This document discusses the chemical investigation of the freshwater bryozoan Pectinatella magnifica. P. magnifica forms large colonies in freshwater environments and little is known about its chemistry. The study aimed to extract, separate, and identify lipophilic compounds from a P. magnifica specimen. Various extraction and fractionation techniques were used to obtain fractions that were analyzed by gas chromatography-mass spectrometry. Some fractions contained fatty acid derivatives and cholesterol. The extracts showed no antibacterial activity against Staphylococcus aureus or antifungal activity against Candida albicans.
This document reports on the isolation and characterization of a denitrifying Acinetobacter baumannii strain H1 that was isolated from shrimp farming ponds. Strain H1 was able to use nitrite as its sole nitrogen source and showed high nitrite removal rates. It was identified as A. baumannii through biochemical testing and phylogenetic analysis of its 16S rRNA gene. PCR analysis detected key denitrification genes in strain H1. In vitro experiments demonstrated that strain H1 was able to remove nitrite effectively over a wide range of pH, temperatures, and cell densities. Tests also showed that strain H1 was non-pathogenic to mammals and shrimp. This study suggests that A. baumannii strain H
ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALGERIAN POPULUS NIGRA L. BUDS EX...bioejjournal
his study is part of a goal to investigate chemical composition, antibacterial, antifungal and antioxidant activities of the flower buds extracts from the Algerian Polulus nigra L., which were collected from Djarifet - mansourah at Tlemcen city in the West Northern of Algeria. In organic extracts, tanins, flavonoïds, coumarins, alkaloids and terpenoïds were the principals secondary metabolites identified from the flower buds of black poplar. Antibacterial and antifungal activities of extracts were tested using agar-well diffusion method and micro-well determination of MIC assay against eleven bacteria and two Candida species. It was found that extracts of black poplar buds exhibit antibacterial and anticandidal activities with agar disk diffusion (7 to 43mm) and MIC methods (MIC= 90.33 µg/ml against several strains of bacteria and MIC=45.16 µg/ml against Candida albicans). The antioxidant effect of hydroalcoholic extract was evaluated using DPPH and FRAP assays. It was showed good and similar activity than ascorbic acid and BHA by DPPH method: IC50= 220µg/mL for hydroethanol extract.
Antioxidant and Antimicrobial Activities Of Algerian Populus Nigra L. Buds Ex...bioejjournal
This study is part of a goal to investigate chemical composition, antibacterial, antifungal and antioxidant activities of the flower buds extracts from the Algerian Polulus nigra L., which were collected from Djarifet - mansourah at Tlemcen city in the West Northern of Algeria. In organic extracts, tanins, flavonoïds, coumarins, alkaloids and terpenoïds were the principals secondary metabolites identified from the flower buds of black poplar. Antibacterial and antifungal activities of
extracts were tested using agar-well diffusion method and micro-well determination of MIC assay against
eleven bacteria and two Candida species. It was found that extracts of black poplar buds exhibit
antibacterial and anticandidal activities with agar disk diffusion (7 to 43mm) and MIC methods (MIC=
90.33 µg/ml against several strains of bacteria and MIC=45.16 µg/ml against Candida albicans). The
antioxidant effect of hydroalcoholic extract was evaluated using DPPH and FRAP assays. It was showed good and similar activity than ascorbic acid and BHA by DPPH method: IC50= 220µg/mL for hydroethanol extract.
Bioactive potential of sea urchin temnopleurus toreumaticus from devanampatti...Alexander Decker
This document summarizes a study that investigated the bioactive potential of the sea urchin Temnopleurus toreumaticus. Biochemical analysis found proteins, carbohydrates, and lipids in the aqueous extract. Hemolytic assays showed the extract had hemolytic activity against various blood cells. Cytotoxicity assays found the extract was toxic to brine shrimp at certain concentrations. Antimicrobial assays indicated the extract inhibited some bacteria and fungi. Fourier transform infrared spectroscopy identified functional groups in the extract. Overall, the results suggest the sea urchin extract has hemolytic, cytotoxic, and some antimicrobial activity.
This document summarizes the extraction and characterization of polyhydroxybutyrates (PHB) from Bacillus thuringiensis KSADL127 isolated from mangrove environments in Saudi Arabia. 50 bacterial strains from mangrove samples were screened for PHB production, with one strain producing 137 mg/L of PHB. This highest producing strain was identified as B. thuringiensis KSADL127 based on phenotypic and genetic analysis. PHB was extracted from the strain and characterized using various analytical techniques, confirming it was intracellularly accumulated PHB.
An Investigation Into The Mechanisms Underlying Enhanced Biosulphidogenesis I...iosrjce
Anthropogenic activities like mining, processes of metallurgy and other chemical industries lead to
the discharge of a high amount of sulphate into the environment that causes serious problems to human health.
This paper illustrates the employment of thermophilic sulphate reducing bacteria for biosulphidogenesis. Two
different species have been isolated from hot water spring of Vajreshwari and Ganeshpuri,Thane, Maharashtra,
INDIA.The mechanism involved in biosulphidogenesis includes production of specific protein as well as
liberation of some extracellular polymeric compound (EPS) e.g. proteins, carbohydrate, acids etc. that are
produced during the microbial cell metabolism. These compounds plays an important role in the faster
reduction of sulphate and decrease in production rate of sulphide.The isolate was found to be of genus
Bacillusand type strain was found to be subtilis Zankar and licheniformis Sonali. The strain sequence were
deposited in NCBI database with accession number KJ939324 and KJ939325 respectively. The result highlights
the potential use of these organism in biosulphidogenesis.
This document summarizes a research study that investigated the molluscicidal properties of two plant species, Cestrum nocturnum and Cestrum diurnum, against the freshwater snail Lymnea accuminata. Aqueous and alcoholic extracts of the leaves of both plants, as well as an isolated saponin compound, were tested for toxicity against L. accuminata. The alcoholic extracts proved most toxic, with 100% mortality occurring within 24-48 hours at concentrations of 2.5-4.0 mg/L. The isolated saponin compound also showed strong molluscicidal effects. The study identifies saponins as active compounds and suggests Cestrum species may be a potential source
Phylogenetic Analysis of the Potential Microorganism for Remediation of Heavy...CSCJournals
The present research work has been carried out to study the waste disposal contaminated site for its physico chemical and microbial characterization and identification of potential microorganism capable of bioaccumulation and biodegradation of heavy metals. The ambient conditions present in the metal contaminated environment shows the values: pH(5.4),temperature(30°C), moisture(11.71%), nutrients; Nitrogen(0.2mg/l), phosphorus(22.65mg/l) and sulphur(559.3mg/l) respectively. The biological parameters studied indicate Dissolved oxygen (7.4mg/l), Biological oxygen demand (3.8 mg/l), Chemical oxygen demand (64.6 mg/l). The microbial consortium identified was found to survive and multiply in the present environmental conditions. Microbial consortium was sequenced and compared using Bioinformatics tools like BLAST, ClustalW and PHYLIP. In order to identify potential microorganism, microbial consortium was exposed to increasing concentrations of heavy metals viz 5mg/l, 25mg/l, 50mg/l, 100mg/l up to 800mg/l with special reference to Iron. At a concentration of 500mg/l, only one microorganism was found survived and multiplied. This shows that potential microorganism was only survived at higher concentration of iron. The 16SrRNA sequence and phylogenetic tree characterized the organism as Klebsiella pneumoniae, which was also confirmed by biochemical tests. The potential microorganism identified by BLAST technique can be used for remediation of the heavy metal from contaminated environment.
This document reports on the isolation and characterization of a denitrifying Acinetobacter baumannii strain H1 that was isolated from shrimp farming ponds. Strain H1 was able to use nitrite as its sole nitrogen source and showed high nitrite removal rates. It was identified as A. baumannii through biochemical testing and phylogenetic analysis of its 16S rRNA gene. PCR analysis detected key denitrification genes in strain H1. In vitro experiments demonstrated that strain H1 was able to remove nitrite effectively over a wide range of pH, temperatures, and cell densities. Tests also showed that strain H1 was non-pathogenic to mammals and shrimp. This study suggests that A. baumannii strain H
ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALGERIAN POPULUS NIGRA L. BUDS EX...bioejjournal
his study is part of a goal to investigate chemical composition, antibacterial, antifungal and antioxidant activities of the flower buds extracts from the Algerian Polulus nigra L., which were collected from Djarifet - mansourah at Tlemcen city in the West Northern of Algeria. In organic extracts, tanins, flavonoïds, coumarins, alkaloids and terpenoïds were the principals secondary metabolites identified from the flower buds of black poplar. Antibacterial and antifungal activities of extracts were tested using agar-well diffusion method and micro-well determination of MIC assay against eleven bacteria and two Candida species. It was found that extracts of black poplar buds exhibit antibacterial and anticandidal activities with agar disk diffusion (7 to 43mm) and MIC methods (MIC= 90.33 µg/ml against several strains of bacteria and MIC=45.16 µg/ml against Candida albicans). The antioxidant effect of hydroalcoholic extract was evaluated using DPPH and FRAP assays. It was showed good and similar activity than ascorbic acid and BHA by DPPH method: IC50= 220µg/mL for hydroethanol extract.
Antioxidant and Antimicrobial Activities Of Algerian Populus Nigra L. Buds Ex...bioejjournal
This study is part of a goal to investigate chemical composition, antibacterial, antifungal and antioxidant activities of the flower buds extracts from the Algerian Polulus nigra L., which were collected from Djarifet - mansourah at Tlemcen city in the West Northern of Algeria. In organic extracts, tanins, flavonoïds, coumarins, alkaloids and terpenoïds were the principals secondary metabolites identified from the flower buds of black poplar. Antibacterial and antifungal activities of
extracts were tested using agar-well diffusion method and micro-well determination of MIC assay against
eleven bacteria and two Candida species. It was found that extracts of black poplar buds exhibit
antibacterial and anticandidal activities with agar disk diffusion (7 to 43mm) and MIC methods (MIC=
90.33 µg/ml against several strains of bacteria and MIC=45.16 µg/ml against Candida albicans). The
antioxidant effect of hydroalcoholic extract was evaluated using DPPH and FRAP assays. It was showed good and similar activity than ascorbic acid and BHA by DPPH method: IC50= 220µg/mL for hydroethanol extract.
Bioactive potential of sea urchin temnopleurus toreumaticus from devanampatti...Alexander Decker
This document summarizes a study that investigated the bioactive potential of the sea urchin Temnopleurus toreumaticus. Biochemical analysis found proteins, carbohydrates, and lipids in the aqueous extract. Hemolytic assays showed the extract had hemolytic activity against various blood cells. Cytotoxicity assays found the extract was toxic to brine shrimp at certain concentrations. Antimicrobial assays indicated the extract inhibited some bacteria and fungi. Fourier transform infrared spectroscopy identified functional groups in the extract. Overall, the results suggest the sea urchin extract has hemolytic, cytotoxic, and some antimicrobial activity.
This document summarizes the extraction and characterization of polyhydroxybutyrates (PHB) from Bacillus thuringiensis KSADL127 isolated from mangrove environments in Saudi Arabia. 50 bacterial strains from mangrove samples were screened for PHB production, with one strain producing 137 mg/L of PHB. This highest producing strain was identified as B. thuringiensis KSADL127 based on phenotypic and genetic analysis. PHB was extracted from the strain and characterized using various analytical techniques, confirming it was intracellularly accumulated PHB.
An Investigation Into The Mechanisms Underlying Enhanced Biosulphidogenesis I...iosrjce
Anthropogenic activities like mining, processes of metallurgy and other chemical industries lead to
the discharge of a high amount of sulphate into the environment that causes serious problems to human health.
This paper illustrates the employment of thermophilic sulphate reducing bacteria for biosulphidogenesis. Two
different species have been isolated from hot water spring of Vajreshwari and Ganeshpuri,Thane, Maharashtra,
INDIA.The mechanism involved in biosulphidogenesis includes production of specific protein as well as
liberation of some extracellular polymeric compound (EPS) e.g. proteins, carbohydrate, acids etc. that are
produced during the microbial cell metabolism. These compounds plays an important role in the faster
reduction of sulphate and decrease in production rate of sulphide.The isolate was found to be of genus
Bacillusand type strain was found to be subtilis Zankar and licheniformis Sonali. The strain sequence were
deposited in NCBI database with accession number KJ939324 and KJ939325 respectively. The result highlights
the potential use of these organism in biosulphidogenesis.
This document summarizes a research study that investigated the molluscicidal properties of two plant species, Cestrum nocturnum and Cestrum diurnum, against the freshwater snail Lymnea accuminata. Aqueous and alcoholic extracts of the leaves of both plants, as well as an isolated saponin compound, were tested for toxicity against L. accuminata. The alcoholic extracts proved most toxic, with 100% mortality occurring within 24-48 hours at concentrations of 2.5-4.0 mg/L. The isolated saponin compound also showed strong molluscicidal effects. The study identifies saponins as active compounds and suggests Cestrum species may be a potential source
Phylogenetic Analysis of the Potential Microorganism for Remediation of Heavy...CSCJournals
The present research work has been carried out to study the waste disposal contaminated site for its physico chemical and microbial characterization and identification of potential microorganism capable of bioaccumulation and biodegradation of heavy metals. The ambient conditions present in the metal contaminated environment shows the values: pH(5.4),temperature(30°C), moisture(11.71%), nutrients; Nitrogen(0.2mg/l), phosphorus(22.65mg/l) and sulphur(559.3mg/l) respectively. The biological parameters studied indicate Dissolved oxygen (7.4mg/l), Biological oxygen demand (3.8 mg/l), Chemical oxygen demand (64.6 mg/l). The microbial consortium identified was found to survive and multiply in the present environmental conditions. Microbial consortium was sequenced and compared using Bioinformatics tools like BLAST, ClustalW and PHYLIP. In order to identify potential microorganism, microbial consortium was exposed to increasing concentrations of heavy metals viz 5mg/l, 25mg/l, 50mg/l, 100mg/l up to 800mg/l with special reference to Iron. At a concentration of 500mg/l, only one microorganism was found survived and multiplied. This shows that potential microorganism was only survived at higher concentration of iron. The 16SrRNA sequence and phylogenetic tree characterized the organism as Klebsiella pneumoniae, which was also confirmed by biochemical tests. The potential microorganism identified by BLAST technique can be used for remediation of the heavy metal from contaminated environment.
IJPCBS 2012, 2(1), 110-116 Kavya et al. ISSN: 2249-9504
110
INTERNATIONAL JOURNAL OF PHARMACEUTICAL, CHEMICAL AND BIOLOGICAL SCIENCES
Available online at www.ijpcbs.com
ISOLATION AND SCREENING OF STREPTOMYCES SP. FROM
CORINGA MANGROVE SOILS FOR ENZYME PRODUCTION AND
ANTIMICROBIAL ACTIVITY
M. Kavya Deepthi1*, M. Solomon Sudhakar1 and M. Nagalakshmi Devamma2 1Department of Biotechnology, Rajalakshmi Engineering College, Thandalam, 2Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pr Taadmesihln, aInddui,a I.n dia.
International Journal of Biometrics and Bioinformatics(IJBB) Volume (3) Issue...CSCJournals
The document summarizes a study on identifying potential microorganisms from a contaminated waste disposal site capable of remediating heavy metals. Physicochemical analysis of the site showed presence of metals like iron. Microbial consortium from the site was exposed to increasing concentrations of iron up to 800mg/l. A microorganism survived and grew at 500mg/l iron concentration. 16S rRNA sequencing and phylogenetic tree analysis identified the organism as Klebsiella pneumoniae, which was confirmed by biochemical tests. Bioinformatics tools like BLAST, ClustalW and PHYLIP were used to characterize the potential microorganism for bioremediation of heavy metals at the contaminated site.
8- IJRANSS-ANTIMICROBIAL POTENTIAL OF MARINE ACTINOMYCETESRavindragouda Patil
- 47 actinomycetes were isolated from mangrove swamp samples in Tamil Nadu, India, with most isolated from sediment.
- 34 isolates exhibited antagonism against shrimp pathogens Vibrio alginolyticus, V. harveyi, and V. parahaemolyticus.
- Isolate A10 showed the strongest inhibitory activity against all three pathogens, inhibiting growth by ≥20mm.
- Isolate A10 was identified as Streptomyces spp. based on chemotaxonomic and microscopic analysis.
Isolation and Growth Kinetic Studies Of Bacillus Methylotrophicus P10 & P11 ...theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
The papers for publication in The International Journal of Engineering& Science are selected through rigorous peer reviews to ensure originality, timeliness, relevance, and readability.
Theoretical work submitted to the Journal should be original in its motivation or modeling structure. Empirical analysis should be based on a theoretical framework and should be capable of replication. It is expected that all materials required for replication (including computer programs and data sets) should be available upon request to the authors.
The International Journal of Engineering & Science would take much care in making your article published without much delay with your kind cooperation.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Use of stable and radio isotopes to understand the plant physiological processRAHUL GOPALE
Introduction
what is isotope ?
Types of Isotopes
Isotopic Labelling
ADVANTAGES AND DISADVANTAGES OF ISOTOPIC STUDY
APPLICATIONS OF ISOTOPES IN AGRICULTURE
Principle isotopes used in plant-soil studies
Case studies
FUTURE THRUSTS OF ISOTOPIC STUDY
CONCLUSIONS
REFERENCES
9-IJABR-OCCURRENCE AND ANTIBACTERIAL ACTIVITY OF ACTINOMYCETES against fish p...Ravindragouda Patil
This document summarizes a study on the isolation and characterization of actinomycetes from marine environments in Thoothukkudi, Tamil Nadu, India and their antibacterial activity against fish pathogens. Marine samples were collected from 3 sites and 87 actinomycete strains were isolated. The highest number of antagonistic actinomycetes came from Thermal Beach samples. 8 highly antagonistic isolates were tested against fish pathogens; isolate A55 showed the highest inhibitory activity. A55 was identified as Streptomyces spp. Crude extracts from A55 inhibited the growth of fish pathogens. The results suggest marine environments can be a source of novel antimicrobial compounds effective against fish pathogens.
The document summarizes a study that isolated and screened marine actinomycetes for antagonistic activity against food-borne human pathogens. A total of 133 actinomycetes were isolated from 129 marine samples collected from 5 stations along the coast of Thoothukkudi, Tamil Nadu, India. The highest number of isolates (45) came from Thermal Beach samples. 104 isolates exhibited antagonism against Salmonella typhi, S. typhimurium, and Escherichia coli in a cross-streak assay. The Thermal Beach samples had the highest percentage (86.67%) of antagonistic isolates. The study indicates that antagonistic marine actinomycetes can be a source of novel
Profiling and Characterization Antioxidant Activities in Anoectochilus formos...Cây thuốc Việt
Phytochemical characteristics and antioxidant activities of the crude and fractionated plant extracts of Anoectochilus formosanus were evaluated using five different assay systems. An acid-treatment (2 N HCl in 95% ethanol) was employed to treat a butanol fraction (BuOH), creating an acid-hydrolyzed
BuOH fraction. The IC50 values for DPPH radicals in the BuOH and acid-hydrolyzed BuOH fractions were 0.521 and 0.021 mg/mL, respectively. The acid-hydrolyzed BuOH exhibited approximately 5-fold higher activity in scavenging superoxide anion than catechin. The acid-hydrolyzed BuOH fraction
also effectively protected φ x174 supercoiled DNA against strand cleavage induced by H2O2 and reduced oxidative stress in HL-60 cells. Metabolite profiling showed that the aglycones of flavonoid glycosides in BuOH were produced after acid hydrolytic treatment, and this resulted in a significant increase in antioxidant activities of acid-hydrolyzed BuOH. One new diarylpentanoid, kinsenone, and three known flavonoid glycosides and their derivatives were identified for the first time from A. formosanus, with strong antioxidant properties
2008 - Molecular microbial and chemical investigation of the bioremediation o...WALEBUBLÉ
The document describes a study that used laboratory-scale bioreactors to investigate the biodegradation of two-phase olive mill waste (TPOMW) by its indigenous microbiota under different conditions. The effects of nutrient addition and aeration on bioremediation and microbial community changes were evaluated. Analysis found that nutrient addition and aeration led to greater decreases in polyphenolic content and increases in the fungal to bacterial ratio. Molecular identification of bacteria and fungi in the bioreactors identified several genera present, with fungi like Penicillium and Candida dominant.
Secondary Metabolites of the Entomopathogenic Fungus, Cladosporium cladospori...Premier Publishers
Cladosporium cladosporioides is one of the promising entomopathogenic fungi acting as insect-pathogenic microorganism or can be used as a source of toxins against insect pests. Ethyl acetate extract of the secondary metabolites of C. cladosporioide was obtained, and its volatile constituents were characterized using GC/MS technique. Also, two major compounds were isolated and identified as 3-phenyl propanoic acid (6) and 3-(4β-hydroxy-6-pyranonyl)-5-isopropylpyrrolidin-2-one (7). It's worthy to mention that this isolated compound (7), is reported from C. cladosporioides for the first time. Also, the toxicity of the ethyl acetate extract of the secondary metabolites of C. cladosporioides against both adults and nymphs of cotton aphid, A. gossypii was determined. Data showed that C. cladosporioides ethyl acetate extract was most effective against nymphs showing LC50 of 24.5827 ppm, LC90 of 128.7385 ppm and toxicity index of 100%, while, it showed LC50 of 36.6959 ppm, LC90 of 154.4394 ppm and toxicity index of 76.69% against adults.
GC/MS analysis and In-vitro Antioxidant activity of methanol extract of Uloth...IOSRJPBS
The determination of phytochemical constituents, total phenol, flavonoid contents and antioxidant assays of methanol extract of Ulothrix flacca and its main constituent dimethyl sulfone was studied. The mass spectra of the compounds were matched with the NIST library. The GC-MS analysis of methanol extracts of Ulothrix flacca showed sixteen peaks. Of all the sixteen chemical compounds revealed from the GC-MS analysis of Ulothrix flacca, Dimethyl Sulfone (C2H6O2S) (RT-8.9), 4-Bromobenzoic Acid, 2-Chlorophenyl Ester (C13H8BrClO2) (RT-12.642), Tetradecanoic Acid, 10,13-Dimethyl-, Methyl Ester (C17H34O2) (RT-18.669) are the three major components. The methanol extracts of Ulothrix flacca possess phenolic and flavonoid content of (5.74 ± 0.45mg Gallic acid equivalent (GAE)/g Wt, and 12.58 ± 1.52mg quercetin eq/g wt) respectively. Antioxidant activity was determined using 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical, for evaluating free radicle scavenging activity, ABTS radical cation scavenging activity, Ferric reducing antioxidant power (FRAP) assay, Phosphomolybdenum assay and Metal chelating activity using BHT, Rutin and Quercetin. The highest radicle scavenging activity was shown by dimethyl sulfone (15.156mg/ml), which is higher than the BHT and Rutin. In vitro antioxidant activity of methanolic extract of Ulothrix flacca and Dimethyl sulfone showed an increase with increasing concentration indicating positive association with the total phenolic and flavonoid contents of the extract, which could be considered for future applications in medicine, dietary supplements ,cosmetics or food industries.
The document discusses Vibrio parahaemolyticus, a halophilic bacterium that can cause foodborne illness. It is found in coastal waters worldwide and can cause gastroenteritis when consumed in raw or undercooked seafood. Key virulence factors include the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH), which are encoded by the tdh and trh genes, respectively. Strains containing these genes are more likely to be pathogenic. The Kanagawa phenomenon (KP) refers to beta-hemolysis observed on specialized media and is correlated with TDH production and pathogenicity. Outbreaks have occurred globally but are especially common in Asia where
ABSTRACT- The development of human civilization throughout history has led to growing disruption of the natural
balance and the occurrence of different types of pollution. Environmental pollution with petroleum and petrochemical
products has been recognized as significant and serious problem. Diesel engine oil, which is one of the major products of
crude oil, constitutes a major source of pollution in our environment. Therefore diesel engine oil can enter into the
environment through wrecks of oil tankers carrying diesel oil, cleaning of diesel tanks by merchants, war ships carrying
diesel oil and motor mechanics. In present study the microorganisms utilising petrol and diesel oil as carbon source were
isolated and investigation of their characteristics towards the production of polyhydroxyalkanoates (PHA), which is now a
days well known as biodegradable polymer.
Key Words- Petrol and Diesel oil contamination, Bioremediation, Biodegradable bacterial polymer, Sudan
Black B staining, 16sr RNA sequencing
This document summarizes research on using the bacteria Acinetobacter jenuii to decolorize the azodye Direct Red. Azodyes are commonly used in dyeing but 10-50% are wasted and pollute the environment. The bacteria can effectively reduce the azodye through the enzyme azoreductase and reduce sludge levels compared to other decolorization methods. However, further study of Acinetobacter jenuii is needed because it has been linked to some pathological conditions in humans. The methodology describes growing the bacteria and measuring its ability to decolorize a Direct Red solution. Results showed the bacteria could successfully decolorize the dye while reducing sludge.
The document summarizes a study that investigated the effect of different pH regimes on the growth and micro-sclerotial formation of Phoma tropica, a fungus that causes leaf spot disease in Indian bean. The fungus was able to grow across a wide pH range of 4.0 to 8.0, but growth and micro-sclerotial production were significantly higher under acidic conditions compared to alkaline conditions. Optimum pH for growth and micro-sclerotial production was found to be 6.0. Near acidic pH levels were more conducive to fungal growth and micro-sclerotial formation than alkaline pH levels.
This study isolated actinomycetes from marine samples in Thoothukkudi, Tamil Nadu, India and tested their inhibitory activity against fish pathogens. Higher bacterial populations were found in sediment than water samples. 46 actinomycete strains were isolated, of which 36 showed antagonism against E. coli in a screening assay. Nine highly antagonistic isolates were further tested against fish pathogens Aeromonas hydrophila, A. sobria, Vibrio fischeri, V. vulnificus, Edwardsiella tarda and Pasteurella spp. using a cross-streak assay. Most isolates inhibited all pathogens. Isolate A15 strongly inhibited all pathogens and was identified as Streptomy
The document summarizes research into what makes certain natural clays antibacterial. Key points:
- Antibacterial clays contain nano-scale illite-smectite and reduced iron phases that buffer water pH and oxidation state, promoting Fe2+ solubility.
- E. coli exposed to an antibacterial clay's leachate accumulate high intracellular concentrations of Fe and P, supporting a role for polyphosphate or phospholipids in regulating Fe2+.
- Excess Fe2+ overwhelms cell membrane regulatory proteins, then oxidizes inside cells to Fe3+, producing lethal hydroxyl radicals through the Fenton reaction.
- High-resolution SEM images show the nano-scale crystals and Fe
Emerging Diversity Within Well-known Heterotrophic Flagellates Groups Reveal...Javier del Campo
During the last ten years, an overwhelming amount of data has been generated from culturing independent techniques to study the diversity of marine protists. These studies show a large diversity and the existence of new groups, such as for MAST (Marine Stramenopiles) or MALV (Marine Alveolates). However, a large part of these sequences has not been analysed together, and constitutes a potentially important source of information related with protists diversity and distribution. Using sequences from our studies and from GenBank and CAMERA, we have been able to define several novel clades in three important marine representative groups such are Choanoflagellates, Chrysophytes and Bicosecids. Most of the novel clades correspond to uncultured organisms. Analyzing all sequences together permits to observe this diversity, which was already presented by generally ignored. Only an important data mining work developed using GenBank allows this novel diversity hidden inside well know groups to emerge from the enormous sea of data generated.
This document summarizes a study examining the toxicity and antioxidant activity of two extracts from the brown seaweed Fucus vesiculosus. Both extracts were found to lack relevant toxic effects in acute and 4-week toxicity tests in rats. The extracts exhibited antioxidant activity in non-cellular systems by reducing oxidative stress and scavenging free radicals. They also showed antioxidant effects in activated macrophages and in the plasma and erythrocytes of rats treated with the extracts for 4 weeks, indicating their compounds may be absorbed and act as antioxidants in vivo. The findings support the view that daily consumption of one extract could benefit humans by reducing oxidative stress.
IJPCBS 2012, 2(1), 110-116 Kavya et al. ISSN: 2249-9504
110
INTERNATIONAL JOURNAL OF PHARMACEUTICAL, CHEMICAL AND BIOLOGICAL SCIENCES
Available online at www.ijpcbs.com
ISOLATION AND SCREENING OF STREPTOMYCES SP. FROM
CORINGA MANGROVE SOILS FOR ENZYME PRODUCTION AND
ANTIMICROBIAL ACTIVITY
M. Kavya Deepthi1*, M. Solomon Sudhakar1 and M. Nagalakshmi Devamma2 1Department of Biotechnology, Rajalakshmi Engineering College, Thandalam, 2Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pr Taadmesihln, aInddui,a I.n dia.
International Journal of Biometrics and Bioinformatics(IJBB) Volume (3) Issue...CSCJournals
The document summarizes a study on identifying potential microorganisms from a contaminated waste disposal site capable of remediating heavy metals. Physicochemical analysis of the site showed presence of metals like iron. Microbial consortium from the site was exposed to increasing concentrations of iron up to 800mg/l. A microorganism survived and grew at 500mg/l iron concentration. 16S rRNA sequencing and phylogenetic tree analysis identified the organism as Klebsiella pneumoniae, which was confirmed by biochemical tests. Bioinformatics tools like BLAST, ClustalW and PHYLIP were used to characterize the potential microorganism for bioremediation of heavy metals at the contaminated site.
8- IJRANSS-ANTIMICROBIAL POTENTIAL OF MARINE ACTINOMYCETESRavindragouda Patil
- 47 actinomycetes were isolated from mangrove swamp samples in Tamil Nadu, India, with most isolated from sediment.
- 34 isolates exhibited antagonism against shrimp pathogens Vibrio alginolyticus, V. harveyi, and V. parahaemolyticus.
- Isolate A10 showed the strongest inhibitory activity against all three pathogens, inhibiting growth by ≥20mm.
- Isolate A10 was identified as Streptomyces spp. based on chemotaxonomic and microscopic analysis.
Isolation and Growth Kinetic Studies Of Bacillus Methylotrophicus P10 & P11 ...theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
The papers for publication in The International Journal of Engineering& Science are selected through rigorous peer reviews to ensure originality, timeliness, relevance, and readability.
Theoretical work submitted to the Journal should be original in its motivation or modeling structure. Empirical analysis should be based on a theoretical framework and should be capable of replication. It is expected that all materials required for replication (including computer programs and data sets) should be available upon request to the authors.
The International Journal of Engineering & Science would take much care in making your article published without much delay with your kind cooperation.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
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Use of stable and radio isotopes to understand the plant physiological processRAHUL GOPALE
Introduction
what is isotope ?
Types of Isotopes
Isotopic Labelling
ADVANTAGES AND DISADVANTAGES OF ISOTOPIC STUDY
APPLICATIONS OF ISOTOPES IN AGRICULTURE
Principle isotopes used in plant-soil studies
Case studies
FUTURE THRUSTS OF ISOTOPIC STUDY
CONCLUSIONS
REFERENCES
9-IJABR-OCCURRENCE AND ANTIBACTERIAL ACTIVITY OF ACTINOMYCETES against fish p...Ravindragouda Patil
This document summarizes a study on the isolation and characterization of actinomycetes from marine environments in Thoothukkudi, Tamil Nadu, India and their antibacterial activity against fish pathogens. Marine samples were collected from 3 sites and 87 actinomycete strains were isolated. The highest number of antagonistic actinomycetes came from Thermal Beach samples. 8 highly antagonistic isolates were tested against fish pathogens; isolate A55 showed the highest inhibitory activity. A55 was identified as Streptomyces spp. Crude extracts from A55 inhibited the growth of fish pathogens. The results suggest marine environments can be a source of novel antimicrobial compounds effective against fish pathogens.
The document summarizes a study that isolated and screened marine actinomycetes for antagonistic activity against food-borne human pathogens. A total of 133 actinomycetes were isolated from 129 marine samples collected from 5 stations along the coast of Thoothukkudi, Tamil Nadu, India. The highest number of isolates (45) came from Thermal Beach samples. 104 isolates exhibited antagonism against Salmonella typhi, S. typhimurium, and Escherichia coli in a cross-streak assay. The Thermal Beach samples had the highest percentage (86.67%) of antagonistic isolates. The study indicates that antagonistic marine actinomycetes can be a source of novel
Profiling and Characterization Antioxidant Activities in Anoectochilus formos...Cây thuốc Việt
Phytochemical characteristics and antioxidant activities of the crude and fractionated plant extracts of Anoectochilus formosanus were evaluated using five different assay systems. An acid-treatment (2 N HCl in 95% ethanol) was employed to treat a butanol fraction (BuOH), creating an acid-hydrolyzed
BuOH fraction. The IC50 values for DPPH radicals in the BuOH and acid-hydrolyzed BuOH fractions were 0.521 and 0.021 mg/mL, respectively. The acid-hydrolyzed BuOH exhibited approximately 5-fold higher activity in scavenging superoxide anion than catechin. The acid-hydrolyzed BuOH fraction
also effectively protected φ x174 supercoiled DNA against strand cleavage induced by H2O2 and reduced oxidative stress in HL-60 cells. Metabolite profiling showed that the aglycones of flavonoid glycosides in BuOH were produced after acid hydrolytic treatment, and this resulted in a significant increase in antioxidant activities of acid-hydrolyzed BuOH. One new diarylpentanoid, kinsenone, and three known flavonoid glycosides and their derivatives were identified for the first time from A. formosanus, with strong antioxidant properties
2008 - Molecular microbial and chemical investigation of the bioremediation o...WALEBUBLÉ
The document describes a study that used laboratory-scale bioreactors to investigate the biodegradation of two-phase olive mill waste (TPOMW) by its indigenous microbiota under different conditions. The effects of nutrient addition and aeration on bioremediation and microbial community changes were evaluated. Analysis found that nutrient addition and aeration led to greater decreases in polyphenolic content and increases in the fungal to bacterial ratio. Molecular identification of bacteria and fungi in the bioreactors identified several genera present, with fungi like Penicillium and Candida dominant.
Secondary Metabolites of the Entomopathogenic Fungus, Cladosporium cladospori...Premier Publishers
Cladosporium cladosporioides is one of the promising entomopathogenic fungi acting as insect-pathogenic microorganism or can be used as a source of toxins against insect pests. Ethyl acetate extract of the secondary metabolites of C. cladosporioide was obtained, and its volatile constituents were characterized using GC/MS technique. Also, two major compounds were isolated and identified as 3-phenyl propanoic acid (6) and 3-(4β-hydroxy-6-pyranonyl)-5-isopropylpyrrolidin-2-one (7). It's worthy to mention that this isolated compound (7), is reported from C. cladosporioides for the first time. Also, the toxicity of the ethyl acetate extract of the secondary metabolites of C. cladosporioides against both adults and nymphs of cotton aphid, A. gossypii was determined. Data showed that C. cladosporioides ethyl acetate extract was most effective against nymphs showing LC50 of 24.5827 ppm, LC90 of 128.7385 ppm and toxicity index of 100%, while, it showed LC50 of 36.6959 ppm, LC90 of 154.4394 ppm and toxicity index of 76.69% against adults.
GC/MS analysis and In-vitro Antioxidant activity of methanol extract of Uloth...IOSRJPBS
The determination of phytochemical constituents, total phenol, flavonoid contents and antioxidant assays of methanol extract of Ulothrix flacca and its main constituent dimethyl sulfone was studied. The mass spectra of the compounds were matched with the NIST library. The GC-MS analysis of methanol extracts of Ulothrix flacca showed sixteen peaks. Of all the sixteen chemical compounds revealed from the GC-MS analysis of Ulothrix flacca, Dimethyl Sulfone (C2H6O2S) (RT-8.9), 4-Bromobenzoic Acid, 2-Chlorophenyl Ester (C13H8BrClO2) (RT-12.642), Tetradecanoic Acid, 10,13-Dimethyl-, Methyl Ester (C17H34O2) (RT-18.669) are the three major components. The methanol extracts of Ulothrix flacca possess phenolic and flavonoid content of (5.74 ± 0.45mg Gallic acid equivalent (GAE)/g Wt, and 12.58 ± 1.52mg quercetin eq/g wt) respectively. Antioxidant activity was determined using 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical, for evaluating free radicle scavenging activity, ABTS radical cation scavenging activity, Ferric reducing antioxidant power (FRAP) assay, Phosphomolybdenum assay and Metal chelating activity using BHT, Rutin and Quercetin. The highest radicle scavenging activity was shown by dimethyl sulfone (15.156mg/ml), which is higher than the BHT and Rutin. In vitro antioxidant activity of methanolic extract of Ulothrix flacca and Dimethyl sulfone showed an increase with increasing concentration indicating positive association with the total phenolic and flavonoid contents of the extract, which could be considered for future applications in medicine, dietary supplements ,cosmetics or food industries.
The document discusses Vibrio parahaemolyticus, a halophilic bacterium that can cause foodborne illness. It is found in coastal waters worldwide and can cause gastroenteritis when consumed in raw or undercooked seafood. Key virulence factors include the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH), which are encoded by the tdh and trh genes, respectively. Strains containing these genes are more likely to be pathogenic. The Kanagawa phenomenon (KP) refers to beta-hemolysis observed on specialized media and is correlated with TDH production and pathogenicity. Outbreaks have occurred globally but are especially common in Asia where
ABSTRACT- The development of human civilization throughout history has led to growing disruption of the natural
balance and the occurrence of different types of pollution. Environmental pollution with petroleum and petrochemical
products has been recognized as significant and serious problem. Diesel engine oil, which is one of the major products of
crude oil, constitutes a major source of pollution in our environment. Therefore diesel engine oil can enter into the
environment through wrecks of oil tankers carrying diesel oil, cleaning of diesel tanks by merchants, war ships carrying
diesel oil and motor mechanics. In present study the microorganisms utilising petrol and diesel oil as carbon source were
isolated and investigation of their characteristics towards the production of polyhydroxyalkanoates (PHA), which is now a
days well known as biodegradable polymer.
Key Words- Petrol and Diesel oil contamination, Bioremediation, Biodegradable bacterial polymer, Sudan
Black B staining, 16sr RNA sequencing
This document summarizes research on using the bacteria Acinetobacter jenuii to decolorize the azodye Direct Red. Azodyes are commonly used in dyeing but 10-50% are wasted and pollute the environment. The bacteria can effectively reduce the azodye through the enzyme azoreductase and reduce sludge levels compared to other decolorization methods. However, further study of Acinetobacter jenuii is needed because it has been linked to some pathological conditions in humans. The methodology describes growing the bacteria and measuring its ability to decolorize a Direct Red solution. Results showed the bacteria could successfully decolorize the dye while reducing sludge.
The document summarizes a study that investigated the effect of different pH regimes on the growth and micro-sclerotial formation of Phoma tropica, a fungus that causes leaf spot disease in Indian bean. The fungus was able to grow across a wide pH range of 4.0 to 8.0, but growth and micro-sclerotial production were significantly higher under acidic conditions compared to alkaline conditions. Optimum pH for growth and micro-sclerotial production was found to be 6.0. Near acidic pH levels were more conducive to fungal growth and micro-sclerotial formation than alkaline pH levels.
This study isolated actinomycetes from marine samples in Thoothukkudi, Tamil Nadu, India and tested their inhibitory activity against fish pathogens. Higher bacterial populations were found in sediment than water samples. 46 actinomycete strains were isolated, of which 36 showed antagonism against E. coli in a screening assay. Nine highly antagonistic isolates were further tested against fish pathogens Aeromonas hydrophila, A. sobria, Vibrio fischeri, V. vulnificus, Edwardsiella tarda and Pasteurella spp. using a cross-streak assay. Most isolates inhibited all pathogens. Isolate A15 strongly inhibited all pathogens and was identified as Streptomy
The document summarizes research into what makes certain natural clays antibacterial. Key points:
- Antibacterial clays contain nano-scale illite-smectite and reduced iron phases that buffer water pH and oxidation state, promoting Fe2+ solubility.
- E. coli exposed to an antibacterial clay's leachate accumulate high intracellular concentrations of Fe and P, supporting a role for polyphosphate or phospholipids in regulating Fe2+.
- Excess Fe2+ overwhelms cell membrane regulatory proteins, then oxidizes inside cells to Fe3+, producing lethal hydroxyl radicals through the Fenton reaction.
- High-resolution SEM images show the nano-scale crystals and Fe
Emerging Diversity Within Well-known Heterotrophic Flagellates Groups Reveal...Javier del Campo
During the last ten years, an overwhelming amount of data has been generated from culturing independent techniques to study the diversity of marine protists. These studies show a large diversity and the existence of new groups, such as for MAST (Marine Stramenopiles) or MALV (Marine Alveolates). However, a large part of these sequences has not been analysed together, and constitutes a potentially important source of information related with protists diversity and distribution. Using sequences from our studies and from GenBank and CAMERA, we have been able to define several novel clades in three important marine representative groups such are Choanoflagellates, Chrysophytes and Bicosecids. Most of the novel clades correspond to uncultured organisms. Analyzing all sequences together permits to observe this diversity, which was already presented by generally ignored. Only an important data mining work developed using GenBank allows this novel diversity hidden inside well know groups to emerge from the enormous sea of data generated.
This document summarizes a study examining the toxicity and antioxidant activity of two extracts from the brown seaweed Fucus vesiculosus. Both extracts were found to lack relevant toxic effects in acute and 4-week toxicity tests in rats. The extracts exhibited antioxidant activity in non-cellular systems by reducing oxidative stress and scavenging free radicals. They also showed antioxidant effects in activated macrophages and in the plasma and erythrocytes of rats treated with the extracts for 4 weeks, indicating their compounds may be absorbed and act as antioxidants in vivo. The findings support the view that daily consumption of one extract could benefit humans by reducing oxidative stress.
Assessment of the Plankton Biodiversity,Bay of Bengal, Cox's Bazar, BangladeshAbuMusa51
I am Abu Musa. This is my Internship Presentation. This is for partial fulfillment of the 4th-year final examination of the Department of Fisheries, University of Dhaka. This is based on my findings from one month of research on the Coxs Bazar coast. The research is done in the live feed lab of BFRI Cox's Bazar.
The document presents information on marine natural products. It discusses how marine organisms like algae, sponges and corals produce biologically active compounds with properties like anti-cancer, anti-inflammatory and antimicrobial effects. It describes methods used to isolate and purify compounds from marine sources, as well as challenges in marine drug research like taxonomic identification and chemical screening. Recent advances in marine drugs for conditions like cancer, inflammation and infections are also summarized. Finally, it briefly discusses marine toxins produced by some marine species that can cause illnesses in humans.
A Review on the Antimicrobial Activity of Sesuvium Portulacastrumijtsrd
The document summarizes a study on the antimicrobial activity of Sesuvium portulacastrum, a mangrove plant. The study found that ethanol extracts of S. portulacastrum leaves contained phytochemicals like steroids and showed strong antibacterial activity against pathogens like Staphylococcus aureus and E. coli. Gas chromatography-mass spectrometry identified compounds in the ethanol extract including 22, 23-Dihydrostigmasterol, Benzoic acid, Epicatechin, and Capsaicin that were responsible for the antimicrobial properties. The presence of these phytochemicals supports the potential of S. portulacastrum as a source of antimicrobial agents.
This document presents information on marine pharmacognosy. It defines marine pharmacognosy as the study of medicinally active natural substances obtained from marine species such as bacteria, viruses, algae, fungi and sponges. It provides examples of commercially used marine drugs and discusses the chemical diversity of compounds isolated from marine organisms. It also outlines proper procedures for collecting, handling, and storing marine organisms to prevent decomposition and chemical degradation.
Chemical communications among plant and animal components are fundamental elements for the functioning and the connectivity of ecosystems. In particular, wound-activated infochemicals trigger specific reactions of invertebrates according to evolutionary constraints, permitting them to identify prey cues, escape predators and optimize their behaviors according to specific life strategies.
In order to assessing whether algae can reduce the pollution concentration of the effluents by
absorbing the nutrients, it is found that effluents can effectively be treated by employing algal organisisms such
as Oscillatoria and Stigeoclonium species and these organisms are frequently found in the polluted waters and
they were recorded as pollution tolerant forms. In the laboratory procedures out of the several media tested
Modified CHU No. 10 medium was found to be quite suitable for both the test organisms. It was found that up to
87% and 85% of phosphate uptake was achieved by Oscillatoria and Stigeoclonium respectively with 13% and
16% increase of D.O. in the effluents by the tenth day. In case of organic matter Oscillatoria removed 73% and
Stigeoclonium 70% up to tenth day
This document summarizes a study on meiofaunal organisms found in Songculan Lagoon in the Philippines. Eleven meiofaunal taxa were identified, with nematodes being the most dominant. Sampling at three stations found varying abundances of taxa. Nematodes, copepods, ostracods, and turbellarians were the most relatively abundant taxa overall. The study provides a baseline analysis of meiofaunal composition and abundance in Songculan Lagoon.
This study aimed to identify and analyze meiofaunal organisms in Songculan Lagoon, Philippines. Eleven meiofaunal taxa were identified, including nematodes, copepods, ostracods, turbellarians, gastropods, flatworms, gastrotrichs, polychaetes, oligochaetes, rotifers, and tardigrades. Nematodes and gastropods were present at all sampling stations. Analysis found that nematodes made up 34% of organisms, making them the most abundant taxa overall. Physicochemical properties of the water and sediments were also measured. This baseline data on meiofaunal composition and environmental conditions can inform further studies
This document reviews methods for sampling, isolating, and identifying microplastics ingested by fish and invertebrates. It discusses how organisms can be collected from both field and laboratory settings. Common isolation techniques examined include dissection of digestive tracts, depuration to examine feces, and digestion of tissues using chemicals or enzymes. Visual identification under microscopes and chemical analysis are evaluated for categorizing extracted microplastics. The discussion highlights the need for standardized protocols while allowing flexibility. Concluding remarks note that most studies focus on uptake occurrence rather than risks or ecosystem impacts.
This document discusses the selection of biological methods for assessing the quality of industrial effluents. It identifies existing bioassays for evaluating acute and chronic toxicity of wastewater. Biological methods can be divided into microbiological, limnological, and ecotoxicological tests. Common ecotoxicological tests identified include those using algae, microcrustaceans like Daphnia, fish, and bacteria to analyze effluent toxicity. The document compares different test methods and variables for routinely monitoring effluents and researching their impacts.
Isolation of typical marine bacteria by dilution culture - growth, maintenanc...ITSON
Isolation of Typical Marine Bacteria by Dilution Culture: Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions - FRITS SCHUT
This document describes a study that used bioassay-guided fractionation to isolate active metabolites from the green alga Caulerpa sertularioides that inhibit or promote the growth of marine bacteria. The crude extract was separated into chloroform and methanol/water partitions which were further separated by liquid chromatography. A fraction from the methanol/water partition promoted the growth of Vibrio sp., a bacterium isolated from C. sertularioides. NMR analysis identified aromatic, aldehyde, and methoxy functional groups in the active compound. The goal is to determine the full structure and test activity against human pathogens.
A preliminary study on the toxic potentials of shea butter effluent using Cla...IOSR Journals
This study was conducted purposely to evaluate the effects of shea butter effluent (SBE) on the
freshwater inhabitant using Clarias gariepinus as a biological model. A prominent Local factory of shea butter
at Tede, ATISBO Local Government was chosen because the effluent flows directly into a near-by stream that
ends up at a popular Dam in the Local Government on which more than 120,000 people depend for domestic
use.Static bioassay was conducted to determine the LC50 of shea butter effluent to Clarias gariepinus. Ten fishes
each were exposed to 0.05, 0.06, 0.07, 0.08, and 0.09ppt (lethal concentration) of SBE in separate water plastic
bowl of (40cmX29cmX28cm) of 60litres capacity.The lethal Concentration (LC50) value of SBE was 0.057ppt for
96hrs of exposure. Total mortality occurred in the concentrations of 0.08 and 0.09ppt within 24hours of
exposure period. Behavioural reactions exhibited by the fish include erratic movement, air gulping, loss of
reflex, molting, barbell deformation, hemorrhage, and excessive mucus secretion in fish exposed to higher
concentration of shea butter effluent.
The appreciable increase in the mean value of heavy metal, such as Manganese, Nickel, Cadmium,
Zinc, Copper and Lead revealed that the increase in the concentration of shea butter effluent leads to
bioaccumulation of the aforementioned heavy metals in the test organisms. The values for all the metals exceed
the permissible Criteria of the national and international regulatory body. Therefore, Shea butter effluent is
highly toxic to freshwater fishes, its discharged directly into water bodies, new fish farms or in areas close to
aquatic environment should not be encouraged.
This document summarizes a study that analyzed the diversity of rRNA genes in the guts of adult and fingerling Mugil cephalus (flathead grey mullet) fish inhabiting an Egyptian Mediterranean estuary. Bulk DNA was extracted from the guts and the eukaryotic 18S rRNA gene, bacterial 16S rRNA gene, and archaeal 16S rRNA gene were amplified via PCR, cloned, and sequenced. Rarefaction analyses identified 11, 18, and 13 phylotype groups of rRNA genes for eukaryotes, bacteria, and archaea, respectively, in adult guts, and 6 and 11 phylotype groups for eukaryotes and bacteria in fingerling guts (archaea were not detected in
Chemical characterization of Cinachyrella tarentina: Sponge of Atlantic Moroc...journal ijrtem
ABSTRACT : Currently, marine organisms are a very important source of new molecules in pharmacology and thus in the development of new bioactive products. Sponges, in particular, given their very primitive origin and persistence during evolution, have developed a chemical defense system.The chemical study of Cinachyrella tarentina, marine sponge recognized by its antibacterial and antifungal activity was investigated for the first time in Morocco. The screening of Cinachyrella tarentina revels that it contains different levels of primary and secondary metabolites. The dosage of polyphenols was carried out using the reagent Foulin-Ciocalteu. The antioxidant activity was evaluated by the DPPH test. The fatty acid composition determined by Gas chromatography-mass spectrometry (GC/MS) showed a predominance of palmitic and stearic acids. Furthermore, we found the presence of several sterols which cholesterol and sitosterol are the most abundants. Keywords: Antioxydant activity, Chemical composition, Cinachyrella tarentina, Marine sponge, Polyphenols.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Marine natural products are drugs obtained from marine organisms that have been studied since ancient times. The oceans cover most of the earth's surface and are home to a vast diversity of species, many of which are still unknown. Marine organisms produce unique biochemical adaptations for survival in extreme conditions that could provide benefits for pharmacology. However, issues like limited supply, taxonomic identification challenges, and screening large numbers of microbes associated with invertebrates present obstacles for drug development from marine sources. Improved genetic engineering, databases, and activity-based screening methods may help address these problems and unlock the potential of marine organisms for new pharmaceuticals.
Phenotype and genotype of lactic acid bacteria (LAB) isolated from the tiger ...UniversitasGadjahMada
Lactic acid bacteria (LAB) have been isolated successfully from the tiger grouper Epinephelus fuscoguttatus intestine. However, their genus or species have not been identified. Therefore, the objective of the present study was to characterize the three isolated LAB (KSBU-12C, KSBU-5Da, and KSBU-9) based on their phenotype and genotype. The LAB phenotype was examined by observing morphological features including cell morphology, spore production and motility. The physiological tests were performed in 6.5% NaCl at the temperatures of 10 C and 45 C, and the biochemical tests were evaluated based on the production of enzymes catalase, oxidase and arginine dehydrolase, following the Standard Analytical Profile Index, API 50 CH kit. The genotype was examined based on 16S rDNA gene sequence analysis , and the products were analyzed using the BLAST (Basic Local Alignment Search Tool) NCBI database. The three isolates (KSBU-5Da, KSBU-12C, and KSBU-9) were categorized into the genus Enterococcus. 16S rDNA sequence analysis indicated that the isolates had 99% similarity to E. hirae ATCC 9790, registered in GenBank with accession number NR_075022.1. It was concluded that the three LAB isolates taken from the tiger grouper Epinephelus fuscoguttatus are E. hirae.
Phenotype and genotype of lactic acid bacteria (LAB) isolated from the tiger ...
tesi finale Giulio Scalzotto
1. 1
UNIVERSITA’ DEGLI STUDI DI PADOVA
DIPARTIMENTO DI SCIENZE DEL FARMACO
CORSO DI LAUREA SPECIALISTICA IN CHIMICA E TECNOLOGIA
FARMACEUTICHE
TESI DI LAUREA:
CHEMICAL INVESTIGATION ON PECTINATELLA MAGNIFICA
(Leidy 1851), A FRESHWATER BRYOZOAN
RELATORE: Prof.ssa GABBRIELLA INNOCENTI
CORRELATORE: Assoc. Prof. KAREL ŠMEJKAL
LAUREANDO: GIULIO GAETANO SCALZOTTO
ANNO ACCADEMICO 2013/2014
4. 4
1 ABSTRACT
2
SUMMARY
8
3
Introduction
9
3.1
PECTINATELLA
MAGNIFICA
11
3.1.1
DESCRIPTION
AND
BIOLOGICAL
ASPECTS
12
3.1.2
HABITAT
15
3.1.3
ISOLATION
OF
BIOLOGICALLY
ACTIVE
NATURAL
COMPOUNDS
17
4
AIM
OF
WORK
23
5
EXPERIMEntal
part
24
5.1
MATERIAL
24
6
APPLIED
METHODS
26
6.1.1
Extraction
and
liquid-‐liquid
extraction
26
6.1.2
Separation
of
hexane
part
of
extract
28
6.1.3
Fractionation
of
group
C
obtained
from
hexane
extract
30
6.1.4
Fractionation
of
group
D
obtained
from
hexane
extract
31
6.1.5
Fractionation
of
group
G
obtained
from
hexane
extract
33
6.1.6
Fractionation
of
group
I
obtained
from
hexane
extract
35
6.1.7
The
evaluation
of
purity
of
fractions
obtained
and
possible
identification
of
components
36
6.1.8
Antimicrobial
and
antifungal
activity
of
different
P.
magnifica
extracts
42
7
RESULTS
AND
DISCUSSION
44
7.1
Preliminary
GC-‐MS
analysis
of
selected
fraction
from
n-‐hexane
extract:
44
7.2
Identification
of
fraction’s
component
by
GC-‐MS
47
7.3
Derivatives
of
fatty
acid
54
7.4
Cholesterol
and
derivatives
56
8
CONCLUSION:
63
9
REFERENCES:
64
10
THANKS
TO:
67
5. 5
LIST OFABBREVIATIONS
P.magnifica Pectinatella magnifica
pH - log10 [H3O+
]
Ext X Primary extract from PM
Kr Control of growth (pure bacteria/fungus)
Ks Blank (RPMI only)
Solv DMSO + NaCl 0,9%
A.B/A.F Positive Control.
Antibiotic: Ciprofloxacin
Antifungal: Fungistatic-5-flucytosine
PBS Phosphate Buffered Saline
MHB Mueller Hinton Broth
CFU Colony Forming Units
PTS Petri’s Capsule
RPMI Medium1640
6. 6
1. ABSTRACT:
Il lavoro di tesi è stato svolto, nell’ambito del programma Erasmus, presso il
Department of Natural Drugs, della Facoltà di Farmacia dell’University of
Veterinary and Pharmaceutical Sciences di Brno, Repubblica Ceca, sotto la
supervisione del Prof. Karel Smejkal
Il progetto proposto ha riguardato l’indagine chimica di un organismo di acqua
dolce, nello specifico un briozoa che vive nelle acque del sud della Bohemia
(Repubblica Ceca), chiamato Pectinatella magnifica descritto per la prima volta
da Leidy nel 1851. Poche sono le note disponibili in letteratura riguardanti questo
organismo.
Pertanto si è ritenuto interessante svolgere indagini chimiche su questo organismo
poco conosciuto e studiato con lo scopo di isolare composti a potenziale attività
biologica.
Nello specifico il lavoro di tesi si è basato principalmente sull’estrazione,
separazione ed identificazione di composti lipofili da un esemplare di Pectinatella
magnifica, raccolto nel lago Hnevkovice, nel sud della Boemia.
Per quanto riguarda la fase di estrazione, è stata impiegata la tecnica della
macerazione, successivamente l’estratto ottenuto è stato estratto con solventi a
polarità crescente, al fine di ottenere una prima separazione dei componenti in
funzione della loro diversa lipofilia/idrofilia.
L’estratto lipofilo (n-esano) è stato successivamente frazionato mediante varie
tecniche cromatografiche. Le numerose frazione ottenute sono state esaminate per
GC-MS.
Inizialmente è stato utilizzato un gascromatografo disponibile presso il
Dipartimento del Prof. Smejkal dell’Università di Brno; in seguito, a causa dei
vari problemi sorti durante le analisi è stata chiesta la collaborazione del Prof.
Emil Svajdlenka, docente della Facoltà di Farmacia di Bratislava, Slovacchia. Le
analisi gas cromatografiche preliminari eseguite a Brno, sono state utilizzate a
scopo orientativo per selezionere le frazioni più interessanti da analizzare con il
GC-MS di Bratislava. Attraverso le analsi GC-MS è stato possibile identificare i
7. 7
composti mediante confronto degli spettri di massa con quelli presenti nella
libreria dello strumento.
Successivamente, in collaborazione con la Prof.ssa Marcela Nejezchebová del
Dipartimento di Microbiologia dell’Università di Brno, è stata valutata l’attività
microbiologica dei diversi estratti ottenuti da P. magnifica, contro lo
Staphylococcus Aureus e Candida Albicans,
Gli estratti non hanno dimostrato alcuna attività antibatterica.
8. 8
2 SUMMARY
The objective of my work was to carry out a initial research, directed to the
detailed study on Pectinatella magnifica. The experiments were carried out during
my Erasmus studies at Department of Natural Drugs, Faculty of Pharmacy,
University of Veterinary and Pharmaceutical Sciences Brno, in Brno. The
supervisor specialist of this research was Dr. Karel Šmejkal, Ph.D.
I followed a step by step procedure typical for the primary chemical screening of
unknown species: 1. the extraction to obtain all the substances from an organism,
2. several chromatographic purification steps and consequently 3. characterization
and identification of possible active compounds by chromatographic techniques.
This organism has not been studied previously from the chemical investigation’s
view. There is very little knowledge about the life cycle and biology of this
species. P. magnifica could be possible thread for sweet potable water sources and
therefore the information about composition, secondary metabolites and possible
toxicity should be elucidated.
9. 9
3 INTRODUCTION
The Bryozoa, also known as Polyzoa, Ectoprocta or commonly as moss animal,
are a phylum of aquatic invertebrate animals that almost always live in colonies.
Typically about 0,5 millimetres (0,020 in) long, they are filter feeders that sieve
food particles out of water using a retractable lophopore, a “crown“ of tentacles
lined with cilia. Most marine species live in tropical waters, but few occur in
oceanic trenches, and others are found in polar waters. One class lives only in a
variety of freshwater environments, and few members of a mostly marine class
prefer brackish water. Over 4000 living species are known. One genus is solitary
and the rest colonial.
Individuals in bryozoan (ectoproct) colonies are called zooids, since they are not
fully independent animals. All colonies contain autozooids, which are responsible
for feeding and excretion. Colonies of some classes have various types of non-
feeding specialist zooids, some of which are hatcheries for fertilized eggs, and
some classes also have special zooids for defense of the colony.
Colonies take a variety of forms, including fans, bushes and sheets, and maybe
mistaken for hydroids, corals, or even seaweeds.
Predators of marine bryozoans include nudibranchs (sea slugs), fish, sea urchins,
pycnogonids, crustaceans, mites and starfish. Freshwater bryozoans are preyed on
by snails, insects, and fish. In Thailand, many populations of one freshwater
species have been wiped out by an introduced species of snail. A fast-growing
invasive bryozoan off the northeast and northwest coasts of the USA has reduced
kelp forests so much that it has affected local fish and invertebrate populations.
Bryozoans have spread disease to fish farms and fishermen.
Yet bryozoans produce a remarkable variety of chemical compounds, some of
which uses in medicine.
Specially several alcaloids were isolated from the marine species such as
alogenated indole alkaloids, amide derivatives, isoquinolines, diterpene and
polichetides derivatives compounds. (Blunt et al, 2005,2006,2007)
10. 10
Were isolated and studied some indole alkaloids derivated from marine
bryozoans.
One compound produced by a common marine bryozoan (Bugula neritina), the
drug bryostatin 1 is currently under serious testing as an anticancer drug even if is
still in phase II clinical trials. (Mendola, 2000).
Figure 1: molecule structure of bryostatine 1.
11. 11
3.1 PECTINATELLA MAGNIFICA
Pectinatella magnifica is the Latin name of organism that is part of Bryozoan
Phylum and is an invasive freshwater organism with the ability to produce large
colonies. Particularly Bryozoans are marine organisms, although freshwater forms
are moderately common.
This organism it has been discovered and described by Leidy in 1851 and it is a
rare kind of Bryozoa.
There are just nineteen types of freshwater Bryozoa and one of those is P.
magnifica found in a freshwater environment but there are even other kind as
Plumatella repens, Plumatella rugosa, Plumatella emarginata and Fredericella
sultana.
P. magnifica is able to form the biggest colonies among other kinds of Bryozoans.
The only thing common for P. magnifica and marine Bryozoans is the life in
similar ecological system that it has been described in the Coral Reefs.
Experimental group observed in Connecticut increasing of clear water and the
explanation is very easy, because the individual zooid are able to remove particles
from the water, the immediate result of their greater occurrence in non-native
waters is to increase water quality.
During a preliminary study by Alice W.Wilcox in 1906 she discovered that young
colonies in a early stages has an independent motion system.
When two or more young P.magnifica organisms are moving in the same
direction there is possibility to fuse together forming what we called “moss
animal” or just a colony. (Alice W.Wilcox, 1906)
Therefore the average measure is around 20 centimeter for the normal one, fused
together they could be bigger than one alone.
12. 12
3.1.1 DESCRIPTION AND BIOLOGICAL ASPECTS
Pectintella magnifica is well known through un-academic names as magnificent
bryozoan or “moss animal” or “the blob”. It is permanent organism showing some
degree of growth during all the year, it does not need particular condition to grow,
the location only on a branches of willow tree in fresh water to grab it. The
viscosity of exterior skin is similar to viscosity of gelatine, slurry, or mucilage as
well. It has a translucent body with a lot of star-stuff (white) along with the
outside. It can grow up till 2 meter of diameter with a increase of percentage of
external-surface covered with white star-stuff. The gelatinous skeleton of a young
colony hardens, colonies may fuse their masses together and form mosaic colonies
from more than one genotype.
To illustrate how Pectinatella colonies look like, please see the real photos that
are attached:
Figure 2: P. magnifica colony (zoids on the surface)
13. 13
Figure 3: P. magnifica colony
Figure 4: P. magnifica colony floating by the branch
14. 14
Figure 5: P. magnifica colony, single zooid are perfectly visible.
15. 15
3.1.2 HABITAT
This organism can be found locations of South Bohemia (Czech Republic), in
Connecticut (North America), in Michigan (United States) and Aichi (Japan).
They grow up in lakes or in rivers, demanding the fresh water. It grows in water
and takes advantage of the branches of plant around it, grabs it and floats, what is
the mechanism of P. magnifica life.
The water temperature is evidently an important factor affecting the seasonal
dynamics of occurrence; in fact, P. magnifica grows up from +16 to +20 °C.
The conditions important for expansive growth are different in accordance with
the place where they are, in Michigan's lake and river were found in the water P.
magnifica together with Fredericella sultana, Plumatella repens, in water richly
supplied with Oxygen (O2, 6.3-6.7 centiliter dissolved O2 per liter), with a less
amount of carbon dioxide (CO2) and with a neutral/basic pH between 7,6-8,4.
The warm water is essential to grow well overall for statoblasts, they were
germinated well in reduced presence of Oxygen but the statoblast keep in the
water with a small amount of Oxygen was not affected. With the H-Ion
concentration on the statoblast's germination they got unsatisfactory results.
(Brown and Claudeous J.D. 1933).
The differences in nitrogen and phosphorous content between the water outside
and inside of the colonies were statistically significant. The colonies also
accumulate other elements, including micro-elements. The tentacles are able to
actively catch the particles and remove it from the surrounding water. This is the
only useful function that has been described till present - to clean and remove
particles from water system. The particles caught could be even elements, during
an experiments the different approach of three kind of Bryozoans to the metallic
particles was observed.
Lophodella carteri, Plumatella emarginata and Pectinatella magnifica have been
tested under conditions of different metals spectrum and concentrations. The
experiment was carried out 96 hours to establish lethal concentration (LC50) of
Copper (Cu), Cadmium (Cd), Chromium (Cr) and Zinc (Zn). The final LC50 for
Cu, Cd, Cr and Zn are Lophodella carteri 0.51, 0.15, 1.56 and 5.63 mg/L, for
16. 16
Plumatella emarginata 0.14, 1.09, 0.65 and 5.30 mg/L and for P. magnifica 0.14,
0.70, 1.44 and 4.31mg/L, respectively. The conclusion of this test was that the
bryozoan organisms are more sensitive to the metals than many other
invertebrates, plants and fishes. (Pardue, W.Jeffrey,1980)
Figure 6: P. magnifica feeding ciliated system and zooids
17. 17
3.1.3 ISOLATION OF BIOLOGICALLY ACTIVE NATURAL
COMPOUNDS
Isolation of active compounds is one of the main targets we follow at the
beginning of an organism study. If we want to analyze and test the activity of a
single compound, we need to be sure about the material quality.
Extraction, isolation, characterization and chemo-taxonomic studies, are four
basic phases to find new active compounds that could be used as a disease's
treatment or remedy.
Secondary metabolites play roles different and various. Between primary and
secondary metabolites there are consequent relations because the secondary
metabolites are based and derived only and uniquely from the primary
metabolites, such as sugars, fatty acids, amino acids and others, not the inverse
even if secondary metabolites are not necessary for plants life. They are present in
lower quantities than primary metabolites. The specific conditions which are
necessary for production of the secondary metabolites are not totally clearly
elucidated.
Secondary metabolites we usually distinguish are terpenoids, alkaloids and other
nitrogen containing compounds and sterols.
Each metabolite does their mansion, but it is difficult to establish the role of
secondary metabolite in an organism because it is not necessary for the organism
life. Moreover, secondary metabolites are multifunctional.
Probably first and most important function of secondary metabolites is to protect
and defend the cellular organism from offenders.
18. 18
3.1.3.1 Extraction process
The extraction process is an important point of obtaining of natural compounds
and every mistake could affect the further results.
The purpose of the proper extraction procedures for crude drug is to obtain the
therapeutically active portion and eliminate the inactive material by treating them
with a selective solvent known as the Menstruum.
To make an extraction there are a lot of possibilities, changing in accordance with
type of extracted material.
During the 19th
century there was no progress and knowledge of extraction from a
natural material although it has been used for a long time to make medicinal
drugs.
Techniques of extraction as I mentioned have been traditionally used for making
galenics from medicinal and aromatic plants.
There are different factor that could influence the choice of an extraction process,
as the nature and the stability of the drug (crude), type of solvent and the
concentration of the analytes in product.
Maceration was the suitable compatible technique in accordance with the
physical characteristics of P. magnifica, and due to the fact there is no information
available about chemical composition of the species.
The general maceration process consist of placing the crude and crushed material
into a vessel and adding the selected solvent to soak the material totally with an
excess of solvent. Shaking and checking it if the material is under solvent is
necessary. The liquid just extracted by squeezing the material may be cloudy with
small particles so to improve the quality of the extraction process we have to use a
filter.
Repeated process may be more efficient than a single process because some
residual substances may be left behind during the first process.
The liquid-liquid extraction main advantage is the good capacity and the
impossibility to have an irreversible adsorption. It is easy to apply without special
equipment and the solvent could be changed as possible in accordance with
19. 19
character of compounds. It has a disadvantage - operation is manual, not
automatic, and the ability of separation is limited.
There are several combination for the solvent could used for a liquid/liquid
extraction, to make this better we can check the most suitable solvent just with a
thin layer chromatography. With TLC, making a comparison with different
solvents.
20. 20
3.1.3.2 Separation process
Following the extraction phase it is necessary to continue with separation. The
principal separation process is chromatography, word derives from Greek: χρῶµα
chroma means "color" and γράφειν graphein means "to write".
The history of chromatography began in Russia, in the early of XX century,
Mikhail Semyonovich Tswett described on 30 December 1901 the first
chromatographic technique during research carried out on chlorophyll.
He described the process based on liquid adsorption column containing calcium
carbonate to separate plant pigments in 1903.
The term “chromatography” appeared for the first time in 1906 in two papers
about chlorophyll he published in the German Botanical Journal. (Touchstone,
Joseph C., 1993)
Nobel Prize in Chemistry arrived in 1952 for Archer John Portor Martin and
Richard Laurence Millington Synge for their invention of chromatography
(partition). After the award, this technique became well known and the progress
advanced very quickly.
There are a lot of chromatographic techniques. I worked with two different
chromatographies: a gravity column chromatography and thin layer
chromatography.
The principles of chromatographic separation could be based on adsorption
techniques, ionic exchange, affinity, or size exclusion. Different techniques are
using different ways of interactions: for example dipole/dipole interactions,
hydrogen bonds etc.
The components of chromatographic technique are mobile phase and stationary
phase. The stationary phase could has two different interaction that is attractive or
not attractive permeation, so the first one mean adsorption, partition or electronic
attraction and the second one separation by the form and dimension (measure).
There are some critical point which operator has to respect and pay attention on
them to prevent the instability of compounds and degradation of column material
because we could find some contaminated particles of material from degradation
of the packaging's material.
21. 21
The mobile phase is a chromatographic component that passes through the
stationary phase and serves as a carrier for separated compounds. In dependence
on the type of chromatography, the proper mobile phase could be based on the
polarity. The list of various solvents in order of elution power for a given
adsorbent is called “elutropics series”.
The following table (Table 1) shows a typical elutropics series:
Dipole Parameter Proton Acceptor Proton Donor
Pentane 0 0 0
Hexane 0 0 0
Isopropylether 0,5 0,5 0
Isopropylchloride 3 0 0
Toluene 0 0,5 0
Benzen 0 0,5 0
Chloroform 3 0,5 3
Acetone 5 2,5 0
Ethyl-acetate 3 2 0
Pyridine 4 5 0
Propanol 2,5 4 4
Ethanol 4 5 5
Methanol 5 7,5 7,5
Water Large Large Large
Table 1: List of different solvent’s polarity
22. 22
An important factor to have a good results of separation is the length of the
separation path (column or plate), the longer represents higher number of
theoretical plates of column and improves the process.
The retention time is value representing the time which compound spend in the
system, calculated from the time of injection of a sample to the record of the peak
(band) maximum of the component in the chromatogram.
The most commonly used stationary phase for chromatography is silicagel.
Silicagel is polar substance based on silicate polymer. It is usually used for normal
phase chromatography. It is relatively cheap. It is an inert material that shouldn't
make interaction with the sample, but sometimes some irreversible adsorption or
oxidation occurs.
The one important thing to have a good separation is to assure the solubility of the
sample in mobile phase. The sample should not change in mobile phase. Samples
could degrade under influence of oxygen and light.
The proper mobile phase for column chromatography is selected using TLC.
There is possibility of usage of preparative TLC to control the quality of
separation process, or for direct separation of small amounts of samples.
23. 23
4 AIM OF WORK
Approximately one third of today´s best selling drugs are either natural products
or have been developed based on lead structures provided by nature. Traditionally
higher plants used to be the most prolific sources of drugs from nature. Looking at
drugs from nature it is surprising that up to now almost all medicinally used
natural products or derivatives thereof were obtained from terrestrial organisms
rather than from those inhabiting the sea or lake. Their diversity remains largely
unknown and underexploited.
In recent years aquatic organisms, have been emerging as an important source for
the production of bioactive compounds.
The aim of this work was carried out chemical examination on Pectinatella
magnifica extracts. P. magnifica is a bryozoan species invading the sweet waters
of central Europe (Czech Republic, south Bohemia). There were not present
specific note in the consulted literature therefore it has been interesting looking
for biologically active compounds.
The work thesis was carried out during my Erasmus program in Brno (Czech
Republic) under supervision of Professor Karel Smejkal.
In this thesis work is reported the identification, by GC-MS, of some occurring
compounds in a lipophilic extract (n-hexane) from P.magnifica. Furthermore, the
antimicrobical activities of four main fractions were also evaluated.
24. 24
5 EXPERIMENTAL PART
5.1 MATERIAL
P. magnifica colonies used in our experiments were obtained from sweet water
lakes of Southern Bohemia (Hnevkovice lake). The material was combined to get
mixed sample. P. magnifica was identified by Associated Professor Josef
Rajchard, PhD. (University of South Bohemia in České Budějovice, Faculty of
Agriculture).
The solvent used were methanol, ethanol, ethylacetate, aceton, benzen,
chloroform, diethylether of p. a. quality (Penta, Czech Republic) hexane of HPLC
quality (SIGMA-ALDRICH, USA).
The precise pipettes were two different with different pipetting range:
• 100-1000 µL LLG Micropipette (Germany)
• 10-100 µL BIOHIT m100 (Finland)
The consumable material like plastics vials and tubes were from EPPENDORF
A.G (Germany).
For the preparative chromatography was used typically two different silicagel
particle sizes:
• Si-40 (25-40 µm)
• Si-60 (40-63 µm)
Two different TLC plates were used:
1 - TLC Preparative Plates 20×20 cm 500 microns, UV F254 (ANALTECH,
Germany).
25. 25
2 - Analytical TLC (Aluminum Sheets) 20×20 cm SilicaGel 60 F254 200 microns
(MERCK, Germany).
Gas Chromatograph in Brno (CZ) is from Finnigan (USA):
• GCQ MAT EI Ion Trap
Gas Chromatograph in Bratislava (SVK) is from Agilent Technologies (USA):
• GC 7890A
• Autosampler 7683B
• MS 5975C VL MSD with Triple-Axis Detector
Gas Chromatography's service offered by Bratislava University curated by
Professor Emil Svadjlenka.
Microbiology experiments were done on PST (Petri’s capsule) microtiter plate
with 96 flat bottom wells (ROLL s.a.s., Italy), using Staphylococcus aureus
(Mueller-Hinton Broth, OXOID), Candida albicans and RPMI-1640 Medium,
(SIGMA ALDRICH, Germany).
26. 26
6 APPLIED METHODS
6.1.1 Extraction and liquid-liquid extraction
There is probably no difference between different parts of P. magnifica “bodies”,
therefore we used whole mass. The external jelly skin is composed of
approximately from 99 % of water.
The zooids could probably contain the active substance of the organism, but we
are not sure about that. It is not possible to clarify for now, where the possible
substances are localized, therefore the maceration method was used and all parts
of the organism were macerated together.
The first process was an extraction procedure carried out on Pectinatella
magnifica. I prepared a solution composed of 90 % of MeOH and 10 % H2O. The
solution was added to the extracted material. The process of extraction was
repeated three times for 24 hours to obtain an exhaustive extraction. The solvent
was each time used fresh.
The extracts were combined than filtered and concentrated under vacuum. The
residual water was removed by lyophilization.
The dry extract was dissolved again in 90 % of MeOH and 10 % H2O and using
separatory funnel was extracted with hexane in overall ratio 1:1. The hexane layer
was removed; the process was repeated three times. The combined hexane
portions were evaporated to give hexane part of extract ( Extract A).
The methanol portion was concentrated using rotavapor to remove large part of
methanol. The residue was diluted with water and repeated extraction with
chloroform (3 times) was used to obtain combined chloroform portion (Extract C).
The water part was then three times repeatedly extracted with ethyl-acetate. The
combined ethyl-acetate extracts after removing of solvent on Rotavapor yielded
ethyl-acetate portion (Extract E).
The water from residue was removed by lyophilization and later water portion
was yielded (Extract F).
27. 27
Scheme 1: extraction phase from the crude P. magnifica.
Percentage yield has been calculate as a fraction’s amount of initial extract than a
lyophilized extract.
28. 28
6.1.2 Separation of hexane part of extract
The separation works started with the material of hexane extract. I have carried
out several attempts using TLC to discover the most suitable mobile fraction for
the later preparation of column chromatography on silicagel.
First I have checked various combinations with accurate method taking advantage
of TLC while testing different mobile phase combination, till find the most
suitable mixture. The tested mixtures have been following:
chloroform/ethylacetate 50:50 (v/v); chloroform/acetone 60:40 (v/v);
hexane/acetone 60:40 (v/v); hexane/acetone 50:50 (v/v); benzene/acetone 50:50
(v/v); benzene/acetone 60:40 (v/v); benzene/acetone 70:30 (v/v); benzene/acetone
80:20 (v/v); benzene/ethylacetate 70:30 + 0,1 mL formic acid (v/v);
benzene/ethylacetate 80:20 + 0,1 mL formic acid (v/v); benzene/ethylacetate
70:30 (v/v); benzene/ethylacetate 80:20 (v/v); benzene/ethylacetate 50:50 (v/v).
To increase the possibility to find a proper and most suitable mixture we can add
the formic acid increasing the polarity of mobile phase.
The best mobile phase for separation of hexane extract was found to be
hexane/acetone 60:40 (v/v). To remove all residues of material from the silica gel
at the end of separation it is good use the polar solvent, in this case it was pure
acetone.
The size of the column was:
• Length: 63 cm
• Diameter: 40 mm
To prepare the column, it is usual to use the relation between mobile phase and
stationary phase is 1:1 but it depends on the consistence and viscosity of the
slurry. The amount of S.P used was almost 320 g. After application of sample to
the column, I collected sixty one fractions, named from the hexane extract
(fraction A). Later, TLC analysis using proper mobile phase was used to combine
fractions with the same content, using UV detection (λ 254 and 365 nm). To
improve the detection, I used spraying of TLC plate with a reactive mixture
29. 29
composed of sulfuric acid and diethylether, TLC later heated for three minutes at
100 °C. The spots were than observable under normal light.
A 7-10 N 34 and 35
B 11-13 O 36
C 14-16 P 37-41
D 17 R 42 and 43
E 18 S 44-48
F 19 T 49-51
G 20-23 U 52-53
H 24-26 V 54
I 27-29 Z 55-57
L 30-32 Z' 58-61
M 33
Table 2: Combined groups obtained from hexane extract
The hexane extract yielded 21 combined groups (A-Z’), based on TLC analysis.
Groups C, D, G, and I were selected for further study.
30. 30
6.1.3 Fractionation of group C obtained from hexane extract
The amount of material of group C materials (derived from hexane fraction) was
not sufficient to prepare a separation using column chromatography, therefore a
repeating preparative TLC on silica using glass plate was carried out. The extract
amount was 0,782 g.
The proper mobile phase for this TLC separation was mixed up from hexane :
acetone in ratio 80:20 (v/v). The TLC was carried out on plates of 20 × 20 cm.
The sample was applied to the column using the capillary.
After development and drying time, the spots (strips) of separated compounds
were observed and marked using detection under UV (λ 366 nm and 254 nm). The
silicagel containing separated compounds was scratched from TLC plate, was
transferred into vials and extracted with MeOH : CHCl3 (1:1, v/v). The process
was repeated for three times and each time we will use new vials to maintain
quality of the sample.
The amount of final material obtained at the end of repeated separation was
relatively small, the fractions were later analyzed using GC-MS to identify the
compounds. This separation was co-worked with student Patricia Slotová.
31. 31
6.1.4 Fractionation of group D obtained from hexane extract
Group D materials from hexane extract was sufficient enough to prepare a
chromatographic column.
The size of the column was:
• Length: 72 cm
• Diameter: 26 mm
The stationary phase was silicagel 40-63 mm (almost 105 g) and almost the same
amount of mobile phase was used to prepare a slurry.
First I have checked various combinations taking advantage of TLC while testing
different mobile phase combination, till find the most suitable mixture. Following
the tested mixture has been chloroform/ethylacetate 50:50 (v/v);
chloroform/ethylacetate 60:40 (v/v); hexane/acetone 60:40 (v/v); hexane/acetone
50:50 (v/v); hexane/acetone 70:30 (v/v); benzene/acetone 60:40 (v/v);
benzene/acetone 60:40 + 0,1 mL formic acid (v/v); benzene/acetone 70:30 (v/v);
benzene/acetone 90:10 (v/v); benzene/ethylacetate 50:50 + 0,1 mL formic acid
(v/v); benzene/ethylacetate 60:40 + 0,1mL formic acid (v/v); benzene/ethylacetate
80:20 + 0,1 mL formic acid (v/v); benzene/ethylacetate 90:10 (v/v).
To increase the possibilities to find a property and most suitable mixture we can
add the formic acid increase the polarity of mobile phase.
The best one for this fraction it has been found to be benzen/ethylacetate 90:10
(v/v).
To remove all residues of material from the silica gel at the end of separation it is
good use the polar solvent, in this case it was pure methanol.
After the application of sample’s powder extract into column, the fraction of 150
ml volume were collected and later combined according to the TLC analyses and
similarity of present spots position and color.
32. 32
We collected 57 fractions and they were combined according to the following
table (3):
D-A 4-5 D-M 21
D-B 6 D-N 22
D-C 7 D-O 23
D-D 8-10 D-P 24-25
D-E 11 D-Q 26
D-F 12 D-R 27
D-G 13-14 D-S 28
D-H 15-16 D-T 29-30
D-I 17-18 D-U 31-57
D-L 19-20
Table 3: Combined groups obtained from fraction D (hexane extract)
33. 33
6.1.5 Fractionation of group G obtained from hexane extract
The amount of material of group G of hexane extract was sufficient enough to
prepare a chromatographic column.
The size of the column was:
• Length: 83 cm
• Diameter: 23 mm
The stationary phase was silicagel 40-63 mm (almost 120 g) and almost the same
amount of mobile phase was used to prepare a slurry.
It's not simple to find a correct mobile phase, and the mean of “correct” is the
mobile phase as similar as well to the perfect mobile phase.
First I have checked various combinations taking advantage of TLC while testing
different mobile phase combination, till find the most suitable mixture. Following
the tested mixture has been chloroform/ethylacetate 50:50 (v/v);
chloroform/ethylacetate 60:40 (v/v); hexane/acetone 60:40 (v/v); hexane/acetone
50:50 (v/v); hexane/acetone 70:30 + 0,1 mL formic acid (v/v); benzene/acetone
50:50 + 0,1 mL formic acid (v/v); benzene/acetone 60:40 + 0,1 mL formic acid
(v/v); benzene/acetone 70:30 + 0,1 mL formic acid (v/v); benzene/acetone 90:10 +
0,1 mL formic acid (v/v); benzene/ethylacetate 50:50 + 0,1 mL formic acid (v/v);
benzene/ethylacetate 60:40 + 0,1mL formic acid (v/v); benzene/ethylacetate 80:20
+ 0,1 mL formic acid (v/v); benzene/ethylacetate 90:10 + 0,1mL formic acid
(v/v).
To increase the possibilities to find a property and most suitable mixture we can
add the formic acid increase the polarity of mobile phase.
The best one for this group it has been found to be chloroform/ethylacetate 50:50
(v/v).
To remove all residues of material from the silica gel at the end of separation it is
good use the polar solvent, in this case it was pure ethylacetate.
After the application of sample’s powder extract into column, fractions of 150 ml
volume were collected and later combined according to the TLC analyses and
similarity of present spots position and color.
34. 34
We collected 28 fractions and they were combined according to the following
table (4):
G-A 1
G-B 2
G-C 3
G-D 4
G-E 5
G-F 6 and 7
G-G 8 - 15
G-H 16 - 28
Table 4: Combined groups obtained from fraction G (hexane extract)
35. 35
6.1.6 Fractionation of group I obtained from hexane extract
The amount of material of group I was not sufficient to prepare a separation using
column chromatography, therefore a repeating preparative TLC on silica using
glass plate was carried out. The weight of the fraction was 0,686 grams.
We used the similar procedure as for the fraction C.
First I have checked various combinations with accurate method taking advantage
of TLC while testing different mobile phase combination, till find the most
suitable mixture. Following the tested mixture has been chloroform/ethylacetate
50:50 (v/v), chloroform/aceton 50:50(v/v); hexane/aceton 60:4(v/v);
hexane/aceton 50:50(v/v); benzen/aceton 50:50(v/v); benzen/aceton 60:40(v/v);
benzen/aceton 70:30(v/v); benzen/aceton 80:20(v/v); benzen/ethylacetate 70:30 +
0,1mL formic acid(v/v); benzen/ethylacetate 80:20 + 0,1mL formic acid(v/v);
benzen/ethylacetate 70:30(v/v); benzen/ethylacetate 80:20(v/v);
benzen/ethylacetate 50:50(v/v).
To increase the possibilities to find a property and most suitable mixture we can
add the formic acid increase the polarity of mobile phase.
The best mobile phase suitable for separation of this fraction it has been found to
be benzene/acetone 50:50 (v/v).
The best mobile phase used to make a preparative TLC (glass-silica) for the group
I it has been benzen/ethylacetate 70:30(v/v) + 0,1mL formic acid.
36. 36
6.1.7 The evaluation of purity of fractions obtained and possible
identification of components
There are several ways how to analyze the final purity of obtained material, or
possibly to make the identification of compounds isolated. The simplest way is to
use a TLC to separate the obtained fractions, if one spot only is detected, the
material could be pure. Different mobile phases and systems for detection could
be used to improve the value of this evaluation. My results were not clear and
even mostly of the spot on the TLC were not so defined for that reason we
decided to do step by step.
Furthermore, a HPLC analyses could be used, if there are compounds separable.
There is one condition; the compounds must be observable using the present
technique of detection (in our case UV/Vis). In the case of our separations, there
were no compounds visible under UV, therefore we did not decide to use a
HPLC/DAD.
The one of remaining techniques is a gas chromatography.
Gas chromatography is a branch of chromatography where the mobile phase is
vapor/gas. Specifically this involves a sample being vaporized and injected into
the head of the chromatographic column, where the sample is transported through
the column by the flow of inert, gaseous mobile phase. To maintain a close and
pressurized system we have to inject, manually or automatically, only with a
syringe through a plastic septum.
The column itself contains a liquid stationary phase which is adsorbed onto the
surface of an inert solid.
The main requirement to do it is the volatility of the substances understudied. The
substance inject into the machine is heated and been volatile before get in a glass
column, where the separation is.
The type of career used depends just for the detector in the GC, in my case, I used
only mass spectral detector.
Two different sets of measurements were carried out using GC-MS. First was the
preliminary analysis to determine the purity of analyzed material.
37. 37
Secondly, fractions were analyzed to compare the obtained MS spectra with
spectral database to identify the compounds present in fractions.
Method 1:
Method 2:
Three different variants with variable injection and time of analysis were used for
obtaining chromatograms to read MS spectra of main chromatogram peaks:
Method: KAREL_SPLIT10.M
GC:
Injector: T ij. 260 °C, Pressure 107.29 kPa, Septum purge flow 3 ml/min, Total
flow 14 ml/min, Split ratio 1:10.
Oven: T oven 80 °C, 1 min hold time, 15 °C/min, 320 °C, 3 min final time, Run
time 20 min, He, Vacuum compensation ON, Solvent delay time 4 min,
Equilibration time 0.25 min. Column: J &W 19091S-433N: 001, HP-5MS
BATCH: USA592536H, Max. temp. 325 °C: 30 m × 250 µm × 0.25 µm film
thickness.
Detector MS: Scan 29-1000 m/z, T trans. line to MS 280 °C, MS source 230 °C,
MS Quad 150 °C.
Method: KAREL_SPLIT100_0-2_LONG.M
GC:
Injector: T ij. 260 °C, Pressure 107.29 kPa, Septum purge flow 3 ml/min, Total
flow 14 ml/min, Split ratio 1:100
Oven: T oven 80 °C, 1 min hold time, 15 °C/min, 320 °C, 3 min hold time, 15
°C/min, 325 °C, 9.667 min final time, Run time 30 min, He, Vacuum
compensation ON, Solvent delay time 4 min, Equilibration time 0.25 min
Column: J &W 19091S-433N: 001, HP-5MS BATCH: USA592536H, Max. temp.
325 °C: 30 m × 250 µm × 0.25 µm film thickness.
Detector MS: Scan 29-1000 m/z, T trans. line to MS 280 °C, MS source 230 °C,
MS Quad 150 °C
38. 38
Method: KAREL_SPLIT10_0-2_LONG.M
GC:
Injector: T ij. 260 °C, Pressure 107.29 kPa, Septum purge flow 3 ml/min, Total
flow 14 ml/min, Split ratio 1:10
Oven: T oven 80 °C, 1 min hold time, 15 °C/min, 320 °C, 3 min hold time, 15
°C/min, 325 °C, 9.667 min final time, Run time 30 min, He, Vacuum
compensation ON, Solvent delay time 4 min, Equilibration time 0.25 min
Column: J &W 19091S-433N: 001, HP-5MS BATCH: USA592536H, Max. temp.
325 °C: 30 m × 250 µm × 0.25 µm film thickness.
Detector MS, Scan 29-1000 m/z, T trans. line to MS 280 °C, MS source 230 °C,
MS Quad 150 °C.
The chromatography's analysis could be improved in different ways; of course we
have to respect and follow Van Deemter equation. According to those parameters
to have a better changes we can modify the dimension of column's material or
using one column more packed. The length of the column is fundamental, because
the separation happen along it, so as long as possible it means better separation of
single structures contained in the extract analyzed. 60 meters is the length of the
column used in Bratislava and I took advantages from this useful component.
As I wrote above, as long as better resolution, I obtained several accurate data.
39. 39
Every substances studied and analyzed in Bratislava are matched with the
database named NIST05 and Wiley7 library working on software Enhanced
ChemStation MSD E.02.00.493 Copyright 1989-2008 Agilent Technologies, Inc
The identified compounds in the different fractions were reported in table 5.
Table 5: identified compounds by their mass spectra through to the comparison
with a database.
Sample's Name Retention Time Compound % of accuracy
G-B 11,05 Tetradecanoic acid 99
G-C 11,75 Pentadecanoic acid 99
12,50 Hexadecanoic acid 99
13,06 Heptadecanoic acid 96
13,60 Octadecanoic acid 99
G-D 12,42 Hexadecanoic acid 99
13,10 Heptadecanoic acid 99
13,66 Octadecanoic acid 99
15,02 Unidentified compound 0
16,48 7-oxo-cholesterol 96
G-F 14,88 Unidentified compound
16,44 7-oxo-cholesterol 93
17,77 Unidentified compound
18,10 Unidentified compound
18,36 Unidentified compound
I-C2 11,03 Tetradecanoic acid 96
11,34 Unidentified compound
42. 42
6.1.8 Antimicrobial and antifungal activity of different P. magnifica
extracts
The last part of the work was touching the antifungal and antibacterial activities of
P. magnifica extracts. There are several methods to test the ATB or antifungal
activity; we decided to use microplate dilution method.
Extracts after being dissolved in dimethyl sulfoxide, were serially diluted using
0,9% saline and transferred in quadruplicates to 96-well flat-bottom micro plates.
Extract tested were four main extracts called respectively extract A, C, E and F.
6.1.8.1 Antifungal activity
• Candida albicans inoculum was prepared by picking 1 to 2 colonies from
agar plates and re-suspending them in ∼5 ml RPMI 1640. The optical
density of the suspension was adjusted to the 1,5 × 108
CFU.ml-1
.
• The culture was grown at 37 °C in a rotary shaker for12 hours.
• Then culture was diluted using 0,9% saline to afford final target inoculum
of 4,5 × 108
CFU.ml-1
.
• 20 µl of this inoculum was added to 0,98 ml RPMI and shaken.
• 0,5 ml of this inoculum was added to 9,5 ml RPMI.
The fungal inoculum was re-suspended with a multichannel pipette to the samples
to achieve a final volume of 100 µl. The highest extract concentration was 128
µg.ml-1
. Other concentrations were 64 µg.ml-1
, 32 µg.ml-1
, 16 µg.ml-1
. Control of
growth (Candida albicans and RPMI) and blank (RPMI only) were included on
each test plate. Fungistatic 5-Flucytosine (1 µg.ml-1
) was included as positive
control. Growth was monitored by measuring the absorbance at 600 nm in micro
plates reader (BMG Reader Labtech, Germany) at 37 °C for 0 to 48 hours. Control
measurement of growth of C. albicans after 120 hours (5 days) was performed
using absorbance at 600 nm in micro-plate reader too. Quantitative comparison of
absorbance gives the relative percent growth related to control of growth. Data
points are averages of quadruplicates with S.D.
43. 43
6.1.8.2 Antimicrobial activity
• Staphylococcus aureus ATTC 29213 inoculum was prepared by picking 1
to 2 colonies from agar plates and re suspending them in ∼5 ml MHB. The
optical density of the suspension was adjusted to the 1.5 × 108
CFU.ml-1
.
• 100 ul of this inoculum was added to 5 ml MHB and shaken.
• The culture was grown at 37o
C in a rotary shaker for 3-3,5 hours.
• Culture was diluted using PBS to afford final target inoculum of 3 × 108
CFU.ml-1
.
• 1 ml of this inoculum was added to 4 ml MHB and shaken (6 × 107
CFU.ml-1
).
•
S. aureus ATTC 29213 inoculum was re-suspended with a multichannel pipette to
the samples to achieve a final volume of 100 µl. The highest concentration was
256 µg.ml-1
. Other concentrations were 128 µg.ml-1
, 64 µg.ml-1
and 32 µg.ml-1
.
Control of growth (S. aureus ATTC 29213 and MHB) and blank (MHB only)
were included on each test plate. Bactericide Ciprofloxacin (1 ug.ml-1
) was
included as positive control. Growth was monitored by measuring the absorbance
at 600 nm in micro-plate reader (BMG Reader Labtech, Germany) at 37°C for 0
to 24 hours. Control measurement of growth of S. aureus ATTC 29213 after 144
hours (6 days) was performed using absorbance at 600 nm in micro-plate reader
too. Quantitative comparison of absorbance gives the relative percent growth
related to control of growth. Data points are averages of quadruplicates with S.D.
44. 44
7 RESULTS AND DISCUSSION
As visible from text above, I performed the extraction, liquid/liquid extraction and
column chromatographic separation of hexane part of P. magnifica extract. Later,
I performed other columns chromatography and preparative TLC with aim to
isolate pure content compounds.
7.1 Preliminary GC-MS analysis of selected fraction from n-hexane
extract:
I analyzed, by GC-MS, fifty three samples of fractions obtained from column
chromatography and preparative TLC of P. magnifica hexane portion of extract. I
obtained chromatograms of each one, later I decided to report here that showing
one or maximally two peaks, so I wasted that that contained several and confusing
mass of peaks. These chromatograms represent fractions with low purity to be
used for analyses or identification, or for the biological activity testing.
As an example I attach some clear chromatograph from my samples.
Chromatograms are showing at axis X retention time and at axis Y total ion
current (showing highest peak as 100 %).
45. 45
Figure 7: obtained from fraction C sample 4.
Unfortunately, in several cases it was possible to obtain chromatograms showing
the same “peaks-situation”, because has the same compound composition of
analyzed fraction.
Figure 8: obtained from fraction D sample E.
46. 46
Figure 9: obtained from fraction G sample B.
Figure 10: obtained from fraction I sample A2.
47. 47
7.2 Identification of fraction’s component by GC-MS
I analyzed by GC-MS in collaboration with University of Bratislava, twenty five
fractions, chosen from fifty three fractions analyzed in the preliminary screening.
The purity check and identification was performed using GC-MS and comparison
of obtained mass spectra with a library.
Six decanoic acids were identified with five cholesterol derivatives.
The identified compounds are reported in the table 5.
As an example I report the related chromatograms on fraction C-C-3’.
Figure 11: obtained from fraction C sample C-3’
4.00 6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.00
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
Time-->
Abundance
TIC: 211.Ddata.ms
17.765
19.219
19.551
20.015
20.262
20.700
48. 48
The separated peaks in the chromatogram has been identified compare their mass
spectrum obtained (black) with the mass spectrum contained on the instrument’s
library (blue).
Peak at R.T 19,219 min.
Figure 12: mass spectra correspond to cholesterol
Peak at R.T 19,551 min
0 50 100 150 200 250 300 350 400 450
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
m/z-->
Abundance
Average of 19.195 to 19.238 min.: 211.Ddata.ms (-)
386
275
301
145105
35343 213
81
255
173
326
233
431 470407 491
0 50 100 150 200 250 300 350 400 450
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
m/z-->
Abundance
#544919: CHOLEST-5-EN-3-OL $$ CHOLEST-5-EN-3-OL (3.BETA.)- $$ 17-(1,5-DIMETHYLHE
386
275
55 81 301107
145
213 353
178 247
32632
419441 499469
54. 54
7.3 Derivatives of fatty acid
Lipids are a large group of natural compounds which includes fats, waxes, sterols,
fat-soluble vitamins (such as vitamins A, D, E and K), phospolipids and other.
They play many biological functions including energy storage, structural
components of cell membranes, and signalling molecules.
Although humans and other mammals use various byosynthetic pathways to both
break down and synthesize lipids, some essential lipids can be obtained only from
diet (Bernal,J. Et al, 2011).
Decanoic acid or commonly called capric acid is a saturated fat acid and is
naturally contained in coconut oil or palm kernel oil.
There are several derivatives of n-decanoic acid in P. magnifica as proved by the
analysis done
From the latin name's derivation, deca-noic it means ten carbons molecule.
55. 55
Figure 24: Decanoic Acid(10)
The derivatives are tridecanoic acid (13 carbons), tetradecanoic acid (14 carbons),
pentadecanoic acid (15 carbons), hexandecanoic acid (16 carbons), heptadecanoic
acid (17 carbons), octadecanoic acid (18 carbons).
Figure 25: Octadecanoic acid (18)
All the derivatives of decanoic acid are contained in a plant or animal as so called
fatty acids and they don't have specific activity.
56. 56
7.4 Cholesterol and derivatives
Cholesterol is very important molecule, it is a compound contained in animal cell
membranes required to create and maintain permeability and fluidity and is
present within lipid particles. Cholesterol is a very important component of
biological membranes as well as precursor of steroid hormones, bile acid salts and
vitamin D. This component is necessary for the life of the normal organism.
Figure 26: Cholesterol
Different sterol derivatives were isolated from P. magnifica. Theoretically there
are around 256 isomers although just 10% has activity at all.
Campesterol, stigmasterol, cholesterol-7-oxo and other molecules are found
during the experiment, each molecule has different activity for example
campesterol is a sterol contained in vegetable and fruit as banana, cucumber,
onion, potato.
57. 57
Campesterol compete with cholesterol to reduce the quantitative of cholesterol
absorbed in human body (intestine).
Figure 27: Campesterol
Stigmasterol has a similar structure than an animal cholesterol contained in soya
seeds, rape seed, calabar been. This sterol could be useful in prevention of
ovarian, prostate, colon and breast cancer, but is not certified yet. By the way,
stigmasterol is an organic compound widely in the vegetal fat, particularly in
Calabar’s seeds.
Figure 28: Stigmasterol
58. 58
I found even another sterol named Crinosterol.
Figure 29: Crinosterol
These three sterols has been already found in marine sponges from the indian
ocean. (Gauvin,A, 1998)
The last similar structure than a normal animal cholesterol is a Cholesterol-7-oxo.
Figure 30: Cholesterol-7-oxo
Cholesterol-7-oxo is a oxygenated sterol previously isolated from two marine
sponges (Riccardis et al, 1993) and more recently from CCl4 extract of the marine
59. 59
bryozoan Cryptosula pallasiana together several new and known sterol
derivatives. (Tian et al, 2011)
Tian and coll (2011) evaluated the cytotoxicity of isolated sterols against HL-60
human myeloid leukemia cell line and all the studied compounds exhibited a
moderate toxicity to HL-60 cells except for the cholesterol-7-oxo.
60. 60
7.4.1.1 Results of antibacterial and antifungal activity assays
Using the methodology described above, we analyzed possible antibacterial and
antifungal activity of different P. magnifica extracts. Extracts have been measured
using series of decreasing concentration to obtain MIC. As visible from Figures
X-Y, we did not discovered promising antibacterial or antifungal activity of P.
magnifica in concentration tested. All plate wells treated with P. magnifica
showed absorption similar to positive control of growth, it means there is no
visible growth inhibition of bacterial/fungal growth.
The diagram showing optical density of antibacterial assay well plat
Figure 31: Candida albicans (show absorption versus time of C.A. Touching
Fungistatic 5-Flucytosin)
61. 61
Figure 32: showing absorption versus time of incubation of S. aureus. Positive
control is ciprofloxacin showing no growth of bacteria.
Figure 33: Candida albicans: (show disposition of each compounds in the growth
plate for C.A)
Figure 34: Staphylococcus aureus: (show disposition of each compounds in the
growth plate for S.A)
62. 62
In the graphs above, we can observe there is no interaction between extract from
P. magnifica and Staphylococcus aureus/Candida albicans.
Theoretically if there is bacterial/fungal growth it means there is absorption and if
there is, when we make an addition of antibacterial compounds, the bacteria do
not grow, should be arrested.
In case the antibacterial/fungal block the bacteria growth they do not make
absorption, it means no visible absorption, no colonies and no light ray
disturbance. To make a comparison, is possible compare the bacterial/fungal with
growth control (solvent only, or untreated wells) or with positive control
(antibiotic). As we can see in the figure 31, 32, 33 and 34 there is absorption
(Optical Density, logarithmic ratio of the radiation falling upon a material), it
means there are not activities with the extract from Pectinatella magnifica.
These results of bacterial inactivity could be in accordance with what observed by
Okamura (2001).
Proliferative Kidney Disease (PKD) cause the disease in salmonid fish and
making an idea of the parasite called Tetracapsuloides bryosalmonae.
This parasite is included in the natural host of the myxozoan parasites.
T. bryosalmonae is a complete name of this myxozoan parasite of salmonid
fishes, that used a bryozoan organism as host to replicate and live instead of an
alternative host in a oligochaete or orpolychaete worm. That is an unusual parasite
but is one of the most parasitic cause of disease on salmonid population in North
America and in Europe with causes of, approximately, 90% infected fishes.
T. bryosalmonae is able to infect all the oganism, from primative to recent
bryozoan as Plumatella repens, Plumatella rugosa, Plumatella emarginata,
Fredericella sultana. This kind of organism could has picked up in different
habitat, cold, warm, dry, humid and in eutrophic lakes. Seeing P. Magnifica and
bryozan species are an invasive organisms we have to pay attention if is growing
in a freshwater containing salmonid fish for the reason cited above.
(Okamura,Beth;Anderson, Cort L., 2001)
I want to thanks for avaibility of Professor Marcela Nejezchebová, she followed
me during this experiment and she explain me everything i needed as well.
63. 63
8 CONCLUSION:
This work was carried out on analysis of Pectinatella maginifica hexane extract.
P. magnifica is a bryozoan species invading the sweet waters of central Europe
(Czech Republic, south Bohemia). The work was carried out during the Erasmus
studies at Dept. of Natural Drugs, Faculty of Pharmacy, UVPS Brno, Czech
Republic.
Several chromatographic methods, including column chromatography, TLC,
preparative TLC and gas chromatography in tandem with mass spectrometry were
applied to isolate and identify compounds present in hexane part of crude extract
of P. magnifica.
I used common sequence of methods: TLC to obtain good mobile phase for
separation, column for separation of higher amounts of material, and preparative
TLC for final purification. GC-MS was used to analyze the purity of obtained
fractions and with help of MS to elucidate the identity of present compounds.
Main content compounds found in P. magnifica are cholesterol and sterol
derivatives, and plant sterols like stigmasterol and crinosterol. Furthermore, a
series of fatty acids was also isolated.
Besides, the antimicrobial activity of the main extracts (hexane, chloroform,
ethylacetate, water) was analyzed using Staphylococcus aureus and Candida
albicans. As showed above, no antimicrobial activity of extracts of P. magnifica
was proved using micro-dilution assay.
The isolation and identification of P. magnifica compounds will continue with
aim to identify present secondary metabolites.
64. 64
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