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Evaluation of pre-clinical safety and toxicology of Althaea officinalis extracts as naturopathic medicine for common carp (Cyprinus carpio)

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The current study was done to investigate the preclinical safety and toxicology of Althaea officinalis extract as naturopathic medicine in common carp (Cyprinus carpio). Specimens were treated with 0 (control), 2.5, 5 and 10 g of marshmallow extract for 45 days. Plasma biochemical parameters were measured after 15 and 45 days. Total protein, albumin and globulin levels (p<0.05) were significantly higher in the fish fed with 10 g A. officinalis extract than that in control groups on day 45. Although, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities significantly decreased (p<0.05) in fish fed with A. officinalis extract on day 15. The use of the A. officinalis extract (10 g) led to a significant increase in AST, lactate dehydrogenase (LDH) and ALP activities and cholesterol levels on day 45 (p<0.05) and a significant decrease in plasma glucose and cholesterol levels on day 15 (p<0.05). There was no significant difference in glucose levels and creatine kinase (CK) activity between all treatments and the control group on day 45 (p>0.05). During the experimental period, triglyceride levels noticeably decreased in fish fed with 2.5 g of A. officinalis extract (p<0.05). Although, administration of marshmallow extract up to 5 g per kg of feed did not show any side effect on fishes, the use of the A. officinalis extract (10 g) led to cytotoxicity and modifications in blood biochemical parameters of fish. Therefore, we recommend the use of the lower concentrations than 10 g A. officinalis extract in prospective clinical studies.
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Iranian Journal of Fisheries Sciences 15(2) 613-629 2016
Evaluation of pre-clinical safety and toxicology of Althaea
officinalis extracts as naturopathic medicine for common
carp (Cyprinus carpio)
Soleimany V.; Banaee M.*; Mohiseni M.;
Nematdoost Hagi B.; Mousavi Dehmourdi L.
Received: April 2015 Accepted: September 2015
Abstract
The current study was done to investigate the preclinical safety and toxicology of
Althaea officinalis extract as naturopathic medicine in common carp (Cyprinus carpio).
Specimens were treated with 0 (control), 2.5, 5 and 10 g of marshmallow extract for 45
days. Plasma biochemical parameters were measured after 15 and 45 days. Total
protein, albumin and globulin levels (p<0.05) were significantly higher in the fish fed
with 10 g A. officinalis extract than that in control groups on day 45. Although, alanine
aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase
(ALP) activities significantly decreased (p<0.05) in fish fed with A. officinalis extract
on day 15. The use of the A. officinalis extract (10 g) led to a significant increase in
AST, lactate dehydrogenase (LDH) and ALP activities and cholesterol levels on day 45
(p<0.05) and a significant decrease in plasma glucose and cholesterol levels on day 15
(p<0.05). There was no significant difference in glucose levels and creatine kinase
(CK) activity between all treatments and the control group on day 45 (p>0.05). During
the experimental period, triglyceride levels noticeably decreased in fish fed with 2.5 g
of A. officinalis extract (p<0.05). Although, administration of marshmallow extract up
to 5 g per kg of feed did not show any side effect on fishes, the use of the A. officinalis
extract (10 g) led to cytotoxicity and modifications in blood biochemical parameters of
fish. Therefore, we recommend the use of the lower concentrations than 10 g A.
officinalis extract in prospective clinical studies.
Keywords: Biochemical parameters; Common carp; Marshmallow; Medicinal plants,
pre-clinical study
Natural Resources and the Environment Faculty, Behbahan Khatam Alanbia University of
Technology, Iran
*Corresponding author's Email: mahdibanaee@yahoo.com
614 Soleimany et al., Evaluation of pre-clinical safety and toxicology of Althaea officinalis
Introduction
Medical plants are used in folk
veterinary medicine for treating or
preventing many diseases in
domesticated animals (Viegi et al.,
2003; Suresh Kumar and Mishra,
2004). Herbal products provide an
important source of potential medicines
from which humans have produced
phytomedicines and herbal remedies.
Herbal medicines often contain organic
chemical compounds with different
roles; including chemotherapeutic,
immune-stimulant, bacteriostatic,
bactericidal, antifungal and anti-
parasitical functions (Banaee, 2010;
Ahmadi et al., 2012; Asadi et al.,
2012). In the last two decades, many
studies have been conducted to
determine the feasibility of using herbal
medicine in prevention and curing of
aquatic animals’ diseases (Yin et al.,
2006; Divyagnaneswari et al., 2007;
Ardó et al., 2008; Yin et al., 2009).
There are several basic methods for
the selection of medicinal plants
including a random selection of plant
species, a choice based on plants’
therapeutic properties and their
ethnomedical use, examining literature
on phytochemical compounds and
pharmacological activities as well as
using chemotoxonomic approaches.
Traditionally, it is being thought that
plants and plant products are non-toxic
and do not have any adverse side
effects, however; phytochemical
ingredients of plant extracts are
chemicals that are similar to those
found in synthetic drugs with the same
potential to cause serious adverse
effects. Therefore, choosing plant
species is the first step in evaluating
medicinal properties of plants and the
assessment of a crude medicine is an
essential part of establishing its correct
identity. Prior to including any crude
medicine in herbal pharmacopoeia;
pharmacognostical parameters must be
established. Thus, in introducing herbal
medicine the pharmacognostical
parameters by pre-clinical
pharmacology and toxicology studies
must be determined. In these studies,
evaluating the changes in biochemical
parameters of blood is a common
clinical procedure in evaluating the
medicinal effects on specific organs,
especially in the case of existing
difficulties in studying the changes in
tissues. Measurement of biochemical
markers offers comprehensive
information on the physiological state
of cells and tissues, particularly the
liver, as a target issue. This information
can be used in the pharmaceutical and
toxicological evaluation to determine
non-toxic doses of the drug (Banaee et
al., 2010).
In this study, marshmallow was
chosen as a candidate drug to be used in
the aquaculture industry. Marshmallow
(A. officinalis) is a medicinal plant, the
roots, leaves and flowers of which are
usually used in traditional medicines in
many countries all over the world
(Wynn and Fougère, 2007). The
flowers of marshmallow are in three
colors: white, pink, and pinkish red.
Sadighara et al. (2012) found out that
Iranian Journal of Fisheries Sciences 15(2) 2016 615
antioxidant activities and the amount of
flavonoids in marshmallow extract are
heavily dependent on the color of the
flowers. A. officinalis L. are rich in
mucilage polysaccharides, antioxidants,
flavonoids, terpenes, and several
terpenoids, sterols, saturated and
unsaturated fatty acid, the amino acid
asparagines, coumarins, kaempferol,
phenolic acids, quercetin, sugars,
tannins, and volatile oil (Elmastas et al.,
2004; Sartoratto et al., 2004; Teśević et
al., 2012; Puri et al., 2014).They could
be found in some other medicinal
plants, too. Some of these compounds
such as carvacrol, thymol, linalool, β-
bisabolene, terpinene, and 8,1- cineole,
p-cymene, eugenol, furfural and
camphor have antibacterial, antifungal
and antiviral properties (Sartoratto et
al., 2004; Burt et al., 2005; Jung et al.,
2007; Astani et al., 2010; Abbaszadeh
et al., 2014; Bilia et al., 2014; Kubiça et
al., 2014) or demonstrate antioxidant
activity (Jun et al., 2014; Puri et al.,
2014; Ramos et al., 2014). Aristatile et
al. (2009a;b) verified the liver-
protective properties of carvacrol
against the toxicity of D-glucosamine in
rats. Similarly, Ezhumalai et al. (2014)
reported that the activity of
aminotransferase which had increased
because of a diet rich in fats, decreased
to a normal level after carvacrol
administration. Considering the
aforementioned properties,
marshmallow may be a good alternative
in the prevention and cure of bacterial,
viral and fungal infections and
oxidative stress.
Nevertheless, marshmallow’s
ingredients may have adverse side
effects on the physiology and biology
of fishes. Therefore, preclinical
evaluation and safety evaluation of the
marshmallow for fish are necessary.
Hence, this study was done to collect
basic information on the effects of
marshmallow extract on biochemical
parameters of blood in common carp.
Specimens of common carp, C.
carpio were selected for the present
study based on the following criteria:
The family Cyprinidae is well
represented amongst the fish inhabiting
the freshwaters of Iran. This species can
be found in abundant quantities
throughout the year, and is of great
commercial importance to people in
developing countries. This species is
voraciously omnivorous and is easily
adapted to artificial diets. Also, this fish
is hardy in nature for handling and
transport. Therefore, common carp is a
suitable aquatic animal for bioassay
testing.
Materials and methods
Fish
Common carp, C. carpio, (with the
average weight of 37.65 ± 4.40 g and
total length of 14.15 ± 0.8 cm), were
used in this study according to National
Ethical Framework for Animal
Research in Iran (Mobasher et al.,
2008). Fishes were purchased from a
commercial farm in Shush, Khuzestan
Province, Iran, and they were
transferred to the aquaculture laboratory
of Natural Resources Faculty,
616 Soleimany et al., Evaluation of pre-clinical safety and toxicology of Althaea officinalis
Behbahan Khatam Alanbia University
of Technology. Fishes were randomly
distributed in 12 fiberglass tanks (200
L) and acclimatized in aerated
freshwater (24 ± 2°C; pH, 7.4 ± 0.2; 16
L/8D; 40% water exchange rate/day)
for two weeks before use. During the
acclimatization period, specimens were
fed two times daily with commercial
diet from Beyza Feed Mill, Shiraz, Iran.
Phytochemical analysis of
Marshmallow (A. officinalis) flower
Essential oil extraction of A. officinalis
Marshmallow (A. officinalis) flower
was selected from a list of herbs
commonly used as medicinal plants in
Iran. The hydro-distillation was done
using a Clevenger-type apparatus.
Dried flowers of A. officinalis were
purchased from the local market. In the
experiment, 100 g of dried flower of A.
officinalis (as powder) was distilled
with 1000 mL of water for 3 hours. The
amount of the essential oil obtained was
expressed in mL/100 g calculated on
the basis of dry matter content.
Characterization of the oil extract by
Gas Chromatography (GC-MS)
The analytical GC-MS system used was
an Agilent GC-MSD system (Agilent
Technologies-5975C-MS, 7890A-GC)
with helium as the carrier gas at a
constant linear velocity of 1 ml/min.
The transfer, source and quadrupole
temperatures were 280°C, 230°C and
150°C respectively, operating at
ionization energy of 70 eV. A HP-5MS
capillary column (30.0 m × 0.25 mm ID
× 0.25 µm film thickness) was used and
programmed from 60 to 240°C at
8.5/min. Oil samples (20 µL) were
diluted with acetone (1000 µL). The
injection volume was 0.2 µL, the split
ratio was 1:50 and the injector
temperature was 280°C. Composition
values were recorded as percentage area
based on the total ion current
chromatogram.
Extract of A. officinalis
The powder of dried flower A.
officinalis was mixed with distilled
water and ethanol (1:1), and the mixture
was put on the shaker for 24 hours at
room temperature. The resulting
hydroalcoholic extract was filtered
through Whatman filter paper and
evaporated to dryness on a rotary
evaporator until it became creamy, and
was then dried in an oven at 50°C that
finally gave 8 g (8% of initial amount)
of dried powder. The concentration
used in the experiment was based on
the dry weight of the extract.
Fish diet preparation
The formulated fish feed was prepared
in the laboratory using the powder of a
commercial feed obtained from Beyza
Feed Mill, Shiraz, Iran. In order to
enrich the normal diet, the 2.5, 5 and 1
g of A. officinalis extracts were mixed
with 1 kg feed powder. Each
supplemented diet was mixed with
distilled water (1mL/g) until a
homogenous mixture was obtained.
This mixture was passed through a meat
grinder, producing extruded string
Iranian Journal of Fisheries Sciences 15(2) 2016 617
shapes, which were dried in an oven at
55°C for 12 h and then were broken to
produce pellets approximately 10 mm
in length. The pellets were packed and
stored at -20°C in a freezer until used.
Although no supplement was added to
the feed, the control diet was prepared
using the same process.
The final experiment
The final experiment was done in a
completely randomized design with 3
treatments and 1 control. The fishes
were fed the commercial diet enriched
with the hydroalcoholic extract of
flower A. officinalis 0.25% (2.5 g),
0.5% (5 g), and 1% (10 g) of the
commercial diet and each was repeated
3 times. Common carp were fed a diet
supplemented with marshmallow
extract for 45 days. During
experiments, specimens were monitored
for appetite. Fish appetite was evaluated
based on the weight of intestinal
contents and their feeding behavior.
After 15 and 45 days, 9 fish were
captured randomly from each group and
then anesthetized with clove powder
solution (200 mg/L). Then, the fish
blood was collected from their tail
stem, and stored at 4 °C in sterilized
glass vials with heparin as
anticoagulant. The blood was
centrifuged for 15 min at 4000 g, at 4
°C. Plasma samples were immediately
stored at -21 °C until biochemical
analysis.
Biochemical parameters of blood
Determination of the biochemical
parameters was done using the kits
supplied by Pars Azmoon Company
and a UV/ VIS spectrophotometer
(model UNICO 2100). Each blood
biochemical parameter was determined
by a certain method. Total plasma
protein was measured at 540 nm by the
Biuret reaction. The albumin assay is
based on the dye-binding properties of
plasma albumin with bromocresol
green. An increase in the blue-green
color was measured at 630 nm. The
plasma globulin was based on the ratio
of albumin versus total protein
(Johnson et al., 1999). Plasma glucose
was measured by the glucose-oxidase
method at 500 nm (Sacks, 1999).
Plasma cholesterol levels by the
CHOD-PAP enzymatic method at 510
nm, triglyceride levels by GPO-PAP
enzymatic method at 546 nm (Rifai et
al., 1999) and creatinine by the JAFFE
method and at 510 nm (Foster-Swanson
et al., 1994). The activity of aspartate
aminotransferase (AST) and alanine
aminotransferase (ALT) in plasma was
determined by NADPH consumption
and its conversion to NAD+ at 340 nm.
Lactate dehydrogenase (LDH) in
plasma was determined based on the
conversion of pyruvate to lactate at 340
nm, alkaline phosphatase (ALP) based
on converting nitro phenol phosphate
into nitrophenol and phosphate at 405
nm, creatinine phosphokinase (CK)
based on the conversion of creatinine
phosphate into creatinine at 340 nm and
based on optical density (OD)
618 Soleimany et al., Evaluation of pre-clinical safety and toxicology of Althaea officinalis
absorption and the formula presented in
the kits' manual (Moss and Henderson.
1999). All biochemical parameters were
determined according to the instructions
provided in the kit’s manual.
Data analysis
A significant difference in the
biochemical parameters of specimens
treated with the different concentrations
of A. officinalis extracts was examined
using one-way ANOVA. All the data
were examined for normality (Shapiro-
wilk test). Means were compared by
Tukey’s test and a p<0.05 was
considered statistically significant.
Statistical analyses were performed
using SPSS (IBM, 19) software. Data
are presented as mean ± SD.
Results
The phytochemical analysis of the
marshmallow flower sample used in
this study identified 43 compounds,
representing 97.823 % of the total oil
content (Fig. 1 and Table 1). Table 1
represents major constituents as follows
6,10,14-trimethyl-2-Pentadecanone
(47.31%), Carvacrol (17.65%), 2-
Pentadecanone (11.22%), Dodecanoic
acid (2.322%), n-Tetradecanoic acid
(4.917%), n-Tetradecanol (1.978%), n-
Nonanoic acid (1.176%), Thymol
(1.073%), Methyl hexadecanoate
(1.312%).
The plasma AST activities
significantly decreased in fish which
were fed for 15 days with feeds
supplemented with 5 and 10 g of A.
officinalis extract (p<0.05). The highest
AST activity was observed in fish fed
with 10 g A. officinalis extract-
supplemented feed for 45 days.
No significant difference was
distinguished in AST activity after 15
days of treatment. When fish were fed
with diets supplemented with 5 g A.
officinalis extract for 45 days, the ALT
activities significantly decreased
(p<0.05).
Dietary intake of A. officinalis
extract had significantly decreased the
plasma ALP activity after 15 days of
feeding (p<0.05). ALP activity
decreased significantly in fish fed with
diets supplemented with 5 g A.
officinalis extract for 45 days (p<0.05).
In fish that received 2.5 g A.
officinalis extract-supplemented feeds
for 15 days, there was significant
difference (p<0.05) in the CPK activity.
However, after 45 days of feeding with
extract supplemented diet, there was no
significant alteration in the CPK
activity.
In the present study, although no
significant change in the LDH activity
was observed in the fish that were fed
with different doses of A. officinalis
extract for 15 days, this activity
increased significantly in fish which
were fed diets supplemented with 10 g
marshmallow extract for 45 days
(p<0.05).
When administered as a diet
supplement for 15 days, all the doses of
the A. officinalis extract tested
significantly decreased the plasma
glucose levels (p<0.05). However, there
was no significant difference in glucose
levels between the treatments and the
control group on day 45 (p>0.05).
Iranian Journal of Fisheries Sciences 15(2) 2016 619
Figure 1: Gas chromatography analysis of essential oil of the marshmallow (Althaea
officinalis) flower.
No significant changes were observed
in the total protein and globulin levels
on day 45 and 15 (p>0.05). However,
after 15 days of feeding on A. officinalis
extract-supplemented feed, a significant
decrease was observed in plasma
albumin. Fish that were fed for 45 days
with 5 and 10 g of A. officinalis extract-
supplemented diet exhibited significant
increase of albumin (p<0.05). The
cholesterol significantly decreased in
the fish fed for 15 days with feed
supplemented with different doses of A.
officinalis extract (p<0.05). However,
no significant changes were observed in
cholesterol levels in fish after being fed
A. officinalis extract on day 45
(p>0.05).
The triglyceride levels significantly
decreased in fish fed with 2.5 g of A.
officinalis extract-supplemented diet for
15 and 45 days (p<0.05). No significant
changes were observed in creatinine
levels of fish (p>0.05).
620 Soleimany et al., Evaluation of pre-clinical safety and toxicology of Althaea officinalis
Table 2: Enzymatic activities in plasma of common carp fed with Althaea
officinalis extract as supplement.
Biochemical
parameters
Concentration of
A. officinalis
extract in diet
(g/Kg)
Sampling time
15th day
45th day
AST (U.L-1)
Control (0.0 g)
59.89±15.45c
38.15±10.47ab
2.5 g
48.97±5.04bc
32.45±8.17a
5.0 g
28.61±5.23a
54.11±12.74bc
10.0 g
36.06±7.09ab
60.86±17.30c
ALT (U.L-1)
Control (0.0 g)
11.69±2.39a
15.55±1.86b
2.5 g
13.45±1.23a
11.36±3.77ab
5.0 g
15.44±5.87a
10.15±2.64a
10.0 g
15.11±4.53a
15.11±2.02b
LDH (U.L-1)
Control (0.0 g)
166.54±21.22a
247.77±7.38a
2.5 g
185.24±21.55a
210.17±46.17a
5.0 g
164.75±46.76a
224.42±61.20a
10.0 g
157.63±19.34a
300.12±20.94b
ALP (U.L-1)
Control (0.0 g)
56.83±18.79b
48.71±6.18b
2.5 g
27.88±2.45a
45.80±10.95b
5.0 g
27.57±1.84a
29.87±8.15a
10.0 g
33.70±3.11a
58.21±8.35b
CPK (U.L-1)
Control (0.0 g)
821.27±244.54a
1206.93±280.03a
2.5 g
1432.07±269.30b
956.11±330.41a
5.0 g
1191.33±578.86ab
1200.30±293.98a
10.0 g
1126.44±261.23ab
972.19±105.10a
Significant differences between data were characterized by alphabet letters when
compared with control data in the same test group along time (One way ANOVA, p<
0.05). Values sharing the same superscript letter indicate no significant differences
compared with control value during the same experiment time (p > 0.05). Data are
expressed as means ± S.D. Enzyme activity is expressed as units per liter (U.L-1 or
U/L).
Table 3: Biochemical parameters in plasma of common carp fed with
Althaea officinalis extract as supplement.
Biochemical
parameters
Concentration of A.
officinalis extract in
diet (g/Kg)
Sampling time
15th day
45th day
Glucose
(mg.dL-1)
Control (0.0 g)
95.79±10.79b
68.19±10.05a
2.5 g
53.65±11.35a
56.64±10.74a
5.0 g
61.24±4.35a
66.60±19.78a
10.0 g
59.27±9.74a
66.86±9.34a
Total Protein
(g.dL-1)
Control (0.0 g)
3.72±0.90a
3.70±0.63a
2.5 g
3.15±0.16a
4.70±0.64a
5.0 g
3.41±0.55a
4.10±0.93a
10.0 g
3.13±0.31a
4.73±0.68a
Iranian Journal of Fisheries Sciences 15(2) 2016 621
Significant differences between data were characterized by alphabet letters when
compared with control data in the same test group along time (One way ANOVA, p
< 0.05). Values sharing the same superscript letter indicate no significant differences
compared with control value during the same experiment time (p> 0.05). Data are
expressed as means±S.D.
Discussion
Dodecanoic acid and n-Tetradecanoic
acid are the major saturated fatty acid
found in the essential oil of A.
officinalis. Monoterpenes have various
biological properties including
anticancer (Astani et al., 2010),
antimicrobial, antifungal, anti-
genotoxic, anti-inflammatory,
insecticidal and antioxidant (Aydın and
Türkez, 2014). P-Cymene (1-isopropyl-
4-methylbenzene) is a natural
hydrocarbon that is a component of
essential oils, which are natural
products extracted from marshmallow.
P-Cymene has anti-bacterial properties.
Borneol and camphor are bicycle
monotrpenes found in marshmallow
extract. α-Terpineol (TPN), a volatile
monoterpene alcohol, is relatively non-
toxic and one of the major components
of the essential oils of marshmallow.
Benzaldehyde, n-decanal, is mainly
used as a food and flavoring additive
and can be found in many fruit and
plants. Menthol is a cyclic terpene
Continued Table 3:
Biochemical
parameters
Concentration of A.
officinalis extract in
diet (g/Kg)
Sampling time
15th day
45th day
Albumin
(g.dL-1)
Control (0.0 g)
2.24±0.31b
1.58±0.58a
2.5 g
1.47±0.23a
1.43±0.41a
5.0 g
1.67±0.41a
2.73±0.38b
10.0 g
1.69±0.31a
3.60±0.49c
Globulin
(g.dL-1)
Control (0.0 g)
1.48±0.68a
2.12±1.12ab
2.5 g
1.68±0.26a
3.28±0.30b
5.0 g
1.74±0.83a
1.37±1.19a
10.0 g
1.45±0.46a
1.13±0.76a
Cholesterol
(mg.dL-1)
Control (0.0 g)
59.03±2.37c
73.04±14.35ab
2.5 g
49.86±4.45b
66.37±9.76a
5.0 g
36.69±6.69a
67.20±16.57a
10.0 g
35.52±7.12a
99.05±24.76b
Triglyceride
(mg.dL-1)
Control (0.0 g)
239.44±59.28b
261.74±25.08b
2.5 g
153.05±13.05a
180.05±65.39a
5.0 g
192.49±43.84ab
223.47±35.49ab
10.0 g
208.45±23.63ab
261.50±63.47b
Creatinine
(mg.dL-1)
Control (0.0 g)
0.57±0.26a
0.30±0.06a
2.5 g
0.45±0.22a
0.35±0.05a
5.0 g
0.38±0.13a
0.30±0.06a
10.0 g
0.36±0.19a
0.31±0.09a
622 Soleimany et al., Evaluation of pre-clinical safety and toxicology of Althaea officinalis
alcohol detected in marshmallow
extract. Therefore, marshmallow has
the potential to be used for prevention
and treatment of bacterial, viral, and
fungal infections.
During the experimental periods, no
mortalities or changes in the appetite of
the fish were observed. Platel et al.
(2002) evidenced the favorable effect of
medicinal plants on digestion and a
stimulating effect on bile secretion and
the activity of pancreatic enzymes.
Moreover, adding plants extracts to the
diet can affect the ability of the fish to
find food by stimulating their sense of
smell and can encourage them to eat
more (Adams, 2005).
In this study, oral administration of
the A. officinalis extract significantly
decreased the plasma ALT activity at a
dose of 5 g compared to the control
group on day 45 (p<0.05).
Supplementation of A. officinalis
extract to fish significantly decreased
the activity of alkaline phosphatase on
day 15 (p<0.05). Treatment of the fish
with 2.5 g A. officinalis extract
decreased activities AST, and LDH in
plasma compared to the control group.
Although, increased creatine kinase
(CK) activity was observed in the
plasma of fish fed 2.5 g A. officinalis
extract on day 15 (p<0.05), CK activity
returned to the normal levels in fish
treated with A. officinalis extract at the
end of the experimental period (Table
2). More hydrophilic flavonoids
interacting at the membrane surface
through hydrogen bonding; may reduce
the access of oxidants and per-oxidants,
thus protecting the structure and
function of cellular membranes (Oteiza
et al., 2005). Oral administration of
thymol and carvacrol activated
antioxidant enzymes and protected liver
cells against severe damages
(Hashemipour et al., 2013). Carvacrol
supplement reduced AST, ALT, and
LDH activity in plasma of the fish
treated with D-glucosamine (Jayakumar
et al., 2012). The effects of
marshmallow extract on the activity of
plasma enzymes can be compared with
the effects of other extracts such as
Allium cepa and A. sativum (Al-Salahy,
2002), Curcuma longa (Deshpande et
al., 2003) Cyperus rotundus (Suresh
Kumar and Mishra, 2004), Pterocarpus
santalinus (Palanisamy et al., 2007),
Aloe vera, Clematis hirsute, Cucumis
prophetarum (Alqasoumi et al., 2008),
Hybanthus enneaspermus
(Premalakshmi and Thenmozhi, 2011).
The useful effect of A. officinalis in
decreasing ALP activity might be due
to the presence of minerals, particularly
calcium levels in the A. officinalis
extract. A low concentration of
silymarin (Banaee et al., 2011), yarrow
extract (Nafisi Bahabadi et al., 2014)
and garlic (Al-Salahy, 2002) in diet of
fish are proven to regulate the plasma
activities of AST, ALT, ALP, CK and
LDH.
The results show that AST and LDH
activities increased in plasma of fish fed
a diet supplemented with 10 g/Kg of A.
officinalis extract on day 45 which may
be due to leakage of these enzymes
from the liver cytosol into blood. Some
Iranian Journal of Fisheries Sciences 15(2) 2016 623
monoterpenes such as d-limonene, α-
pinene, myrcene and linalool, stylosin
could have cytotoxic effects (Aydın and
Türkez, 2014). P-Cymene is
metabolized by the liver and the major
p-cymene metabolite is cuminyl alcohol
which can cause mitochondrial toxicity
by affecting the process of energy
production in hepatocytes (Custódio et
al., 2011). Toxicological studies show
that safranal, and furfural, are other
toxic compounds found in plants such
as marshmallow. Dimethyl glutarate is
an ester that may cause olfactory
toxicity (Morris et al., 1991). Furfural
turns into the toxic pyromucic acid in
liver after oxidation. Therefore, furfural
may indirectly cause oxidative stress
and damage the liver cells (Veićković et
al., 2011). Increased activity of AST,
ALT and ALP was reported in the rats
treated with furfural during 7 days
(Veićković et al., 2011).
A significant lower concentration of
glucose was noted in fish fed a diet
enriched with A. officinalis extract as
compared to control fishes on day 15
(p<0.05). The probable mechanism of
A. officinalis extract may be partly due
to the stimulation of insulin secretion or
may be attributed to the activation of
glycogen synthesis and healthy hepatic
function (Ji et al., 2007). Moreover, this
was probably because of the flavonoids
in the plants which reduce glucose
uptake in the intestine via the inhibition
of sodium-dependent glucose transport
(Song et al., 2002). Decreased glucose
levels in the blood of rainbow trout fed
with silymarin extract (Banaee et al.,
2011), yarrow extract (Nafisi Bahabadi
et al., 2014) and African catfish fed
with onion and garlic extracts are
reported (Al-Salahy, 2002).
Since there is a close relationship
between the rate of protein synthesis in
liver and total protein concentration in
plasma (Banaee et al., 2011), the
increased concentration of total protein
in plasma of the fish fed with
marshmallow extract may indicate
increased protein synthesis rate in the
liver. Amino acids present in the A.
officinalis extract may increase protein
synthesis in the liver and other tissues.
Although, there was no significant
difference in plasma globulin level on
days 15, oral administration of 2.5 g A.
officinalis extract significantly
increased plasma globulin level on day
45 (p<0.05). Oral administration of
marshmallow extract decreased
albumin level on day 15, while the
albumin level in the fish fed with
marshmallow extract (5 and 10 g)
increased significantly on day 45
compared with the control group
(p<0.05). An increased level of total
protein, albumin and globulin in plasma
of the fish fed with diets enriched with
Echinacea purpurea and Silybum
marianum was reported by Bohlouli
Oskoii et al. (2012) and Banaee et al.
(2011). No significant changes were
reported in the levels of albumin and
total protein in plasma of fish fed with
diets enriched with onion and garlic
extract (Al-Salahy, 2002).
Supplementation of A. officinalis
extract in the diet significantly reduced
624 Soleimany et al., Evaluation of pre-clinical safety and toxicology of Althaea officinalis
the cholesterol levels in the plasma
(p<0.05). Interference with cholesterol
absorption from intestine due to
administration of A. officinalis might
have played a role in decreasing plasma
cholesterol levels. The beneficial
effects of A. officinalis on cholesterol
might be due to pectin and mucilage
(Ezhumalai et al., 2014) and
phytosterols (Marinangeli et al., 2006;
Jones et al., 2007). Triglycerides or
triacylglycerol are neutral fats, major
energy reserves for the body stored in
the adipose tissue. Therefore, decrease
in triglyceride levels in plasma of fish
fed with 2.5 g A. officinalis extract in
diet may be due to increased
lipogenesis and decrease lipolysis and
ketogenesis. In the lipogenesis process,
triglycerides and fatty acids are
synthesized and stored in adipose
tissue. Decreases in cholesterol and
triglyceride levels were also reported in
blood of rainbow trout and catfish
respectively fed with silymarin extract
(Banaee et al., 2011), yarrow extracts
(Nafisi Bahabadi et al., 2014) and onion
and garlic extract (Al-Salahy, 2002).
This work has demonstrated that A.
officinalis is by far relatively non-toxic
(2.5 and 5 g) in terms of biochemical
parameters. Other findings obtained in
the present study are that A. officinalis
extract exhibited moderate cytotoxicity
in high dose (10 g), while its extract has
moderate antioxidant properties at
doses of 2.5 and 5 g. Indeed, the
potential in the plant to influence
several clinically important parameters
could be linked to various levels of
pharmacological advantages. So, we
recommend the use of these dosages in
prospective clinical study to further
evaluate the efficacy and safety of the
A. officinalis extract as naturopathic
medicine for fishes.
Acknowledgments
The authors gratefully acknowledge the
support offered (8/1/T/244) from the
Behbahan Khatam Alanbia University
of Technology. Also, the authors are
grateful to Maryam Banaee, our English
editor, for proofreading the manuscript.
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... Blood levels of these enzymes significantly elevated in fishes living in heavy metal-contaminated environments (Zikic et al., 2001;Gabriel and George, 2005;Yousafzai and Shakoori, 2011;Abalaka, 2013;Gholizadeh et al., 2018;Ugbomeh et al., 2019). This elevation may be due to the toxic effects of these metals on fishes, and refers to histological damage in some organs, particularly the liver (Yousafzai and Shakoori, 2011;Soleimany et al., 2016;Gholizadeh et al., 2018;Ugbomeh et al., 2019). Blood liver enzymes are normally predominantly contained within hepatocytes and are spilled into the bloodstream following cellular damage (Sallie et al., 1991;Mayne, 2002;Palanivelu et al., 2005;Abalaka, 2013), since toxicants such as toxic heavy metals increase the permeability of hepatocyte plasma membrane, and consequently liver enzymes can leak from hepatocytes into the bloodstream and elevate to higher levels, causing many disease, such as anorexia and fatigue (Nordlie et al., 1999;Coz-Rakovac et al., 2008). ...
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Exposure to microorganisms such as Yersinia ruckeri can significantly affect bacterial infections in fish. Microplastics (MPs) may predispose fish to infection and act as carriers in pathogen transmission. Therefore, this study is designed to evaluate MPs’ effect on damage caused by exposure to Y. ruckeri in rainbow trout. In this study, blood biochemical parameters and hepatic oxidative biomarkers as clinical signs were measured in the fish co-exposed to Y. ruckeri (5 and 10% the median lethal dose (LD50)) and MPs (500 and 1000 mg Kg⁻¹) for 30 days. There were no significant changes in the creatinine, triglyceride, cholesterol levels, and glutamic-pyruvic transaminase activity in the blood of fish infected with Y. ruckeri. In contrast, exposure to MPs had a significant effect on most clinical parameters. The total protein, albumin, globulin, total immunoglobulins, high-density lipoprotein, low-density lipoprotein, cholesterol levels, and γ-glutamyltransferase activity decreased, whereas glucose, triglyceride, and creatinine levels, and glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, alkaline phosphatase, and lactate dehydrogenase activities increased in the plasma of fish after co-exposure to MPs and Y. ruckeri. Dietary MPs combined with a Y. ruckeri challenge decreased catalase and glutathione peroxidase activities, and total antioxidant levels. However, superoxide dismutase activity and malondialdehyde contents increased in the hepatocyte of fish co-exposed to MPs and Y. ruckeri. This study suggests that fish exposure to MPs and simultaneous challenge with Y. ruckeri could synergistically affect clinical parameters.
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The outbreak of COVID-19 has become a global health concern. The vaccines against SARS-CoV-2 are unable to barricade the reinfection in fully vaccinated individuals. Considering this dilemma, the recent research strategies are focused on the new candidates having antiviral potential with significant and consistent efficacies as well as the least side effects. In this study, we have screened plant-derived phytochemicals, antiviral compounds from PubChem, and natural compounds from the Hamdard products for identification of antiviral therapeutics against Spike (S) glycoprotein and main protease (M pro ) of SARS-CoV-2. All these compounds were screened based on their binding affinities as predicted by molecular docking analysis and compounds having binding affinity values ≤ -10 kcal/mol were considered for analysis. Furthermore, from physicochemical assessment, drug-likeness initially nine compounds were identified as the antiviral targets for the selected viral proteins. Finally, after ADMET analysis and MD simulations, the compound 9064 with the lowest Root Mean Square Deviations (RMSD), Coul-SR interaction energy (-71.53 kJ/mol), and LJ-SR energy (-95.32 kJ/mol) was selected as the most stable drug candidate against COVID-19 main protease M pro . The selected antiviral compound 9064 is an antioxidant flavonoid (Catechin or Cianidanol), which is previously known to have significant immunomodulatory, anti-inflammatory, and antioxidant properties.
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Aquatic ecosystems have become a place for accumulating microplastics (MPs). MPs can directly or indirectly damage organisms. Although studies of the toxicity of MPs, there are insufficient literature reports on the effects of MPs on freshwater aquatic life. Therefore, this study aimed to evaluate the effect of MPs toxicity on Cyprinus carpio. In this study, biochemical parameters, oxidative biomarkers, and gene expression were assayed in fish exposed to 0, 175, 350, 700, and 1400 μg L⁻¹ of MPs for 30 days. MPs were detected in the liver and intestine of fish using FTIR-analysis. Mt1, Ces2, and P450 mRNA expression were enhanced in the hepatocytes of fish exposed to MPs, while Mt2 gene expression was significantly decreased. After exposure to MPs, MDA and carbonyl protein levels were higher than those of the reference group. The antioxidant capacity and glycogen contents in the hepatocytes significantly declined. MPs significantly inhibited glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), and catalase (CAT) activities. However, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities increased. MPs decreased the total protein, globulin levels, and butyrylcholinesterase (BChE) activity in blood. In contrast, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and creatine phosphokinase (CPK) activities increased in treated-fish with MPs. Glucose, creatinine, cholesterol and triglyceride concentrations in fish exposed to MPs were significantly higher than that of the reference group. Consequently, MPs exposure could disrupt biochemical homeostasis, oxidative stress and alter the expression of genes involved in detoxification.
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Introduction: Nootropic agents, including cholinesterase inhibitors are being used to improve memory, mood and behavior, but the side-effects associated with these agents have made their use limited. The present study has therefore been undertaken to assess the synergistic effects of Celastrus paniculatous and Angelica glauca on scopolamine induced dementia in rats. Materials and Methods: Rats were treated with scopolamine (1 mg/kg body weight, i.p.) alone and with donepezil (2 mg/kg body weight p.o.), C. paniculatous (150 mg/kg body weight, p.o) and A. glauca (150 mg/kg body weight, p.o.). The changes in behavioral and biochemical parameters were assessed in rats. Results: Scopolamine treated rats showed impaired learning and memory, increased activity of acetylcholinesterase (AChE) (50%), lipid peroxidation (60%), protein carbonyls (47%) and decreased levels of reduced glutathione (GSH) (35%), activity of superoxide dismutase (34%) and catalase (42%) in hippocampus as compared with control. Simultaneous treatment of C. paniculatous and A. glauca with scopolamine also caused an improvement in the learning and memory activity associated with AChE activity in hippocampus of rats as compared to those treated with scopolamine alone. Combined treatment of C. paniculatous, A. glauca and scopolamine significantly improved the learning and memory function and AChE activity (50%) associated with decreased lipid peroxidation (33%), protein carbonyls (27%) and increased levels of antioxidant enzymes like reduced GSH (46%), activity of superoxide dismutase (50%) and catalase (62%) in hippocampus of rats as compared with those treated with scopolamine alone. Conclusion: The results of the present study exhibit protective efficacy of combined treatment of C. paniculatous and A. glauca in scopolamine induced dementiaand promising as a memory enhancing agents that is associated with its strong antioxidant potential.
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The bovine viral diarrhoea virus (BVDV) is suggested as a model for antiviral studies of the hepatitis C virus (HCV). The antiviral activity of the essential oil of Ocimum basilicum and the monoterpenes camphor, thymol and 1,8-cineole against BVDV was investigated. The cytotoxicities of the compounds were measured by the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) test, and the antiviral activities were tested by the plaque reduction assay. The oil or compounds were added to the assay in three different time points: a) pre-treatment of the virus (virucidal assay); b) pre-treatment of the cells; or c) post-treatment of the cells (after virus inoculation). The percentage of plaques inhibition for each compound was determined based on the number of plaques in the viral control. The results were expressed by CC50 (50% cytotoxic concentration), IC50 (inhibitory concentration for 50% of plaques) and SI (selectivity index = CC50/IC50). Camphor (CC50 = 4420.12 μg mL(-1)) and 1,8-cineole (CC50 = 2996.10 μg mL(-1)) showed the lowest cytotoxicities and the best antiviral activities (camphor SI = 13.88 and 1,8-cineol SI = 9.05) in the virucidal assay. The higher activities achieved by the monoterpenes in the virucidal assay suggest that these compounds act directly on the viral particle.
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The antifungal effect of pine needle extract prepared by a distinguishable extraction method and the dry distillation method, was examined. The effect of this extract itself was insignificant. The chemical components of pine needle extract were then investigated by gas chromatographic analysis, and four chemical components, acetol, furfural, 5-methyl furfural, and terpine-4-ol, were identified. The antifungal effects of those four chemical components against Alternaria mali (A. mali), an agent of Alternaria blotch of apple, were then examined. It was observed that the minimum inhibitory concentrations (MICs) were 6.25, 0.78, 0.78, and 12.5 (mg/ml) of acetol, furfural, 5-methyl furfural, and terpine-4-ol, respectively. MICs of furfural and 5-methyl furfural had the same order of magnitude as that of an antifungal agrochemical, chlorothalonil. Although furfural itself can not be completely substituted for an antifungal agrochemical, a partial mixture of furfural and antifungal agrochemical may be used as a substitute. The use of agrochemicals for the prevention of plant disease caused by pathogenic fungus such as A. mali could be partially reduced by the application of this mixture.
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Background: One of the domains of scientific activities is working on animals. Performing experiments on animals is permissible only with the purpose of obtaining necessary information for saving and improving life of human beings or animals. Principally, all religions believe that human life is more valuable than animal life and humans have a God-given authority over animals, but they should not be cruel to animals and cause their pain or suffering. Based on Islamic view points, although Allah has put the Man as the lord of all creatures, he has not the right to use other creatures for any conditions and does not respect their real statues. Because of the widespread use of experimental animals in our country, special ethical codes should be redefined for living conditions of experimental animals based on the present regulations in Iran and also other countries. Therefore, all our researchers should have enough information about ethical codes of treating experimental animals as well as Islamic principles in this regard.
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At present, dyslipidemia is most commonly treated with lipid-altering pharmacological therapies. However, safety concerns regarding the use of these agents have prompted the need for safe and efficacious nonpharmacological lipid-altering interventions. One such natural therapy is the combination of plant sterols and endurance training. This combination lifestyle intervention has been shown to decrease total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride concentrations while increasing high-density lipoprotein (HDL) cholesterol concentrations. However, the mechanisms that underlie these positive lipid alterations have yet to be clarified. Thus, the purpose of this review is to evaluate individual effects of plant sterols and exercise training on lipid levels while attempting to elucidate the possible independent and synergistic mechanisms of action responsible for these modulations. Results reveal that plant sterols decrease both total and LDL cholesterol levels by reducing exogenous cholesterol absorption by way of cholesterol displacement in the intestinal lumen. Additionally, the intestinal membrane transport proteins, ABCG5, ABCG8, as well as NPC1L1, have also been implicated in plant sterol-mediated cholesterol lowering. Conversely, exercise decreases triglyceride levels by reducing hepatic very low-density lipoprotein secretion and increasing skeletal lipoprotein lipase activity. In addition, endurance training was shown to increase HDL cholesterol levels by way of HDL subfraction alterations, in conjunction with changing reverse cholesterol transport enzyme activities. Moreover, plant sterols and exercise may work synergistically to alter lipid levels by modulating lipoprotein transport, composition, release and metabolism. In sum, the present review lends further insight as to the metabolic benefits of adopting a healthy lifestyle, including plant sterols and endurance training, in the treatment of dyslipidemia.
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This full-color reference offers practical, evidence-based guidance on using more than 120 medicinal plants, including how to formulate herbal remedies to treat common disease conditions. A body-systems based review explores herbal medicine in context, offering information on toxicology, drug interactions, quality control, and other key topics. More than 120 herbal monographs provide quick access to information on the historical use of the herb in humans and animals, supporting studies, and dosing information. Includes special dosing, pharmacokinetics, and regulatory considerations when using herbs for horses and farm animals. Expanded pharmacology and toxicology chapters provide thorough information on the chemical basis of herbal medicine. Explores the evolutionary relationship between plants and mammals, which is the basis for understanding the unique physiologic effects of herbs. Includes a body systems review of herbal remedies for common disease conditions in both large and small animals. Discusses special considerations for the scientific research of herbs, including complex and individualized interventions that may require special design and nontraditional outcome goals.
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Inhalation exposure of the male and female rat to high concentrations of a mixture of the dibasic esters dimethyl succinate (DMS), dimethyl glutarate (DMG), and dimethyl adipate (DMA) results in mild olfactory toxicity. This response is thought to be due to the in situ formation of acidic metabolites via nasal carboxylesterases. The current study was designed to provide inhalation dosimetric information for these vapors. Deposition of DMS, DMG, and DMA was measured in the surgically isolated upper respiratory tracts (URT) of ketamine-xylazine-anesthetized male and female rats under constant velocity flow conditions at a flow rate of 100 ml/min. Deposition of acetone was measured in both genders for comparative purposes. URT deposition efficiencies in excess of 98.3% were observed for DMS, DMG, and DMA in animals exposed to each vapor individually. No gender differences in deposition efficiency were observed for these vapors or for acetone. Deposition of DMS, DMG, and DMA was also measured in animals exposed to all three vapors simultaneously. Deposition efficiency under simultaneous exposure conditions ranged between 97.3 and 98.5%. These values were slightly lower (about 1%) than those obtained under individual exposure conditions (p less than 0.0001). The reduced deposition efficiency may have resulted from competitive inhibition of nasal metabolism due to the simultaneous presence of all three carboxylesterase substrate vapors in nasal tissues. If so, inhalation of dibasic ester vapors would be expected to inhibit the uptake of other carboxylesterase substrate vapors without influencing uptake of vapors which are not substrates for this enzyme. Such was observed in studies using DMS, ethyl acetate (the substrate vapor), and isoamyl alcohol (the nonsubstrate vapor). Specifically, simultaneous exposure to DMS markedly inhibited uptake of ethyl acetate without altering uptake of isoamyl alcohol. Gender differences were not observed in URT deposition of any of the six vapors used in the current study, DMS, DMG, DMA, ethyl acetate, isoamyl alcohol, or acetone, suggesting that gender differences in URT deposition may not be widespread among vapors. The high URT deposition efficiencies of the dibasic esters are consistent with the olfactory toxicity resulting from inhalation exposure to these vapors.