ATIDEPRESSAT ACTIVITY OF COR SILK

Abstract

Corn silk (CS) is widely used in Iranian traditional medicine. The aim of present study was to investigate antidepressant activity of CS by forced swimming (FST) and tail suspension tests (TST). Phenol and flavonoid contents were measured by Folin Ciocalteu and AlCl 3 assays, respectively. Hydro alcoholic extract of Corn silk (125, 250, 500 and 1000 mg kg -1) was studied for its antidepressant activity by FST and TST. Immobility period during the experiments were noted. Phenol and flavonoid contents of the extract were determined as gallic acid and quercetin equivalents from a calibration curve, respectively. Extract showed good antidepressant activity in FST and TST. The extract shortened remarkably the immobility period during the FST and TST and exhibited a dose dependent activity. All test groups were significantly different form control group (P<0.001) in both tests. Extract at 1500 mg kg -1 showed similar activity as imipramine 10 mg kg -1 (p> 0.05) in TST. CS extract contained a significant amount of phenol and flavonoids. No mortality has been observed up to 4000 mg/kg. These results introduced CS aerial parts as an easily accessible and edible source of natural antidepressant and antioxidants.

Full text

Pharmacologyonline 3: 647-652 (2009) Ebrahimzadeh et al.
647
ATIDEPRESSAT ACTIVITY OF COR SILK
Ebrahimzadeh M. A.
1
, Mahmoudi M.
2
*, Ahangar N.
3
, Ehteshami S.
2
, Ansaroudi F.
1
,
Nabavi S. F
1,4
and Nabavi
S. M.
1,5
1
Pharmaceutical Sciences
Research Center, School of Pharmacy, Mazandaran University of
Medical Sciences, Sari, 48189, Iran.
2
Department of pharmacology, School of Medicine, Mazandaran University of Medical
Sciences, Sari, 48189, Iran.
3
Department of Toxicology & pharmacology, School of Pharmacy, Mazandaran University
of Medical Sciences, PO Box 48175-861, Sari, Iran.
4
Student Research Development Committee, Mazandaran University of Medical Sciences,
Sari, Iran.
5
Department of Biology, University of Mazandaran, Babolsar, Iran
*Corresponding author: Email: mtmhmit@yahoo.com; Tel: +98-151-3543081-3; Fax:
+98-151-3543084,
Summary
Corn silk (CS) is widely used in Iranian traditional medicine. The aim of present study was to
investigate antidepressant activity of CS by forced swimming (FST) and tail suspension tests (TST).
Phenol and flavonoid contents were measured by Folin Ciocalteu and AlCl
3
assays, respectively. Hydro
alcoholic extract of Corn silk (125, 250, 500 and 1000 mg kg
-1
) was studied for its antidepressant
activity by FST and TST. Immobility period during the experiments were noted. Phenol and flavonoid
contents of the extract were determined as gallic acid and quercetin equivalents from a calibration curve,
respectively. Extract showed good antidepressant activity in FST and TST. The extract shortened
remarkably the immobility period during the FST and TST and exhibited a dose dependent activity. All
test groups were significantly different form control group (P<0.001) in both tests. Extract at 1500 mg
kg
-1
showed similar activity as imipramine 10 mg kg
-1
(p> 0.05) in TST. CS extract contained a
significant amount of phenol and flavonoids. No mortality has been observed up to 4000 mg/kg. These
results introduced CS aerial parts as an easily accessible and edible source of natural antidepressant and
antioxidants.
Keywords: Antidepressant, Corn silk, Forced Swimming Test, Tail Suspension Test.

Pharmacologyonline 3: 647-652 (2009) Ebrahimzadeh et al.
648
Introduction
Depression constitutes the second most common chronic condition in clinical practice and
will become the second leading cause of disease burden worldwide by the year 2020 (1).
Approximately two-thirds of the anxious or depressed patients respond to the currently
available treatments but the magnitude of improvement is still disappointing (2). Although
there are many effective antidepressants available today (3,4), the current armentarium of
therapy is often inadequate with unsatisfactory results in about one third of all subjects
treated. This necessitates the development of newer and more effective antidepressants
from traditional medicinal plants whose psychotherapeutic potential has been assessed in a
variety of animal models (5). In recent years, considerable attention has been directed
towards the identification of plants with antioxidant ability that may be used for Human
consumption (6-8). Diuretic, as well as antilithiasic, uricosuric, and antiseptic, properties
are traditionally attributed to Corn silk (CS), stigma/style of Zea mays Linne (Poaceae/
Gramineae), which has been used in many parts of the world for the treatment of edema as
well as for cystitis, gout, kidney stones, nephritis, and prostatitis (9,10). CS contains
proteins, vitamins, carbohydrates, Ca
2+
, K
+
, Mg
2+
and Na
+
salts, volatile oils, and steroids
such as sitosterol and stigmasterol, alkaloids, saponins, tannins, and flavonoids (9,10).
Phenolic compounds present in CS are anthocyanins, p-coumaric acid, vanillic acid,
protocatechuic acid, derivatives of hesperidin and quercetin, and bound hydroxycinnamic
acid forms composed of p-coumaric and ferulic acid (11). There are also reports about
antioxidant activity of CS (10,12). The constituents in the volatile extract and petroleum
ether, ethanol, and water extract of CS exhibited clear antioxidant activities (13). To the
best of the author’s knowledge, antidepressant activity of CS has not been reported to date
and nothing was found about mechanism /or antidepressant activity of CS. Therefore, the
aim of the present work is to determine the antidepressant activity by forced swimming test
(FST) and tail suspension test (TST) in order to understand the usefulness of this plant as a
foodstuff as well as in medicine.
Materials and methods
Animals: Swiss albino mice (20 ± 2 g) of either sex were randomly housed in groups of six
in polypropylene cages at an ambient temperature of 25± 1
o
C and 45–55% relative
humidity, with a 12 h light: 12 h dark cycle (lights on at 7 a.m.). The animals had free
access to standard pellet and water ad libitum. Each animal was tested once. All of the
experiment conducted between 8:00 and 14:00 h. Mice were divided into five different
groups (n = 8 per group) and tested in FST and TST. The experiments were conducted in
accordance to the ethical guidelines regarding investigation with laboratory animals (NIH
guidelines of the Care and Use of Laboratory animals) and were also approved by the
Ethical Committee for Animal Experimentation of Mazandaran University of Medical
Sciences.
Plant material: CS (dried cut stigmata of Zea mays L, Poaceae flowers) used for this
investigation was collected in January 2008 and authenticated by Dr. Bahman Eslami
(Department of Biology, Islamic Azad University of Qhaemshahr, Iran) and the voucher
specimen was deposited in the Sari School of Pharmacy herbarium (No. HS280). CS was
dried at room temperature and an ethanol-water (1:1) extraction was performed using
maceration method by soaking in the solvent mixture. The extract was collected after
removing the solvent and lyophilization.

Pharmacologyonline 3: 647-652 (2009) Ebrahimzadeh et al.
649
Determination of total phenolic and flavonoid content: Total phenolic compound
content was determined by the Folin-Ciocalteau method (14-16). The extract sample (0.5
ml of different dilutions) was mixed with 2.5 ml of 0.2 N Folin-Ciocalteau reagent for 5
min and 2.0 ml of 75 g/l sodium carbonate were then added. The absorbance of reaction
was measured at 760 nm after 2 h of incubation at room temperature. Results were
expressed as gallic acid equivalents. Total flavonoid was estimated according to method of
our recent paper (17-19). Briefly, 0.5 ml solution of extract in methanol were mixed with
1.5 ml of methanol, 0.1 ml of 10% AlCl
3
, 0.1 ml of 1 M potassium acetate, and 2.8 ml of
distilled water and left at room temperature for 30 minutes. The absorbance of the reaction
mixture was measured at 415 nm with a double beam spectrophotometer (Perkin Elmer).
Total flavonoid contents were calculated as quercetin from a calibration curve.
Forced swimming test: The mouse was dropped into a glass cylinder (20 cm in height and
12 cm in diameter) containing 8-cm-deep water at 24-25 ºC and left there for 6 min. The
duration of immobility during the final 4-min interval of the swimming test was measured
(2-4). Control group was treated with solvent. The other groups of mice received an
interperitoneal (i.p.) injection of extract (125, 250, 500 and 1000 mg kg
-1
) in Tween 80 plus
0.9% (w/v) saline solution and imipramine (5 and 10 mg kg
-1
), 1 h before the experiment.
Imipramine was utilized as positive control of the test.
Median lethal dose: LD
50
was assumed using 50% deaths within 72h after i.p.
administration of the extract at different doses. Male Swiss mice weighing 20-25 g (10 per
group) were used in this experiment (20).
Tail suspension test: This test is a variant of the behavioural despair test in which
immobility is induced by suspending a mouse by its tail. On day 3, 1 h after the extract
treatment, mice were hung individually on a wire in an upside down posture so that its
nostril just touches the water surface in a container. After initial vigorous movements, the
mouse assumes an immobile posture and the period of immobility during a 5 min
observation period were noted (21). This test is reliable and rapid screening method for
antidepressants including those involving the serotonergic system (22).
Statistical analysis: Experimental results are expressed as means ± SD. All measurements
were replicated three times. The data were analyzed by an analysis of variance (p < 0.05)
and the means separated by Duncan's multiple range test.
Results
Total phenol and flavonoid contents: Total phenol compounds, as determined by Folin
Ciocalteu method, are reported as gallic acid equivalents by reference to standard curve (y
= 0.0063x, r
2
= 0.987). The total phenolic content of CS was 118.94 ± 2.78 mg gallic acid
equivalent/g of extract. The total flavonoid contents was 58.22 ± 1.34 mg quercetin
equivalent/g of extract, by reference to standard curve (y = 0.0067x + 0.0132, r
2
= 0.999).
Effect of CS on immobility in FST: The result of effect of hydroalcoholic extract of CS
on the duration of immobility during forced swimming test is shown in Table 1. The extract
shortened remarkably the immobility period during the forced swimming test in comparison
with negative control and exhibited a dose dependent antidepressant activity. ANOVA
analysis followed by Newman–Keuls multiple comparisons test shows that all test groups
(except for lowest dose) were significantly different form control group (P<0.001).

Pharmacologyonline 3: 647-652 (2009) Ebrahimzadeh et al.
650
Imipramine 5 mg/kg, had shortest immobility period that was not comparable with CS
extract (P<0.001). No mortality has been observed up to 4000 mg/kg.
Effect of CS on immobility time in TST: CS (500 and 1500 mg kg
-1
) significantly (P<
0.001) and dose dependently decreased the immobility time as compared to control mice
(Table 1). The extract at the dose of 1500 mg kg
-1
showed the same activity as imipramine
at 10 mg kg
-1
(P> 0.05), in decreasing immobility period.
Table1. Effect of hydroalcoholic extract of Corn silk on the duration of immobility during
forced swimming test and Tail suspension test.
Group Dose (mg/kg) Duration of
immobility (s), FST
Duration of
immobility (s), TST
Control - 150.2 ± 8.9 157.8 ± 12
Corn silk 125 146.8 ± 6.2* ---------
Corn silk 250 120.2 ± 7.6** ---------
Corn silk 500 72.0 ± 4.3** 134.4 ± 7.2*
Corn silk 1000 57.6 ± 3.1** 97.2 ± 6.7**
Corn silk 1500 --------- 79.2 ± 3.4**
Imipramine
5 21.6 ± 0.7** ---------
Imipramine
10 14.4 ± 0.5** 73.8 ± 4.2*
ANOVA followed by Newman–Keuls multiple comparisons test shows that all test groups
are significantly different from control group (*P<0.01, **P<0.001). Values are Mean ± SD
(N = 8).
Discussion
Total phenol compounds were determined as gallic acid and the total flavonoid contents as
quercetin equivalent/g of extract. Phenols and polyphenolic compounds, such as flavonoids,
are widely found in food products derived from plant sources, and they have been shown to
possess significant antioxidant activities (23). Studies have shown that increasing levels of
flavonoids in the diet could decrease certain human diseases (24, 25). CS extract showed
high level of Total phenol and flavonoids contents. The swimming test has been widely
employed to evaluate the effect of various agents on the central nervous system (CNS),
such as CNS depressants, antidepressants, sedative-hypnotics, psychostimulants, euphorics,
nootropics, adaptogens, etc. The immobility seen in rodent during swimming reflects
behavioral despair as seen in human depression (3). The swimming test has also been used
extensively to assess the anti stress activity of plants in mice (26). The forced swimming
test is a classic animal model for antidepressant drug screening (3,4). CS extract shortened
remarkably the immobility period during the FST in comparison with negative control. The
effect was dose dependent. Tail suspension test represents the behavioral despair model,
claimed to reproduce a condition similar to human depression. The test is based on the
observation that animals, following initial escape oriented movements, develop an
immobile posture when placed in an inescapable chamber. The immobility is thought to
reflect either a failure of persistence in escape-directed behavior (i.e. behavioral despair) or
the development of passive behavior that disengages the animal from active forms of
coping with stressful stimuli. It has been argued that the TST is less stressful than FST and
has greater pharmacological sensitivity (27).

Pharmacologyonline 3: 647-652 (2009) Ebrahimzadeh et al.
651
Remarkably, TST detects the anti-immobility effects of a wide array of antidepressants,
including tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRI),
monoamine oxidase inhibitors (MAOI), electro-convulsive shock (ECS), and even atypical
antidepressants. Thus, the activity of CS could involve one of the mechanisms of the
established agents as described above. CS extract significantly (P< 0.001) and dose
dependently decreased the immobility time as compared to control mice. The extract at the
dose of 1500 mg kg
-1
showed the same activity as imipramine at 10 mg kg
-1
(P> 0.05), in
decreasing immobility period. Also, extract was safe and no mortality has been observed up
to 4000 mg/kg.
Conclusions
The finding of the present investigation suggests the antidepressant activity of CS in FST
and TST models of depression. CS significantly reduced the immobility period in both FST
and TST. The extract also had high level of phenol and flavonoids and was so safe at least
up to 4000 mg/kg. However, further studies are necessary for complete understanding the
antidepressant activity of CS. Such identified potential and natural constituents could be
exploited as cost effective food additives for human and animal health.
Acknowledgements
This study has been supported by a grant from Pharmaceutical Sciences Research Center.
This work was a part of M.D. thesis in Sari school of medicine
References
1. An overview of a strategy to improve the mental health of underserved
populations. Social Change and Mental health, WHO, (May 1977, revised May 1999).
Available from: http://www.who.int/entity/mental_health/media/en/46.pdf. Accessed
August 17, 2009.
2. Ebrahimzadeh MA, Hadizadeh F, Hosseinzadeh H, Hajipoor M, Nabavi SM.
Antioxidant and antidepressant effects of four novel Bupropion analogues.
Pharmacologyonline 2009; 2: 317-322.
3. Hadizadeh F, Ebrahimzadeh MA, Hosseinzadeh H, Motamed-Shariaty V, Salami
S, Bekhradnia AR. Antidepressant and antioxidant activities of some 2-benzoxazolinone
derivatives as Bupropion analogues. Pharmacologyonline 2009; 1: 331-335.
4. Mahmoudi M, Ebrahimzadeh MA, Ansaroudi F, Nabavi SF, Nabavi
SM,Antidepressant and antioxidant activities of Artemisia absinthium L. at flowering stage.
Afr J Biotechnol in press.
5. Zhang ZJ. Therapeutic effects of herbal extracts and constituents in animal
models of psychiatric disorders. Life Sci 2004; 75: 1659-1699.
6. Ebrahimzadeh MA, Nabavi SM, Nabavi SF. Correlation between the in vitro iron
chelating activity and poly phenol and flavonoid contents of some medicinal plants. Pak J
Biol Sci 2009; 12(12): 934-938.
7. Ghasemi K, Ghasemi Y, Ebrahimzadeh MA. Antioxidant activity, phenol and
flavonoid contents of 13 Citrus specis peels and tissues. Pak. J. Pharm. Sci., 2009; 22 (3):
277-281.
8. Ebrahimzadeh MA, Nabavi SF, Nabavi SM, Eslami B. Antihypoxic and
antioxidant activity of Hibiscus esculentus seeds. Grasas Y Aceites 2010; 61(1): 30-36.

Pharmacologyonline 3: 647-652 (2009) Ebrahimzadeh et al.
652
9. Grases F, March JG, Ramis, M, Costa-Bauza A. The influence of Zea mays on
urinary risk factors for kidney stones in rats. Phytother Res 1993; 7:146-149.
10. Ebrahimzadeh MA, Pourmorad F, Hafezi S. Antioxidant Activities of Iranian
Corn Silk. Turk J Biol 2008; 32: 43-49.
11. Kaur G, Alam MS, Jabbar Z, Javed K, Athar M. Evaluation of antioxidant
activity of Cassia siamea flowers. J Ethnopharmacol 2006; 108: 340-348.
12. Maksimovic ZA, Kovacevic N. Preliminary assay on the antioxidative activity
of Maydis stigma extracts. Fitoterapia 2003; 74: 144-147.
13. El-Ghorab E, El-Massry KF, Shibamoto T. Chemical composition of the volatile
extract and antioxidant activities of the volatile and nonvolatile extracts of egyptian Corn
Silk (Zea mays L.) J Agr Food Chem 2007; 55(22): 9124- 9127.
14. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Bahramian F. In vitro antioxidant
activity of Phytolacca americana berries. Pharmacologyonline 2009; 1: 81-88.
15. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Fazelian M, Eslami B. In vitro
antioxidant and free radical scavenging activity of Diospyros lotus and Pyrus boissieriana
growing in Iran. Phcog Mag 2009; 4(18):122-126.
16. Dehpour AA, Ebrahimzadeh
MA, Nabavi SF, Nabavi SM. Antioxidant activity
of methanol extract of Ferula assafoetida and its essential oil composition, Grasas y
Aceites 2009; 60(4): 405-412.
17. Ebrahimzadeh MA, Nabavi SM, Nabavi SF, Eslami B. Antioxidant activity of
aqueous extract of Pyrus boissieriana fruit. Pharmacologyonline 2009; 1: 1318-1323.
18. Ebrahimzadeh MA, Bahramian F. Antioxidant activity of Crataegus pentaegyna
subsp. elburensis fruits extracts used in traditional medicine in Iran. Pak J Biol Sci 2009;
12(5): 413-419.
19. Ebrahimzadeh MA, Nabavi SF, Nabavi SM. Antioxidant activities of methanol
extract of Sambucus ebulus L. Flower. Pak J Biol Sci 2009; 12 (5): 447-450.
20. Morteza-Semnani K, Mahmoudi M, Riahi G. Effects of essential oils and
extracts from certain Thymus species on Swimming Performance in Mice. Pharm Boil
2007; 45(6): 464-467.
21. Steru L, Chermat R, Thierry B, Simon P. The tail suspension test: a new method
for screening antidepressants in mice. Psychopharmacology 1985; 85: 367-370.
22. Bhattacharya SK, Satyan KS, Ramanathan M. Experimental methods for
evaluation of psychotropic agents in rodents: II- antidepressants. Indian J Exp Biol 1999;
37: 117–123.
23. Ebrahimzadeh MA, Nabavi SF, Nabavi SM. Antihemolytic and antioxidant
activity of Hibiscus esculenus leaves Pharmacologyonline 2009; 2: 1097-1105.
24. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Hamidinia A. Bekhradnia AR.
Determination of antioxidant activity, phenol and flavonoids content of Parrotia persica
Mey. Pharmacologyonline 2008; 2: 560-567.
25. Ebrahimzadeh MA, Pourmorad F, Bekhradnia AR. Iron chelating activity
screening, phenol and flavonoid content of some medicinal plants from Iran. Afr. J.
Biotechnol. 2008; 7 (18): 3188-3192.
26. Maity TK, Mandal SC, Saha BP, Pal M. Effect of Ocimum sanctum roots extract
on swimming performance in mice. Phytother Res 2000; 14: 120-121.
27. Thierry B, Steru L, Simon P, Porsolt RD. The tail suspension test: ethical
considerations. Psychopharmacology 1986; 90: 284–285.