (c) Flavonoids contents produced by of S. obliquus Different small letter(s) on the bars indicate significant differences (p>0.05) between KNO 3 concentrations according to Duncan's multiple range test Values were taken as SE The effect of KNO 3 concentrations on tannins contents of S .obliquus was clear in Fig. 3. Potassium nitrate limitation enhances tannic acid content of S. obliquus . The highest phenolic contents of S. obliquus were recorded when grown at 0.75 g L G 1 KNO 3 of Kuhlʼs medium followed by 1.5, 3.0, 2.25 and 0.12 g L G 1 KNO 3 , respectively. Figure 3 shows that the flavonoids content of S. obliquus was highest when alga cultivated with 1.5 g L G 1 KNO 3 medium and the stress conditions had no effect on flavonoids content. Flavonoids are secondary metabolites and have the ability to act as antioxidant, antibacterial, anti-inflammatory and anti-cancer agent 37 Antioxidant activity (DPPH), cholesterol reduction and antibacterial activity of methanol extract of S. obliquus grown under different concentrations of KNO 3 : Figure 4 represents influence of KNO 3 concentrations on the antioxidant activity, cholesterol reduction and antibacterial activity of S. obliquus . The results revealed that the maximum amounts of antioxidant activity were present in alga grown under low amount of KNO 3 (0.12 and 0.75 g L G 1 ) followed by alga grown under a high amount of KNO 3 (3.0 g L G 1 ). Results clear that the high antioxidant activity present in S. obliquus cultivated under stress conditions with low and high concentrations of KNO 3 . The antioxidant activities of S. obliquus extracts around (59.8-64.3%) was recorded by Ali et al 9 . Biochemical content of Scenedesmus sp. possessed antioxidant properties and used in the neutraceutical industry 38 The results clear that under KNO 3 deprivation, Scenedesmus secondary metabolites have been affected and hence affect the cholesterol reduction. The high levels of cholesterol reduction (80.12 and 77.9%) were observed with culture grown under KNO 3 concentrations at 0.12 and 0.75 g L G 1 KNO 3 , respectively. The low concentrations of KNO 3 in Kuhlʼs medium were caused high lowering of cholesterol. The Scenedesmus acutus -enriched diet prevented an excessive deposition of cholesterol in the liver 39 The methanol extract of S. obliquus showed significant inhibition activities against Staphylococcus aureus and Escherichia coli The highest zone of inhibition was observed at 0.75 g L G 1 KNO 3 . Both concentrations of nitrogen (0.75 and 3.0 g L G 1 KNO 3 ) showed highest algal growth and also highest antibacterial activity against Staphylococcus aureus and Escherichia coli. Salem et al 8 reported that Scenedesmus sp can serve as a potential antibacterial agent against food-borne pathogen of S. aureus 350 300 250 200 150 100 50 0 T a nn ic a c id s (µ g g d ry w t ) G 1 0.12 0.75 1.5 2.25 3 KNO concentration (g L ) 3 G 1 (a) a b d d c 250 200 150 100 50 0 P h en o l c o nt en t ( µ g g d ry w t ) G 1 0.12 0.75 1.5 2.25 3 KNO concentration (g L ) 3 G 1 (b) a b ba ba a 100 90 80 70 60 50 40 30 20 10 0 F la v on oi d s c o n te nt (µ g g d ry w t ) G 1 0.12 0.75 1.5 2.25 3 KNO concentration (g L ) 3 G 1 (c) a b d c ac 762

Int. J. Pharmacol., 14 (6): 758-765, 2018 Fig. 4(a-c): (a) DPPH radical scavenging activity (%), (b) Cholesterol reduction (%) and (c) Antibacterial activity (mm) of S. obliquus grown with different KNO 3 concentrations Different small letter(s) on the bars indicate significant differences (p>0.05) between KNO 3 concentrations according to Duncan's multiple range test Values were taken as SE CONCLUSION AND FUTURE RECOMMENDATIONS KNO 3 concentrations effect on the growth, primary metabolites (lipids, proteins and carbohydrates)and secondary metabolites (phenolic, tannins and flavonoids) of algae. Antibacterial activity, antioxidant activity and lowering cholesterol activity of S. obliquus varied with KNO 3 concentration in the medium. The tannins content, antioxidant activity and also cholesterol reduction of alga are significantly increased at low KNO 3 concentrations. The best concentration of KNO 3 (0.75 g L G 1 nitrogen medium) significantly increases production of phenol and antibacterial activity. 1.5 g L G 1 of KNO 3 medium) was best for flavonoids content. The extraction contents of S. obliquus were determined by GC/MS chromatogram and the major content present is 5-Hydroxymethylfurfural (HMF) that used for the production of various high-volume plastics, foods and treatments followed by palmitic acid (C 16:0) that is display antioxidant That different concentration of nitrogen can enhance primary and secondary metabolites of Scenedesmus obliquus and significantly increases the production of bioactive compounds. Much study still needs to be done in such area SIGNIFICANCE STATEMENT This study discovers the effect of various concentrations of KNO 3 on the growth, production of primary and secondary metabolites of the micro green alga, Scenedesmus obliquus and also the effect of total phenolic content (TPC), antioxidant activities, cholesterol reduction and antibacterial activity against Staphylococcus aureus and Escherichia coli were studied that can be beneficial for studying the contents of S. obliquus extract that was determined by GC/MS analysis This study will help the researchers to uncover the critical areas of that the potassium nitrate limitation effect on the contents of S. obliquus extract, that many researchers were not able to explore. Thus a new theory on the effect of potassium nitrate on the production of primary and secondary metabolites of the green alga, Scenedesmus obliquus may be arrived REFERENCES 1 Priyadarshani, I. and B. Rath, 2012. Commercial and industrial applications of micro algae: A review. J. Algal Biomass Utln., 3: 89-100 60 50 40 30 20 10 0 D P P H r ad ic al s cave n g in g ac tivit y (% ) 0.12 0.75 1.5 2.25 3 KNO concentration (g L ) 3 G 1 (a) a bc bc bc c 84 82 80 78 76 74 72 70 68 C h o les ter o l r ed u cti o n ( % ) 0.12 0.75 1.5 2.25 3 KNO concentration (g L ) 3 G 1 (b) a ab ab ab b 30 25 20 15 10 5 0 Cl e ar z o n e ( m m ) 0.12 0.75 1.5 2.25 3 KNO concentration (g L ) 3 G 1 (c) Staphylococcus aureus Escherichia coli Vancomycin 763

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