The Discovery and Characterization of Riboflavin

Article (PDF Available)inAnnals of Nutrition and Metabolism 61(3):224-30 · November 2012with 3,028 Reads
DOI: 10.1159/000343111 · Source: PubMed
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Abstract
The first observation of a pigment in milk with yellow-green fluorescence can be traced to the English chemist Alexander Wynter Blyth in 1872, but it was not until the early 1930s that the substance was characterized as riboflavin. Interest in accessory food factors began in the latter half of the 19th century with the discovery of the first vitamin, thiamin. Thiamin was water soluble and given the name vitamin B(1). However, researchers realized that there were one or more additional water-soluble factors and these were called the vitamin B-2 complex. The search to identify these accessory food factors in milk, whole wheat, yeast, and liver began in the early 1900s. As there is no classical nutritional disease attributable to riboflavin deficiency, it was the growth-stimulating properties of the food extracts given to young rats that provided the tool with which to investigate and eventually extract riboflavin. Riboflavin was the second vitamin to be isolated and the first from the vitamin B-2 complex; the essential nature of the vitamin as a food constituent for man was shown in 1939.
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Ann Nutr Metab 2012;61:224–230
DOI: 10.1159/000343111
The Discovery and Characterization of
Riboflavin
Christine A. Northrop-Clewes a David I. Thurnham b
a Nutrition Consultant, Cambridge , and
b Northern Ireland Centre for Food and Nutrition, University of Ulster,
Coleraine , UK
Introduction
Riboflavin, also known as vitamin B
2 , is a water-solu-
ble, yellow-orange organic compound in the vitamin B
complex that is required for a number of metabolic ac-
tivities. Plants and many microorganisms are able to syn-
thesize ribof lavin, but animals must get this essential nu-
trient from their diet, e.g. milk, leafy vegetables, whole
grains, liver, egg white, cheese, and fresh meat. Although
needed only in small amounts, riboflavin is essential to
all animals and deficiency is known as ariboflavinosis. In
man, deficiency is associated with cracking of the skin at
the corners of the mouth and fissuring of the lips, swollen
red beefy tongue, corneal vascularization and sensitivity
of eyes to light, itching and scaling of the facial skin.
However, there is no clear associated disease and defi-
ciency has never been fatal.
Riboflavin is the central component of the coenzymes
flavin adenine dinucleotide (FAD) and flavin mononu-
cleotide (FMN), and like the other B vitamins, it plays a
key role in energy metabolism, especially metabolism of
fats, ketone bodies, carbohydrates, and proteins. Almost
all of the flavin coenzyme released by enzyme turnover
is reutilized. It is involved in the support of the immune
Key Words
Riboflavin Lactoflavin Vitamin B
2 Growth promoting
Abstract
The first observation of a pigment in milk with yellow-green
fluorescence can be traced to the English chemist Alexander
Wynter Blyth in 1872, but it was not until the early 1930s that
the substance was characterized as riboflavin. Interest in ac-
cessory food factors began in the latter half of the 19th cen-
tury with the discovery of the first vitamin, thiamin. Thiamin
was water soluble and given the name vitamin B
1 . However,
researchers realized that there were one or more additional
water-soluble factors and these were called the vitamin B-2
complex. The search to identify these accessory food factors
in milk, whole wheat, yeast, and liver began in the early
1900s. As there is no classical nutritional disease attributable
to riboflavin deficiency, it was the growth-stimulating prop-
erties of the food ex tracts given to young rats that provided
the tool with which to investigate and eventually extract ri-
boflavin. Riboflavin was the second vitamin to be isolated
and the first from the vitamin B-2 complex; the essential na-
ture of the vitamin as a food constituent for man was shown
in 1939. Copyr ight © 2012 S. Karger AG, Ba sel
Publish ed online: November 26, 2012
Dr. Chr istine Clewes
46 High Street, Little Wilbra ham
Cambridge CB21 5JY (UK)
E-Mail christinaclewes @ btinternet.com
© 2012 S. Ka rger AG, Basel
0250–6807/12/0613–0224$38.00/0
Accessible online at:
www.karger.com/anm
Stroke Note Cerebrovasc Dis
3
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Riboflavin Ann Nutr Metab 2012;61:224–230
225
and nervous systems, formation of red blood cells, cell
reproduction, and activation of folate and pyridoxine (vi-
tamin B
6 ).
Riboflavin is one of the more stable vitamins, but can
be readily destroyed by ultraviolet rays or sunlight.
H i s t o r y
Accessory Factors in Food and Deficiency Diseases
The need for the major food groups of protein, carbo-
hydrate, and fat to nourish our bodies and of minerals to
maintain the skeleton was appreciated by the latter half
of the 19th century. However, recognition of essential mi-
cronut rients only starte d to u nfold when research started
to unravel the causes of the major deficiency diseases.
Christiaan Eijkman (1858–1930) demonstrated in 1897
that a paralytic condition closely resembling the polyneu-
ritic symptoms of beriberi could be produced in chickens
by feeding them both stale as well as freshly cooked pol-
ished rice
[1] . Following this discovery, Casimir Funk
(1884–1967), in 1911, was the first person to report the
isolation of a substance in rice polishings that had anti-
beriberi properties. As this substance could be shown to
have an amino group within its structure, he called it a
‘vital amine’. His work introduced the word ‘vitamine’ to
describe a substance present in the diet in small amounts
but essential for life.
No nutritional deficiency disease like beriberi, pella-
gra or xerophthalmia led to the research and discovery of
riboflavin. Even today, riboflavin deficiency is probably
one of the commonest nutritional deficiencies in the de-
veloping world, yet little attention is paid to it as it is not
linked to any serious clinical condition
[2] . In man, early
symptoms are usually mild disturbances of the skin and
mucous membranes, but experimentally, deficiency of ri-
boflavin in animals brings about a rapid restriction of
growth. The effects on growth may equally apply in man
and delay the development of serious clinical symptoms
by lowering requirements. However, experimentally, it
was the impairment of growth when animals were placed
on semi-synthetic diets that initiated the research leading
to the characterization of riboflavin.
In 1879, Alexander Wynter Blyth, an English chemist,
is credited as being the first person to isolate a water-sol-
uble material from cow milk whey that glowed with a
yellow-green fluorescence when exposed to light and
gave it the name lactochrome
[3] , ‘lacto’ from the milk
and ‘chrome’ meaning color because of the yellow pig-
ment. However, at that time Blyth was not able to deter-
mine the chemical composition or properties of lacto-
chrome, and it was not until the 20th century that work-
ers began to look more closely at the yellow pigment when
research on accessory factors really began ( table1 ).
Nutritional Completeness of Individual Foods
In the early 1900s, a number of workers were investi-
gating what was necessary to maintain growth in rats.
Among them were Frederick Gowland Hopkins (1861–
1947) and Elmer McCollum (1879–1967). Hopkins is re-
ported as suggesting that purified diets lack an organic
nutrient that could be supplied by a small quantity of
milk but did not follow up the observation
[4] . McCol-
lum, on the other hand, describes how, in 1907, he re-
viewed the literature starting from 1870 and realized that
animals (mostly mice) reared on purified food stuffs such
as protein, fats, carbohydrate, and inorganic salts failed
rapidly and died
[5] . During the course of this work he
too noted the protective properties of milk and realized
that no one had attempted to determine the completeness
of individual natural food substances as a sole source of
nutrition for an animal. To this end, he started a series of
experiments with rats to determine which foods con-
tained essential elements to supplement the purified diet.
Subsequently, McCollum and assistant Marguerite Davis
(1887–1967)
[6] produced three papers in 1915 which
showed a diet containing 2% of wheat embryo or milk
powder with polished rice, casein, salts, and butter fat
provided enough of anessential accessory to support
growth of young rats
[6–8] . The accessory substance was
soluble in water and in alcohol and was stable to heat. The
authors concluded that ‘unidentified substances’ were in-
dispensable for growth and prolonged maintenance in
young rats
[7] . They also showed that the water-soluble
accessory factor in milk whey and wheat embryo could
be heated by autoclave at 15 pounds pressure for 1 h with-
out loss of activity
[8] . By 1916, McCollum and his gradu-
ate student Cornelia Kennedy thought that there were
two accessory factors in the diet: water-soluble B, found
in milk, egg yolk and wheat embryo, and fat-soluble A,
found in foods like butter fat, egg yolk, and fish oil. At the
time, McCollum thought that his water-soluble B was the
same factor as the anti-beriberi substance described ear-
lier by Christiaan Eijkman
[5] .
Confusion concerning Vitamin B-2 Complex and
Anti-Pellagra Properties
Overlapping in time with McCollum, Joseph Gold-
berger (18741929) was put in charge of investigating the
cause of pellagra in the southern states of the USA.
Northrop-Clewes /Thurnham
Ann Nutr Metab 2012;61:224–230
226
Sufferers developed severe skin eruptions on parts of
their body exposed to strong sunlight. There were mental
changes, which put people into asylums, and there was a
high death rate. Sufferers consumed corn (maize), and
the cause was popularly believed to be an infection from
insects in the corn. Goldberger noted, however, the pov-
erty and monotony of the diets consumed by sufferers
and initially believed that the disease was due to inferior
dietary protein. However, prevention of human pellagra
with proteins proved disappointing, but supplements of
eggs and milk, meat
[9] and later yeast were able to pre-
vent and cure pellagra. Experimentally, he also showed
that the substance in yeast was able to maintain health in
young rats and prevent the decline and death, which was
preceded by severe skin lesions. Goldberger called the
substance the pellagra-preventative or P-P dietary factor,
and in 1927, he thought his factor was the same as the
water-soluble vitamin B
2 identified by McCollum and co-
workers.
In 1927, the British Committee on Accessory Food
Factors defined vitamin B
2 as ‘t he more heat-st able, wat er
soluble dietary factor recently described and named pel-
lagra-preventive (P-P) factor by Goldberger and col-
leagues in 1926’. The committee noted that the latter
workers found the factor necessary for the maintenance
and p reventi on of cha racteris tic sk in lesio ns i n r ats ra ised
on semi-synthetic diets which they likened to the lesions
in human pellagra
[10] . However, at the same time, Har-
riette Chick (1875–1977) and Margaret Honora Roscoe
(born 1903)
[11, 12] reviewed the literature to date and
suggested that the water-soluble B, identified by McCol-
lum and Goldberger, was not one substance but at least
two: (1) an anti-neuritic (anti-beriberi) vitamin B
1 and (2)
vitamin B
2 , another water-soluble vitamin. They pointed
out that some foods were rich in anti-neuritic properties
but poorly supported growth and vice versa. For example,
yeast was a rich source of both B vitamin properties, but
wheat embryo was rich in the anti-neuritic component
but poor in the water-soluble vitamin B/P-P factor
[11] .
Identification of Riboflavin and Separation from
Anti-Pellagra Factor
By the end of 1932, Paul György (1893–1976) reported
that three separate components had been identified in the
vitamin B complex as needed by the rat: (1) vitamin B
1 ,
the anti-neuritic factor; (2) vitamin B
4 which prevented
loss of coordination and ataxia, and (3) vitamin B
2 , the
anti-pellagra factor
[13] . However, at the same time,
György noted that vitamin B
2 may be further divisible
into two: one factor that was predominantly growth pro-
moting and the second, the anti-pellagra factor
[12, 13] .
Vitamin B
2 was noted because of its high thermostability
Tab le 1. M ilestones in the discovery of riboflavin
Date Researcher Observation
1897 Blyth Isolation of lactochrome, a water-soluble, yellow fluorescent material from milk whey
1906 Hopkins Synthetic diets lacked an organic nutrient present in minute amounts in milk that stimulated appetite,
food consumption and growth in rats and mice
1915 McCollum Proposed that there were two accessory factors in diet: a water-soluble B and a fat-soluble A. Believed
that B was the anti-beriberi vitamin B1 identified by Eijkman
1927 Goldberger Proposed there was an anti-pellagra factor in eggs, milk, etc., and it was the same substance as
‘water-soluble B’ identified by McCollum
1927 Chick and Roscoe Proposed the water-soluble B identified by McCollum was two factors: anti-beriberi B1 and the B-2
factor
1932 György Suggested B-2 factor comprised an anti-pellagra factor and growth-promoting factor
1932 Warburg and
Christian
Extracted a yellow enzyme from yeast and demonstrated the yellow color was dialyzable and could be
recoupled to the enzyme
1933 Kuhn Isolation of lactoflavin from milk. Lactoflavin was the growth component in B-2 factor and had the
same characteristics as the factor isolated by Blyth
1934–35 Kuhn and Karrer Independently described the structure of riboflavin
1935 Birch, György,
and Harris
Differentiated the anti-pellagra factor from the growth-promoting vitamin B-2 factor (riboflavin)
1935 Theorell Riboflavin in the yellow enzyme was in the form of FMN
1939 Sebrell and Harris Demonstrated riboflavin was essential in man
1968 Glatzle Proposed the use of the glutathione reductase test as a functional measurement of riboflavin status
Riboflavin Ann Nutr Metab 2012;61:224–230
227
compared to vitamins B
1 and B
4 , but all three showed
growth-promoting properties under ‘suitable circum-
stances’ [13] . Ch ick and Alice Mary Copping (1906 –1996),
in 1930, had shown that an alkali- and heat-stable, water-
soluble, growth-promoting factor called factor Y proba-
bly existed, but its place in the B complex was not deter-
mined
[14] . It was subsequently noted by Thomas Wil-
liam Birch et al.
[15 ] that vitamin B
4 deficiency was
probably due to a chronically deficient thiamin state
since the signs disappeared when thiamin was given in a
sufficiently large dose.
The extraordinary difficulties faced by workers in this
field to carry out reproducible experiments is illustrated
in the paper of Chick and Roscoe
[1 2] who were trying to
deprive young rats of vitamin B
2 and obtain reproducible
skin lesions. In early experiments, young rats fed a puri-
fied diet plus a source of vitamin B
1 failed to grow and,
within 7 weeks, developed dermatitis. In later experi-
ments, skin symptoms were absent or irregular. Limiting
the supply of water-soluble vitamins to the dam produced
no improvement in consistency of clinical outcome when
the pups were used, and it was not until a rigorous puri-
fication procedure of the milk protein was introduced
that a consistent inhibition of growth was achieved and
skin symptoms gradually developed from the 5th week
[12] .
Paul György began his intensive work on the vitamin
B
2 factor with two objectives: (1) to chemically isolate the
growth-promoting factor, which he did in collaboration
with Richard Kuhn (1900–1967) and Julius Wagner-Jau-
regg (1857–1940) at the University of Heidelberg, and (2)
to show that the pellagra-like dermatitis was due to a dif-
ferent factor. The fact that many workers had difficulty in
reproducing the skin lesions which Goldberger attributed
to an anti-pellagra factor suggested to György that the
growth restriction and dermatitis were due to different
factors
[13] . For all these studies, they used a synthetic
diet originally devised by Anne Bourquin (born 1897)
and Henry Clapp Sherman (1875–1955) in 1931, which
contained thiamin but lacked the B-2 complex
[16] . On
that diet, the weight curves of young rats soon flattened
out or showed a decline, and addition of crude extracts of
yeast, rice bran, liver, milk concentrates or whey (all au-
toclaved to destroy thiamin) all restored normal growth.
To test each extract from each of these foods for growth-
promoting activity required a period of 3–4 weeks and a
constant supply of prepared animal s. Fortunat ely, an ear-
ly observation established that one of the active growth-
promoting components (riboflavin) in the growth exper-
iments had a yellow-green fluorescence which was visible
in near-neutral solution and was destroyed by exposure
to visible light for 6–24 h
[10] . Using these characteristics,
they were able to identify lactof lavin, wh ich w as f irst pre-
pared in crystalline form from milk.
Active Yeast’
In the course of the above work, György and colleagues
found that when the diet of Bourquin and Sherman was
used and growth in rats became stationary, it was not
possible to restimulate growth by addition of lactoflavin
alone. György and his colleagues then used the Bourquin
and Sherman diet plus ‘active yeast’ (which was yeast
from which all lactoflavin had been removed), but it still
produced no growth. However, providing the Bourquin
and Sherman diet plus the ‘active yeast’ and lactoflavin to
the growth-stationary rats did produce a normal growth
response. György therefore used the Bourquin and Sher-
man diet plus the ‘active yeast’ to test their food extracts
for growth promoting activity
[13] .
During the chemical isolation of lactoflavin, not a sin-
gle rat developed the pellagra-like dermatitis but, as the
experiments were too short for the appearance of clinical
symptoms, it was still possible that lactoflavin had anti-
pellagra properties
[13] . Around this time, however, com-
mercially prepared crystalline thiamin became available,
and to overcome some of the shortcomings in the diet of
Bourquin and Sherman, in which the vitamin B
1 content
was largely uncontrolled, an additional amount of thia-
min was added to the experimental diet. Having removed
an element of uncertainty about the vitamin B
1 status of
the animals, the addition of ‘active yeast’ was no longer
necessary, and György found that the appearance of the
pellagra-like dermatitis reported by Goldberger could be
reproducibly obtained by a B
2 -deficient diet whether or
not lactoflavin was present. It appeared that the pellagra-
like dermatitis was a feature of a vitamin B
2 -deficient rat,
but its appearance was erratic when rats had marginally
adequate thiamin status (the Bourquin and Sherman
diet) and independent of the growth-promoting proper-
ties of lactoflavin
[13] .
A lack of riboflavin in young rats manifests itself first
by retardation of growth and later complete cessation of
growth. Effects on the skin are not very striking or spe-
cific. They do have a definite seborrheic quality but are
certainly not characteristic of a pellagra-like disease
[10] .
However, working with Birch and Leslie Julius Harris
(born 1898), György and colleagues were able to show in-
dependent of and simultaneously with Conrad Elvehjems
(1901–1962) group in Wisconsin that riboflavin was dif-
ferent from the specific pellagra-preventing factor of
Northrop-Clewes /Thurnham
Ann Nutr Metab 2012;61:224–230
228
Goldberger and colleagues. Elvehjem’s group went on to
identify nicotinic acid (niacin) as the pellagra-preventing
component in the vitamin B-2 complex
[15 , 17] .
Riboflavin and Role in Tissues
In papers written by György, Kuhn, and Wagner-Jau-
regg (1933, 1934)
[18] two new pieces of information were
reported: (1) vitamin B
2 was not a single substance, and
(2) it was possible to identify one of the accessory food fac-
tors in crystalline form prepared from milk. The first iso-
lation of the colored component was from milk in 1933
[19] , and it was termed lactoflavin and corresponded to
the lactochrome of Blyth. Similar crystalline compounds
termed ovaflavin (from egg)
[20] and hepatoflavin (from
liver) soon followed. Kurt Günter Stern (born 1904) was
first to suggest that the structure of the substance was a
derivative of isoalloxazine
[21, 22] , a nd this was conf irmed
independently by Kuhn
[23, 24] and Paul Karrer (1889–
1971)
[25] when it was realized that all three compounds
were chemically identical. The biologically active ‘flavin
was found to be a derivative of isoalloxazine with two
methyl groups and a sugar (pentose) radical attached. As
the sugar was ribose, the name riboflavin was proposed
and adopted in 1937 by the Council of Pharmacy and
Chemistry of the American Medical Association ( fig.1 )
[10] . However, riboflavin still retains the name vitamin B
2
as it was the first vitamin isolated from the B-2 complex.
The isolation and identification of lactoflavin at the
University of Heidelberg under the guidance of Kuhn and
György makes interesting reading. György initially asked
Kuhn to isolate vitamin B
2 from the livers of rats, but they
quickly expanded their efforts, purifying flavin pigments
from plants and animal materials. Extraction with tradi-
tional solvents proved ineffective, and co-worker Edgar
Lederer (1908–1988) began to develop new chromato-
graphic techniques to purify the materials. Even with the
new methods, purification was an immense undertaking.
Although flavins are widespread pigments in nature, they
are only present in extremely small concentrations. In fact,
the isolation and purification of 1 g of the ‘beautiful yellow
substance’, as Kuhn called it, required 1 5,000 liters of milk
or the dried albumin from 34,000 eggs
[18] . Lederer was
forced to flee to France in March of 1933, and Wagner-
Jauregg took over the collaboration with György. He iso-
lated flavins in yeast, heart muscle, and a variety of plant
materials. The physics director, Karl Wilhelm Hausser
(1887–1933), also in Heidelberg at the Kaiser Wilhelm In-
stitute for Medical Research, worked closely with the Kuhn
group on the spectral analysis of the purified substances,
and Friedrich Weygand (19111969), Hermann Rudy, and
later Rudolf Ströbele and Pierre Desnuelle (1911–1986)
made significant contributions as Kuhn expanded the
scope of the investigation to include clarification of the
chemistry and functions of these molecules
[18] .
From the beginning, Kuhn assumed a fixed relation-
ship between the growth-promoting components of vita-
min B
2 and the flavins, especially after they were able to
correlate the absorption patterns of the flavin pigments to
the absorption characteristics of vitamin B
2 . Determining
if the f lavins were precursors, or the vitamin itself was dif-
ficult because crystallizing the lactoflavin, hindered the
structural analysis. However, the team discovered a break-
down product of lactoflavin, lumif lavin, in which the ri-
bose residue was removed by irradiation with ultraviolet
light, which they were able to crystallize. After determin-
ing the structure of lumiflavin, they were able to extrapo-
late enough information to synthesize lactoflavin and de-
termine its composition. Finally, they had proof that lac-
toflavin was riboflavin, not a precursor of the growth
factor in vitamin B
2 . Indeed, in experimental tests with
rats, Kuhn’s crystallized flavins proved to be the most ac-
tive preparation of vitamin B
2 yet discovered [18] .
Riboflavin Coenzymes
The question remained as to how riboflavin stimu-
lated growth and other bioactivity. The key to finding the
answer was once again the breakdown product lumif la-
vin. In 1932, Otto Heinrich Warburg (1883–1970) and
Walter Christian extracted a yellow enzyme (‘Gelbes
CH3
CH3
N
N
N
O
NH
O
OH
OH
HO
OR
Fig. 1. Riboflavin and its coenzymes FMN and FAD. The com-
pound shown is an isoalloxazine molecule linked to a molecule of
ribose in its reduced form (ribityl) through the nitrogen atom at
position number 10. ‘R’ represents hydrogen, phosphate or ade-
nosine diphosphate linked through its phosphate group; the re-
spective compounds are riboflavin, FMN, and FAD.
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    Background The fungus Ashbya gossypii is an important industrial producer of the vitamin riboflavin. Using this microbe, riboflavin is manufactured in a two-stage process based on a rich medium with vegetable oil, yeast extract and different precursors: an initial growth and a subsequent riboflavin production phase. So far, our knowledge on the intracellular metabolic fluxes of the fungus in this complex process is limited, but appears highly relevant to better understand and rationally engineer the underlying metabolism. To quantify intracellular fluxes of growing and riboflavin producing A. gossypii, studies with different ¹³C tracers were embedded into a framework of experimental design, isotopic labeling analysis by MS and NMR techniques, and model-based data processing. The studies included the use ¹³C of yeast extract, a key component used in the process. Results During growth, the TCA cycle was found highly active, whereas the cells exhibited a low flux through gluconeogenesis as well as pentose phosphate pathway. Yeast extract was the main carbon donor for anabolism, while vegetable oil selectively contributed to the proteinogenic amino acids glutamate, aspartate, and alanine. During the subsequent riboflavin biosynthetic phase, the carbon flux through the TCA cycle remained high. Regarding riboflavin formation, most of the vitamin’s carbon originated from rapeseed oil (81 ± 1%), however extracellular glycine and yeast extract also contributed with 9 ± 0% and 8 ± 0%, respectively. In addition, advanced yeast extract-based building blocks such as guanine and GTP were directly incorporated into the vitamin. Conclusion Intracellular carbon fluxes for growth and riboflavin production on vegetable oil provide the first flux insight into a fungus on complex industrial medium. The knowledge gained therefrom is valuable for further strain and process improvement. Yeast extract, while being the main carbon source during growth, contributes valuable building blocks to the synthesis of vitamin B2. This highlights the importance of careful selection of the right yeast extract for a process based on its unique composition. Electronic supplementary material The online version of this article (10.1186/s12934-018-1003-y) contains supplementary material, which is available to authorized users.
  • Riboflavin (RF) has been found to be a promising antioxidant and/or anti-inflammatory agent in several studies. However, the effect of RF against acetic acid (AA)-induced colonic injury is currently unknown. This study aimed to investigate the potential antioxidant and protective effects of RF in a rat model of ulcerative colitis. Starting immediately after the colitis induction (AA+RF group) or 1 week before the colitis induction (RF+AA+RF group), the rats were treated with RF (25 mg/kg/d;p.o.) for 3 days. The control and AA groups received saline (1 ml;p.o.) whereas AA+SS group (positive control) received sulfasalazine (100 mg/kg/d;p.o.) for 3 days. Colonic samples were taken for the biochemical and histological assessments on the 3rd day. High damage scores, elevated tissue wet weight index (WI), tissue myeloperoxidase (MPO) activity, 8-hydroxy-2′-deoxyguanosine levels and chemiluminescence values, and a pronounced decrease in antioxidant glutathione (GSH) levels of the AA group were all reversed by RF pretreatment (RF+AA+RF group) and SS treatment (AA+SS group) (p<0.05-0.001). Tissue WI, MPO activity and GSH levels were not statistically changed in the AA+RF group. Western blot analysis revealed that the decreased protein expressions of tissue collagen (COL) 1A1, COL3A1 and transforming growth factor-β1 in the AA group were elevated in all the treatment groups (p<0.05-0.001). In conclusion, RF exerts both the antioxidant and anti-inflammatory effects against AA-induced colonic inflammation by suppressing neutrophil accumulation, inhibiting reactive oxidant generation, preserving endogenous glutathione, improving oxidative DNA damage and regulating inflammatory mediators, suggesting a future potential role in the treatment and prevention of ulcerative colitis. This article is protected by copyright. All rights reserved.
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