ARTICLE IN PRESS

Pulmonary Pharmacology & Therapeutics 21 (2008) 668– 674

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Pulmonary Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/ypupt

A comparative study of grape seed extract and vitamin E effects

on silica-induced pulmonary fibrosis in rats
Ali A. Hemmati , Zahra Nazari, Mehdi Samei
Department of Pharmacology and Toxicology, School of Pharmacy, Physiology Research Center, Ahwaz Jundishapur University of Medical Sciences, Ahwaz 61357, Iran

a r t i c l e in f o a b s t r a c t

Article history: Due to the production of reactive oxygen species (ROS), oxidative stress has been implicated in the
Received 18 June 2007 pathogenesis of silica-induced lung fibrosis. So it is hypothesized that grape seed extract (GSE) or
Received in revised form vitamin E (Vit E) as antioxidants may ameliorate some symptoms of the disease. Male Wistar albino rats
13 April 2008
were divided into 7 groups: rats in group I instilled intratracheally (IT) with a single dose of silica
Accepted 16 April 2008
suspension (50 mg/rat) as positive control (PC). Treatment groups (II–IV) received Vit E (20 IU/kg/day),
GSE (150 mg/kg/day), or Vit E+GSE simultaneously orally 1 day after instillation of silica. Groups V and
Keywords: VI were given oral GSE or Vit E after instillation of the equivalent volume of saline (IT) as controls for
Silica
GSE or Vit E. Rats of group VII only instilled saline (IT) as negative control. After 90 days animals were
Grape seed extract
sacrificed and plasma-malondialdehyde (p-MDA) and lung tissue hydroxyproline (HP) were quantified.
Vitamin E
Pulmonary fibrosis The lungs were also investigated for histopathological changes. The mean concentrations of p-MDA and
Lipid peroxidation HP in studied groups (I–VII) were 1.95, 2.77, 0.72, 0.81, 0.64, 0.94, 1.02 mmolMDA/Lplasma and 28.476,
27.85, 22.83, 22.64, 15.40, 18.31, 18.51 mgHP/gtissue, respectively. Silica caused a significant increase in
HP content of lungs and MDA levels in the plasma except in GSE-treated groups (III and IV). According to
the results of this study GSE could reduce the fibrogenic effect of silica. However; no synergistic effect
was observed after co-administration of GSE and Vit E.
& 2008 Elsevier Ltd. All rights reserved.

1. Introduction can fill with inflammatory fluid, causing severe shortness of

Silicosis, of all pneumoconiosis, has probably claimed the
largest number of victims, either alone or in combination with
tuberculosis. Classically it is a chronic progressive disease of
pulmonary parenchyma [1]. The crystalline silica, alpha quartz, is
the major cause of silicosis worldwide. The incidence of overt
disease is greatly increased in industrial operations by the
mechanization and use of sand blasting, drilling, pulverizing,
cutting, grinding tools and other pneumatic equipments [2].
Clinically there are three types of silicosis: (1) Simple chronic
silicosis which results from long-term exposure (more than 20
years) to low amounts of silica dust. Inflammatory responses
caused by the silica dust form in the lungs and chest lymph nodes.
This disease may cause people to have trouble breathing and may
be similar to chronic obstructive pulmonary disease (COPD). (2)
Accelerated silicosis usually occurs after exposure to larger
amounts of silica over a shorter period of time (5–15 years).
Swelling in the lungs and symptoms occur faster than in simple
silicosis. (3) Acute silicosis results from short-term exposure to
very large amounts of silica. The lungs become very inflamed and
 Corresponding author. Tel.: +98 611 3336318; fax: +98 611 3340988.
E-mail address: hemmati_aa@yahoo.com (A.A. Hemmati).
breath and low blood oxygen levels. Progressive massive fibrosis
can occur in either simple or accelerated silicosis, but is more
common in the accelerated form. Progressive massive fibrosis is
caused by severe scarring and destroys normal lung structures [3].
The molecular basis for cell injury by silica has been the
subject of considerable studies. Destabilization of the cell’s
plasma membrane and membranes of the phagosomes containing
silica particles can provoke the generation of reactive oxygen
species (ROS) and the release of hydrolytic enzymes. However, the
actual molecular mechanisms of cell injury in silicosis have not
yet been defined [4,5]. The fibrogenic features of silica in lung is
unique and it is different from other types of pulmonary fibrosis,
e.g. due to bleomycin. It excites a foreign body reaction with
marophages, giant cells, etc., which is later, followed by dense
fibrosis. The fibrotic nodules seperated by emphysematouse lung
produce the typical picture of silicosis.
There is an exquisite balance between production and
destruction of ROS. When this equilibrium is destroyed, ROS are
produced excessively and all tissues are exposed to oxidative
injury [4]. Antioxidants, including vitamins, carotenoids and
tannins provide protection against oxidative damage. Such agents
are now gaining big attention as potential chemo-preventive
agents. Grape seeds are rich in antioxidant compounds, including
phenolic compounds (predominantly tannins), and it has been

1094-5539/$ - see front matter & 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pupt.2008.04.004
 ARTICLE IN PRESS
A.A. Hemmati et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 668–674
demonstrated that these compounds reduce the risk of chronic
disease by protecting against free radical-mediated damage [4].
Grapes (Vitis vinifera) are one of the most widely consumed fruits
in the world. Grape seeds are rich in dimers, trimers and other
oligomers of flavan-3-ols (the major are catechin, epicatechin and
epicatechin-3-O-gallate), named proanthocyanidins (PAs) [6].
There is a growing interest in the utilization of PAs for their
dietary and pharmacological properties, especially positive effects
on vascular injury [7], capillary protective action [8], free radical
scavenging [9,10] and antimutagenic activity [11]. PAs present in
grape seeds are known to exert anti-inflammatory, anti-arthritic
and anti-allergic activities, prevent skin aging, scavenge oxygen-
free radicals and inhibit UV radiation-induced peroxidation
activity [11]. Oral administration of grape seed PAs at a dose of
2 mg/kg three times daily for 6 days inhibits carrageenin or
dextran-induced hind paw edema, stabilizes the capillary wall and
prevents the increase in capillary permeability caused by local
cutaneous application of xylene [12]. The antioxidative activities
of PAs were found to be much stronger than Vitamin C or E (Vit C
or E) in aqueous systems [13]. The potential influence of grape
seed extract (GSE) on the silica-induced lung fibrosis has not been
previously reported. The aim of the present study was to examine
the effects of orally administered GSE in a rat model of lung injury
produced by endotracheal silica by comparing it with that of Vit E.
In the present study crystalline quartz used in the ferrosilicon
production factory in Iran was investigated. We investigated the in
vivo effect of GSE or Vit E on silica-induced lung toxicity in rats.
The alterations in state of oxidative stress were determined by
measuring the plasma levels of malondialdehyde (MDA), and
hydroxyproline (HP) as an index of collagen synthesis were
determined in rat lung tissue and histopathological evaluation
of lung tissue was performed.
2. Materials and methods
2.1. Quartz particle samples
The quartz stones were obtained from the Iranian Ferrosilicon
Factory (Semnan-Iran). The stone samples were delivered to
the Iranian Geological and Mine Exploring Organization, which
crushed, processed and then milled them in a rotary ball mill to
produce respirable silica particles (o5 mm). The surface character-
istics of the particles were determined with an Oxford scanning
Fig. 1. SEM images of the quartz particle used in the study. Black bar represents
2 mm.
669
electron microscope (Fig. 1). The particles were angular or
rounded.
2.2. Hydroalcoholic extract of grape seed
Grape seeds were a gift from Sasan Shahd Dietary Industries
(Urommia, Iran). Seeds were obtained from red grapes (V. vinifera)
in the process of grape juice production. Grape seeds were dried in
drying oven at 50 1C for 72 h. Then, dried seeds were ground to
fine powder by a grinder. About 500 g of the powder was mixed
with 1000 ml of 70% ethanol in distilled water and kept for 3 days
at room temperature. The extract was then filtered through a
Büchner funnel. Solvent (ethanol/water) was removed using
rotary evaporator under vacuum at 50 1C. The residue (dried
extract) obtained and kept in refrigerator for further experiments.
Enough amounts of the dried extract were suspended in water and
administered orally to rats for 90 days (150 mg/kg/day).
2.3. Vitamin E
DL-a-tocopherol (Darmstadt, Germany) was dissolved in liquid
oil and administered at a dose of 20 IU/kg/day for 90 days.
2.4. Animals
Male Wistar albino rats weighing 180–200 g were purchased
from the Animal house and research center, Jundishapur Uni-
versity of Medical Sciences, Ahwaz, Iran. The animals were kept on
standard food pellet and tap-water ad libitum. The rats were
housed in polycarbonate cages (5 animals per cage) and kept in an
air-conditioned animal room at a temperature of 2373 1C, with a
relative humidity of 5075%. The animal room was on a 12 h
photo-period cycle.
2.5. Intratracheal instillation of silica
The rats were anesthetized by IP injection of ketamine
hydrochloride (50 mg/kg) and instilled intratracheally (IT) with
silica suspension (50 mg in 0.1 ml saline/rat), while normal
animals received an equal volume of sterilized saline instead
of silica suspension. The rats were sacrificed 90 days after silica
injection. Pulmonary fibrosis and silicosis were assessed as
described in the following sections.
2.6. Experimental groups
Thirty five rats were randomly divided into the 7 groups as
follows; group I: a single IT instillation of silica (positive control
(PC)); group II: single IT silica and oral Vit E (20 IU/kg/day) [32];
group III: single IT silica and oral GSE (150 mg/kg/day) [32]; group
IV: single IT silica and GSE+Vit E at doses mentioned above
simultaneously for probable synergistic effects; group V: single IT
saline and oral GSE (control for GSE); group VI: single IT saline and
oral Vit E (control for Vit E); and group VII: only single IT saline
(negative control). The day of IT injection of silica or saline was
designated as day 0.
2.7. Biochemical measurements
The plasma MDA levels were determined by the method described
by Buege and Aust [14] using fluorescent measurements as described
by Yagi [15]. Values obtained were compared with a series of standard
solutions (1,1,3,3-tetraethoxypropane). The plasma samples were
obtained by collecting 1 ml blood from left ventricle, centrifuging at
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670 A.A. Hemmati et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 668–674

1000g for 5 min, and diluting 20 times with phosphate buffer solution + slight
(pH ¼ 7.4). Results were expressed as mmol/L. ++ moderate
The left lobe of lung was used for HP determination. HP +++ severe
content of lung tissue was determined by the colorimetric method ++++ very severe
as described by Edwards and O’Brien [16]. HP was extracted from
collagen and oxidized to pyrrole by chloramine-T, it can then
produce color with para-dimethylbenzaldehyde. Tissue samples
were homogenized and processed according to the earlier
2.9. Statistical analysis
discussed method. The absorbance was measured at 500 nm to
determine the HP content (mg/g tissue).
Statistical comparison was made by one-way ANOVA. Sig-
nificant F-values were tested with Tukey’s test. Data are presented
2.8. Histological studies as mean7SEM. In all cases Po0.05 was considered significant.

When the animal was sacrificed the lung was removed, the left
lobe was kept for HP determination. Right lobes were perfused
with 10% formalin in an inflation pressure not more than 20 cm of
3. Results
H2O. Then the whole tissue was placed in formalin for at least
48 h. Lung tissues were embedded in a paraffin block and 5-mm-
3.1. Lipid peroxidation
thick sections were made and stained with hematoxylin and eosin
(H&E). The sections were examined by light microscopy and
The MDA levels in the lung of each experimental group at 90th
assessed for the presence of fibrosis and severity of lesion. The
day are summarized in Fig. 2. MDA levels in the plasma of the GSE
structural alterations of tissue due to silica or treatment with GSE
or GSE/Vit E treatment groups were not significantly different
and Vit E were assessed based on the degree of cellular
from the control groups, while the levels for positive or Vit E-
proliferation, alveolar wall thickening, inflammatory lesions and
treated groups were very significant (po0.001) (Fig. 2). Interest-
collagen deposition or fibrosis. Such changes were graded in terms
ingly, MDA levels in plasma samples of Vit E-treated group was
of severity and distribution. The grading system adopted is as
also significant in comparison to the PC group (p ¼ 0.005) (Fig. 2).
follows and was utilized for each group of animals.
For distribution of lesion over the tissue

0 absent 3.2. Hydroxyproline

7 rare/occasional
+ sparse/limited Collagen production in the pulmonary tissue was assessed
++ moderate by the HP content of the lung at 90th day of treatment. The HP
+++ extensive/widespread contents of the lungs obtained from each experimental group are
++++ very extensive/ predominant summarized in Fig. 3. A significant increase in HP of the lung
tissue was observed in the group that received silica (PC) and the
For severity of lesions Vit E-treated group in comparison to the control groups
(po0.001); however, no significant decrease was observed in
0 nothing/zero HP contents in GSE treated and GSE+Vit E-treated groups in
7 marginal comparison to the GSE, Vit E or negative controls.

3.00

2.50
MDA concentration (µmol/lit
plasma) (mean ± SEM)

2.00

1.50

1.00

0.50

0.00
VitE-treated GSE/VitE-treated Control for VitE
Pos. control GSE-Treated Control for GSE Neg control
Rat groups

Fig. 2. Comparison of plasma MDA levels (mmol/L) of silica-treated, silica+Vit E-treated, silica+GSE-treated, silica+GSE/Vit E-treated, saline+GSE-treated, saline+Vit E-
treated and saline-treated rats. (K) A significant difference between the silica (pos. control) and silica+Vit E groups and the other control groups (neg. control, controls for
Vit E or GSE) (po0.001). (m) A significant difference between the silica (pos. control) group and silica+Vit E group (p ¼ 0.005).
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A.A. Hemmati et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 668–674 671

Hydroxyproline concentration (mg/g tissue)

* *
30.0

(mean±SEM)
20.0

10.0

0.0
Pos. control GSE-treated Control for GSE Neg. control
VitE-treated GSE/VitE-treated Control for VitE
Rat groups

Fig. 3. The hydroxyprolin concentration (mg/gtissue) in the lungs of rats treated with IT silica, IT silica+Vit E (20 IU/kg; po), IT silica+GSE (150 mg/kg; po), IT silica+GSE/Vit E
simultaneously, IT saline+ GSE, IT saline+Vit E and IT saline alone at 90th day. (*) A significant difference between the silica (pos. control) and silica+Vit E groups and the
other control groups (Neg. control, controls for Vit E or GSE) (po0.001). GSE-treated groups (GSE or GSE/Vit E) showed significant difference neither with pos. control and
Vit E-treated groups nor with the other controls.

Fig. 5. Photomicrograph of rat control lung tissue treated with IT saline+ po GSE
Fig. 4. Photomicrograph of rat control lung tissue treated with IT saline only showing normal lung structure (H&E, 100).
showing normal lung structure. No inflammatory cells are evident (H&E,  100).

3.3. Histopathology

Histopathological evaluation of the pulmonary tissue was

performed with light microscopy for the seven different experi-
mental groups 90 days after treatment. Lungs of rats in control
group, which received IT saline and GSE, Vit E, or none showed
normal lung structure and no lesion was evident (Figs. 4–6). After
90 days of instillation, in silica group, there were mainly
perivascular and peribronchilal fibrosis associated with silicotic
nodules and distributed hyalinized collagen fibers were observed
in the nodules (Figs. 7 and 8). The Vit E-treated group also showed
a considerable change in tissue structure due to interstitial
inflammation and fibrosis (Fig. 9). At 90 days after instillation,
in GSE/Vit E-treated groups, cellular nodules were observed,
composed mainly of mononuclear phagocytes, fibroblasts and tiny Fig. 6. Photomicrograph of rat control lung tissue treated with IT saline+ po Vit E
collagen fibers (Fig. 10). In the GSE-treated group such events showing normal lung structure. There are fine alveolar walls (H&E,  100).
were less pronounced and the numbers of nodules were reduced
(Fig. 11). changes include the area of lung damages and their locations,
The severity and distribution of histological changes in the mainly around bronchial tree and vascular bed. Vit E treatment
silica-treated group (PC) was highest among all the groups. Such could reduce the severity of damages 11 (+). However the
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672 A.A. Hemmati et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 668–674

Fig. 7. Photomicrograph of rat lung tissue 90 days after IT silica administration. A

typical silicotic nodule is present (arrow);(H&E, 100). Fig. 10. GSE/Vit E-treated rat lung 90 days after instillation. Two cellular nodules
(arrows) are evident (H&E, 100  ).

Fig. 8. Photomicrograph of rat lung 90 days after IT silica administration

illustrating perivascular fibrosis (arrow) (H&E,  400).

Fig. 11. GSE-treated group, at 90 days after instillation. Number of nodules was
much less than other groups. The intensity of fibrosis has been reduced (H&E,
100  ).

Table 1
Severity and distribution of histological changes in silica or treatment groups

Name of group Severity of lesion Distribution

Saline treated 0 0–7

Silica (positive control) ++++ ++++
Vit E treated +++ ++++
GSE treated + +
Vit E+GSE treated ++ ++

Fig. 9. Photomicrograph of rat lung 90 days after IT silica+ po Vit E administration

illustrating diffused fibrosis. Deposition of collagen are evident (arrow heads)
(H&E, 100).
distribution of changes was similar to the PC group. GSE caused a
big reduction in severity and distribution of lung damages.
Combination of Vit E and GSE could diminish the severity of
damages but their effect was less than GSE alone (Table 1)
4. Discussion
Pulmonary fibrosis is a frequent response to different insults or
injuries to the lung. Although there are various initiating
mechanisms, the terminal phases of fibrosis are characterized by
proliferation and progressive accumulation of connective tissue
replacing normal functional parenchyma. The pathogenesis of
pulmonary fibrosis includes endothelial and epithelial cell injury,
influx of the inflammatory cells and production of their chemical
mediators leading to the proliferation and activation of the
fibroblasts [17–19]. Sililica-induced pulmonary toxicity is some-
how different from other types of pulmonary fibrosis, e.g. by
bleomycin. Silica particles are insoluble, once deposited in lung
parenchyma are subsequently ingested by macrophages. Further
damage may results from internal reactions in these cells. A
hypothesis by Heppleston and Styles [20] claims that on the
surface of silica particles, silicic acid groups interact with amide
groups in proteins and phospholipide in the cell membrane of
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macrophages and eventually destroy the cell membrane. As a
result, the digesting enzyme and silica (quartz) crystals are
released. Other macrophages proliferate and migrate to the
reaction site, where they may release inflammatory mediators
which stimulate the synthesis and accumulation of collagen fibers
[20]. It is well known that exogenous agents such as fibrogenic
minerals can cause pulmonary fibrosis through production of ROS
in animal models [21]. Also it has been suggested that the
generation of ROS by dusts intrinsically or via the inflammatory
process is important in the initiation of the disease [22–24] In
silica toxicity, cell damage by oxidant radical is mediated by iron
ions. Sianol groups (SiOH), which are common to all silicates, react
with ferric ions forming a complex which is referred to as a
silicato-iron complex. Iron in the complex can mediated an
electron exchange when it is reduced from ferric (fe3+) to ferrous
(Fe2+) state by superoxide anions. Hydroxyl radicals which may be
produced by this reaction at the surface of silica are potent
oxidizing agents. They can initiate lipid peroxidation of the cell
membrane and oxidatively inactive cell proteins. The generation
of oxidants initiated by silica particles, leads to activation of the
immune system and interaction with other cells, e.g. T and B
lymphocytes. Macrophages and lymphocytes seem to have a close
co-operation in the process of silicosis. Histological study of silica-
exposed lung tissue shows a large increase in the numbers of
these cells. Interaction between these cells may lead to the
production of a number of mediators such as, platelet-derived
growth factor (PDGF), interleukin 1 and 6 (IL1, Il6), transforming
growth factor-beta (TGFb) and tumor necrosis factor-alfa (TNFa).
Such mediators may provoke the proliferation of fibroblasts and
myofibroblasts which produce collagen fibers and finally cause
fibrosis [25].
It has been suggested that tetrandrine an alkaloid from
Stephania tetrandra has antisilicotic effects [26]. But other studies
state that this alkaloid has toxic effect on macrophages accom-
panied by overproduction of prostaglandins [27,28]. In many
experimental studies GSE components as antioxidants have been
employed [29–31]. The present study has been designed based on
the free-radical scavenging actions of GSE or Vit E (a-tocopherol)
on the ROS generated by silica particles intrinsically or via the
inflammatory process that has not been investigated previously.
The microscopic and biochemical findings of this study
indicate that GSE treatment after silica instillation rendered
relatively therapeutic effect against silica toxicity in rat. MDA
value in the GSE treated group has decreased significantly
compared to PC Vit E-treated group. This means that GSE
components are able to suppress the rate of lipid peroxidation
due to silica in the lung tissue. GSE caused a big reduction in
severity and distribution of lung damages. The exact mechanisms
of GSE compounds cannot be verified by this work but in
association with other reports we may give some suggestions.
Based on the oxidative stress hypothesis of pulmonary fibrosis, it
is offered that GSE, a potent antioxidant, can exert anti-fibrosis
effects by scavenging ROS; although it cannot suppress the
progression of the disease it probably delays the process of
silicosis subchronically. Therefore some other mechanisms, such
as immunological cells and factors, should be involved in the
progression of silicosis in rat.
In this work Vit E was used as a standard antioxidant agent.
Our previous work on bleomycin-induced pulmonary fibrosis, Vit
E showed a significant antifibrotic effect [32]. However this study
depicted the higher potency of GSE. Interestingly Vit E in
a-tocopherol form, a potent breaking free radical chain reaction,
did not ameliorate the fibrogenic effect of silica in the lung but
exacerbated lipid peroxidation in the plasma. This finding may be
attributed to the nature of the mechanism silicosis. Our result is
similar to the recent suggestions that introduce it as an oxidant or
673
a preoxidant [33,34]. In accordance with the results of their study,
in low fluxes of aqueous radicals a-tocopherol plays a role as an
oxidant which oxidizes low-density lipoproteins in plasma. So it is
assumed that there is mild fluxes of aqueous and relatively stable
radicals (such as hydroxyl radical) from lung tissue to the blood
and increase of MDA. The results of this study did not prove any
synergistic effect between GSE compounds and Vit E. The values
of MDA and HP in negative control group which received normal
saline were slightly increased. Such increase may be due to the
weight gain of animals or as the saline effect.
There were limitations in this study which may be considered
in future studies. Since silicosis is a chronic respiratory disease,
we may administer silica in an inhalable route on a daily basis for
a certain period of time. Results of such work give better image of
silica impact on lung tissue. We may also look at the function of
other organs such as kidney and liver which might respond to
foreign substances.
In conclusion, inspite the limitations of this study, we believe
that the use of GSE can provide a protective action against
the harmful effect of silica on lung. However, more studies are
required to verify whether GSE or other antioxidants are effective
chronically in the immunological process of silicosis or other
types of pulmonary fibrosis.
Acknowledgments
This work was funded by the Research Deputy of Jundishapur
University of Medical Sciences, Ahwaz, Iran
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