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Purification and characterization of hyaluronic acid from chicken combs

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Hyaluronic acid (HA) is an important macromolecule in medical and pharmaceutical fields. The umbilical cord and the chicken comb are some of the tissues richest in this polysaccharide. The profit from obtaining HA from the combs of slaughtered animals is particularly attractive. This work aimed to extract, purify, and characterize HA. The glycosaminoglycan concentration in the chicken comb was found to be about 15µg of hexuronic acid mg-1 of dry tissue. Fractionation using ion exchange chromatography and subsequent identification of the fractions by agarose gel electrophoresis showed that HA corresponded to 90% of the total amount of extracted glycosaminoglycans.
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1682
Rosa et al.
Ciência Rural, v.42, n.9, set, 2012.
Ciência Rural, Santa Maria, v.42, n.9, p.1682-1687, set, 2012
ISSN 0103-8478
Claudia Severo da Rosa
I
Ana Freire Tovar
II
Paulo Mourão
II
Ricardo Pereira
II
Pedro Barreto
III
Luiz Henrique Beirão
III
Purification and characterization of hyaluronic acid from chicken combs
ABSTRACT
Hyaluronic acid (HA) is an important
macromolecule in medical and pharmaceutical fields. The
umbilical cord and the chicken comb are some of the tissues
richest in this polysaccharide. The profit from obtaining HA
from the combs of slaughtered animals is particularly attractive.
This work aimed to extract, purify, and characterize HA. The
glycosaminoglycan concentration in the chicken comb was
found to be about 15 g of hexuronic acid mg
-1
of dry tissue.
Fractionation using ion exchange chromatography and
subsequent identification of the fractions by agarose gel
electrophoresis showed that HA corresponded to 90% of the
total amount of extracted glycosaminoglycans.
Key words: glycosaminoglycans; chicken combs, hexuronic acid.
RESUMO
O ácido hialurônico (AH) é uma importante
macromolécula nas áreas médica e farmacêutica. O cordão
umbilical e a crista de frango constituem uns dos tecidos mais
ricos nesse polissacarídeo. O aproveitamento das cristas dos
animais abatidos para a obtenção de HA é particularmente
atraente. O presente trabalho teve como objetivo a extração,
purificação e caracterização do AH. A concentração de
glicosaminoglicanos encontrada na crista de frango foi ao redor
de 15 g de ácido hexurônico mg
-1
de peso seco. O fracionamento
por cromatografia de troca iônica e a subsequente identificação
das frações por eletroforese de gel de agarose mostrou que o AH
corresponde a 90% do total de glicosaminoglicanos extraídos.
Palavras-chave: glicosaminoglicanos, co-produtos de frangos,
ácido hexurônico.
INTRODUCTION
Poultry production is one of the most
important industries in Brazil. In the first trimester of
2011, Brazil produced 1.306 billion chickens (IBGE, 2012).
Among the countries in the area, Brazil is the third
largest chicken producer in the world market. To
maintain success, it is necessary to invest in ways to
support low-cost productivity. Moreover, it is
necessary to pay special attention to the environment,
highlighting the importance of profiting from using the
remains from the poultry industry.
The chicken comb is rich in hyaluronic acid
(HA) and, being a part of the remains, is discarded with
the head to make grease. HA belongs to the
glycosaminoglycan (GAG) group, which consists of
anionic heteropolysaccharides composed of long, non-
ramified and repetitive disaccharide units. HA contains
a hexosamine (N-acetyl-D-glucosamine) and uronic acid
(D-glucuronic acid). It differs from other GAGs because
it does not have N-/O- sulfate groups distributed in its
disaccharide units and is not a proteoglycan (HANDEL
et al., 2005).
HA is an essential component of the
extracellular matrix of vertebrates, and it is also
produced by viruses, bacteria and mushrooms. It has
several functions, such as joint lubrication and
extracellular matrix hydration, and is involved in tumor
I
Departamento de Tecnologia e Ciência dos Alimentos, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil. E-
mail: claudiasr37@yahoo.com.br. *Autor para correspondência.
II
Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho (HUCFF), Universidade Federal do Rio de
Janeiro (UFRJ), Rio de Janeiro, RJ, Brasil.
III
Departamento de Ciência dos Alimentos, Universidade Federal de Santa Catarina (UFSC), Florianópolis, SC, Brasil.
μ
μ
Purificação e caracterização do ácido hialurônico obtido da crista de frango
Received 11.23.10 Approved 06.05.12 Returned by the author 07.04.12
CR-4426
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Purification and characterization of hyaluronic acid from chicken combs.
Ciência Rural, v.42, n.9, set, 2012.
progression, inflammation and regeneration
(ALMOND, 2007).
Studies have shown that the hyaluronic acid
is not only a lubricant with dermatologic and
ophthalmologic applications but also can be used in a
control system for drug release, as in anesthesia
prolongation in bones and joints (GOLDENHEIN et al.,
2001), arthropathy treatment (SUZUKI et al., 2001),
chemotherapeutic agents in surgical implants
(AEBISCHER et al., 2001), drug release in dental caries
(SUHONEN & SCHUG, 2000), controlled antigen release
for immunotherapy (PARDOLL et al., 2001), and contact
lenses (BEEK et al., 2008), and as a copolymer with
anti-thrombotic properties in vascular applications (XU
et al., 2008).
The main dermatological application of
hyaluronic acid is growing soft tissues through
intradermic injections to correct skin problems caused
by wrinkles, scars, lip enlargement or other defects
(MANNA et al., 1999; INGLEFIELD, 2011).
HA was first isolated from vitreous humor
by Meyer and Palmer in 1934. It is a high molecular
weight polysaccharide (10
6
-10
7
Da) with quite a high
turnover rate as a component of the cellular matrix. It is
catabolized by the enzyme hyaluronidase. Its principal
natural sources include the chicken comb, umbilical
cord, vitreous humor, and synovial fluid (DEVLIN, 2000;
ALMOND, 2007). This work aimed to extract, purify
and characterize hyaluronic acid from the chicken comb
of 48-day-old male and female chickens.
MATERIALS AND METHODS
Source material
Chicken combs were provided by the Pena
Sul slaughterhouse (Caxias do Sul, RS). Forty kilograms
of combs were collected from a 50:50 population of 48-
day-old male and female chickens. The combs were
submerged in hot water and then frozen at -18
o
C until
the experiments were performed. The combs were
analyzed without gender distinction. The trials were
conducted at the Laboratory of Connective Tissue at
the University Hospital Fraga Filho at the Federal
University of Rio de Janeiro.
Extraction of the total glycosaminoglycans from the
chicken combs
The combs were crushed and placed in
acetone for dehydration and delipidation.
Subsequently, they were dried and weighed (100g) for
each extraction (n
o
3). As a first step in the extraction,
delipidation was conducted in a chloroform and
methanol solution (2:1, v/v) for 24h at 25ºC. The tissues
were dried and hydrated in digestion buffer (100mM
sodium acetate pH 5.0, 5.0mM cysteine and 5.0mM
disodium-EDTA) in a ratio of 2.0mL of buffer to 100mg
of dry tissue. After hydration (24h at 4ºC), a solution of
papain in digestion buffer (20mg mL
-1
) was added in
the ratio of 0.5mL to 100mg of dry tissue. The mixture
was incubated (24h at 60ºC), centrifuged at 3200rpm
for 30min, and the supernatant was removed. The pellet
was discarded. Then, 10% CPC was added to the
supernatant in the ratio of 0.2mL to 100mg of dry tissue
and left for 24h at 25ºC. The sample was centrifuged
(3200rpm / 30min), the supernatant was discarded, and
the pellet washed with 3.0mL of 2.0M NaCl and absolute
ethanol (100:15 v/v). Absolute ethanol (2:1, v/v) was
added, and the mixture was incubated (24h at -16ºC).
Next, centrifugation was performed (3200rpm / 30min),
the supernatant was discarded, and the pellet was
washed once with 10mL of 80% ethanol. The solution
was centrifuged again (3200rpm / 30min), the
supernatant was discarded and the pellet was dried
(24h at 25ºC). The final solid was re-suspended in 5mL
of distilled water, and the total content of the GAGs
was measured by the hexuronic acid percentage in the
solution using a carbazole reaction (DISCHE, 1946).
Fragmentation of the glycosaminoglycans
The glycosaminoglycans from the chicken
comb (~500 g in hexuronic acid) were applied to a
Mono-Q column coupled to a FPLC system, equilibrated
using 20mM Tris-HCl (pH 8.0) and submitted to a NaCl
(0 to 1.5M) linear gradient in the same buffer. The column
had a flow of 1mL min
-1
, and 0.5mL fractions were
collected. They were evaluated by the content of
hexuronic acid (carbazole reaction) and the
metachromasia produced by the glycosaminoglycans
sulfated in the presence of 1.9-dimethylmethylene blue
(FARNDALE et al., 1986). The salt concentration was
measured by the conductivity. The fractions containing
glycosaminoglycans, as indicated by the uronic acid
dosage, were gathered and precipitated with 3 volumes
of absolute ethanol.
Agarose gel electrophoresis
The total glycosaminoglycans samples
from the chicken comb and the fractions obtained
through the ionic exchange chromatography were
applied (~5 g in hexuronic acid) to a 0.5% agarose gel
prepared in a 50mM diaminopropane (pH 9.0) buffer
and submitted to 110V for almost 1h (DIETRICH &
DIETRICH, 1976). The GAGs in the gel were fixed with
0.1% cetavlon (N-cetyl-N,N,N-trimethylammonium
bromide in water) and then dyed with 0.1% toluidine
blue in acetic acid/ethanol/water (0.1:5:5,v/v/v) to reveal
μ
μ
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Rosa et al.
Ciência Rural, v.42, n.9, set, 2012.
the sulfated glycosaminoglycans. After identifying the
metachromatic fractions, the gel was dyed with 0.005%
Stains-All in 50% ethanol (VOLPI et al., 2005; VOLPI &
MACCARI, 2006). The standards were human thoracic
aorta GAGs and hyaluronic acid (Sigma-Aldrich, USA).
13
C-NMR spectroscopy
The
13
C-NMR spectra were obtained using
a Bruker DPX 400MHz spectrometer. A D
2
O solvent
was used to acquire the
13
C-NMR spectra (reference:
=0ppm, 4-dimethyl-4-silapentane-1-sulfonate).
The experimental parameters used to acquire the
spectra were as follows
Bruker DPX-400: SF 400.13MHz
spectrometer for
1
H and 100.23MHz for
13
C; pulse width
90°: 8.0 s (
1
H) and 13.7 s (
13
C); acquirement time 6.5s
(
1
H) and 7.6s (
13
C); spectral window 965Hz (
1
H) and
5000Hz (
13
C); scanning number 8-32 for
1
H and 2000-
20000 for
13
C, depending on the compost; number of
points: 65536 with digital resolution Hz/point
1
H equal
to 0.677065 (
1
H) and 0.371260 (
13
C); temperature: 50ºC.
RESULTS AND DISCUSSION
GAG concentration in the chicken combs
The powder obtained from the chicken combs
corresponded to ~16% of the net weight. The total
glycosaminoglycan concentration was 15 g of hexuronic
acid mg
-1
of dry tissue. This value is much lower than
that reported by NAKANO & SIM (1989) and NAKANO
et al. (1994), which was 42.1 g of hexuronic acid mg
-1
of
dry tissue. However, in that study, the
glycosaminoglycans were extracted from 52-week-old
animals, while in this study, they were obtained from 48-
day-old animals. NAKANO et al. (1994) reported that
hexuronic acid in the wattle of 52-week-old chickens was
19.1 g mg
-1
of dry tissue. This value is closer to the
amount we found in the chicken combs.
According to NAKANO et al., (1994), the
combs of older males possess greater amounts of
hyaluronic acid. In addition, scalding the combs may
decrease the HA concentration (SZIRMAI, 1956;
BALAZS et al., 1958; SWANN, 1968). Nevertheless,
HA extraction may be worthwhile due to the number of
chickens slaughtered in slaughterhouses.
In the first trimester of 2011, 1.306 billion
chickens were slaughtered in Brazil (data from IBGE
2012). Considering that each chicken comb has an
average of 3 grams of humid weight, the amount of
combs generated by the poultry industry would be
3918 tons. In this study, we obtained 2.0 g of hexuronic
acid mg
-1
of humid tissue extracted from chicken combs.
Because hyaluronic acid corresponded to 90% of the
extracted hexuronic acid, an estimated 7.05 tons of
hyaluronic acid could have been extracted during the
first trimester of 2011. It must be highlighted that
hyaluronic acid has a high market value (US $65.00
100mL
-1
) and is not commercially produced in Brazil
(OGRODOWSKI, 2006). The chicken comb, which is
part of the remains of the poultry industry, is potentially
a great source of hyaluronic acid for use in the medical,
pharmaceutical and cosmetic industries.
Fragmentation of the GAGs extracted by ion exchange
chromatography
The total GAG extract from the chicken
combs was fractionated using a Mono-Q column. The
results are shown in figure 1. We observed a large peak
corresponding to ~90% of the total hexuronic acid in
the sample that eluted with ~400mM NaCl without
showing any significant metachromasia. These
properties are characteristic of HA when it is applied to
this column. We also observed a fraction with
metachromatic properties when eluted with a NaCl
concentration greater than 1M, which indicates the
presence of sulfated GAGs in the analyzed sample.
The non-sulfated glycosaminoglycans did
not change color in the presence of DMB due to the
deprotonation of the carboxyl groups. This finding
shows that other specific factors beyond polymer
charge density, such as sulfate groups, are required
for metachromasia in the presence of DMB. In solutions
with balance between monomer and dimer colorants,
the position of balance does not affect the presence of
sulfated glycosaminoglycans, but the interaction with
monomer and dimer colorants with polyanions
produces a new species of absorption and removes
the solution color. HA interacts with the dimer colorant
to build a new species of absorption or extinguish the
monomers and dimers, creating a new balance with
metachromasia (TEMPLETON, 1988).
Qualitative analysis of the extracted GAGs
To identify the GAG species present in the
fractions recovered using ion exchange
chromatography, the samples and the total extract were
gathered and analyzed using agarose gel
electrophoresis. Figure 2a shows the gel dyed with
toluidine blue, which is used to identify the presence
of sulfated species. For the total extract, we observed
the presence of two bands with metachromatic coloring
and electrophoretic mobility that were similar to
dermatan sulfate and chondroitin sulfate. We also
observed a non-metachromatic fraction migrating
between dermatan sulfate and the heparan sulfate
δ
μ
μ
μ
μ
μ
μ
1685
Purification and characterization of hyaluronic acid from chicken combs.
Ciência Rural, v.42, n.9, set, 2012.
standard. The latter had a strong blue color when using
Stains All, (Figure 2b), indicating that it is hyaluronic
acid. When analyzing the fractions obtained through
ion exchange chromatography, we eluted a greater
quantity of hyaluronic acid using 0.4M NaCl. The
remaining 10% was mainly composed of dermatan
sulfate and chondroitin sulfate chains. Similar results
were found by LAGO et al. (2005) when isolating large
amounts of hyaluronic acid from human umbilical cords;
thus, both umbilical cords and chicken combs are the
main sources of HA.
13
C-NMR spectroscopy
The total amount of GAG extracted from the
chicken combs was identified using
13
C NMR
spectroscopy. The
13
C-NMR spectra were acquired at
100.23MHz in a Bruker DPX400 spectrometer at 50ºC.
The sample was prepared by dissolving 5mg of the solid
in 0.5mL of D
2
O (pH=6.0). The chemical shifts were
measured in relation to the internal standard 4,4-
dimethyl-4-silapentane-1-sulfonate. In the
13
C-NMR
spectra of this sample, we found signals similar to those
previously reported by BOCIEK et al. (1980) and VOLPI
Figure 1 - Fractionation of the GAGs extracted from the chicken combs using ion
exchange chromatography. The fractions were analyzed through
metachromasia ( ) and hexuronic acid content ( ). The horizontal bars
show both fractions.
Figure 2 - Agarose gel electrophoresis of total GAG and fractions recovered
through ion exchange chromatography. (a) Stained with toluidine
blue; (b) Stained with toluidine blue and Stains-All. P – standard,
A
1
– total GAG, A
2
– fraction I, A
3
– fraction II, CS – chondroitin
sulfate, DS – dermatan sulfate.
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Rosa et al.
Ciência Rural, v.42, n.9, set, 2012.
& MACCARI (2003) for HA from other sources under
similar conditions, including pH. LAGO et al. (2005),
working with human umbilical cord, using
13
C NMR
spectroscopy identified carboxyl and acetamide groups
at 173.4 and 144.5ppm, respectively, two anomeric
carbons at 100.1 and 102.7ppm and an acetamide carbon
at 22.1ppm. The signals at 53.9 and 60.4ppm are from the
C2 and C6 carbons of the glucosamine residue. All of
the resonances described support the presence and
purity of HA from the umbilical cord, confirming the
results found in this experiment under similar conditions.
In the
13
C-NMR spectra (Figure 3), the
chemical shifts were determined for the carbons from
the -D-glucuronic acid units, and a signal was observed
at 176.96ppm for COO
-
. The signals were verified at
106.06ppm for C-1, at 83.0ppm for C-4, and at 79.41ppm
for C-5. Signals corresponding to C-3 at 76.70ppm and
C-2 at 75.59ppm were also observed. For the 2-
acetamide-deoxy- -D-glucopyranoside units, a signal
was observed at 177.87ppm for the C-2 carbons, and
we identified signals at 103.44, 85.68, 78.45, 71.62, 63.73
and 57.33ppm corresponding to the C-1, C-3, C-5, C-4,
C-6 and C-2 carbons, respectively. At 25.56ppm, a
characteristic shift for the methylic carbon was
observed. The other signals found in the spectra were
classified as matrix impurities.
CONCLUSION
The results obtained shows that the applied
methodology is effective for extracting and purifying
hyaluronic acid. The quantitative and qualitative
analysis of the GAGs showed that the chicken comb is
predominately composed of HA (90%), with the
presence of sulfated GAGs in a lower concentration
(10%). Thus, the hyaluronic acid obtained from chicken
combs may be used as a co-product from the poultry
industry for research and clinical applications.
Figure 3 -
13
C-NMR {H} spectra of hyaluronic acid in D
2
O at 100MHz.
β
β
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Ciência Rural, v.42, n.9, set, 2012.
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... HA was first isolated from the vitreous bovine humor by Karl Meyer and John Palmer [46], and extracted from rooster combs in subsequent years [47][48][49][50]. Currently, most HA is produced by microbial fermentation using Streptococci bacteria [51][52][53][54][55][56][57][58][59][60], especially Streptococcus zooepidemicus [61][62][63][64][65]. ...
... In addition to purification, ion exchange is reported to be used in fractioning for HA characterization. Rosa et al. [48] used a Mono-Q column to fractionate glycosaminoglycans (GAGs) derived from rooster combs, in which HA corresponded to 90 % of the total amount. Lago et al. [69] characterized HA from umbilical cord, using a Mono Q HR 5/5 Column after a HA purification sequence involving precipitations with CTAB, ethanol and acetone. ...
Article
Hyaluronic acid (HA), or hyaluronan, is a natural polyelectrolyte, ubiquitous in human tissues. Exogenous HA has been a valuable material due to its wide range of medical applications, such as in osteoarthritis treatment, ophthalmic surgery, adhesion prevention after surgeries and wound healing, as well as cosmetic applications. However, to ensure the physicochemical and biological properties, a purity near to 99% is a primary requirement, aiming clinical applications. To achieve this goal, various downstream operations have been used, aiming HA concentration, separation and purification. Precipitation with organic solvents has been a common operation in most purification processes, combined with other downstream operations such as precipitation with quaternary salts, filtration, adsorption and ion exchange. This work presents an updated review of HA purification, emphasizing the performance of the main downstream operations used to achieve highly purified HA, in the period from 1970 to 2019. We conclude that, in the majority of the published works, there is a lack of studies regarding the operational conditions, as well as an absence of the purification percentage development during the processes.
... Moreover, CS was isolated from thornback skate (Raja clavata) by ED using papain combined with chemical hydrolysis using an alkaline hydroalcoholic solution (Murado, Fraguas, Montemayor, Vázquez, & González, 2010). In addition, papain was also employed to extract HA from mollusc bivalve, rooster and chicken combs and wattle (Nakano et al., 1994;Rosa et al., 2012;Volpi & Maccari, 2003). In the isolation of HA from the terrestrial by-products, the tissues were defatted using ethanol followed by delipidation with chloroform and methanol, prior to the hydrolysis using papain. ...
... % (w/w of different fish bones), 14.84 % (dry weight of crocodile cartilage) and 15.05 % (dry weight of shark fins) were obtained when applying this enzyme in the extraction process (Garnjanagoonchorn et al., 2007;Maccari et al., 2015). In contrast, organic solvents such as chloroform and methanol were used prior to the application of papain for the extraction of HA from chicken combs for the separation of proteins and lipids (Rosa et al., 2012). Chloroform was also used without the use of enzyme, as a solvent in the extraction of HA from rooster combs (Boas, 1949;Kulkarni et al., 2018). ...
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Hyaluronic acid (HA) and chondroitin sulfate (CS) are valuable bioactive polysaccharides that have been highly used in biomedical and pharmaceutical applications. Extensive research was done to ensure their efficient extraction from marine and terrestrial by-products at a high yield and purity, using specific techniques to isolate and purify them. In general, the cartilage is the most common source for CS, while the vitreous humor is main used source of HA. The developed methods were based in general on tissue hydrolysis, removal of proteins and purification of the target biopolymers. They differ in the extraction conditions, enzymes and/or solvents used and the purification technique. This leads to specific purity, molecular weight and sulfation pattern of the isolated HA and CS. This review focuses on the analysis and comparison of different extraction and purification methods developed to isolate these valuable biopolymers from marine and terrestrial animal by-products.
... Over a period of 10 years, an academic effort to experimentally improve the extraction performance of HA was noted; however, not all livestock residues were found to have significant volumes and have been used for the extraction of hyaluronic acid. [133] The biomedical applications of hyaluronic acid include its use in ocular and plastic surgery, the treatment of osteoarthritis, corneal xerosis in anti-aging products, and tissue culture, as well as in carriers for different osteo-inductive or osteogenic components [123,124,132]. HA has been also applied for a wide range of pharmaceutical purposes, such as the design of nanoparticles, microparticles, microspheres, gels, polyplexes, liposomes, micelles, and implants [124,132]. ...
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The large-scale industrial use of polysaccharides to obtain energy is one of the most discussed subjects in science. However, modern concepts of biorefinery have promoted the diversification of the use of these polymers in several bioproducts incorporating concepts of sustainability and the circular economy. This work summarizes the major sources of agro-industrial residues, physico-chemical properties, and recent application trends of cellulose, chitin, hyaluronic acid, inulin, and pectin. These macromolecules were selected due to their industrial importance and valuable functional and biological applications that have aroused market interests, such as for the production of medicines, cosmetics, and sustainable packaging. Estimations of global industrial residue production based on major crop data from the United States Department of Agriculture were performed for cellulose content from maize, rice, and wheat, showing that these residues may contain up to 18%, 44%, and 35% of cellulose and 45%, 22%, and 22% of hemicellulose, respectively. The United States (~32%), China (~20%), and the European Union (~18%) are the main countries producing cellulose and hemicellulose-rich residues from maize, rice, and wheat crops, respectively. Pectin and inulin are commonly obtained from fruit (~30%) and vegetable (~28%) residues, while chitin and hyaluronic acid are primarily found in animal waste, e.g., seafood (~3%) and poultry (~4%).
... Hyaluronic acid is an acidic polysaccharide of animal and bacterial origin that serves various physiological purposes, for example, in connective tissues of vertebrates and bacterial capsules [1][2][3][4]. It is an alternating co-polymer of 1,4-linked β-D-glucuronic acid and 1,3linked N-acetyl-β-D-glucosamine. ...
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Hyaluronic acid, together with collagen, vitamins or plant extracts, is a part of many cosmetic and food preparations. For example, this polysaccharide is used in formulation of many food supplements due to its protective effects on human health. In this work, the screening of the chemical composition of three chosen dietary supplements (powder, tablets and capsules) containing hyaluronic acid was carried out using Fourier-transform infrared spectroscopy. Because of the low amount of analyte in all these samples, it was isolated or concentrated prior to the analysis using a suitable sequential fractionation protocol. Individual isolation procedures were established for each sample based on their declared composition. Firstly, the major components such as collagen or vitamins were removed to obtain polysaccharide fractions by the enzymatic treatment and/or washing out with the appropriate solvents. In some cases, the water insoluble part was removed from the rest dissolved in water. Then, hyaluronic acid was precipitated with copper(II) cations and thus separated from the other polysaccharides. Finally, the analyte was identified in the enriched fractions by the characteristic vibrational bands. The amount of hyaluronic acid in the purified fractions was determined in three ways: gravimetrically, spectrophotometrically, and using isotachophoresis. The combination of the appropriate preparative and analytical steps led to the successful evaluation of chemical composition, finding and quantification of hyaluronic acid in all the studied samples.
... However, 6 h digestion period may be adjusted if other sources are to be used. In similar studies, digestion time of 24 h was used in the hydrolysis of the chicken crest (Da Rosa et al., 2007), chicken combs (Da Rosa et al., 2012), with the use of cysteine; and liver of marine stingray (Sadhasivam et al., 2013) with the use of papain. The results of the present study could be reasonable since those abovementioned sources were hard tissues with higher amount of protein component and were therefore expected to have longer digestion period (Calatroni et al., 1969). ...
... Adeyinka et al. (2006) reportaron que los pollos homocigotos NaNa presentan reducción de 40% en la cobertura del plumaje en comparación con las aves normales (nana), mientras que en heterocigotos (Nana) esta reducción es de 20% (Fathi et al., 2013). La cresta es un crecimiento carnoso, rico en ácido hialurónico que algunas especies de aves presentan en la parte superior de la cabeza (Severo da Rosa et al., 2012), y en los gallos criollos es un indicador de la madurez sexual. Navara et al. (2012) observaron que los gallos con cresta colorida y brillante presentaron mejor calidad reproductiva. ...
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Objective: To evaluate the distribution of plumage [naked neck (Na) or normal (NN)] and the type of comb [simple (SC) or rose (RC)] in the progeny of creole roosters (Gallus gallus domesticus L.) with Na and RC. Design/methodology/approach: A completely random design was used, two groups, each one consisting of a rooster with Na and RC, and eight hens NN with SC were used to evaluate the type of neck and comb, and initial weight of the progeny. Results: The 53% of the progeny presented Na and 47% NN, while 81% showed RC and 19% SC. The initial live weight was similar in Na than in NN chickens and similar in RC and SC birds. Limitations on study/implications: In the revised of the literature, little information was found on the progeny of creole chickens with naked neck and rose comb. Findings/conclusions: The results of the present work showed that when crossing roosted naked neck and rose comb with hens normal neck and simple crest in their progeny, can be observed both types of neck and a greater presence chickens with rose comb, without presenting a difference in weight initial. RESUMEN Objetivo: Evaluar la distribución del plumaje [cuello desnudo (CD) o cuello normal (CN)] y tipo de cresta [cresta simple (CS) o cresta rosa (CR)] en la progenie de gallos criollos (Gallus gallus domesticus L.) con cuello desnudo CD y CR. Diseño/metodología/aproximación: Se utilizó un diseño completamente al azar, dos grupos, cada uno conformado por un gallo con CD y CR, y ocho gallinas CN con CS fueron utilizados para evaluar la distribución del plumaje, tipo de cresta, y peso inicial de la progenie. Los datos fueron analizados con una prueba de Chi-cuadrada. Resultados: El 53% de la progenie presentó CD y el 47% CN, mientras que, el 81% mostró CR y el 19% CS. El peso vivo inicial fue similar en pollos CD comparados con CN y similar con CR y CS.
... Previously, HA was extracted from rooster comb. According to a study conducted by Rosa et al. (2012), they concluded that rooster comb was composed of about 90% HA, making it suitable for extraction. However, use of animal-derived biochemical can be risky due to zoonosis and unpremeditated infection transfer. ...
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Cloning and expression of hyaluronan synthase (hasA) in recombinant Escherichia coli BL21 and its hyaluronic acid production in shake flask culture Lai, Z. W., Teo, C. H. Abstract × Aims: Hyaluronic acid (HA) is a high molecular weight polymer and a major component of mucoid capsule in bacteria and extracellular matrix (ECM) of vertebrate tissue. Due to its unique characteristics, HA is used extensively in medical and cosmetic field. However, because of the exotoxins production from animal tissues extraction and Streptococcus zooepidemicus, HA production by recombinant microorganisms has gained interest. The present study was aimed at cloning of hasA gene in Escherichia coli and optimization of the medium components for HA production. Methodology and results: A fragment of an approximate size of 1.5kb that encodes the hyaluronan synthase (hasA) gene from S. zooepidemicus ATCC 39920 was amplified by PCR using hasA-specific primers. The hasA gene was ligated into the bacterial expression vector pLbADH and transformed into the expression host, Escherichia coli BL21 strain. Then, genetically engineered E. coli strain BL21 was used for the production of HA by fermentation using different glucose concentration (10-50 g/L) and different IPTG concentration (0.1, 0.5 and 1.0 mM) in shake flask culture. Amongst varying glucose concentrations, results showed that 50 g/L glucose with nutrient rich media containing nitrogen source was able to produce the highest HA concentration (0.115 ± 0.002 g/L). With addition of 1.0 mM IPTG, HA production reached a peak 0.532 ± 0.026 g/L which is around fivefold higher compared to without IPTG. Conclusion, significance and impact of study: The hasA gene was cloned from S. zooepidemicus and successfully expressed in recombinant E. coli BL21 cells. This low molecular weight HA is gaining more importance in medical and cosmetic application due to possess pronounced free radical scavenging and antioxidant activities.
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Hyaluronic acid (HA), a unique polysaccharide with excellent physicochemical properties, is broadly used in pharmaceutical, biomedical, and cosmetic fields. It is widely present in all vertebrates, certain bacterial strains, and even viruses while it is not found in plants, fungi, and insects. HA is naturally synthesized by a class of integral membrane proteins called Hyaluronic acid synthase (HAS). Thus far, industrial production of HA is carried out based on either extraction from animal sources or large-scale microbial fermentation. The major drawbacks to using these systems are contamination with pathogens and microbial toxins. Recently, the production of HA through recombinant systems has received considerable attention. Plants are eco-friendly ideal expression systems for biopharmaceuticals production. In this research, the optimized human hyaluronic acid synthase2 (hHAS2) sequence was transformed into Nicotiana tabacum using Agrobacterium rhizogenes. The highest hHAS2 production in the tobacco hairy roots was 65.72 ng/kg (wet weight). The extracted HA was verified and quantified by the HA ELISA kit. The DPPH radical scavenging activity of HA with the highest concentration of 0.56 g/kg (wet weight) showed the maximum activity of 46%. Gel Permeation Chromatography (GPC) analyses revealed the high molecular weight HA with about > 0.8 MDa.
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Glycosaminoglycans (GAG) were isolated from the rooster comb and wattle by digestion with papain and analyzed by cellulose acetate electrophoresis and enzymatic digestion. The results indicated that the concentration of GAG uronic acid is approximately two-fold greater (P<.01) in the comb than in the wattle. In both tissues, hyaluronic acid was the major GAG, with a small proportion of dermatan sulfate.
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1. Hyaluronic acid has been prepared quantitatively from the rooster comb under mild conditions, with a yield of 6% of the acetone-dried combs. 93% of this hyaluronic acid was extracted with distilled water, while stronger treatments, 1 M NaCl extraction and digestion with pronase, were required to remove the remainder of the hyaluronic acid from the comb tissues.
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A simple rapid method of quantitative analysis of hyaluronic acid (HA) from comb and wattle tissues is described. The technique involves short-time acetone drying and proteolysis of tissues, cellulose acetate electrophoresis of the digests without deproteinization, and measurement of absorbance of the solution of Alcian Blue extracted from the HA band. With this method, the determination of HA, which may take 1 week or longer with other methods, could be made within a day.
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The main objective of this paper is to discuss new procedures of the isolation of Hyaluronan. Hyaluronic acid can be obtained from human umbilical cord residual, which is obtained from other biopharmaceutical productions. The route involves treatment of human umbilical cord residuals with sodium chloride solution, followed by ammonium quaternary salt solution precipitation; the solid is re-suspended in calcium chloride solution in order to dissociate the hyaluronan ammonium quaternary salt complex followed by ethanol-induced precipitation to give a product. The product was purified four times by chloroform extraction, and characterized by chemical methods such as the Blumenkrantz and Asboe-Hansen uronic technique for uronic acid determination, Elson Morgan qualitative tests for hexosamines, intrinsic viscosity, ion-exchange chromatography, and 13C NMR spectroscopy. The results showed that the product might be used in the formulation of ointment, lotion and cream for the treatment of skin diseases.
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An increasing number of women are seeking minimally-invasive procedures to enhance the shape and volume of their breasts. Early, limited use of Macrolane™ suggests it is a promising agent for non-surgical breast enhancement. To assess the safety and efficacy of Macrolane™ in non-surgical breast enhancement. A prospective report of 194 women presenting for non-surgical breast enhancement at London Bridge Plastic Surgery between November 2007 and August 2009. Safety: Adverse events were reported in a total of 21.1% of patients. Minor adverse events (12%) were mainly confined to product migration, lumpiness, scar pigmentation and breast pain. All events were of mild to moderate intensity and resolved promptly without any additional treatment. Major adverse events (8.7%) included infection, capsular contracture, early resorption and product removal. Efficacy: Efficacy of treatment was assessed by patients using the five-point Global Esthetic Improvement Scale (GEIS). Patient satisfaction with treatment was consistently high with a mean score≥3.3 at all time points during follow-up. Patient-assessed GEIS indicated that some degree of improvement was seen by all (100%) patients at all time points up to and including 12 months irrespective of whether they had been re-treated. At the time of analysis, follow-up data are available for 45% of patients at 12 months, with 19% of all patients presenting for re-treatment with Macrolane™ to date and 5.7% going on to have breast implants. This review represents the largest European clinical experience with Macrolane™ for breast enhancement. It shows that Macrolane™ can provide satisfactory improvement in breast shape. It is associated with high patient satisfaction, and provides a long-lasting result. Follow-up to data have been adequate to identify early complications; however, further follow-up is required to monitor long-term outcomes. The impact of HA on breast cancer remains inconclusive to date.
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The dimethylmethylene blue assay for sulphated glycosaminoglycans has found wide acceptance as a quick and simple method of measuring the sulphated glycosaminoglycan content of tissues and fluids. The available assay methods have lacked specificity for sulphated glycosaminoglycans in the presence of other polyanions, however, and have not discriminated between the different sulphated glycosaminoglycans. We now describe a modified form of the dimethylmethylene blue assay that has improved specificity for sulphated glycosaminoglycans, and we show that in conjunction with specific polysaccharidases, the dimethylmethylene blue assay can be used to quantitate individual sulphated glycosaminoglycans.