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.
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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
9 REFERENCES:
Alice W. Wilcox, Locomotion in young colonies of Pectinatella magnifica, 1906
Anderson C.L., Canning E.U., Okamura B., “Molecular Data implicate
bryozoansas hosts for PKX(phylum Myxozoa) and identify a clade of bryozoan
parasites within the Myxozoa”, “Parasitology”, 199(pt 6),555-61, (1999).
Bernal, J.; Mendiola, J.A.; Ibanez, E.Cifutens, Advanced analysis of
nutraceuticals. J.Pharm. Biomed. Anal. 2011
Black, Robert E.;Proud, Virginia K., “Biochemical changes temperature-activated
statoblasts of an ectoproct bryozoan”, “Journal of Experimental Zoology”, 197(1),
141-7, (1976).
Brown, Claudeous J. D., “A limnogical study of certain fresh-water Polyzoa with
special reference to their statoblast”, “Trans. Am. Microscop. Soc., 52,217-316
(1933)
Chandler M. Brooks, Notes on the statoblast and polypoids of Pectinatella
magnifica, 1929
Desser, Sherwin S., Koehler, Anne; Barta, John R.; Kamyab, Jubin; Ringuette,
Maurice J., “Trichonosema Algoquinensis n. sp. (phylum microsporidia) in
Pectinatella Magnifica (bryozoa: phylactolaemata) from Algoquin Park, Ontario
Canada”, “Journal of Eukaryotic Microbiology, 51(4), 389-393, (2004).
Gauvin,A ; Smadja, J; Aknin, M; Gaydou, E-M, “Study of five marine sponges
from the indian ocean”, “Journal of Nature”,10(1), 3-10, 1998.
65. 65
Heinrich, Barnes, Gibbons, M.Williamson – Fundamentals of Pharmacognosy and
phytotherapy, (2004)
John W. Blunt, Brent R. Copp, Murray H. G. Munro, Peter T. Northcote and
Michèle R. Prinsep, “Nat. Prod. Rep”, “Marine natural product”, 2005 ,22, 15-61
John W. Blunt, Brent R. Copp, Murray H. G. Munro, Peter T. Northcote and
Michèle R. Prinsep, “Nat. Prod. Rep”, “Marine natural product”, 2006,23, 26-78
John W. Blunt, Brent R. Copp, Murray H. G. Munro, Peter T. Northcote and
Michèle R. Prinsep, “Nat. Prod. Rep”, “Marine natural product”, 2007,24, 31-86
Liang & Fang, Medicinal chemistry of bioactive natural products, 2006
Onuma, Junichi; Hattori, Yoshiji; Sakai, Shoko, Yoshida, Kyoji; Niwa, Tomoko;
Tanaka, Yasua, “Occurence of Pectinatella Magnifica in Iruka Pound”, “Aichi-
ken Kankyo Chosa Senta Shoho, 29,111-114, (2001).
Mendola, D. Aquacultural production of bryostatin 1 and ecteinascidin 743,
“Drugs from the sea”, 120-133, 2000.
Okaumura, Beth; Anderson, Cort L.; Longshaw, Matthew: Feist, Stephen W;
Canning, Elizabeth U, “Patterns of accurance and 18S rDNA sequence variation
of PKD (Tetracapsula bryosalmonae) the causative agent of salmonid proliferative
kidney disease”, “Journal Of Parasitology”, 87(2), (2001)
P. Brandwein, The culture of Pectinatella magnifica Leidy, 1938
Pardue, W. Jeffrey; Wood, Timothy S., “Baseline toxicity data for freshwater
bryozoa exposed to copper, cadmium,chromium and zinc”, “Journal Of Tennessee
Academy Of Science (55)1, (1980)
66. 66
Riccardis,F.; Minale, L.; Iorizzi, M;Debitus, C; Levi, C. Marine sterols. Side-
chain oxygenated sterols. Possibly of abiotic origin from the New Caledonian
sponge Stelodoryx Chlorofilla. J.Nat.Prod, 56, 282-287, 1993.
S.M.Colegate and R.J.MOLYNEUX, Bioactive natural products - Detection,
isolation & structural determination 2ed, 2007
S.W. Geisnek, Pectinatella magnifica on occasiona river-pest in Iowa, 1937
Sarker, Latif & Gray, Natural products isolation 2ed – TLC In Phytochemistry,
2008
Shinzuo Mawatari, New occurrence of Pectinatella Magnifica Leidy in a
Japanese lake, 1973
Touchstone, Joseph C. ,History of chromatography – Journal of liquid
chromatography, vol 16, no.8 (1993).
Trease & Evans, Pharmacognosy, 2000
Walton and Brown, Chemicals from Plants – Perspectives on Plant Secondary
Products,1999
Xiang-Rong Tian, Hai-Feng Tang, Yu-Shan Li, Hou-Wen Lin, Xiao-Li Chen,
Ning Ma, Min-Na Yao and Ping-Hu Zhang, “New Cytotoxic Oxygenated Sterols
from the Marine Bryozoan Cryptosula Pallasiana”, “Marine Drugs”, 2011.
