International Research Journal of Vol. 2(1), 35-40, January (201 3 International Science Congress Association The Effect of Di- Ethylstilbestriol (DES), Oxytocin and Testosterone on the Content of Carbohydrate, Chlorophyll and Protein in Green Algae Meghwal Rajani , Shekhawat 1 Department of Microbiology, M.D.S. University, Ajmer, Rajasthan, INDIA 2 Department of Zoology, University College of Science, Mohanlal Sukhadia University, Udaipur, Rajasthan, INDIA Received 26th October Abstract The main aim of this study was to check the influence of sex steroids: Testosterone, Di Oxytocin on changes of proteins, chlorophyll and carbohydrate contents of in BG-11 medium for 15- days period, in 4 different concentrations (1 µg/ml, 2.5 µg/ml, steroids. It has been ascertained that the best possible stimulation of protein was caused by Di (DES) in the range from 148.55%, testosterone 64.59% and the effect of oxytocin algae was negative (decreased up to 13.33% )in comparison to a 100% control. Carbohydrate content was best encouraged by testosterone in range from 42.62% in comparison to oxytocin (15.94%) whi carbohydrate content of green algae was negative (decreased up to 26.70%). Chlorophyll content was stimulated most by testosterone in range from 94.09%, followed by oxytocin 56.69% and DES 34.39% i control during the whole period of 15 days of green algae cultivation. The effect on Protein, Carbohydrate a Chlorophyll by Testosterone (p‹0.05, F (p‹0.05, F3.88= 491.5, r =0.9879) is statistically significant. Keywords: Carbohydrate, c hlorophyll, Di Introduction Algae encompasses many different groups of living organisms that capture light energy through photosynthesis and convert inorganic substances into simple sugar. algae have plastids, the bodies with chlorophyll that carry out photosynthesis, which are present in different combinations of chlorophyll molecules in varied algal strains. Some have only Chlorophyll a, some with a and b, while other strains possess a and c. Algal biomass contains t hree main components: proteins, carbohydrates, and natural oil. While the percentages vary with the type of algae, there are algae types that are comprised of up to 40% of their overall mass by fatty acids. It is this fatty can be extracted and converted into biodiesel a terpenoid lipid, characterized by its sterane core and additional functional group. The core is a carbon structure of four fused rings: three cycloh exane rings and one cyclopentane ring. Exogenous usage of steroidal hormones in specific concentrations would enable selective growth control of plants and produced biomass could be utilized as a fertilizer in agriculture. A dditional growth stimulation through exogenous and selectively used steroidal hormones seems to be advantageous in the usage of highly trophic biomass as an additive to animal feed. Typical male steroid hormones (androgens) testosterone and its derivatives, stimulated only the Journal of Biological Sciences __________________________ _________ 3 ) International Science Congress Association Ethylstilbestriol (DES), Oxytocin and Testosterone on the Content of Carbohydrate, Chlorophyll and Protein in Green Algae , Shekhawat Deependra Singh1*, Soni Naveen Kumar and Bhatnagar Chhaya Department of Microbiology, M.D.S. University, Ajmer, Rajasthan, INDIA Department of Zoology, University College of Science, Mohanlal Sukhadia University, Udaipur, Rajasthan, INDIA Available online at: www.isca.in October 2012, revised 6th November 2012, accepted 25th November 2012 The main aim of this study was to check the influence of sex steroids: Testosterone, Di - Ethylstilbestriol (DES) and on changes of proteins, chlorophyll and carbohydrate contents of green algae. Green algae were days period, in 4 different concentrations (1 µg/ml, 2.5 µg/ml, 5 µg/ml and 10 µg/ml) of steroids. It has been ascertained that the best possible stimulation of protein was caused by Di (DES) in the range from 148.55%, testosterone 64.59% and the effect of oxytocin on protein content of green algae was negative (decreased up to 13.33% )in comparison to a 100% control. Carbohydrate content was best encouraged by testosterone in range from 42.62% in comparison to oxytocin (15.94%) whi carbohydrate content of green algae was negative (decreased up to 26.70%). Chlorophyll content was stimulated most by testosterone in range from 94.09%, followed by oxytocin 56.69% and DES 34.39% i control during the whole period of 15 days of green algae cultivation. The effect on Protein, Carbohydrate a Chlorophyll by Testosterone (p‹0.05, F 3.88= 61.66, r=0.9113), DES (p‹0.05, F3.88 = 41.04, r =0.9879) is statistically significant. hlorophyll, Di -Ethylstilbestriol (DES), green algae, oxytocin, protein, t Algae encompasses many different groups of living organisms that capture light energy through photosynthesis and convert inorganic substances into simple sugar. All algae have plastids, the bodies with chlorophyll that carry out photosynthesis, which are present in different combinations of chlorophyll molecules in varied algal strains. Some have only Chlorophyll a, some with a and b, while other strains possess a and c. Algal biomass contains hree main components: proteins, carbohydrates, and natural oil. While the percentages vary with the type of algae, there are algae types that are comprised of up to 40% of their overall mass by fatty acids. It is this fatty acid that can be extracted and converted into biodiesel . A steroid is a terpenoid lipid, characterized by its sterane core and additional functional group. The core is a carbon structure exane rings and one cyclopentane ring. Exogenous usage of steroidal hormones in specific concentrations would enable selective growth control of plants and produced biomass could be utilized dditional growth stimulation through exogenous and selectively used steroidal hormones seems to be advantageous in the usage of highly trophic Typical male steroid hormones (androgens) , mainly testosterone and its derivatives, stimulated only the germination of seeds and the development of plants embryos in the beginning of the heterotrophic period. Androgens promoted the formation of stamens and spermato zoid production in the process of flower development. While the vegetative development of small plants begins with its photosynthesis process, it is inhibited under the influence of androgens 3 Oxytocin is best acknowledged for its roles in reproduction , particularly during and after Oxytocin is a peptide of nine amino acids Its systematic name is cysteine glutamine-asparagine-cysteine- proline and its chemical formula is (C Mass- 1007.19 g/mol). Testosterone from the androgen group. It is the principal hormone and an anabolic steroid is (C1928 , Molecular Mass Diethylstilbestrol (DES ) is a synthetic non steroidal estrogen that was first synthesized in 1938 Molecular Mass- 268. 35 g/mol). Structure of DES _________ ISSN 2278-3202 Int. Res. J. Biological Sci. 35 Ethylstilbestriol (DES), Oxytocin and Testosterone on the Content of Carbohydrate, Chlorophyll and Protein in Green Algae and Bhatnagar Chhaya 2 Department of Zoology, University College of Science, Mohanlal Sukhadia University, Udaipur, Rajasthan, INDIA 2012  Ethylstilbestriol (DES) and Green algae were grown 5 µg/ml and 10 µg/ml) of steroids. It has been ascertained that the best possible stimulation of protein was caused by Di -Ethylstilbestriol on protein content of green algae was negative (decreased up to 13.33% )in comparison to a 100% control. Carbohydrate content was best encouraged by testosterone in range from 42.62% in comparison to oxytocin (15.94%) whi le the effect of DES on carbohydrate content of green algae was negative (decreased up to 26.70%). Chlorophyll content was stimulated most by testosterone in range from 94.09%, followed by oxytocin 56.69% and DES 34.39% i n contrast to 100% control during the whole period of 15 days of green algae cultivation. The effect on Protein, Carbohydrate a nd = 41.04, r =0.8724) and oxytocin t estosterone. germination of seeds and the development of plants embryos in the beginning of the heterotrophic period. Androgens promoted the formation of stamens and zoid production in the process of flower development. While the vegetative development of small plants begins with its photosynthesis process, it is inhibited 3 -15. is best acknowledged for its roles in sexual , particularly during and after childbirth. amino acids (a nonapeptide). Its systematic name is cysteine -tyrosine-isoleucine- proline -leucine-glycine-amine (C 43661212, Molecular Testosterone is a steroid hormone group. It is the principal male sex anabolic steroid , and its chemical formula , Molecular Mass - 288.4 g/mol). ) is a synthetic non steroidal that was first synthesized in 1938 (C18202, 35 g/mol). Structure of DES International Research Journal of Biological Sciences Vol. 2(1), 35-40, January (2013) International Science Congress Association Structure of Testosterone Structure of oxytocin Material and Methods The work was carried out on homogenous culture of green algae. The algae were cultivated for 15 days in 20 ml test tubes with cotton pluck stopper, containing 10 ml BG medium under controlled condition (Temp. 23ºc ±1ºc, photoperiod- 8 hrs. dark + 16 hrs. light). In the BG medium test tube, possess the different concentration of different steroids. The pH of BG- 11 medium was set to 7.0. In the experiment the steroids compound within BG applied in concentration ranges 1 µg/ml to 10 µg/ml on the 15th day of culture, when algae had reached the maximal l evel of development and metabolism 15 working days, these algae were utilized the nutrients and steroids which present in the medium for growth. We were used the 4 different concentrations of 3 types stero ids in this experiment, according to table 1 we have made the different concentrations of steroids by mix specific amount of steroid stock solution with BG inoculum. The total content of biological parameters Carbohydrate, Protein and Chlorophyll were determined by specific methods such as: Porra method for Chlorophyll, Lowry method for Protein and Thio- Urea Anthrone method for c arbohydrate content determination. A was used for all measurements. International Research Journal of Biological Sciences ________________________________________________ International Science Congress Association Structure of Testosterone The work was carried out on homogenous culture of green algae. The algae were cultivated for 15 days in 20 ml test with cotton pluck stopper, containing 10 ml BG -11 medium under controlled condition (Temp. 23ºc ±1ºc, 8 hrs. dark + 16 hrs. light). In the BG -11 medium test tube, possess the different concentration of 11 medium was set to 7.0. In the experiment the steroids compound within BG -11 were applied in concentration ranges 1 µg/ml to 10 µg/ml on day of culture, when algae had reached the evel of development and metabolism 6,16,17. During 15 working days, these algae were utilized the nutrients and steroids which present in the medium for growth. We were used the 4 different concentrations of 3 types ids in this experiment, according to table 1 we have made the different concentrations of steroids by mix -up the specific amount of steroid stock solution with BG -11 and inoculum. The total content of biological parameters Carbohydrate, Protein and Chlorophyll were determined by specific methods such as: Porra method for Chlorophyll, Urea Anthrone method arbohydrate content determination. A spectrophotometer Stock Solution Preparation: We have required the maximum 10 µg/ml concentration of different steroids, thus we mixed the 1 ml amount of steroid into 20 ml, 0.1% Tween- 80. After that we take the 1 ml of above mixer and mixed in to 99 ml steril e BG this sample has become 10 µg/ml stock solution and this was further used for preparation of another different concentration according to table 1. Table- 1 Different Concentrations of stock Concentration Steroid (stock solution) Control 0 ml 1 µg/ml 0.10 ml 2.5 µg/ml 0.25 ml 5 µg/ml 0.5 ml 10 µg/ml 1 ml Statistical Analysis: Calculations of cluster analysis for effect of three steroids on protein, chlorophyll and carbohydrate content of green algae at 15th day (% similarity) were performed using Other statistical analysis was performed using Prism 5.0 software. We performed one- way analysis of variance on the effect of t estosterone, DES, carbohydrate, protein and c hlorophyll content. Results and DiscussionApplied steroid show the greatest biological activity in green algae between 7th and 15 th Testosterone and Oxytocin were more active biochemically than DES. In this experiment, four different concentrations (1.0 µg/ml, 2.5 µg/ml, 5 µg/ml and 10 µg/ml) of steroids were used for cultivation of algae and after 15 days cultivation measured the algal growth according to chlorophyll, protein and c arbohydrates content in algae in comparison to control table 2. 1.0 µg/ml concentration of testosterone was negatively (decreased) effected only protein content of green algae while other concentration (2.5 µg/ml, 5 µg/ml and 10 µg/ml) were positively increased the protein, and chlorophyll content of green algae in comparison to control table 2. 1.0 µg/ml concentration of DES was negatively correlated (decreased) the chlorophyll content of green algae whiles other concentration (2.5 µg/ml, 5 µg/ml and 10 µg/ml) were positively increased the chlorophyll content and carbohydrate content concentration significantly decreased at all concentrations (1.0 µg/ml, 2.5 µg/ml, 5 µg/ml and 10 µg/ml), and protein c ontent concentration significantly increased at all concentrations (1.0 µg/ml, 2.5 µg/ml, 5 µg/ml and 10 µg/ml) table 2. ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 36 We have required the maximum 10 µg/ml concentration of different steroids, thus we mixed the 1 ml amount of steroid into 20 ml, 0.1% 80. After that we take the 1 ml of above mixer e BG -11 medium. Right now this sample has become 10 µg/ml stock solution and this was further used for preparation of another different concentration according to table 1. 1 Different Concentrations of stock Steroid (stock BG-11 Inoculum 9.0 ml 1 ml 8.9 ml 1 ml 8.75 ml 1 ml 8.50 ml 1 ml 8.00 ml 1 ml Calculations of cluster analysis for effect of three steroids on protein, chlorophyll and carbohydrate content of green algae at 15th day (% similarity) were performed using biodiversity pro software. was performed using Prism 5.0 way analysis of variance on estosterone, DES, and oxytocin on hlorophyll content. greatest biological activity in th day of cultivation, where Testosterone and Oxytocin were more active biochemically than DES. In this experiment, four different concentrations µg/ml, 5 µg/ml and 10 µg/ml) of steroids were used for cultivation of algae and after 15 days cultivation measured the algal growth according to arbohydrates content in algae in 1.0 µg/ml concentration of testosterone was negatively (decreased) effected only protein content of green algae while other concentration (2.5 µg/ml, 5 µg/ml and 10 µg/ml) were positively increased the protein, carbohydrate and chlorophyll content of green algae in comparison to 1.0 µg/ml concentration of DES was negatively correlated (decreased) the chlorophyll content of green algae whiles µg/ml, 5 µg/ml and 10 µg/ml) were positively increased the chlorophyll content and carbohydrate content concentration significantly decreased at all concentrations (1.0 µg/ml, 2.5 µg/ml, 5 µg/ml and 10 ontent concentration significantly increased at all concentrations (1.0 µg/ml, 2.5 µg/ml, 5 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 35-40, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 37 Protein content of green algae was significantly decreased at all concentration of oxytocin (1.0 µg/ml, 2.5 µg/ml, 5 µg/ml and 10 µg/ml) and 1.0 µg/ml concentration of oxytocin was negatively affected the carbohydrate content in comparison to control table 2. DES was maximum increased the protein content in green algae in comparison to testosterone and oxytocin, Testosterone was more increased the chlorophyll and carbohydrate content than DES and oxytocin. The effect on protein, carbohydrate and chlorophyll by testosterone (p‹0.05, F3.88= 61.66, r=0.9113), DES (p‹0.05, F3.88= 41.04, =0.8724) and oxytocin (p‹0.05, F3.88= 491.5, r=0.9879) is statistically significant. According to Bray- Curtis cluster analysis (single link), oxytocin and testosterone were around 82.58% similar affected (either positive or negative) the protein content of green algae, DES and other two steroid were only 73.91% similar affected figure 1. Oxytocin and testosterone were around 91.80% similarly affected the chlorophyll content of green algae, although DES and other two steroid were only 89.93% similarly affected figure 2. Oxytocin and testosterone were around 91.09% similarly affected the carbohydrate content of green algae while DES and other two steroid were only 88.70% similarly affected figure 3. It has been ascertained that the best possible stimulation of protein was caused by Di-Ethylstilbestriol (DES) in the range from 148.55%, testosterone 64.59% and the effect of oxytocin on protein content of green algae was negative (decreased up to 13.33% )in comparison to a 100% control. Carbohydrate content was encouraged the strongest by testosterone in range from 42.62%, oxytocin 15.94% and effect of DES on carbohydrate content of green algae was negative (decreased up to 26.70%). Chlorophyll content was stimulated mostly by testosterone in range from 94.09%, oxytocin 56.69% and DES 34.39% in contrast to 100% control during the whole period of 15 days of green algae cultivation. Previous studies show that steroid harmones used in research were a derivative of pregnane and their work mechanics affected mainely the nucleus cell and ribosomes, chiefly the process of transcription and translation but above results in table 1 shows that only DES and Testosterone are positively influenced protein content by affecting the process of transcription and translation, but oxytocin hormone inhibit the protein synthesis in comparison to control. In this case, the research was focused on changes of total content of proteins, carbohydrate, and chlorophyll From among used hormones, testosterone sex steroids showed stronger stimulation of carbohydrate and chlorophyll content in green algae cultivation, in comparison to analyzed DES and oxytocin. However, the content of protein was the most intensely stimulated by DES, in comparison to testosterone and oxytocin. The obtained results are some similar to data obtained from a few publications relating to research of exogenously applied sex steroids and corticosteroids on growth and biochemical changes in vascular plants: seeds, peas, beans and lentils5,7,15,16,17,18Table-2 The influence of different concentrations of three steroids on analyzed metabolites in green algae in 7th and 15th day of cultivation Names of steroid hormones Applied concentration (µg/ml) Protein concentration (mg/ml) Chlorophyll concentration (µg/ml) Carbohydrate concentration (mg/ml) 7 th day 15 th day th day 15 th day 7 th day 15 th day Control 35.25 66.66 .447 .878 35.41 69.35 Testosterone 1 µg/ml 33.04 64.12 .609 1.213 39.21 77.31 2.5 µg/ml 38.01 73.03 .681 1.352 41.05 81.85 5 µg/ml 49.58 97.57 .759 1.504 45.27 88.68 10 µg/ml 56.24 109.57 .851 1.698 49.97 98.91 DES 1 µg/ml 65.35 127.00 .399 .795 32.89 64.35 2.5 µg/ml 72.51 140.33 .473 .938 31.35 61.47 5 µg/ml 78.64 153.33 .541 1.09 28.17 55.33 10 µg/ml 84.08 165.66 .587 1.18 25.79 50.84 Oxytocin 1 µg/ml 34.01 65.94 .540 1.072 33.10 65.35 2.5 µg/ml 31.25 62.71 .598 1.193 35.98 71.47 5 µg/ml 30.58 60.13 .642 1.26 37.01 73.24 10 µg/ml 28.49 57.80 .692 1.365 40.87 80.41 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 35-40, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 38 Figure-1 Effect of three steroids on protein content of green algae at 15th day (% similarity) Figure-2 Effect of three steroids on Carbohydrates content of green algae at 15th day (% similarity) Figure-3 Effect of three steroids on Chlorophyll content of green algae at 15th day (% similarity) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 35-40, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 39 Figure-4 Comparison of three steroids effect on protein content of green algae 0.001.002.505.0010.00 0 1 Testosterone DES oxytocin concentration of steroids(µg/ml)Chlorophyllconcentration(µg/ml)Figure-4 Comparison of three steroids effect on chlorophyll content of green algae 0.001.002.505.0010.00 0 25 50 75 100Testosterone DES oxytocin concentration (µg/ml)Carbohydrateconcentration (mg/ml)Figure-5 Comparison of three steroids effect on carbohydrate content of green algae 0.01.02.55.010.0 0 100 200Testosterone oxytosine DES Concentration of steriod (µg/ml)Concentration ofprotein (mg/ml) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(1), 35-40, January (2013) Int. Res. J. Biological Sci. International Science Congress Association 40 ConclusionFrom the above results it can be concluded that the protein content is more increase by DES. Chlorophyll and carbohydrate content are more increase by testosterone steroid. So testosterone steroid is much better for algal growth compare than DES and Oxytocin. AcknowledgementsWe are greatly thankful to respected Noble teacher Dr. Ashish Bhatnagar (HOD, Dept. of Microbiology, MDSU, Ajmer) for important suggestion and guidance for this experiment, and also thankful to Dr. Prabhu Dan Charan, Dr. Hemraj Chippa, Mr. Pramod Jangir, Miss. Charul, Miss Sapna for supporting and helping to us in this project and we are also endless thanks to Dr. Vijay Kumar Koli (Assistant Professor, Department of Zoology, MLSU, Udaipur) and Mr. Devendra Kumar Goyal for supporting in making this manuscript. 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