DARU Journal of Pharmaceutical Sciences
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Angiogenic effect of the aqueous extract of Cynodon dactylon on human umbilical
vein endothelial cells and granulation tissue in rat
DARU Journal of Pharmaceutical Sciences (2015)3: Sample
doi:10.1186/s40199-015-0093-x
Hamid Soraya (soraya.h@umsu.ac.ir)
Milad Moloudizargari (Miladmoludi@gmail.com)
Shahin Aghajanshakeri (Shahin.aghajanshakeri@yahoo.com)
Soheil Javaherypour (Soli1998@gmail.com)
Aram Mokarizadeh (arammokarizadeh@yahoo.com)
Sanaz Hamedeyazdan (hamedeyazdans@hotmail.co.uk)
Hadi Esmaeli Gouvarchin Ghaleh (H.smaili69@yahoo.com)
Peyman Mikaili (Peyman_mikaili@yahoo.com)
Alireza Garjani (garjania2002@yahoo.com)
Sample
ISSN
Article type
2008-2231
Research article
Submission date
27 July 2014
Acceptance date
12 January 2015
Article URL
http://dx.doi.org/10.1186/s40199-015-0093-x
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Angiogenic effect of the aqueous extract of Cynodon
dactylon on human umbilical vein endothelial cells
and granulation tissue in rat
Hamid Soraya1*
*
Corresponding author
Email: soraya.h@umsu.ac.ir
Milad Moloudizargari2
Email: Miladmoludi@gmail.com
Shahin Aghajanshakeri2
Email: Shahin.aghajanshakeri@yahoo.com
Soheil Javaherypour2
Email: Soli1998@gmail.com
Aram Mokarizadeh3
Email: arammokarizadeh@yahoo.com
Sanaz Hamedeyazdan4
Email: hamedeyazdans@hotmail.co.uk
Hadi Esmaeli Gouvarchin Ghaleh5
Email: H.smaili69@yahoo.com
Peyman Mikaili1
Email: Peyman_mikaili@yahoo.com
Alireza Garjani6
Email: garjania2002@yahoo.com
1
Department of Pharmacology, Faculty of Pharmacy, Urmia University of
Medical Sciences, Urmia, Iran
2
Student of Veterinary Medicine, Faculty of Veterinary Medicine, Urmia
University, Urmia, Iran
3
Department of Immunology, Faculty of Medicine, and Cellular & Molecular
Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran
4
Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of
Medical Sciences, Tabriz, Iran
5
Department of Microbiology, Faculty of Veterinary Medicine, Urmia
University, Urmia, Iran
6
Department of Pharmacology & Toxicology, Faculty of Pharmacy, Tabriz
University of Medical Sciences, Tabriz, Iran
Abstract
Background
Cynodon dactylon, a valuable medicinal plant, is widely used in Iranian folk medicine for the
treatment of various cardiovascular diseases such as heart failure and atherosclerosis.
Moreover, its anti-diabetic, anti-cancer and anti-microbial properties have been also reported.
Concerning the critical role of angiogenesis in the incidence and progression of tumors and
also its protective role in cardiovascular diseases, we investigated the effects of the aqueous
extract prepared from the rhizomes of C. dactylon on vascular endothelial growth factor
(VEGF) expressions in Human Umbilical Vein Endothelial Cells (HUVECs) and also on
angiogenesis in carrageenan induced air-pouch model in rats.
Methods
In the air-pouch model, carrageenan was injected into an air-pouch on the back of the rats and
following an IV injection of carmine red dye on day 6, granulation tissue was processed for
the assessment of the dye content. Furthermore, in an in vitro study, angiogenic property of
the extract was assessed through its effect on VEGF expression in HUVECs.
Results
Oral administration of 400 mg/kg/day of the extract significantly increased angiogenesis (p <
0.05) and markedly decreased neutrophil (p < 0.05) and total leukocyte infiltration (p <
0.001) into the granulation tissues. Moreover, the extract increased the expression of total
VEGF in HUVECs at a concentration of (100 µl/ml).
Conclusion
The present study showed that the aqueous extract of C. dactylon promotes angiogenesis
probably through stimulating VEGF expression.
Keywords
Cynodon dactylon, Angiogenesis, Air pouch, HUVECs, VEGF
Background
Recently, medicinal plants have been largely considered as the harmless alternate to synthetic
drugs, especially due to their more safety and less side effects as compared to the chemical
drugs. Cynodon dactylon, also known as Bermuda grass, is a perennial grass native to the
warm temperate and tropical regions [1]. In North West of Iran, C. dactylon is known as
“Chayer” and the aqueous extract of its rhizomes is widely used in the treatment of
cardiovascular disorders such as atherosclerosis and heart failure due to its hypolipidemic and
cardiac tonic effects [2,3]. However, despite the presence of several reports on the antidiabetic, anti-microbial [4], hypolipidemic [5], hepatoprotective [6], anti-emetic and antiinflammatory [7] properties of C. dactylon, the probable role of angiogenesis as the
mechanism involved in cardioprotective effect of the plant has remained largely elusive.
Angiogenesis, the formation of new blood vessels from pre-existing capillaries, plays an
important role in many physiologic and also pathologic processes including cancer, ischemic
heart diseases and chronic inflammation [8,9]. Moreover, since the therapeutic interference
with angiogenesis offers a valuable tool for clinical application in several pathological
conditions, much attention has been paid on medications or compounds that alter the gene
expression profile of vascular endothelial cell growth factor (VEGF) and fibroblast growth
factors (FGFs) as two key pro-angiogenic molecules [10]. In some cases such as heart failure
or cardiac ischemia-reperfusion, stimulation of angiogenesis is beneficial and can be
considered as a target to improve the disease condition, however in several other diseases
such as cancer, atherosclerosis, rheumatoid arthritis and diabetic retinopathy, excessive
angiogenesis is part of the pathology and progression of the disease [9-11]. Therefore,
stimulation or inhibition of angiogenesis to reach therapeutic purpose is dependent on the
type of the disease.
Marappan and Subramaniyan [12] assessed anti-tumor effects of the methanolic extract of C.
dactylon leaves against ascetic lymphoma (ELA) in Swiss albino mice. The results showed
that the plant possesses significant anti-tumor effects [12]. Another study by Krishramoorthy
and Ashwini [13] also reported anti-cancer effects of C. dactylon in Swiss albino mice. The
hydroalcoholic extract from rhizomes of C. dactylon has been shown to have strong
protective effect on right heart failure in rats, in part by improving cardiac function and
increasing the contractile force [2]. In another study this effect was also attributed to its antiarrhythmic activity [14]. The plant also increased heart beat rate in a study on zebrafish with
a potency greater than that of betamethasone [15].
As stated above, C. dactylon has been proven to be an effective cardiovascular agent by
several studies; however almost none of the studies have precisely investigated the
underlying mechanisms through which this plant exerts its effects on the cardiovascular
system. The authors speculate that such beneficial effects of C. dactylon might be at least
partly associated with its probable effects on angiogenesis. Since there have been no studies
conducted so far on the possible effects of C. dactylon on angiogenesis, the present study was
carried out to investigate the possible angiogenic activity of the plant in human umbilical vein
endothelial cells (HUVECs) and also in an air-pouch model in rats.
Materials and methods
Extract preparation
C. dactylon was purchased from a traditional herbal market and the genus and species was
authenticated at the Herbarium of Botany, Faculty of Pharmacy, Urmia, Iran. The rhizomes
of the plant were dried in shade and coarsely ground to powder using an automatic grinder.
200 g of the powder was mixed in 2 L of distilled water and placed on a magnet stirrer at a
temperature of 50°C for three days. The mixture was then filtered three times using the
Wattman’s paper. The solution was finally evaporated to dryness for 12 hrs at 70°C. The total
amount of the crude extract obtained was 34 g. The extract was diluted with water in order to
be given orally by gavage needle.
Preliminary phytochemical screening
Qualitative phytochemical analysis of C. dactylon aqueous extract was conducted following
the standard procedures as described by Harborne [16], Sofowora [17] and Trease and Evans
[18].
Alkaloids
Crude extract was mixed with 2 ml of 1% HCl and heated gently. Mayer’s reagent was then
added to the mixture. Turbidity of the resulting precipitate is as evidence for the presence of
alkaloids.
Anthocyanins
2 ml of aqueous extract was added to 2 ml of 2 N HCl and NH3. Manifestation of pink-red
turning blue-violet indicates the presence of anthocyanins.
Coumarins
3 ml of 10% NaOH was added to 2 ml of aqueous extract, formation of yellow color indicates
the presence of coumarins.
Flavonoids
Crude extract was mixed with few fragments of magnesium ribbon and concentrated HCl was
added drop wise. Appearance of pink scarlet color after few minutes indicates the presence of
flavonoids (Shinoda test).
Saponins
Crude extract was mixed with 5 ml of distilled water in a test tube and shaken vigorously to
obtain a stable persistent froth. The frothing is then mixed with 3 drops of olive oil and for
the formation of emulsion which indicates the presence of saponins.
Tannins
Crude extract was mixed with 2 ml of 2% solution of FeCl3. Observed blue-green or black
coloration indicates the presence of tannins.
Assay for in vitro antioxidant activity
The free radical scavenging capacity of the extract was measured from the bleaching of the
purple-colored methanolic solution of 2,2-diphenyl-1-picrylhydrazyl )DPPH), a routinely
practiced material for the assessment of antiradical properties of different compounds [19].
The stock concentration of the C. dactylon aqueous extract (1 mg/mL) was prepared followed
by dilution to reach for concentrations 5 × 10−1, 2.5 × 10−1, 1.25 × 10−1, 6.25 × 10−2, 3.13 ×
10−2 and 1.56 × 10−2 mg/mL of the extract. The acquired concentrations in the same volumes
of 2 mL were added to 2 mL of a 0.08 % of DPPH solution [20-22]. Later than a 30 min of
incubation at 30°C, the absorbance of each solution was read against a blank sample at 517
nm (Shimadzu 2100 spectrophotometer - Japan). The average absorption value was noted for
each sample after the test was carried out in triplicate. Besides, as the positive control the
same procedure was gone over with quercetin. The inhibition percentage of DPPH free
radicals of by the aqueous extract was calculated as follows:
R% 100
Ablank– Asample/Ablank
Herein, “A blank” stands for the absorbance value of the control reaction and “A sample” is
the absorbance value for each sample. Additionally, RC50 value, the concentration of the
extract reducing 50% of the DPPH free radicals, was calculated from the graph of inhibition
percentages versus concentrations of C. dactylon extract in mg/mL.
Assay for total phenolics content
Total phenolic constituents of the C. dactylon aqueous extract was verified by assigning
Folin-Ciocalteu reagent and gallic acid as the standard compound for phenolics, the same
procedure as given in the literature [23-26]. Briefly, 0.5 mL of the extract was mixed with 5
mL of Folin-Ciocalteu reagent (10% v/v in distilled water) with 4 mL of 1 M aqueous
Na2CO3 after 5 min and the mixture was allowed to stand for 15 min with intermittent
shaking. The absorbance of the blue color produced by the reaction was measured using a
UV/visible spectrophotometer (Shimadzu 2100 - Japan) at 765 nm. The standard curve was
prepared using 25–300 µg/mL solutions of gallic acid in methanol: water (50:50, v/v).
Eventually, the value for total phenol content of the C. dactylon extract was represented in
terms of gallic acid equivalent which is a common reference compound.
Animals
Albino Wistar rats (220-250 g) were used in this study. Rats were housed at constant
temperature (20 ± 1.8°c) and relative humidity (50 ± 10%) in standard polypropylene cages,
eight per cage, under a 12 L:12D schedule and were allowed food and water freely. This
study was performed in accordance with the Guide for the Care and Use of Laboratory
Animals of Urmia University of Medical Sciences, Urmia-Iran.
In vivo angiogenesis assay
Rats were divided into 5 groups consisting 8 rats each. Rats in group 1 (carrageenan) received
intra-pouch injection of carrageenan and normal saline as vehicle (0.5 ml) was given orally.
Rats in group 2 received intra-pouch injection of carrageenan and intraperitoneal injection of
dexamethasone. Rats in group 3 to 5 received intra-pouch injection of carrageenan and
cynodon dactylon extract was given orally at doses 100, 200 and 400 mg/kg/day. For
analyzing the effects of the aqueous extract of C. dactylon on in vivo angiogenesis, the airpouch model described by Gosh et al. [8] was used with minor modifications. Briefly, rats (n
= 8) were lightly anesthetized with diethyl ether, the back was shaved and then swabbed with
70% ethanol. Subsequently, 8 ml of sterile air was injected subcutaneously on the back of the
animals to make an air-pouch oval in shape. Twenty four hours later, 4 ml of a 1% (w/v)
solution of carrageenan (Sigma Co; USA) in saline was injected into the air-pouch under light
diethyl ether anesthesia. The carrageenan solution had been sterilized by autoclaving at
121°C for 15 min and supplemented with penicillin G potassium and streptomycin sulfate
(JaberEbne – e- Hayyan, Iran) (0.1 mg/ml of the solution) after cooling to 40–45°C. The
aqueous extract of C. dactylon was administered orally at doses of 100, 200 and 400
mg/kg/day and dexamethasone as a standard anti-inflammatory agent (10 mg/kg/day) was
injected intraperitoneally, a day before and for 6 days after carrageenan injection.
Determination of angiogenesis in granulation tissue
Six days after carrageenan injection, measurement of the angiogenesis in the granulation
tissue was carried out using carmine dye, as an indicator of angiogenesis, according to the
methods described by Gosh et al. [8] with slight modification. Briefly, the rats were
anesthetized by intraperitoneal injection of a mixture of ketamine (60 mg/kg) and xylazine
(10 mg/kg). Then 3 ml of 5% (w/v) carmine dye (Sigma Co; USA) in 5% (w/v) gelatin
(Sigma Co; USA) in saline at 37°C was injected into the jugular vein of each rat. The
carcasses were chilled on ice for 3 hrs and then the entire granulation tissue was dissected and
weighted. After being washed with PBS (PH 7.4) the whole granulation tissue was
homogenized in two volumes of 0.5 mM sodium hydroxide using a T25 basic homogenizer
(IKA labortechnik, Gremany) for 4 min at 10000 g on an ice bed. The tissue homogenate was
centrifuged at 3000 g and 4°C for 45 min and 500 µl of the supernatant was diluted 2-fold
with 0.5 mM sodium hydroxide and centrifuged again. Then 100 µl of the supernatant was
diluted with 900 µl of 0.5 mM sodium hydroxide and the carmine dye content was assessed
spectrophotometrically at a wavelength of 490 nm. For histopathological visualization of the
granulation tissues, the tissues were fixed in 10% (v/v) formalin in PBS for 48 hrs at 4°C.
The samples were dehydrated by continuous immersion in 70% (v/v) ethanol for 48 hrs, 90%
(v/v) ethanol for 48 hrs, and pure ethanol for 48 hrs. After dehydration, the samples were
cleared by their immersion in the cedarwood oil (Sigma Co; USA) for 14 days. Retention of
carmine dye within the vascular bed was observed with a light microscope (40×
magnification).
Determination of pouch fluid accumulation, granulation tissue weight, total
leukocyte infiltration along with lymphocyte and neutrophil percentage in the
pouch exudates
Six days after the carrageenan injection, total pouch fluid was collected, the volume was
measured and the entire granulation tissue was dissected and weighted. The total leukocyte
count was determined in a neubauer chamber and the differential cell count was determined
by microscopic counting of Gimsa stained slides.
In vitro angiogenesis assay
The expression changes in cytoplasmic and surface levels of VEGF in PBS or extract treated
HUVECs were determined using a PAS flow cytometer (Partec GmbH, Germany). Briefly,
human umbilical vein endothelial cells (HUVECs) were cultured at 37°C and 5% CO2 in
low-glucose Dulbecco’s Modified Eagle’s Medium (LG-DMEM) supplemented by
Supplement Mix (PromoCell) and10% fetal bovine serum (FBS). At second passage cells
were treated with PBS (100 µl/ml) and C. dactylon extract (100 µl/ml). After 12 hrs using
trypsin/EDTA (Ethylenediaminetetraacetic acid) solution (0.25%), cells were detached and
then washed twice in PBS. The collected cells were permeablized with 0.1% PBS-Tween for
20 min. After incubation in 1x PBS / 10% normal goat serum / 0.3 M glycine to block nonspecific interactions, unconjugated rabbit anti human VEGF antibody (abcam) was added.
Subsequently, staining was performed using secondary goat anti-rabbit IgG PE antibody. The
total 20000 events for each sample were acquired. Flow max software was used for data
analysis.
Statistical analysis
Data were presented as mean ± standard error of the mean (SEM) and were analysed using
one-way-ANOVA to make comparisons between the groups. If the ANOVA analysis
indicated significant differences, Student–Newman–Keuls post test was performed to
compare the mean values between the treatment groups and the control group. Differences
between groups were considered significant at P < 0.05.
Results
Phytochemical analysis
Phytochemical compounds such as alkaloids, anthocyanins, coumarins, flavonoids, saponins,
tannins and phenolic compounds were screened in the C. dactylon aqueous extract.
Availability of these compounds, important secondary metabolites, has been tabulated in
Table 1. Among the selected compounds alkaloids, coumarins, saponins and some phenolics,
were present in the plant which could be responsible for the medicinal values of the
respective plant. Moreover, the amount of total phenolic compounds in the C. dactylon
aqueous extract established through Folin Ciocalteu method was calculated as gallic acid
equivalent. The equation [Sample absorbance = 0.0067 × gallic acid (µg) + 0.0132, (R2:
0.987)] achieved by the standard gallic acid graph was applied in calculation of the phenolic
compounds concentration. Subsequently, the content for C. dactylon total phenolics showed
the value 39.82 mg of gallic acid equivalent in g of plant extract (Table 1). The findings of
the antioxidant activity for C. dactylon aqueous extract, accomplished by the DPPH method,
revealed sensible values in vitro. Regarding the results for the DPPH radical scavenging
antioxidant assay, C. dactylon extract exhibited pleasant antioxidant activity with RC50 values
of 0.70 mg/ml for the extract and 3 µg/mL for the control quercetin (Table 1). Quantitative
phytochemical analysis revealed that the plant contained phenolic compounds a class of
phytochemicals that could be responsible for the antioxidant and free radical scavenging
effect of the plant material.
Table 1 Phytochemical analysis, DPPH radical scavenging capacity and total phenols
content of C. dactylon aqueous rhizomes extract
Alkaloids Anthocyanins Coumarins Flavonoids Saponins Tannins Total DPPH
phenols (IC50)
+
+
++
39.82 0.70
C.
mg/g mg/ml
dactylon
extract
Effects of the aqueous extract of C. dactylon on angiogenesis in granulation
tissue
Six days after the injection of carrageenan solution into the air-pouch, a dissectible
granulation tissue was formed in the subcutaneous tissue. Following intravenous injection of
carmine red dye to the anaesthetized animals, the dye was accumulated in the granulation
tissue and the amount of the dye was assessed as an index of angiogenesis. As shown in
Figure 1, oral administration of the aqueous extract of C. dactylon (400 mg/kg) produced a
significant (P < 0.05) increase in angiogenesis. In agreement with these findings, vascular
network formation was also stimulated by C. dactylon as shown in Figure 1 (upper trace;
right).
Figure 1 Upper trace: Effects of the aqueous extract of C. dactylon (400 mg/kg) on
angiogenesis in granulation tissue versus positive control (carrageenan; left) in the air
pouch model of angiogenesis in rats. Lower trace: The effect of oral administration of
aqueous extract of C. dactylon on carmine dye content (as an index of angiogenesis) in
granulation tissue in the air pouch model of angiogenesis in rats. Cyno: Cynodon dactylon,
Carrageen: Carrageenan. Data represented as mean ± SEM. N = 6. *P < 0.05 and ***P <
0.001 vs control group (Carrageenan).
Effects of the aqueous extract of C. dactylon on pouch fluid volume, leukocyte
Infiltration and granulation tissue weight
The treatment of rats with oral administration of the aqueous extract of C. dactylon at doses
of 100, 200 and 400 mg/kg which was started one day before the intra-pouch injection of
carrageenan and continued for 6 days, dose dependently reduced neutrophil (p < 0.05) and
total leukocyte (p < 0.001) recruitment whereas increased lymphocyte recruitment into the
exudates (Table 2). In contrast, administration of C. dactylon dose dependently increased
pouch fluid volume and granulation tissue weight in comparison to the carrageenan group
(Figure 2). Dexamethasone was used as a positive control which significantly reduced total
leukocyte accumulation in the exudate, pouch fluid volume and granulation tissue weight
compared to the carrageenan group (Table 2, Figure 2).
Table 2 Effect of the aqueous extract of C. dactylon on leukocytes recruitment into the
pouch exudate
Carrageenan Dexamethasone Carrageen + Cyno Carrageen + Cyno Carrageen + Cyno
100 mg/kg
200 mg/kg
400 mg/kg
12 ± 0.33*
22 ± 1.2
17 ± 3
13 ± 2*
Neutrophil percentage 20 ± 1
58 ± 2.8
50 ± 1.8
57 ± .32
59 ± 4.4
67 ± 3.7
Lymphocyte
percentage
Total leukocyte (105) 895 ± 6.4
498 ± 15.2*** 635 ± 14.3***
629 ± 33***
523 ± 19.1***
Data represented as mean ± SEM. N = 6. *P < 0.05 and *** P < 0.001 vs control group (Carrageenan) using one
way ANOVA with Student-Newman-Keuls post-hoc test. Carrageen: Carrageenan; Cyno: Cynodon dactylon.
Figure 2 Effect of the aqueous extract of C. dactylon at various doses on pouch fluid
volume and granulation tissue weight 6 days after carrageenan injection. Cyno: Cynodon
dactylon, Carrageen: Carrageenan. Data represented as mean ± SEM. N = 6–8. *p < 0.05 and
*** p < 0.001 versus the carrageenan group.
Effects of the aqueous extract of C. Dactylon on VEGF expression in human
umbilical vein endothelial cells (HUVEC)
The results obtained from flow cytometric analysis showed the increased expression of total
VEGF (both in cytoplasmic and surface levels) in HUVECs treated with the aqueous extract
of C. dactylon as compared to those treated with PBS. Three experiments were performed
and 12% increase in expression of VEGF was detected in extract-treated cells as compared to
the PBS-treated ones (Figure 3).
Figure 3 The expression change of VEGF in HUVECs by flow cytometric procedure.
The expression level of VEGF in PBS treated cells set as control and the expression changes
following treatment with C. dactylon extract compared to it. The increased expression of
VEGF in extract treated HUVECs has been shown as compared to the PBS treated ones.
Histograms are representative of three separate experiments.
Discussion
Overall, as far as we know, the long history of use and prevailing reputation of many types of
natural resources, particularly higher plant species, among the nations are impressive. More
recently, herbal medicines have been identified as sources of various phytochemicals, many
of which possess different countless activities. Accordingly, C. dactylon signifying to have
medicinally valuable secondary metabolite types of phytochemicals like alkaloids,
coumarins, saponins and some types of phenolics might have a role in angiogenesis.
Nonetheless, further research is required to meet the challenges of isolation and structural
elucidation of the major active compounds in the plant for identifying an efficient natural
medicine which is reliant on a better understanding of the association between chemical
constituents and biological properties of natural resources. In this time of increasing requisite
for effective, affordable health promotion and treatment strategies for our growing
populations and enlarging health problems, the history and reputation of herbal medicines
must be examined in a rigorous and scientific way which may be translated into clinical
benefit.
C. dactylon has been widely used in the traditional medicine of Iran and other countries for
the treatment of several cardiovascular conditions such as heart failure [2], and arrhythmias
[14]. Moreover, various pharmacological effects of the plant have been proven in several
studies [27]. Examples of these effects include analgesic and antipyretic [28], negative
ionotropic and negative chronotropic effects [29], anti-arthritic, and anti-inflammatory
activities [30]. Although promotion of angiogenesis has been a therapeutic strategy in the
treatment of cardiovascular diseases such as ischemic heart disease; however, it can be part of
the pathogenesis of several other diseases such as cancer [9,10].
The present study was carried out to evaluate the possible effects of C. dactylon on
angiogenesis which thought to mediate the part of beneficial effects of the plant in
cardiovascular conditions. Accordingly, the possible angiogenic effect of the plant was
assessed in both Carrageenan-induced air-pouch model as an in vivo and human umbilical
vein endothelial cells as an in vitro models. The results demonstrated that the constituents
present in the aquatic extract of the plant have the potential to increase angiogenesis probably
through up-regulation of VEGF-gene expression. Other angiogenesis parameters including
exudate volume and granulation tissue weight were increased dose dependently following the
administration of the extract. These results were accompanied with the decreased total
leukocyte and neutrophil infiltration into the air-pouch. Since decreasing total leukocyte and
neutrophil count indicates the anti-inflammatory effect of C. dactylon the obtained results
were parallel to the findings of the previous studies [7]. However, the extract of C. dactylon
exerts an anti-inflammatory effect against acute inflammation, while increasing angiogenesis.
It has been already shown that simultaneous administration of two different substances may
upregulate the angiogenic responses, while downregulating the inflammatory responses [31].
The same effect can be also attributed to different constituents present in the aqueous extract
of the plant.
In vitro study on the effect of C. dactylon on the expression of VEGF in human umbilical
vein endothelial cells revealed that there might be a positive correlation between the
angiogenic effect of the plant and its ability to increase VEGF expression. This might be the
possible underlying mechanism through which the plant exerts its effect. It has been also
shown in several studies that the extract of C. dactylon possesses significant anti-tumor
activities [32,33]. As noted earlier, angiogenesis plays a central role in the pathogenesis of
neoplastic diseases [34], however the exact mechanism(s) responsible for such effect of the
plant is not yet clearly understood. In the mentioned studies, the sole reversal effects of the
plant against undesired consequences of tumors [12] and its anti-oxidant properties [16] have
been partly attributed to this effect of the plant without adequate evaluation of other
mechanisms affecting tumor growth. For instance, none of these studies have evaluated the in
vivo angiogenesis changes of the tumors affected by the administration of the extract. Based
on the findings of the present study which indicate the potential of the plant to promote
angiogenesis at high doses, it has to be taken into consideration that the plant might contain
unsafe agents which may aid in the growth of the tumor through increasing the tumor
angiogenesis. This is probably the first study that reveals an unsafe aspect of this plant in
contrast to its beneficial properties previously shown in anti-cancer studies.
Conclusion
To the best of our knowledge, this is the first study showing the angiogenic property of the
aquatic extract of C. dactylon. Based on these findings, C. dactylon is suggested as a potential
source of angiogenic compounds, the effects of which might be attributable to its increasing
effect on the expression of VEGF, a growth factor mainly involved in angiogenesis.
Accordingly, this plant can be used in the development of novel herbal medicines for the
amelioration of the consequences of conditions such as ischemic heart disease, and other
cardiovascular complications. Our findings are in contrast with the results of previously
conducted studies on the anti-tumor effects of C. dactylon, indicating that in addition to its
beneficial effect as an anti-cancer source, it might be a toxic agent due to increasing tumor
angiogenesis. Therefore, critical care should be taken on the safety of the plant to be
introduced as an anti-tumor agent. Indeed, further studies are required to clarify the precise
underlying mechanisms and to identify the safe aspects of the plant to be employed as a
therapeutic source.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HS: Supervising and directing the project, carried out the data analysis and interpretations
and prepared the manuscript. MM: Carried out angiogenesis experiments. SA: Accompanied
Milad Moloudizargari in angiogenesis experiments. SJ: Animal grouping and handling. AM:
Carried out flow cytometric procedure and interpretation. SH: Performed phytochemical
analysis. HE-G: Carried out inflammation studies. PM: Prepared the plant material and the
extract. AG: Contributed in data analysis, interpretation and preparation of the manuscript.
All authors read and approved the final manuscript.
Acknowledgments
The present study was supported by a grant from the Research Vice Chancellors of Urmia
University of Medical Sciences, Urmia, Iran.
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