全 文 ： 2012 年 7 月 第 10 卷 第 4 期 Chin J Nat Med July 2012 Vol. 10 No. 4 247

Chinese Journal of Natural Medicines 2012, 10(4): 02470254
doi: 10.3724/SP.J.1009.2012.00247
Chinese
Journal of
Natural
Medicines







Achillea millefolium inflorescence aqueous extract
ameliorates cyclophosphamide-induced toxicity
in rat testis: stereological evidences
Ali Shalizar Jalali*, Shapour Hasanzadeh, Hassan Malekinejad
Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
Available online 20 July 2012
[ABSTRACT] Cyclophosphamide (CP) is extensively used for the treatment of various cancers, as well as an immunosuppressive
agent. However, CP is known to cause several adverse effects including reproductive toxicity. Achillea millefolium, a widely distributed
medicinal plant, is highly regarded for its medicinal activities, including antioxidant and anti-inflammatory properties. The present
study was conducted to assess whether Achillea millefolium inflorescences aqueous extract with antioxidant and anti-inflammatory
activities could serve as a protective agent against reproductive toxicity during CP treatment. Male Wistar rats were categorized into
four groups. Two groups of rats were administered CP at a dose of 5 mg·kg-1·d-1 for 28 d by oral gavages. One of these groups received
Achillea aqueous extract at a dose of 1.2 g·kg-1·d-1 orally 4 h after cyclophosphamide administration. A vehicle treated control group
and an Achillea control group were also included. The CP-treated group showed significant decreases in the body, testes and epidi-
dymides weights as well as many histological alterations. Stereological parameters, spermatogenic activities and testicular antioxidant
capacity along with epididymal sperm count and serum testosterone concentration were also significantly decreased by CP treatment.
Notably, Achillea co-administration caused a partial recovery in above-mentioned parameters. These findings indicate that Achillea
millefolium inflorescence aqueous extract may be partially protective against CP-induced testicular toxicity.
[KEY WORDS] Achillea millefolium; Cyclophosphamide; Toxicity; Testis
[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2012)04-0247-08

1 Introduction
Cyclophosphamide (CP), is a widely used cytotoxic al-
kylating agent with antitumor and immunosuppressant prop-
erties. It is used for the treatment of chronic and acute leuke-
mia, multiple myeloma, lymphomas, rheumatic arthritis and
systemic lupus erythematosus and in the preparation for bone
marrow transplantation[1]. Cyclophosphamide undergoes bio-
activation by the hepatic microsomal cytochrome P450
mixed function oxidase system to active metabolites that
enter the circulatory system. Phosphoramide mustard and
acrolein are the two active metabolites of cyclophos-
phamide[2]. The antineoplastic effects of cyclophosphamide

[Received on] 06-July-2011
[Research funding] This project was supported by Urmia University,
Faculty of Veterinary Medicine as a part of Ph. D thesis in histology
[*Corresponding author] Ali Shalizar Jalali: Ph. D, Tel: 98-9111166004,
Fax: 98-4412771926, E-mail: ali_shalizar@yahoo.com
These authors have no any conflict of interest to declare.
are associated with phosphoramide mustard, whereas acrolein
is linked to toxic side-effects like cell death, apoptosis, onco-
sis and necrosis[3]. In spite of its therapeutic importance, a
wide range of adverse effects including reproductive toxicity
has been demonstrated following cyclophosphamide treat-
ment in humans and experimental animals[4]. Adult male
patients treated with CP have demonstrated diminished sperm
counts and an absence of spermatogenic cycles in their tes-
ticular tissue[5]. Previous studies on male rats have confirmed
the potential of CP to cause oligospermia, azoospermia and
histological alterations in the testis and epididymis[6-7]. De-
crease in weight of reproductive organ, impaired fertility,
growth and development of next generation was also ob-
served in cyclophosphamide treated male rats[8]. Although the
precise mechanism by which CP causes testicular toxicity is
poorly understood, numerous studies have shown that CP
exposure can disrupt the redox balance of tissues leading to
oxidative stress[9-11]. It has been reported that oxidative DNA
damage is caused by hydroperoxide derivative of CP through
generation of H2O2[12]. Further, spermatozoa are more sus-
ceptible to peroxidative damage because of high concentra-
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248 Chin J Nat Med July 2012 Vol. 10 No. 4 2012 年 7 月 第 10 卷 第 4 期

tion of polyunsaturated fatty acids and low antioxidant ca-
pacity[13]. Also, acrolein has been found to interfere with the
tissue antioxidant defense system and produces highly reac-
tive oxygen free-radicals that are mutagenic to mammalian
cells[14]. Consequently, from these aforementioned studies,
combination of the drug delivery together with potent and
safe antioxidant may be the appropriate approach to reduce
CP-induced reproductive toxicity.
Achillea millefolium, popularly known as “yarrow”, is a
member of the Asteraceae family that has been used as medi-
cine by many cultures for over 3 000 years[15]. The medicinal
properties of Achillea millefolium are recognized worldwide
and the plant is included in the national Pharmacopoeias of
countries such as Germany, Czech Republic, France and
Switzerland[15-18]. Different preparations of Achillea mille-
folium have been shown to have anti-inflammatory, antitumor,
antimicrobial, liver protective and antioxidant properties[19-24].
In addition, previous studies have reported that infusions
prepared from Achillea species had an antioxidant capacity,
which is consistent with their total flavonoid and phenol con-
tents. It was found that Achillea infusions are good scaven-
gers of active oxygen species, including OH• radical, H2O2
and DPPH•[25]. These findings supported preliminary studies
which had demonstrated that powerful antioxidant properties
of this plant are associated with the presence of flavonoids
such as apigenin, luteolin and rutin[26-27]. Based on the above
findings, the present study was undertaken to assess whether
Achillea millefolium inflorescences aqueous extract with
anti-oxidant and anti-inflammatory properties could serve as
a protective agent against reproductive toxicity during CP
treatment in a rat model.
2 Materials and Methods
2.1 Plant material
Achillea millefolium plants were harvested from its nat-
ural habitat around the city of Urmia in West Azerbaijan
Province, northwestern Iran during the flowering season
(between May and July). The identification of collected
plants was confirmed scientifically at the research laborato-
ries of the Department of Agriculture of West Azerbaijan
province.
2.2 Preparation of the aqueous extract
The aqueous extract of dried inflorescences of the plant
was prepared by infusion of the finely dried material (3 × 30
min) in water at 70 ºC (1 : 10, W/V). The infusion was filtered
and concentrated under vacuum (at 56 ºC) to 1/12 of the
original volume and stored at −20 ºC. The concentrated ex-
tract was diluted in distilled water immediately before use[28].
2.3 Animal model
Adult sexually mature male [4 months of age weighing
(177.75 ± 7.68) g] albino rats of Wistar strain were ob-
tained from animal house of Veterinary School of Urmia
University. They were housed in a specific pathogen-free
environment under standard conditions of temperature [(25 ±
2) °C], relative humidity [(50 ± 10)%] and light (12 h light/12
h dark). They were fed with a standard pellet diet and had
free access to water. Body weights were recorded weekly
during the treatment period. Clinical and behavioral observa-
tions were also recorded throughout the study. Animal work
was conducted in compliance with guidelines for the humane
care and use of laboratory animals using protocols approved
by the university.
2.4 Experimental protocol
After 7 days of acclimation to the environment, the rats
were randomly divided into four groups consisting of six
animals each (n = 6). Group I served as control receiving
saline vehicle throughout the experiment. Group II received
CP (5 mg·kg-1·d-1) dissolved in saline, for a period of four
weeks by gavage. Group III received Achillea millefolium
aqueous extract (1.2 g·kg-1·d-1) dissolved in distilled water
orally. Group IV was given orally Achillea solution (1.2
g·kg-1·d-1) 4 h after CP administration. The protocol for this
study, including doses and duration of treatment for CP and
Achillea, were all designed according to previous studies[9,
28].
2.5 Sampling
Animals were euthanized by CO2 exposure in a special
device following anesthesia with Ketamine 24 h after the last
Achillea treatment. Blood was collected without anticoagu-
lant for serological analyses. Testes and epididymides were
quickly dissected out, cleared of adhering connective tissue
and weighed on a Mattler Basbal scale (Delta Range, Tokyo).
Testes were fixed in Bouin’s fixative (0.2% picric acid/2%
(V/V) formaldehyde in PBS) for histological evaluation.
2.6 Epididymal sperm count
In order to assess the sperm motility, one caudal epidi-
dymis was placed in 1 mL of Ham’s F10 medium. Cauda was
cut into 2–3 pieces and incubated at 37 ºC for 10 min in CO2
incubator to allow sperm to swim out of the epididymal tu-
bules. The epididymal sperm count was determined by he-
mocytometer. After dilution of epididymal sperm to 1 : 20 in
Ham’s medium, approximately 10 μL of this diluted speci-
men was transferred to each of the counting chambers of the
hemocytometer, which was allowed to stand for 5 min in a
humid chamber to prevent drying. The cells sediment during
this time and were counted with a light microscope at 400 ×.
The sperm count was expressed as number of sperm per mil-
liliter[29].
2.7 Determination of serum testosterone concentration
Serum concentration of testosterone was measured by
enzyme-linked immunosorbent assay (ELISA) as described
in the instructions provided by manufacturer’s kit (Demeditec
Diagnostics GmbH, Germany).
2.8 Assessment of testicular antioxidant capacity (TAOC)
To determine the effect of CP on oxidative stress and
consequently the potential beneficial effects of Achillea
aqueous extract, testicular antioxidant capacity was measured.
The assay is based on the assessment of ferric reduction an-
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2012 年 7 月 第 10 卷 第 4 期 Chin J Nat Med July 2012 Vol. 10 No. 4 249

tioxidant power (FRAP) assay[30]. Briefly, at low pH which
was achieved by adding of acetate buffer (300 mmol·L-1, pH
3.6), reduction of FeIII-TPTZ complex to the ferrous form
produces an intensive blue color that could be measured at
593 nm. Aqueous solution of FeII (FeSO4.7H2O) and appro-
priate concentrations of freshly prepared ascorbic acid were
used as blank and standard solutions, respectively. The TAOC
was expressed as mmol·L-1 per mg protein of the samples.
The protein content of the samples was measured according
to the Lowry method [31].
2.9 Histological analysis
After fixation of testes, they were directly dehydrated in
a graded series of ethanol, cleared in xylol and embedded in
paraffin wax. Thin sections (6 μm) perpendicular to the long-
est axis of the testis were cut using a microtome, stained with
hematoxylin and eosin and examined using a light micro-
scope.
2.10 Determination of histological parameters
For each testis, five vertical sections from the polar and
the equatorial regions were sampled[32] and an unbiased nu-
merical estimation of the following histological parameters
was determined using a systematic random scheme.
Seminiferous tubules Diameter (STsD) and germinal ep-
ithelium height (GEH): For measuring of seminiferous tubule
diameter and germinal epithelium height, 200 round or nearly
round cross-sections of seminiferous tubules were randomly
analyzed in each rat (one hundred per testis). Then, two per-
pendicular diameters of each cross-section of seminiferous
tubules were measured using an ocular micrometer of light
microscopy (Olympus Co., Germany) and their means were
calculated. Also, germinal epithelium height was measured in
4 equidistance of each cross-section of seminiferous tubules
and their means were calculated[33].
Cross-sectional area of the seminiferous tubules (AC):
The cross-sectional areas of the seminiferous tubules were
determined from the formula AC = πD2/4, where π is equiva-
lent to 3.142 and D the mean diameter of the seminiferous
tubules[34].
Number of profiles of seminiferous tubules in a unit area
of testis (NA): The number of profiles of seminiferous tubules
per unit area was determined by using the unbiased counting
frame proposed by Gundersen (1977)[35]. Using this frame, in
addition to counting profiles completely inside the frame we
counted all profiles with any part inside the frame provided
they do not touch or intersect the forbidden line (full-drawn
line) or exclusion edges or their extension.
Numerical density of seminiferous tubules (NV): This is
the number of profiles per unit volume and was determined
by using the modified Floderus equation: NV =NA/ (D +T)
Where; NA is the number of profiles per unit area, D is the
diameter and T the average thickness of the section[36].
Sertoli cell index (SCI), repopulation index (RI) and mi-
otic index (MI): Sixty seminiferous tubules per group were
randomly examined for the calculation of Sertoli cell index
(SCI), repopulation index (RI) and miotic index (MI). SCI is
the ratio of the number of germ cells to the number of Sertoli
cells identified by a characteristic nucleus and nucleolus in
all seminiferous tubules[37]. RI is the percentage of tubules
populated with germ cells that had clearly reached the inter-
mediate spermatogonial stage or later[38]. MI, the number of
round spermatids for each pachytene primary spermatocytes,
was calculated for determination of cell loss percentage dur-
ing cell division[39].
Leydig cell nuclear diameter (LCND) and Sertoli cell
nuclear diameter (SCND): These parameters were also de-
termined using calibrated ocular micrometer as described by
Elias and Hyde[40].
2.8 Statistical analysis
Results are expressed as x ± s. Differences between
groups were assessed by the analysis of variance (ANOVA)
using the SPSS software package for Windows. Statistical
significance between groups was determined by Tukey mul-
tiple comparison post hoc test and the P-values less than 0.05
were considered to be statistically significant. In addition,
between and within groups’ degree of freedom were 3 and 20,
respectively.
3 Results
3.1 Clinical signs and body and organ weight changes
All animals survived the experimental period. CP-treated
animals showed general signs of deterioration such as pilo-
erection, hair loss, lethargy, hunched posture, shivers and low
activity. Testes and epididymides weights were significantly
decreased by cyclophosphamide treatment, while it was less
decreased from controls with Achillea coadministration (Fig.
1). The absolute and relative weights of testes and epidi-
dymides were significantly lower than controls after cyclo-
phosphamide treatment, whereas daily administraton of
Achillea caused significant increase in the absolute and rela-
tive weights of testes and epididymides of cyclophos-
phamide-Achillea group in comparison with cyclophos-
phamide group (Table 1).
3.2 Epididymal sperm count
Treatment of male rats with CP caused a significant de-

Fig. 1 Gross appearance of testes from all groups of rats.
Illustrations of representative testes in Control, CP, Achillea
and CP + Achillea groups, respectively. CP group rats have
obviously smaller testes as compared to the other three groups.
Ali Shalizar Jalali, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 247254
250 Chin J Nat Med July 2012 Vol. 10 No. 4 2012 年 7 月 第 10 卷 第 4 期

Table 1 Effect of cyclophosphamide and Achillea millefolium inflorescences aqueous extract on body weight and weights of tes-
tis and epididymis ( x ± s, n = 6)
Control CP Achillea CP + Achillea F-value
Final body weight (BW/g) 226.33 ± 5.35 182.00 ± 6.06a 226.16 ± 9.26b 194.83 ± 10.72a 45.491
Absolute weight (g)
Testes 2.01 ± 0.065 1.49 ± 0.040a 2.04 ± 0.084b 1.69 ± 0.099a,b 72.695
Epididymides 1.15 ± 0.044 0.85 ± 0.010a 1.16 ± 0.039b 1.00 ± 0.081a,b 51.359
Relative weight (per BW/%)
Testes 0.88 ± 0.011 0.82 ± 0.004a 0.90 ± 0.007b 0.86 ± 0.008a,b 5.578
Epididymides 0.50 ± 0.019 0.46 ± 0.023a 0.51 ± 0.017b 0.51 ± 0.031b 104.470
a P <0.05 vs control group, bP <0.05 vs cyclophosphamide group

crease in the sperm concentration, while coadministration of
Achillea millefolium inflorescences aqueous extract caused a
significant increase in epididymal sperm quantity and mini-
mized the toxic effects of CP (Fig. 2).

Fig. 2 Effect of cyclophosphamide and Achillea millefolium
inflorescences aqueous extract on epididymal sperm count.
( x ± s, n = 6). aP < 0.05 vs control group, bP < 0.05 vs cyclo-
phosphamide group. F-value = 94.114
3.3 Serum testosterone level
Administration of CP alone significantly decreased se-
rum level of testosterone as compared to control rats. The
administration of aqueous extract Achillea millefolium inflo-
rescences along with CP significantly restored serum testos-
terone level towards the control value (Fig. 3).
3.4 Testicular antioxidant capacity
Interestingly, treatment with Achillea millefolium inflo-
rescences aqueous extract alone significantly increased anti-
oxidant capacity in the testis of normal rats. CP administra-
tion caused a significant decrease in TAOC level when com-
pared to that of control, while concurrent treatment with
Achillea aqueous extract significantly suppressed this reduc-
tion (Fig. 4).
3.5 Histopathological findings
CP induced drastic morphologic changes in the testis
(Fig. 5B). Atrophied seminiferous tubules showed severe
hypocellularity (reduction in number of germ cells) and in-
traepithelial vacuolization. Rupture, vacuolization, vascular
congestion, inflammatory cells infiltration, oedematous fluid
accumulation and interstitial space widening were also ob-
served in intertubular connective tissue. In these specimens,
Leydig cells were degenerated and appeared with pyknotic
nuclei. Moreover, Sertoli cells lost their junction with germ
cells and looked amorphous with irregular and smaller nuclei.

Fig. 3 Effect of cyclophosphamide and Achillea millefolium
inflorescences aqueous extract on serum concentrations of
testosterone. ( x ± s, n = 6). aP < 0.05 vs control group, bP <
0.05 vs cyclophosphamide group. F-value = 127.198

Fig. 4 Effect of cyclophosphamide and Achillea millefolium
inflorescence aqueous extract on testicular antioxidant ca-
pacity (TAOC). ( x ± s, n = 6). aP < 0.05 vs control group, bP <
0.05 vs cyclophosphamide group. F-value = 25.558
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2012 年 7 月 第 10 卷 第 4 期 Chin J Nat Med July 2012 Vol. 10 No. 4 251


Fig. 5 Photomicrographs of testicular sections of control (A), Cyclophosphamide (B), Achillea (C) and Cyclophosphamide +
Achillea (D) treated rats. Testes from control (A) and Achillea-treated (C) rats exhibit a normal feature of seminiferous epithe-
lium and interstitial tissue with active spermatogenesis. However, a testis from a Cyclophosphamide treated rats (B) reveals
markedly atrophied seminiferous tubules with severe hypocellularity and impaired spermatogenesis. Note Rupture, vacuolization,
vascular congestion (black arrow), oedematous fluid accumulation (white arrows) and interstitial space widening in intertubular
connective tissue. Achillea cotreated animals (D) display nearly normal architecture. Hematoxylin and eosin (× 200).
Administration of Achillea along with CP restored these
changes towards normalcy (Fig. 5D).
3.6 Histological parameters
As seen in Table 2, Cyclophosphamide treatment in-
duced deletion of germ cells during spermatogenesis, which
resulted in a dramatic decrease in SCI. Due to the germ cells
deletion, the number of repopulated seminiferous tubules was
greatly decreased in the CP-treated animals. CP treatment
also caused considerable decrease in miotic index. However,
Achillea coadministration significantly attenuated the CP-in-
duced germ cell loss from seminiferous tubules.
The seminiferous tubules diameters (STsD) and their
epithelial heights (GEH), Cross-sectional area of the seminif-
erous tubules (AC), Number of profiles of seminiferous tu-
bules in a unit area of testis (NA) and Numerical Density of
seminiferous tubules (NV) as well as Leydig cell nuclear di-
ameter (LCND) and Sertoli cell nuclear diameter (SCND)
were reduced by CP treatment. CP-induced testicular dam-
ages were mitigated by Achillea coadministration (Table 3).
4 Discussion
Many drugs used for cancer chemotherapy are known to

Table 2 Effect of cyclophosphamide and Achillea millefolium inflorescences aqueous extract on Sertoli cell index, repopulation
index and miotic index ( x ± s, n = 6)
Control CP Achillea CP + Achillea F-value
Sertoli cell index 25.22 ± 0.85 4.50 ± 0.17a 25.62 ± 0.76b 19.36 ± 0.56a,b 1 406.481
Repopulation index 95.41 ± 2.03 19.58 ± 2.08a 92.41 ± 2.33b 76.41 ± 2.51a,b 1 471.521
Miotic index 2.11 ± 0.027 0.98 ± 0.013a 2.18 ± 0.018a,b 1.63 ± 0.013a,b 5 041.751
a P <0.05 vs control group, bP <0.05 vs cyclophosphamide group


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252 Chin J Nat Med July 2012 Vol. 10 No. 4 2012 年 7 月 第 10 卷 第 4 期

Table 3 Effect of cyclophosphamide and Achillea millefolium inflorescences aqueous extract on histological parameters of testis
( x ± s, n = 6)
Control CP Achillea CP + Achillea F-value
STsD (µm) 256.79 ± 4.94 97.23 ± 4.99a 256.21 ± 9.52b 193.05 ± 5.83a,b 780.530
GEH (µm) 97.90 ± 3.99 37.75 ± 3.14a 99.74 ± 5.48b 69.33 ± 3.49a,b 299.295
AC (×103 µm3) 51.81 ± 2.00 7.44 ± 0.76a 51.62 ± 3.82b 29.29 ± 1.76a,b 483.736
NA (×10-8 µm-2) 33.93 ± 2.96 9.73 ± 0.54a 34.80 ± 2.55b 22.60 ± 1.78a,b 176.287
NV (×10-10 µm-2) 12.91 ± 1.16 9.43 ± 0.55a 13.27 ± 0.94b 11.35 ± 0.96a,b 21.064
LCND (µm) 6.32 ± 0.48 3.48 ± 0.27a 6.36 ± 0.30b 5.45 ± 0.44a,b 72.944
SCND (µm) 9.22 ± 0.75 6.44 ± 0.53a 9.74 ± 0.40b 8.92 ± 0.29b 46.327
STsD, seminiferous tubules diameter; GEH, germinal epithelium height; AC, Cross-sectional area of the seminiferous tubules; NA, Number of
profiles of seminiferous tubules in a unit area of testis; NV, Numerical Density of seminiferous tubulesas; LCND, Leydig cell nuclear diameter;
SCND, Sertoli cell nuclear diameter. a P <0.05 vs control group, bP <0.05 vs cyclophosphamide group

produce toxic side-effects in multiple organ systems includ-
ing the testes. In a clinical context, testicular stem cell dam-
age in patients exposed to chemotherapeutic drugs for a lim-
ited duration could result in long-term infertility or genetic
alterations[41]. A strategy to diminish the side-effects of anti-
cancer drugs with preservation of their chemotherapeutic
efficacy is necessary. Effective anticancer and immunosup-
pressive therapy with CP is severely limited by testicular
toxicity as documented in a variety of species[4]. An oxidant
mechanism may be involved in the reproductive toxicity,
wherein CP and its metabolite acrolein cause inactivation of
microsomal enzymes and result in increased reactive oxygen
species generation and lipid peroxidation[42]. In the present
study, reduction in body weight, weight of the testis and epi-
didymis and histological changes in testis were indicative of
drug toxicity. Because the weight of the testis largely depends
on the mass of the differentiated spermatogenic cells[43], the
marked reduction in organ weight by CP can be explained by
diminished number of germ cells, atrophy of Leydig cells and
a significant lower rate of spermatogenesis as confirmed by
our findings. Reduction in the weight of testes and epidi-
dymides in CP-treated animals reflect the reduced availability
of androgens[44]. Increased generation of free radicals is one
of the possible mechanisms involved in CP-induced Leydig
cell degeneration resulted in marked reduction of serum tes-
tosterone[45]. Chemotherapy can result in long-term or per-
manent azoospermia, the mechanism of which is most likely
the death of germ cells[46] and stereological parameters such
as seminiferous tubules diameters and their epithelial heights,
cross-sectional area of the seminiferous tubules, number of
profiles of seminiferous tubules in a unit area of testis and
numerical density of seminiferous tubules can also give in-
formation about the testicular damage degree as a conse-
quence of germ cell death. In general, massive germ cell loss
caused by anticancer drugs is followed by a sharp decline in
testicular stereological parameters[47]. As shown in present
study, depletion of seminiferous epithelium and the conse-
quent decrease of morphometric and stereological measure-
ments caused by cytotoxic agents were confirmed in our
report.
Structural development and maturation of germ cells and
spermiation are important functions of Sertoli cells[48].
Therefore, a potential explanation for the failure of spermio-
genesis in the CP-treated males is disruption of testoster-
one-dependent junction of Sertoli cells with germ cells lead-
ing to their disorganization and separation. In the present
study, epididymal sperm count decreased by, confirming a
previous report that CP induces an epididymis specific effect
on sperm count[49]. The decreased sperm count clearly shows
the elimination of sperm cells at different stages of develop-
ment and points to free radical attack through CP metabolism.
In fact, oxidative damage to polyunsaturated fatty acids of
cell membranes has long been considered to result in the
impairment of membrane fluidity and permeability. This
results in the damage of germ cells, spermatozoa and mature
sperm[50]. It has also been reported that CP causes an increase
in apoptosis at specific stages of germinal cycle[51]. Hence,
the decrease in epididymal sperm count observed in
CP-treated rats might reflect the spermatogenic cell death.
There are several reports on the benefit of antioxidants in
protecting male reproductive system from deleterious effects
of reactive oxygen species and other free radicals generated
during CP exposure. It was found that ascorbic acid reduces
cyclophosphamide-induced reproductive toxicity[9] as well as
alpha-tocopherol-succinate[52]. There is also evidence that
Yukmijihwang-tang as a multi-herbal medicinal formula can
improve reproductive toxicity of CP through reduction of
oxidative stress[53]. Two studies from the same researchers
indicated that supplementation with lipoic acid as an antioxi-
dant reduces CP-induced reproductive toxicity by the same
mechanism[54-55].
In the present study, it has been shown that Achillea mil-
lefolium inflorescences aqueous extract coadministration was
effective in protection or attenuation of testicular damage
following CP exposure. Increasing evidences support the fact
that Achillea is beneficial where free radicals are known to
play a predominant role in toxicity. Previous studies have
shown Achillea millefolium protected rat stomach against
Ali Shalizar Jalali, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 247254
2012 年 7 月 第 10 卷 第 4 期 Chin J Nat Med July 2012 Vol. 10 No. 4 253

gastric ulcers induced by reactive oxygen species due to its
antioxidant properties[56]. Furthermore, it has been revealed
that Achillea infusions reduce H2O2-induced oxidative dam-
age in human erythrocytes and leucocytes, which is consis-
tent with their total flavonoid and phenol contents[57]. In con-
clusion, the finding of our study indicate that cyclophos-
phamide can adversely damage the testicular tissue through
imposing oxidative stress, while Achillea millefolium inflo-
rescences aqueous extract coadministration could effectively
prevent these adverse effects by effective inhibition of oxida-
tive processes and efficient scavenging of free radicals.
References
[1] Dollery C. Therapeutic Drugs[M]. Edinburgh: Churchill Liv-
ingstone, 1999, 349-354.
[2] Ludeman SM. The chemistry of the metabolites of cyclophos-
phamide[J]. Curr Pharm Des, 1999, 5 (8): 627-643.
[3] Kern JC, Kehrer JP. Acrolein-induced cell death: a caspase
influenced decision between apoptosis and oncosis/necrosis[J].
Chem Biol Interact, 2002, 139 (1): 79-95.
[4] Anderson D, Bishop JB, Garner RC, et al. Cyclophosphamide:
review of its mutagenicity for an assessment of potential germ
cell risks[J]. Mutat Res, 1995, 330 (1-2): 115-181.
[5] Howell S, Shalet S. Gonadal damage from chemotherapy and
radiotherapy[J]. Endocrinol Metab Clin North Am, 1998, 27 (4):
927-943.
[6] Meistrich ML, Parchuri N, Wilson G, et al. Hormonal protec-
tion from cyclophosphamide-induced inactivation of rat stem
spermatogonia[J]. J Androl, 1995, 16(4): 334-341.
[7] Kaur F, Sangha GK, Bilaspuri GS. Cyclophosphamide induced
structural and biochemical changes in testis and epididymidis
of rats[J]. Indian J Exp Biol, 1997, 35(7): 771-775.
[8] Trasler JM, Hales BF, Robaire B. Chronic low dose cyclo-
phosphamide treatment of adult male rats: effect on fertility,
pregnancy outcome and progeny[J]. Biol Reprod, 1986, 34 (2):
275-283.
[9] Das UB, Mallick M, Debnath JM, et al. Protective effect of
ascorbic acid on cyclophosphamide-induced testicular gameto-
genic and androgenic disorders in male rats[J]. Asian J Androl,
2002, 4 (3): 201-207.
[10] Ghosh D, Das UB, Ghosh S, et al. Testicular gametogenic and
steroidogenic activities in cyclophosphamide treated rat: a cor-
relative study with testicular oxidative stress[J]. Drug Chem
Toxicol, 2002, 25 (3): 281-292.
[11] Manda K, Bhatia AL. Prophylactic action of melatonin against
cyclophosphamide-induced oxidative stress in mice[J]. Cell Bi-
ol Toxicol, 2003, 19 (6): 367-372.
[12] Murata M, Suzuki T, Midorikawa K, et al. Oxidative DNA
damage induced by a hydroperoxide derivative of cyclophos-
phamide[J]. Free Radic Biol Med, 2004, 37 (6): 793-802.
[13] Vernet P, Aitken RJ, Drevet JR. Antioxidant strategies in the
epididymis[J]. Mol Cell Endocrinol, 2004, 216 (1-2): 31-39.
[14] Arumugam N, Sivakumar V, Thanislass J, et al. Effects of
acrolein on rat liver antioxidant defense system[J]. Indian J
Exp Biol, 1997, 35 (12): 1373-1374.
[15] Mitich LW. Intriguing world of weeds: yarrow—the herb of
Achilles[J]. Weed Technol, 1990, 4: 451-453.
[16] British Herbal Medicine Association, British Herbal Compen-
dium (Vol. 1) [M]. Bournemouth: Bradley PR (Ed.), 1992,
190-191.
[17] Alonso JR. Milenrama. In: Tratado de Fitomedicina: bases
cl´ınicas y farmacol´ogicas[M]. Isis: Buenos Aires, 1998.
725-729.
[18] Blumenthal M, Busse WR, Goldberg A, et al. (Eds.). Herbal
Medicine: Expanded Commission E Monographs[M]. Newton,
MA: Integrative Medicine Communications, 2000, 419-423.
[19] Goldberg AS, Mueller, EC, Eigen E, et al. Isolation of the anti-
inflammatory principles from Achillea millefolium (Composi-
tae)[J]. J Pharmaceutical Sci, 1969, 58 (8): 938-941.
[20] Tozyo T, Yoshimura Y, Sakurai K, et al. Novel antitumor ses-
quiterpenoids in Achillea millefolium[J]. Chem Pharm Bull,
1994, 42 (5): 1096-1100.
[21] Gagdoli, C, Mishra, SH. Preliminary screening of Achillea
millefolium, Cichorium intybus and Capparis spinosa for anti-
hepatotoxic activity[J]. Fitoterapia, 1995, 66 (4): 319-323.
[22] Tunon H, Olavsdotter C, Bohlin L. Evaluation of anti-inflam-
matory activity of some Swedish medicinal plants. Inhibition of
prostaglandin biosynthesis and PAF-induced exocytosis[J]. J
Ethnopharmacol, 1995, 48 (2): 61-76.
[23] Lin LT, Liu LT, Chiang LC, et al. In vitro anti-hepatoma activ-
ity of fifteen natural medicines from Canada[J]. Phytother Res,
2002, 16 (5): 440-444.
[24] Candan F, Unlu M, Tepe B, et al. Antioxidant and antimicrobial
activity of the essential oil and methanol extracts of Achillea
millefolium subsp. millefolium Afan. (Asteraceae)[J]. J Ethno-
pharmacol, 2003, 87 (2-3): 215-220.
[25] Konyalioglu S, Karamenderes C. Screening of total flavonoid,
phenol contents and antioxidant capacities of Achillea L. spe-
cies growing in Turkey[J]. Acta Pharm Turcica, 2004, 46(3):
163-170.
[26] Giorgi A, Bombelli R, Luini A, et al. Antioxidant and cytopro-
tective properties of infusions from leaves and inflorescences
of Achillea collina Becker ex Rchb[J]. Phytother Res, 2009, 23
(4): 540-545.
[27] Kocevar N, Glavac I, Injac R, et al. Comparison of capillary
electrophoresis and high performance liquid chromatography
for determination of flavonoids in Achillea millefolium[J]. J
Pharm Biomed Ana, 2008, 46 (3): 609-614.
[28] Cavalcanti AM, Baggio CH, Freitas CS, et al. Safety and an-
tiulcer efficacy studies of Achillea millefolium L. after chronic
treatment in Wistar rats[J]. J Ethnopharmacol, 2006, 107 (2):
277-284.
[29] Zambrano E, Rodríguez-González GL, Guzmán C, et al. A
maternal low protein diet during pregnancy and lactation in the
rat impairs male reproductive development[J]. J Physiol, 2005,
563 (1): 275-284.
[30] Benzie IF, Strain JJ. Ferric reducing/antioxidant power assay:
direct measure of total antioxidant activity of biological fluids
and modified version for simultaneous measurement of total
antioxidant power and ascorbic acid concentration[J]. Methods
Enzymol, 1999, 299: 15-27.
[31] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measure-
ment with the Folin phenol reagent[J]. J Biol Chem, 1951, 193
(1): 265-275.
[32] Qin D, Lung MA. Morphometric study on Leydig cells in
capsuletomized testis of rats[J]. Asian J Androl, 2002, 4(1):
49-53.
[33] Vendramini V, Sasso-Cerri E, Miraglia SM. Amifostine reduces
Ali Shalizar Jalali, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 247254
254 Chin J Nat Med July 2012 Vol. 10 No. 4 2012 年 7 月 第 10 卷 第 4 期

the seminiferous epithelium damage in doxorubicin-treated
prepubertal rats without improving the fertility status[J]. Re-
prod Biol Endocrinol, 2010, 8: 3.
[34] Gundersen HJG, Jenson EB. The efficiency of systematic sam-
pling in stereology and its prediction[J]. J Microsc, 1987, 147
(3): 229-263.
[35] Gundersen HJG. Notes on the edge of the numerical density of
arbitrary profiles: the edge effect[J]. J Microsc, 1977, 111:
219-223.
[36] Gilliland KO, Freel CD, Lane CW, et al. Multilamellar bodies
as potential scattering particle in human age-related nuclear
cataracts[J]. Mol Vision, 2001, 7: 120-130.
[37] Russell LD, Ettlin RA, SinhaHikim AP, et al. Histological and
histopathological evaluation of the testis[M]. Florida: Cache
River Press, 1990.
[38] Meistrich ML, van Beek MEAB. Methods in Toxicology[M].
New York: Academic Press, 1993, 106-123.
[39] Kheradmand A, Dezfoulin O, Tarrahi MJ. Ghrelin attenuates
heat-induced degenerative effects in the rat testis[J]. Regul
Peptides, 2011, 167 (1): 97-104.
[40] Elias H, Hyde DM. An elementary introduction to stereology
(quantitative microscopy)[J]. Am J Anat, 1980, 159 (4):
412-446.
[41] Sawada T, Tamada H, Mori J. Secretion of testosterone and
epidermal growth factor in mice with oligozoospermia caused
by doxorubicin hydrochloride[J]. Andrologia, 1994, 26 (3):
151-153.
[42] Lear L, Nation RL, Stupans I. Effects of cyclophosphamide and
adriamycin on rat hepatic microsomal glucuronidation and lipid
peroxidation[J]. Biochem Pharmacol, 1992, 44 (4): 747-753.
[43] Katoh C, Kitajima S, Saga Y, et al. Assessment of quantitative
dual-parameter flow cytometric analysis for the evaluation of
testicular toxicity using cyclophosphamide and ethinylestradiol
treated rats[J]. J Toxicol Sci, 2002, 27 (2): 87-96.
[44] Patil S, Patil S, Londonkar R, et al. Effect of pethidine on
spermatogenesis in albino rats[J]. Indian J Pharmacol, 1998,
30 (4): 249-253.
[45] Debnath D, Mandal TK. Study of quinalphos (an environ-
mental oestrogenic insecticide) formulation (Ekalux 25
E.C.)-induced damage of the testicular tissues and antioxidant
defence systems in Sprague- Dawley albino rats[J]. J Appl Tox-
icol, 2000, 20 (3): 197-204.
[46] Meistrich ML. Relationship between spermatogonial stem cell
survival and testis function after cytotoxic therapy[J]. Brit J
Cancer, 1986, 7: 89-101.
[47] França LR, Russel LD. Male reproduction: a multidisciplinary
overview[M]. Madrid: Churchill Communications, 1998,
198-219.
[48] Mruk DD, Cheng CY. Sertoli-Sertoli and Sertoli-germ cell
interactions and their significance in germ cell movement in the
seminiferous epithelium during spermatogenesis[J]. Endocr
Rev, 2004, 25 (5): 747-806.
[49] Higuchi H, Nakaoka M, Kawamura S, et al. Application of
computer-assisted sperm analysis system to elucidate lack of
effects of cyclophosphamide on rat epididymal sperm mo-
tion[J]. J Toxicol Sci, 2001, 26 (2): 75-83.
[50] Sikka SC. Role of oxidative stress and antioxidants in androl-
ogy and assisted reproductive technology[J]. J Androl, 2004, 25
(1): 5-18.
[51] Cai L, Hales BF, Robaire B. Induction of apoptosis in the germ
cells of adult male rats after exposure to cyclophosphamide[J].
Biol Reprod, 1997, 56 (6): 1490-1497.
[52] Ghosh D, Das UB, Misro M. Protective role of al-
pha-tocopherolsuccinate (provitamin-E) in cyclophosphamide
induced testicular gametogenic and steroidogenic disorders: a
correlative approach to oxidative stress[J]. Free Radic Res,
2002, 36 (1): 1209-1218.
[53] Oh MS, Chang MS, Park W, et al. Yukmijihwang-tang protects
against cyclophosphamide-induced reproductive toxicity[J].
Reprod Toxicol, 2007, 24 (3-4): 365-370.
[54] Selvakumar E, Prahalathan C, Sudharsan PT, et al. Chemopro-
tective effect of lipoic acid against cyclophosphamide-induced
changes in the rat sperm[J]. Toxicology, 2006a, 217 (1): 71-78.
[55] Selvakumar E, Prahalathan C, Sudharsan PT, et al. Protective
effect of lipoic acid on cyclophosphamide-induced testicular
toxicity[J]. Clin Chim Acta, 2006b, 367 (1-2): 114-119.
[56] Potrich FB, Allemand A, da Silva LM, et al. Antiulcerogenic
activity of hydroalcoholic extract of Achillea millefolium L.:
Involvement of the antioxidant system[J]. J Ethnopharmacol,
2010, 130 (1): 85-92.
[57] Konyalioglu S, Karamenderes C. The protective effects of
Achillea L. species native in Turkey against H2O2-induced oxi-
dative damage in human erythrocytes and leucocytes[J]. J Eth-
nopharmacol, 2005, 102 (2): 221-227.