therapeutic potential of seaweed bioactive …
TRANSCRIPT
THERAPEUTIC POTENTIAL OF SEAWEED BIOACTIVE
COMPOUNDS AGAINST Helicobacter pylori
T. VinodhKumar1*, G.Ramanathan2, Henson Jebajose3, B. Ambika4
1 Department of Microbiology AVS College of Arts and Science, Salem, Tamil Nadu, India.
2, 4 Post Graduate Department of Microbiology, Sri Paramakalyani College, Alwarkurichi-
627412.
3 Centre for Marine Science and Technology, Manonmaniam Sundaranar University Rajakkamangalam, Nagercoil, Tamil Nadu, India.
Corresponding Author - T. VinodhKumar1
ABSTRACT
The prevalence of H. pylori is closely tied to socioeconomic conditions and accordingly, this
infection is more common in developing countries than in developed countries such as the
United States. Regardless, it has been estimated that 30–40% of the U.S. population is
infected with H. pylori. Antimicrobial resistance is largely responsible for treatment
failures. Resistance to metronidazole can frequently be overcome by increasing the dose
and duration of treatment with acid suppression. Seaweeds or marine macroalgae are the
renewable living resources which are also used as food, feed and fertilizer in many parts of
the world. Seaweeds are of nutritional interest as they contain low calorie food, but rich in
vitamins, minerals and dietary fibres. In these present investigation metabolites from
seaweeds was evaluated for their antihelicobacterial activity. Two Helicobacter isolates
were found in the samples and have been characterized based on selective biochemical
and morphological characteristics. The isolates were screened for their antibiotics
resistance / sensitivity pattern against twelve commercially availed antibiotics. Among the
antibiotics ten of them exhibit sensitivity to the Helicobacter pylori isolates. But the
isolates showed resistance to Nystatin, omeprazole, metronidazole, ranitidine,
trimethoprim and methicillin. There are eleven marine seaweeds were collected from
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Thondy coastal region ant they were screened for antihelicobacterial activity with crude
metabolites. Among the seaweeds screened against both Helicobacter pylori isolates the
seaweed Padina tetrastomatica (13, 16mm), Halmiola sp (9, 12mm) and Gracilaria edulis
(6, 20mm) exhibit better antihelicobacterial activity in in vitro assay. The mass spectral
data of LC- MS report revealed that the peaks indicated, may be the presence of phenolic
compounds and sulphated polysaccharides which were compared based on earlier reports
on seaweeds.
Keywords – Seaweed, Antibiotics, Antimicrobial resistance, Gracilaria
1.0 INTRODUCTION
Peptic ulcer, lesion that occurs primarily in the mucous membrane of the stomach or
duodenum (the upper segment of the small intestine); Between 10 and 15 percent of the
world’s population suffers from peptic ulcer. Duodenal ulcers, which account for 80 percent of
peptic ulcers, are more common in men than in women, but stomach ulcers affect women more
frequently. The symptoms of gastric and duodenal ulcer are similar and include a gnawing,
burning ache and hunger like pain in the mid-upper abdomen, usually experienced from one to
three hours after meals and several hours after retiring. In the early 1980’s Australian
researcher, Barry Marshall challenged previous theories of ulcer development with evidence
that ulcers could be caused by H. pylori. H. pylori exists the world over and its prevalence in the
population increases with age. In developed countries, prevalence increases about 1% per year
of age where it is rare in children, and reaches 70% in the seventh decade. In developing
countries, more than 50% children acquire the infection by the age of 10 years, and more than
80% of the population gets infected by the age of 20 years. In asymptomatic individuals
prevalence of H. pylori infection varies from 31%-84%.
A recent study of 655 subjects from a teaching hospital in Rome found an overall prevalence of
infection of 40%, with a higher prevalence among nurses and auxiliary employees than among
physicians. Recently, H. pylori were defined as a class 1 carcinogen by the World Health
Organization (Honda et al., 1998). Despite of host’s vigorous immune response, the organisms
evade humoral and cellular immune mechanisms and persist even for decades in the stomach
environment (Baldari et al., 2005). The currently most used eradicating treatment strategies
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for H. pylori infection relies on a triple antibiotic therapy, but the cost of these drugs, poor
patient compliance and emerging of antibiotic-resistant strains are some of the drawbacks of
these therapies. In addition successful eradication therapy does not protect the host from re-
infection (Unge 1999). Vaccination against H. pylori becomes an attractive approach for both
therapeutic and prophylactic purpose. Various antigens which known to be involved in the
pathogenesis of the infection have been proposed as suitable vaccine candidates, such as
urease, the cytotoxin-associated antigen (Cag A), the vacuolating cytotoxin (Vac A), the
Helicobacter pylori adhesion A (Hpa A), and others .The urease which is expressed by almost
all H. pylori isolates is one of the most essential enzymes for virulence and colonization of H.
pylori in the gastric mucosa by neutralizing the microenvironment of H. pylori in the stomach
(Porta et al., 1995). This enzyme consists of two major subunits, UreA (26.5 kD) and UreB (61
kD). The UreB is considered as a reliable vaccine candidate antigen and found to be protective
in mice (Smythies et al., 2005).
The development of safe anti- H. pylori compounds is therefore desirable. Studies have
documented that some medicinal plant extracts have antibacterial activities, including H. pylori
(Cowan, 1999; Isogai et al., 2000; Funtogawa et al., 2004; Ndip et al., 2007). The course of
treatment against helicobacteriosis is usually based on classic triple therapy including proton
pump inhibitors and antibacterial therapy, clarithromycin and amoxicillin. For allergic
patients, amoxicillin could be replaced by metrodinazole. The consumption of foods that
inhibit the growth of bacteria may provide an alternative to current therapies or complement
and expedite current treatments (Keenan et al., 2010). Since seaweeds possess various classes
of chemical constituents with pharmacological importance. The present study was initiated to
screen antihelicobacterial efficacy of seaweeds.
MATERIALS AND METHODS
Sample collection
A total of 5 individuals (2 male; age 50-55years, and 3 female; age 23-45years). Those
individuals who had previously received ulceration symptoms in stomach. Gastric juice as
possible 5 to 10 ml was collected from each individuals during early morning episodes of
intermittent reflux which may facilitate the passage viable organisms into the mouth.
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Bacterial isolates
A total of two clinical isolates of H. pylori obtained from the gastric mucosa of patients. It was
plated on Egg Yolk Emulsion agar medium. Suspicious growth was noted; bacteria were sub
cultured and were incubated for 4 days in a micro aerobic gas environment which was handled
in candle jar method. Bacterial growth was identified as H. pylori on the basis of colony
morphology; positive biochemical reaction for catalase, urease, and oxidase and negative Gram
stain.
Collection of seaweeds
Seaweeds (Gracilaria edulis, Sargassum wightii, Bryopsis sp, Jama sp, Caloroba grapes,
Euchoma sp, Chondrococus sp, Padina tetrastomatica, Caloroba sp, Gracilaria sp, Halmiola sp)
samples which were healthy and fully grown and submerged underwater from the tide pools
were collected from Thondi Coastal region Ramnad District. The samples were washed with
seawater and freshwater to remove salt, epiphytic microorganisms and other suspended
materials. The clean algae were frozen. The dry material was stored.
Extraction of bioactive metabolites from marine algae
Organic solvent methanol was used for extraction. Each powdered sample (5g) was soaked in
about 40 ml of the solvent for three days. The resultant crude extracts were filtered and then
concentrated in a rotatory evaporator at a temperature of less than 400C. The residual water
was removed with a vacuum pump. The crude extracts were weighed and deep frozen (-200C)
until testing.
Analysis of phytochemical constituents
The crude extracts from chosen seaweeds were subjected for the qualitative identify of
different classes of natural compounds, using the methodology of Sofowora (1982). The major
phamaceutically valuable phytochemical compounds viz., alkaloids, flavanoids, steroids,
terpenoids, total sugars, total protein, tannin, phenolic compounds, anthroquinone, and
saponins were investigated in the present study.
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Antibacterial activity of seaweed extract
The helicobacterial activity was determined by well diffusion method. Muller Hinton
Agar (MHA) was (3.1 g/100 ml) weighed and dissolved in 100 ml of distilled water in a sterile
conical flask using test organism. The medium was sterilized by autoclaving and was allowed
to cool at room temperature. The medium was poured into the sterile Petri plate. The
inoculated plates were kept aside for few minutes, using well cutter. Two wells are made in
those plates at required distance. In each step of well cutting, the well cutter was thoroughly
wiped with alcohol, using sterilized micropipette, 100μl of methanol solvents with selected
seaweed extract was added into one well and in another well the same volume of
corresponding controls. The plate was incubated at 37°C for overnight. Inhibition of microbial
growth was determined by measuring the diameters of zone of inhibition.
FTIR spectroscopy analysis
The maximum zone producing seaweeds were selected and it was analyzed by FTIR
Spectroscopy method. The seaweed extract samples (10 mg) was mixed with 100 mg of dried
Potassium bromide (KBr) and compressed to prepare as a salt disc. The disc was then read
spectrometrically. The frequencies of different components present in active sample were
analyzed.
HPLC analysis
Data was obtained at 280 nm by using UV-DAD (UV diode array detector). Data consisted of
various peaks for crude extracts with their respective retention times. Analytical grade HPLC
(high performance liquid chromatography) was performed on a Varian 9021 solvent delivery
system equipped with Varian 9065 Polychrom UV-diode array detector (190-367 nm). Data
was processed by Star Polychrom version 5.2. The system was maintained in a controlled room
temperature at 21±10°C. A flow rate of 1ml/min-1 and injection volume of 10 μl were used.
Sample analysis was performed by gradient elution on a 150 mm x 4.6 mm i.d., 5uM, Luna C-
18(2) column (Phenomenex, Australia) with guard column (Phenomenex, Australia). The
mobile phases were freshly prepared and degassed under vacuum using Phenomenex nylon 45
μm membranes and sonicated in a sanophon ultrasonic bath (Ultrasonic Industries Pty. Ltd,
Sydney, Australia) for 15 minutes prior to HPLC analysis.
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LC-MS (Liquid chromatography mass spectrometry)
Liquid chromatography–mass spectrometry (LC–MS) of the methanolic extracts of selected
seaweed species was performed on a Micromass Quattro micro tandem quadrupole mass
spectrometer (Waters, Manchester, UK). LC separation was attained by a Waters liquid
chromatograph (Waters, Milford, USA), consisting of a 2695 Separation Module and 2487 dual
wavelength UV detector operated at 240 and 280 nm. Columns and gradients were same as
described previously for analytical scale HPLC. An injection volume of 10 μL and a constant
flow of 1 ml/min-1 were used for each analysis. The entire flow from the LC was directed into
the mass spectrometer. Data was acquired by the Masslynx data system for both the MS and
UV data.
Molecular mass from LC-MS data
LC-MS chromatograms obtained for two crude extracts were analysed by Masslynx to get
spectra of various peaks. Spectra were recorded in negative and positive ion mode to estimate
molecular masses of various components. Only major peaks in each chromatogram were
analyzed for determination of molecular masses. Determination of molecular mass from LC-MS
data can be a useful tool to identify components on the basis of molecular masses.
RESULT & DISCUSSION
In this present investigation there are two Helicobacter isolates retrieved from five
patients with the Age group of 23-53 was analyzed. Among them 3 female and 2 male all are
susceptible to ulcer symptoms of intermitted reflux. The isolates were subjected for to screen
against commercially availed antibiotics to evaluate their sensitivity / resistant pattern against
12 antibiotics. Among the two strains adopted, the bacterial isolates 2, was more susceptible
than isolate 1. Helicobacter isolate 1 was resistance to Nystatin, Omeprozole, Metronodazole
and Ranitidine. Su et al., (2013) revealed that the resistance rates to clarithromycin,
metronidazole, levofloxacin, amoxicillin, gentamicin and furazolidone were 21.5, 95.4, 20.6,
0.1, 0.1 and 0.1%, respectively. Double, triple and quadruple antibacterial resistant
percentages were 25.5, 7.5 and 0.1%, respectively. A positive association between the
resistance to levofloxacin and to clarithromycin was found, but there was a negative
correlation in the resistances to levofloxacin and to metronidazole.
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Antihelicobacterial efficacy was accessed by crude seaweed extracts, which was
sourced from Thondy coastal region against Helicobacter isolates 1 and 2. It was found that,
among the seaweed extract screened, the seaweeds Gracilaria, Padina and Halmolia exhibit
better antihelicobacterial activity. Among the Helicobacter isolates, the isolate 2 was more
susceptible with seaweed extract. Time kill assay was also performed; it was found that, higher
concentration of seaweed extract of both Padina tetrastomatica and Gracilaria edulis control
the multiplication of the pathogen with increased concentration at 12 hours.
Antihelicobacterial efficacy was accessed by crude seaweed extracts, which was
sourced from Thondy coastal region against Helicobacter isolates 1 and 2. It was found that,
among the seaweed extract screened, the seaweeds Gracilaria, Padina and Halmolia exhibit
better antihelicobacterial activity. Among the Helicobacter isolates, the isolate 2 was more
susceptible with seaweed extract. Time kill assay was also performed; it was found that, higher
concentration of seaweed extract of both Padina tetrastomatica and Gracilaria edulis control
the multiplication of the pathogen with increased concentration at 12 hours.
The potential seaweed extracts which shows significant helicobacterial efficacy was
further evaluated to characterize the active metabolites by FTIR, HPLC and LC-MS analysis
based on that it was indicated that the active principles may be phenolic, sulphated
polysaccharides which is present as major phytochemical constituents in seaweeds. Time hill
assay was performed against H. pylori isolated by using potential seaweed metabolites with 1x,
4x, and 16 x concentrations for 12 hours. Efficiency of reduction in bacterial population was
collected in log CFU for every 3hours incubation. It was found that 16 x concentrations of all
the four seaweed extracts reduced the population considerably. FTIR analysis was followed to
the potential seaweed extract. The peak value reveals the stretching point of the particular
compound which is present in the seaweed extract. Gracilaria edulis have 29 peaks and Padina
tetrastomactica have 31 peaks
Retention times of all the compounds which are presented in the extracts were
detected. The identification of the peaks is based on the analysis of their retention time. The
Retention time of identified peaks for Gracilaria edulis are 1.400, 2.183, 2.303, and 2.753 and
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for Padina tetrastomatica is 1.427, which indicates the presence of halogenated / sulfated
polysaccharides based on earlier reports on seaweed bioactive metabolites. The peak value of
Gracilaria edulis and Padina tetrastomatica were analyzed and showed in. The major peaks
obtained at four different alternative scans. The mass spectral data revealed that the peaks
indicated, may be the presence of phenolic compounds and sulphated polysaccharides which
were compared based on earlier reports on seaweeds.
Padina tetrastomatica Gracilaria edulis
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Conclusion
In humans, Helicobacter pylori colonizes the stomachs of about half of the world’s
population and highly associated with the number of most important disease of the upper
gastrointestinal tract particularly prevalence in developing countries. Eradication of the
H. pylori primarily dependent on continued application of antibiotics. Although effective,
repeated use of these chemical drugs have sometimes resulted in the development of
resistance and had undesirable effects. These problems have highlighted the need for the
development of new strategies for selective H. pylori eradication. Marine plants may be an
alternative source of materials for the H. pylori eradication because they constitute a rich
source of bioactive chemicals. The present study also found that the seaweeds Gracilaria
edulis, Padina tetrastomatica showed antihelicobacterial activity. The importance of
seaweed for human consumption is well known in many countries, they offer a good source
of recovery of various useful chemicals particularly polysaccharide present in most of the
seaweeds may used for the treatment of intestinal and stomach disorders. In this present
investigation Padina tetrastomatica has proven antihelicobacterial activity based on the
outcome of this investigation the active metabolites derived from seaweeds may be an
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alternative therapeutic agent for the treatment of Helicobacter infection but still further
evaluation and complete structural characterization of active metabolites is warranted
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