The Effects of Hydroalcoholic Extract of Apium Graveolens Leaf On The Number of Sexual Cells and Testicular Structure in Rat
The Effects of Hydroalcoholic Extract of Apium Graveolens Leaf On The Number of Sexual Cells and Testicular Structure in Rat
The Effects of Hydroalcoholic Extract of Apium Graveolens Leaf On The Number of Sexual Cells and Testicular Structure in Rat
*Corresponding author: Esrafil Mansouri, Cellular and Molecular Research Center, Department of Anatomical Sciences, School of Medicine, Ahvaz Jundishapur University of Medical
Sciences, Ahvaz, IR Iran. Tel: +98-9111183028, E-mail: esrafilmansori@yahoo.com
Received: January 12, 2014; Revised: March 11, 2014; Accepted: July 4, 2014
Background: Use of medicinal plants with high antioxidant properties could be effective to increase fertility and improvement of
disorders such as hormonal imbalance, impotency, oligospermia and immotile sperm. Celery (Apium graveolens) is rich in antioxidant
agents. The leaf and stems of celery contain phenols, furanocoumarin and luteolin. Apigenin is one of the main flavonoids of celery leaf.
Objectives: This study aimed to investigate the effects of hydroalcoholic extract of celery on histological properties of testis and number
of sexual cells in male rats.
Materials and Methods: Thirty-two male Wistar rats were divided into four groups of eight rats each. Control, did not receive any
medication; sham, received normal saline; and two groups received celery extract orally in dosages of 100 and 200 mg/kg/BW once every
two days for 60 days. At the end, animals were anesthetized, and caudal part of the right epididymis was used for sperm counting. After
fixation of testis, tissue sections were prepared and studied microscopically to evaluate morphometric (lumen diameter, number of
primary spermatocyte and sertoli cell) and histological changes. Data was analyzed by one-way ANOVA test using SPSS15 software. P < 0.05
was considered as statistically significant.
Results: There was a significant increase in the number of sperms, sertoli cells, and primary spermatocyte (P < 0.05) in groups receiving
extract; however, structural changes were not observed in the groups.
Conclusions: It seems that celery increases spermatogenesis in male rats, but has no destructive effects on testicular tissue.
1. Background
Infertility is one of the most common health problems tial fat. This fat contains mostly limonene (typically 60%),
in the world, which involves approximately 15% of cou- selenin (10%), furocoumarin, furocoumarin glycosides,
ples. Infertility can be present in both genders, but about palmitic acid and federonoid (4). Celery contains vitamin
50% of infertility is associated with male factor. Decreased C and other compounds such as phalides and coumarins,
sperm count and motility and deformity of sperm are which are health-enhancing compounds. Different parts
the most important factors of male infertility (1). Vari- of this plant owe a wide spectrum of biological, pharma-
ous chemical drugs are available to treat infertility; how- cological and therapeutic effects including anti-rheuma-
ever, researchers are looking for drugs with less adverse tism, sedative, blood pressure lowering, antifungal, anal-
effects and toxicity. In developing countries, traditional gesic, anti-inflammatory, detoxification, anti-spasmodic,
medicine is important in maintaining health of popula- anti-bacterial, anti-contractions and antiepileptic (5).
tion (2). Celery is used as food in some countries, which Recently, several studies reported increasing prevalence
also has medicinal properties. On the other hand, due to of infertility. Infertility can be due to consumption of
adverse effects of synthetic drugs, it is necessary to con- natural plant compounds (phytoestrogens) if consumed
duct scientific researches to assess medicinal properties in high amounts. These compounds can affect the repro-
of various plants. Celery (Apium graveolens) belongs to ductive system and reduce fertility (6). Based on available
the parsley (umbelliferae) species from the Apiaceae fam- reports, plants such as celery contain phytoestrogens,
ily, with a height of 100 cm, and a strong scent and fleshy which can be effective in fertility and reproductive sys-
and solid stems (3). Celery seeds contain 2% to 3% essen- tem (7). Plasma membrane of sperm is susceptible to oxi-
Copyright © 2014, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences; Published by DOCS. This is an open-access article distributed under the
terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and
redistribute the material just in noncommercial usages, provided the original work is properly cited.
Kooti W et al.
dative damage due to large amounts of unsaturated fatty of ketamine (60 mg/kg) and xylazine (10 mg/kg) (Al-
acids, finally leading to decreased motility and viability fasan, Holland). Then, testes and epididymis were care-
of sperm. Antioxidant compounds increase sperm func- fully separated. Testes were weighed by a digital scale
tion and can improve fertility. Furthermore, a study indi- (Sartorius-PT120-Germany) and fixed by Bouin’s fixative,
cated that celery has a protective effect on testes against dehydrated through increasing concentrations of etha-
sodium valproate (8) and di (2-ethylhexyl) phthalate (9). nol and embedded in paraffin. Then five sections with
Studies demonstrated that celery protects testes from thickness of 5 µm (an interval of 100 µm) in each animal
functional and structural damages and sperm from tox- were stained with hematoxylin-eosin. The sections were
icity induced by atrazine (10) and quinine sulfate (11). studied regarding morphometric and histopathologic
changes (10 fields in each section) (16) by Motic software
2. Objectives (Micro-Optic Industrial Group CO., LTD., UK ) (17) and light
microscope, respectively (Olympus, 3H-Z-Japan). Sperms
The present study was performed to assess the effects
were extracted from the caudal portion of epididymis by
of hydroalcoholic extract of celery leaf on the number of
fragmentation of epididymis into 1 mL in normal saline.
sperm and testes structure in male rats.
After five minutes incubation at 37˚C, sperm suspension
3. Materials and Methods
was diluted with a ratio of 1:100. This diluted solution was
used for sperm count using Neubauer’s slide. Therefore, a
This experimental study was performed in Physiology drop of solution was poured on the slide, and lamella was
Research Center of Ahvaz Jundishapur University of Med- placed on it; sperms were counted by light microscope in
ical Sciences (AJUMS). Thirty-two male Wistar rats (weight corner areas (18).
range of 170-220 grams) were obtained from the central
animal house of AJUMS. Animals were housed in plastic 3.3. Statistical Analysis
cages with 12/12h light/dark cycle at 21 ± 2˚C. These condi-
All data were expressed as mean ± SE. Data was analyzed
tions were maintained constant throughout the experi-
by One-way ANOVA and LSD tests using SPSS for windows
ments. The study was performed in accordance with the
(version 15, IBM, USA). P < 0.05 was considered as statisti-
principles of laboratory care established by the Ethics
cally significant.
Committee of Ahvaz Jundishapur University of Medical
Sciences, Ahvaz, Iran.
4. Results
3.1. Extraction
To prepare the extract, leaves of celery was prepared 4.1. Testis Index
from agricultural lands of Hamidiye (Khoozestan, Iran) There was no significant difference between the groups
and specified by an expert in Pharmacognosy depart- regarding testis index (P > 0.05) (Table 1).
ment of pharmacy faculty of AJUMS. The leaves were
shade-dried and milled to fine powder using a mechani- Table 1. Testicular Index a
cal grinder. Powder (50 g) was soaked in 200 mL of 70%
Group Control Sham Experimen- Experimen-
ethanol (Merck, Germany) for three days with occasional
tal Group 1, tal Group 2,
100 mg/kg 200 mg/kg
shaking. The solution was passed through filter paper
and evaporated in oven at 40˚C for 48 hours. Extracted
Testicular 0.63 ± 0.01 0.65± 0.01 0.66± 0.01 0.65± 0.01
index, g
powder was kept at 4˚C until use. Concentrations of 100
and 200 mg/kg/BW were prepared from obtained powder a Comparison of Mean ± SE of testis index between the experimental
of celery using normal saline as solvent (Daru Pakhs, Iran) (100 and 200 mg/kg hydro alcoholic extract of celery) and control
(12). Hydroalcoholic extract and solvent administrated by groups.
gavage once every two days for 60 days. The duration of
spermatogenesis in Wistar rats is 52 days according to the
literature (13, 14). 4.2. Morphometric Studies
Figure 1. Comparison of Mean ± SE of Sertoli Cells Between the Ex- Figure 2. Comparison of Mean ± SE of Lumen Diameter Between the
perimental (100 and 200 mg/kg Hydroalcoholic Extract of Celery) and Experimental (100 and 200 mg/kg Hydro-Alcoholic Extract of Celery) and
Control Groups (n = 8) Control Groups (n = 8)
20 120
*
18
100
16 *
*
14
80
Lumen diameter
Sertoli cell
12
10 60
8
40
6
4
20
2
0 0
control sham extract 100 exteract200 control sham extract 100 exteract200
Number of Sertoli cells increased significantly in groups receiving extract Lumen diameter decreased significantly in experimental groups (100 and
(200 mg/kg); * (P < 0.05). 200 mg/kg); * (P < 0.05).
Figure 3. Comparison of Mean ± SE of Primary Spermatocytes Between Figure 4. Comparison of Mean ± SE of Sperm Count Between the
the Experimental (100 and 200 mg/kg Hydro-Alcoholic Extract of Celery) Experimental (100 and 200 mg/kg Hydro-Alcoholic Extract of Celery) and
and Control Groups (n = 8) Control Groups (n = 8)
80 120
* * *
70
100
60 *
80
Sperm (×106)
50
Primary spermatocyte
40 60
30
40
20
20
10
0 0
control sham extract 100 exteract200 control sham extract 100 exteract200
Number of primary spermatocytes increased significantly in experimen- Sperm count increased significantly in experimental groups (100 and 200
tal groups (100 and 200 mg/kg); * (P < 0.05). mg/kg); * (P < 0.05).
ment and structure of epithelial cells in seminiferous tu- cells and primary spermatocytes increased and lumens
bules and interstitial tissue in groups receiving hydroal- diameter reduced in experimental groups. Our result
coholic extract of celery compared to groups І and II; only is similar to those reported in previous studies (10, 11).
the germinal epithelium was thicker in these groups (Fig- A previous study suggested that hydroalcoholic extract
ures 5C and D). of celery seed increased testosterone secretion (22). The
level of testosterone as the most important androgenic
hormone is effective in the evolution and proliferation
Figure 5. Microscopic Slides of Different Groups of germ cells and spermatid differentiation. Likely, celery
directly affects Sertoli cells by stimulating secretion of
testosterone (23). Sertoli cells have a major role in differ-
entiation and development of spermatogenesis. These
cells promote caryokinesis karyokinesis, cytokinesis and
differentiating of spermatozoa with producing growth
factors such as activin in the presence of calcium ion. Fur-
thermore, Sertoli cells secrete tubule fluid, which helps to
nourish sexual cells and plays an important role in their
supporting (24). In addition to the shape and quality of
sex cells, blood concentration of reproductive hormones
is an important parameter to evaluate function of the
reproductive system. Testosterone affects seminiferous
tubules and induces spermatogenesis (25). Our findings
indicated that administration of hydroalcoholic extract
of celery at both dosages increased sperm count. In a sim-
ilar study by Hamza et al. it was shown that celery extract
decreased toxic effects of sodium valproate and increased
all of spermatogenic cells lineages (8). Sperms count is
I, control; II, sham (solvent of normal saline); III and IV, experimental one of the important factors of fertility, and decrease of
(100 and 200 mg/kg hydroalcoholic extracts of celery). Germinal epithe- sperm count can decrease the probability of a successful
lium of seminiferous tubules and interstitial tissue in the control and
sham groups were normal. Germinal epithelium was thicker in groups pregnancy. Researchers suggested that spermatogen-
III and IV. esis and maturation of sexual cells depend on protec-
tion of cytotoxic and pathologic lesions that threaten
these events (26, 27). Previous studies demonstrated that
5. Discussion
induced damage by free radicals and oxidative stress
could cause various disorders, such as infertility (28, 29).
Historically, plants have been considered as a valuable Spermatogenic cells, unlike other cells are very sensitive
source; however, they are mostly used for food consump- to oxidative damage, which is due to high amounts of
tion rather than their therapeutics effects. The current poly-unsaturated fatty acids in their plasma membrane
study showed that hydroalcoholic extract of celery has and very low levels of cytoplasmic antioxidants (30). Per-
useful effects on spermatogenesis and testis in male rats. oxidation of membrane fatty acids leads to loss of mem-
In this study, we observed a slightly increase of testes in- brane fluidity and decreases its enzyme activity and ion
dex in groups ІІІ and IV, but was not statistically signifi- channels leading to gradual loss of the ability of sperm
cant. In addition, we did not observe any changes or de- to bind to the oocyte. The main effect of lipid peroxida-
structive effects on testicular tissue. The results indicated tion in all cells, especially sperm is disturbance of struc-
that used dosages of hydroalcoholic extract of celery had ture and function of organelles or cell membranes (ion
no clear effect on these parameters. Celery contains flavo- transport processes, fluidity and permeability, metabolic
noids and phenolic acids with anti-inflammatory effects; gradients). These changes can affect the structure and
also, apigenin and epiein as main flavonoids of celery function of sperm (31). In normal conditions, a balance
owe anti-inflammatory properties (19). Apigenin as an exists between reactive species oxygen and free radicals
antioxidant inhibits the production of hydrogen perox- production and antioxidant defense system. An imbal-
ide and IgE, which is responsible for inflammation and ance between free radical production and antioxidant
allergic responses (20). Apigenin has inhibitory effects on defenses leads to injuries induced by free radicals. Fla-
cyclooxygenase and lipoxygenase (21). Therefore, celery is vonoids are secondary metabolites of plant compounds
able to reduce harmful effects of free radicals on cells and with powerful antioxidant properties, and cannot be syn-
prevents cell death and loss of weight or tissue volume thesized in the body and must be received through diet.
due to its antioxidant and anti-inflammatory properties. Celery is a strong antioxidant due to flavonoids such as
These results are in agreement with some previous inves- apiein and apigenin (32, 33). Antioxidant compounds are
tigations (9). Besides, our results indicated that Sertoli able to protect cell membranes against damage (34). An-
Acknowledgements
17. Takzaree N, Mortazavi H, Hassanzadeh G, Safaye S, Hossini M.
Male rat spermatogenesis influenced by Achillea millefolium L
18. . Tehran Univ Med J. 2013;70(11):684–90.
We would like to gratitude Mrs. Noorbehbahani, from 19. Khaki A, Prove T. Effect of ciprofloxacin on caudal epididymis
the Laboratory of Biochemistry Department who helped sperm quality and apoptosis. UMJ. 2008;19(1):29–35.
20. Mencherini T, Cau A, Bianco G, Della Loggia R, Aquino RP, Autore
us and the Research Consultation Center (RCC) for their
G. An extract of Apium graveolens var. dulce leaves: structure of
technical support. the major constituent, apiin, and its anti-inflammatory proper-
ties. J Pharm Pharmacol. 2007;59(6):891–7.
Authors’ Contributions
21. Sultana S, Ahmed S, Jahangir T, Sharma S. Inhibitory effect of cel-
ery seeds extract on chemically induced hepatocarcinogenesis:
Study concept, design and critical revision of the manu- modulation of cell proliferation, metabolism and altered hepat-
ic foci development. Cancer Lett. 2005;221(1):11–20.
script for important intellectual content: Esrafil Man- 22. Silvan AM, Abad MJ, Bermejo P, Villar A. Effects of compounds ex-
souri, Wesam Kooti, Maryam Ghasemiboron; drafting of tracted from Santolina oblongifolia on TXB(2) release in human
the manuscript and advisor and conducting the experi- platelets. Inflammopharmacology. 1998;6(3):255–63.
23. Kerishchi P, Nasri S, Amin Gh, Tabibian M. The effects of Apium
ments: Mahmoud Harizi, Reza Afrisham and Damoon
graveolens extract on sperm parameters and H-G hormonal axis
Ashtary-Larky. in mice. Hamadan Iran: Proceedings of the International Con-
gress of Physiology and Pharmacology; 2011.
References 24. McLachlan RI, O'Donnell L, Meachem SJ, Stanton PG, de Kretser
DM, Pratis K, et al. Identification of specific sites of hormonal
1. Karamzade A, Mirzapour H, Kheirollahi M. Genetics Aspects of regulation in spermatogenesis in rats, monkeys, and man. Re-
Male Infertility. J Isfahan Med Sc. 2013;31(246):1149–62. cent Prog Horm Res. 2002;57:149–79.
2. Austin DF. Ipomoea littoralis (Convolvulaceae)—taxonomy, dis- 25. Grootegoed JA, Siep M, Baarends WM. Molecular and cellular
tribution, and ethnobotany. Econ Bot. 1991;45(2):251–6. mechanisms in spermatogenesis. Baillieres Best Pract Res Clin En-
3. Khare CP. Indian Medicinal Plants: An Illustrated Dictionary.New docrinol Metab. 2000;14(3):331–43.
York, USA: Springer Science; 2007. 26. Wang C, Zhang Y, Liang J, Shan G, Wang Y, Shi Q. Impacts of ascor-
4. Kitajima J, Ishikawa T, Satoh M. Polar constituents of celery seed. bic acid and thiamine supplementation at different concentra-
Phytochem. 2003;64(5):1003–11. tions on lead toxicity in testis. Clin Chim Acta. 2006;370(1-2):82–8.
5. Blumental M, Cladbery A, Brinkman J, Newton MA. Herbal medi- 27. Modaresi M, Messripour M, Rajaei R. Effect of cinnamon extract
cine: expanded commission E Monographs. Integ Med Communi- on the number of spermatocyte and spermatozoa cells in mice.
cat. 2000:78–83. IJMAP. 2010;26(1):83–90.
6. Csupor-Loffler B, Hajdu Z, Zupko I, Rethy B, Falkay G, Forgo P, et al. 28. Nikravesh MR, Jalali M, Mohamadi Sh. Effects of Crude Onion
Antiproliferative effect of flavonoids and sesquiterpenoids from Extract on Murine Testis. J Reprod Infertility. 2009;10(41):239–44.
Achillea millefolium s.l. on cultured human tumour cell lines. 29. Santos FW, Graca DL, Zeni G, Rocha JB, Weis SN, Favero AM, et al.
Phytother Res. 2009;23(5):672–6. Sub-chronic administration of diphenyl diselenide potentiates
7. Kooti W, Ghasemiboroon M, Asadi-Samani M, Ahangarpoor M, cadmium-induced testicular damage in mice. Reprod Toxicol.
Zamani M, Amirzargar A, et al. The Effect of Halcoholic Extract of 2006;22(3):546–50.
Celery Leaves on the Delivery Rate (Fertilization and Stillbirths), 30. Allen JA, Diemer T, Janus P, Hales KH, Hales DB. Bacterial endo-
the Number, Weight and Sex Ratio of Rat off Spring. Adv Environ toxin lipopolysaccharide and reactive oxygen species inhibit
Biol. 2014;8(10):824-30. Leydig cell steroidogenesis via perturbation of mitochondria.
8. Hamza AA, Amin A. Apium graveolens modulates sodium valpro- Endocrine. 2004;25(3):265–75.
ate-induced reproductive toxicity in rats. J Exp Zool A Ecol Genet 31. Gebreegziabher Y, Marcos E, McKinon W, Rogers G. Sperm
Physiol. 2007;307(4):199–206. characteristics of endurance trained cyclists. Int J Sports Med.
9. Madkour NK. The beneficial role of celery oil in lowering of di(2- 2004;25(4):247–51.
ethylhexyl) phthalate-induced testicular damage. Toxicol Ind 32. Aitken RJ. Free radicals, lipid peroxidation and sperm function.
Reprod Fertil Dev. 1995;7(4):659–68. spermatocytes reserve in adult male Sprague Dawley rat. Sci Res
33. Fazala SS, Ansarib MM, Singlac RK, Khand S. Isolation of 3-n-Butyl Essays. 2007;2(1):319–24.
Phthalide & Sedanenolide from Apium graveolens Linn. IGJPS. 37. Shittu LAJ, Shittu RK, Adesite SO, Ajala MO, Bankole MA, Benebo AS,
2012;2(3):258–61. et al. Sesame radiatum Phytoestrogens Stimulate Spermatogenic
34. Fazal SS, Singla RK. Review on the Pharmacognostical & Phar- Activity and Improve Sperm Quality in Adult Male Sprague Dawley
macological Characterization of Apium Graveolens Linn. IGJPS. Rat Testis. Inter J Morphol. 2008;26(3).
2012;2(1):36–42. 38. Kooti W, Mansori E, Ghasemiboroon M, Harizi M, Amirzargar A.
35. Bouayed J, Bohn T. Exogenous antioxidants - Double-edged swords Protective effects of celery (Apium Graveolens) on testis and cauda
in cellular redox state: Health beneficial effects at physiologic epididymal spermatozoa in rat. Iran J Reprod Med. 2014; 12(5):365-6.
doses versus deleterious effects at high doses. Oxid Med Cell Lon- 39. Nouri M, Ghasemzadeh A, Farzadi L, Shahnazi V, Ghaffari-Novin M.
gev. 2010;3(4):228–37. Vitamins C, E and lipid peroxidation levels in sperm and seminal
36. Shittu LAJ, Bankole MA, Oguntola JA, Ajala O,, Shittu RK, Ogundipe plasma of asthenoteratozoospermic and normozoospermic men.
OA, et al. Sesame leaves intake improve and increase epididymal Iran J Reprod Med. 2008;6(1):1–5.