Hormonal and Histomorphologic Effects of Azadirachta indica leaf

Int. J. Morphol.,
29(2):441-445, 2011.
Hormonal and Histomorphologic Effects of Azadirachta indica
leaf Extract on the Pars Anterior of Wistar Rats
Efectos Hormonales e Histomorfológicos del Extracto de la Hoja
de Azadirachta indica en la Pars Anterior de Ratas Wistar
*
Amabe O. Akpantah; **Moses B. Ekong; *Kebe E. Obeten; *Mfon I. Akpaso & *Theresa B. Ekanem
AKPANTAH, A. O.; EKONG; M. B.; OBETEN, K. E.; AKPASO; M. I. & EKANEM, T. B. Hormonal and histomorphologic effects
of Azadirachta indica leaf extract on the pars anterior of Wistar rats. Int. J. Morphol., 29(2):441-445, 2011.
SUMMARY: Azadirachta indica is a tree whose medicinal value is unquantifiable. Any part of the tree can be used in the
treatment of malarial infection. Reports have indicated its antifertility effects, and this necessitated this study on the effects of the
methanol leaf extract on serum luteinizing (LH) and follicle stimulating hormones (FSH) levels and the histomorphology of the
pars anterior of Wistar rats. Thirty adult male Wistar rats were equally divided into 3 groups of A, B and C. Group A was the control
and the animals received distilled water orally, while groups B and C were treated with 200mg/kg and 400mg/kg respectively of the
leaf extract by oral gavage for fourteen days. On day fifteen, the animals were sacrificed by chloroform anaesthesia. Blood was
obtained from their hearts, while the skull was opened to assess the hypophysis. Hormonal assay showed that luteinizing (LH) and
follicle stimulating (FSH) hormone levels in the serum were lower in groups B and C treated with 200mg/kg and 400mg/kg
respectively of the leaf extract, while that of LH were significant (P<0.001). Histomorphologic sections of the pars anterior revealed
reduced acidophil and basophil populations, with prominent degranulated chromophobes which were larger in the group treated
with 400mg/kg of A. indica leaf extract. This group also presented hypertrophy of the basophils compared to the control. In conclusion,
methanol leaf extract of A. indica decreases serum LH and FSH and caused histomorphologic changes in the pars anterior of adult
male Wistar rats.
KEY WORDS: Azadirachta indica; Pars anterior; Luteinizing hormone; Follicle stimulating hormone; Wistar rat.
INTRODUCTION
The plant, Azadirachta indica commonly called neem
has been used widely due to its medicinal values (Subaprinya
& Nagini, 2005). Its chemical components includes various
alkaloids where some are; nimbitin, azadirachtin and salanin
(Koul et al., 1990). These alkaloids contribute to the general properties of the plant.
Different parts of this plant have been used as
insecticides and pesticides (Koul et al.; dos Santos et al.,
2004; Siddiqii et al., 2004; Jin et al., 1995), and have
been reported to have inhibitory effects on several ranges
of micro-organisms (Talwar et al., 1997; Garq et al., 1998;
Baswa et al., 2001; Bocke et al., 2004).
A. indica regulates metabolism, and functions as
anti-diabetic, anti-hyperglycaemic and anti-carcinogenic
agents. Immuno-modulatory and anti-inflammatory
*
**
effects have been reported as well (Subaprinya & Nagini;
Chattophyay, 1998; Khosla et al., 2000).
Extracts of A. indica have been reported to have
several anti-fertility effects (Awasthy, 2001; Raji et al.,
2003). These include; reduction in serum testosterone and
luteinizing hormone levels, inhibition of folliculogenesis
and immunocontraceptive activity (Awasthy; Raji et al.;
Roop et al., 2005; Akpantah et al., 2010).
The serum level reduction of the hypophysis
hormones as well as hypophysis induced processes as seen
in previous works raised our concern to carry out this study
on the effect of the leaf extract of A. indica on the
luteinizing and follicle stimulating hormones levels and
histomorphology of the pars anterior of adult male Wistar
rats.
Department of Anatomy, Faculty of Basic Medical Sciences, University of Calabar, Calabar, Nigeria.
Department of Anatomy, Faculty of Basic Medical Sciences, University of Uyo, Uyo, Nigeria.
441
AKPANTAH, A. O.; EKONG; M. B.; OBETEN, K. E.; AKPASO; M. I. & EKANEM, T. B. Hormonal and histomorphologic effects of Azadirachta indica leaf extract on the pars anterior of Wistar
rats. Int. J. Morphol., 29(2):441-445, 2011.
MATERIAL AND METHOD
RESULTS
Thirty adult male Wistar rats were assigned
equally into 3 groups (A, B and C). The animals weighed
110-150g and the experiment commenced after
acclimatizing for two weeks in the Animal House of the
Department of Anatomy, Faculty of Basic Medical
Sciences, University of Calabar, Calabar, Nigeria. They
were cared for in compliance with the international
guidelines for animal research study, and ethical approval
was obtained from the institution. Fresh leaves of A. indica were washed and oven-dried at 60ºC for twenty-four
hours. The leaves were blended into powder by an electric
blender. Soxhlet extractor using methanol was employed
in extracting 220g of the powder. A stock solution of 40g
was arrived at. Distilled water was used to dilute the stock
solution to concentrations of 200mg/kg and 400mg/kg.
Hormonal Assay. The luteinizing hormone (LH) level of groups
B and C treated with 200mg/kg and 400mg/kg respectively of
A. indica leaf extract were significantly (P<0.001) lower than
the control.
The follicle stimulating hormone (FSH) level of groups
B and C treated with 200mg/kg and 400mg/kg respectively of
A. indica leaf extract were not significantly (P<0.05) lower than
the control. These are as presented in Table I below.
Histomorphology of the pars anterior. The control pars anterior section showed normal histomorphology. It presented two
cell types: chromophils and chromophobes irregularly
distributed. The chromophils were further grouped into the
acidophils and the basophils (Fig.1).
Group A animals served as the control and the
animals received distilled water, while groups B and C
were the experimental and were treated with 200mg/kg
and 400mg/kg of the leaf extracts of A. indica
respectively for fourteen days by oral gavage.
On the fifteenth day the animals were sacrificed
by chloroform anaesthesia. Blood was collected from the
heart and stored in heparinized test tubes for hormonal
assay. The hyphophysis was dissected out after excision
of the sphenoid bone, the pars anterior separated and
preserved in Bouins fluid. Routine haematoxylin and
eosin method was used in the histological processing.
Luteinizing (LH) and follicle stimulating (FSH)
hormones in the serum were determined by the EnzymeLinked Immunoabsorbent Assay (ELISA) method using
Microwell’s kits.
One way analysis of variance was employed in
determining the relationship of the hormonal levels
between the control and experimental groups, and
differences at p<0.05 were regarded as significant.
Fig. 1. The control pars anterior section showed normal histology. It
presented two cell types: chromophils and chromophobes (C) irregularly
distributed. The chromophils were further grouped into the acidophils
(A) and the basophils (B). H & E. Mag. x400.
Table I. Serum hormonal levels of luteinizing and follicle stimulating hormones in the control, 200mg/kg and
400mg/kg of A. indica groups.
Hormones
Group A
Group B
Group C
Luteinizing Hormone (LH)
Follicle Stimulating Hormone (FSH)
Control
200mg/kg of A. indica
400mg/kg of A. indica
1.24±0.09
0.62±0.18***
0.34 ±0.24***
0.34±0.11
NS
0.24±0.003
0.26±0.04NS
Results are presented as Mean ± SEM *** Significantly different from control at P<0.001 NS Not significantly different from the
control at p<0.05 A. indica-Azadirachta indica.
442
AKPANTAH, A. O.; EKONG; M. B.; OBETEN, K. E.; AKPASO; M. I. & EKANEM, T. B. Hormonal and histomorphologic effects of Azadirachta indica leaf extract on the pars anterior of Wistar
rats. Int. J. Morphol., 29(2):441-445, 2011.
Group B pars anterior section treated with 200mg/kg of A. indica leaf extract for fourteen days presented numerous
degranulated chromophobes and reduced acidophils and
basophils compared to the control (Fig.2).
DISCUSSION
Fig. 2. Group B pars anterior section treated with 200mg/kg of A. indica leaf extract for fourteen days presented numerous degranulated
chromophobes (DC) and reduced acidophils (A) and basophils (B)
compared to the control. H & E. Mag. x400.
The gonadotrophs are the gonadal cells that
secrete the glycoprotein hormones called the LH and
FSH (Young & Heath). Assay of the serum levels of
these hormones revealed lower levels in the two
treatment groups, with the LH being significantly
(P<0.001) reduced.
Group C pars anterior section treated with 400mg/kg of A. indica leaf extract presented reduced acidophil distributions, less
but hypertrophied basophils and large degranulated
chromophobes compared to the control (Fig. 3).
Fig. 3. Group C pars anterior section treated with 400mg/kg of A. indica leaf extract presented reduced acidophil (A) distributions, less but
hypertrophied basophils (B) and large degranulated chromophobes
(DC) compared to the control. H & E. Mag. x400.
The hypophysis serves as a master gland by
dictating to other glands in the body. It does this by
secreting different types of trophic and neuro-hormones
(Murray et al., 2000). It has two parts; the pars posterior, and the pars anterior. The pars anterior is further
divided into the pars distalis, tuberalis and intermedia
(Bowen, 2006). The different cells of the pars anterior
are divided into chromophils and chromophobes using
haematoxylin and eosin staining technique (Bancroft
& Steven, 1992; Young & Heath, 2004; Bowen). The
chromophils are further divided into the acidophils and
the basophils based on their colour staining to acidic
and basic dyes respectively. The acidophils are made
up of cells called the somatotrophs and the
mammotrophs; these secrete the polypeptide hormones.
The basophils are made up of cells called the
corticotrophs, thyrotrophs and the gonadotrophs; these
secrete the glycoprotein hormones (Bowen).
FSH indirectly stimulates gametogenesis in both
sexes and directly stimulates estrogen synthesis and
follicular development. It also maintains the structure
of the gonads in conjunction with LH (Guyton & Hall,
2001). On the other hand, LH is critical to luteinization
of the ovarian follicles and post-ovulatory follicular
function (Murray et al.). Reduced levels of FSH may
result in the inability of follicular cells to reach
maturation, and hence ovulation and complete
spermatogenesis in females and males respectively may
be hindered. This situation may inhibit the synthesis of
the LH whose maximum effect is felt after ovulation.
The limitation to the normal physiologic process may
be exploited in contraception, hence this extract may
be useful as a contraceptive.
Our report is in line with Raji et al. and Akpantah
et al. who reported a reduction in serum LH level after
treatment with extracts of A. indica. Gbotolorun et al.
(2004) reported partial blockage of ovulation and
alteration in the estrous cycle of Sprague-Dawley rats
treated with seed extract of A. indica. This may have
contributed to the levels of the LH and FSH seen in this
study.
443
AKPANTAH, A. O.; EKONG; M. B.; OBETEN, K. E.; AKPASO; M. I. & EKANEM, T. B. Hormonal and histomorphologic effects of Azadirachta indica leaf extract on the pars anterior of Wistar
rats. Int. J. Morphol., 29(2):441-445, 2011.
Histomorphologically, Groups B and C treated with
200mg/kg and 400mg/kg respectively of the extract showed
reduced acidophil and basophil populations, with prominent
degranulated chromophobes which were larger in group C.
Group C also presented hypertrophy of the basophils
compared to the control.
The reduced presence of acidophils and basophils in
the sections of the groups treated with 200mg/kg of A. indica
leaf extract may indicate a destructive effect of the leaf extract
of A. indica to the somatotrophs, lactotrophs, corticotrophs,
thyrotrophs and the gonadotrophs. This may have caused the
degranulated chromophobes seen in this study. The reduced
basophil population may have resulted in reduced
gonadotrophs which secretes FSH and LH, and this may be a
reason for the reduced hormonal levels in this study.
The hypertrophy of the basophils in the 400mg/kg
extract group may be due to the increased dose of the extract
resulting in their blotted size in order to cope with the dose.
By so doing, they occupy greater spaces and may cluster
together as seen in this study.
The group treated with 200mg/kg and 400mg/kg
of the extract showed degranulated chromophobes which
increased in population in the higher dose group.
Chromophobes have been speculated to probably
represent resting or degranulated chromophil cells (Young
& Heath). Their increase may be due to the loss of the
active neuromolecules of the tissue, since these are
responsible for the tissue’s staining ability. Yajurvedi et
al. (1985) earlier reported the same effect on pars distalis
after treatment of frogs with gonadotropin releasing
hormone.
A. indica has been reported to have antifertility effects
(Awasthy; Raji et al.; Roop et al.), though the degree of
effect by different parts of the plants cannot be ascertained.
The decreased basophils of the pars distalis and the decreased
LH and FSH may be a pointer to already reported antifertility
effects of this plant with other extracts. These results indicate
that the leaf extract’s antifertility effect of the plant may be
a result of hypophysis changes that ultimately cause the
reduction of LH levels.
In conclusion, methanol leaf extract of A. indica
decreases serum LH and FSH levels and causes
histomorphologic changes in the pars anterior of adult male
Wistar rats. Hence the plant may be exploited in
contraception, though with caution.
AKPANTAH, A. O.; EKONG; M. B.; OBETEN, K. E.; AKPASO; M. I. & EKANEM, T. B. Efectos hormonales e histomorfológicos
del extracto de la hoja de Azadirachta indica en la pars anterior de ratas wistar. Int. J. Morphol., 29(2):441-445, 2011.
RESUMEN: Azadirachta indica es un árbol cuyo valor medicinal es invaluable. Cualquier parte del árbol se puede utilizar en el
tratamiento de la infección por malaria. Reportes han indicado su efecto antifertilidad, lo que requirió estudiar los efectos del extracto
metanólico de la hoja sobre los niveles séricos de las hormonas luteinizante (LH) y folículo estimulante (FSH) y la histomorfología de la
pars anterior de ratas Wistar. Treinta ratas Wistar adultas fueron divididas en tres grupos. El grupo A fue utilizado como control y los
animales recibieron agua destilada por vía oral, mientras que los grupos B y C fueron tratados con 200 mg/kg y 400 mg/kg respectivamente, con extracto de hoja mediante una sonda nasogástrica durante catorce días. A los quince días, los animales fueron sacrificados
por anestesia con cloroformo. Se obtuvo sangre desde sus corazones, mientras que el cráneo fue abierto para evaluar la hipófisis. Los
ensayos hormonales mostraron que los niveles en suero de la LH y FSH se redujeron en los grupos B y C, tratados con 200 mg/kg y 400
mg/kg respectivamente, siendo la reducción de LH significativa (p<0,001). Secciones histomorfológicos de la pars anterior revelaron
una reducción de las poblaciones acidófilas y basófilas, con prominentes cromófobos degranulados que fueron mayores en el grupo
tratado con 400 mg/kg del extracto de A. indica. Este grupo también presentó hipertrofia de los basófilos en comparación con el control.
En conclusión, el extracto alcohólico de la hoja de de A. indica disminuye el nivel sérico de LH y FSH y provoca cambios histomorfológicos
en la pars anterior de ratas Wistar adultas.
PALABRAS CLAVE: Azadirachta indica; Pars anterior; Hormona luteinizante; Hormona folículo estimulante; Rata
Wistar.
REFERENCES
Akpantah, A. O.; Ekong, M. B.; Uruakpa, K. C.; Akpaso,
M.; Eluwa, M. A. & Ekanem, T. B. Gonadal histomorphologies and serum hormonal milieu in female rats
treated with Azadirachta indica leaf extract. Iranian J.
Reprod. Med., 8(4):185-90, 2010.
444
Awasthy, K. S. Genotoxicity of a crude leaf-extract of neem
in male germ cells of mice. Cytobios, 106:151-64, 2001.
Bancroft, J. D. & Steven, A. Theory and Practice of Histological
Techniques. 3rd ed. Edinburgh, Churchill Livingstone, 1992.
AKPANTAH, A. O.; EKONG; M. B.; OBETEN, K. E.; AKPASO; M. I. & EKANEM, T. B. Hormonal and histomorphologic effects of Azadirachta indica leaf extract on the pars anterior of Wistar
rats. Int. J. Morphol., 29(2):441-445, 2011.
Baswa, M.; Rath C. C.; Dash, S. K. & Mishra, R.K.
Antibacterial activity of Karanji (Pngamia pinnata) and
neem (Azadirachta indica) seed oil: a preliminary report.
Microbios, 105:153-89, 2001.
Raji, Y.; Udoh, U. S.; Mewoyeka, O. O.; Ononye, F. C. &
Bolarinwa, A. F. Implication of reproductive endocrine
malfunction in male anti-fertility efficacy of Azadirachta
indica extract in rats. Afr. J. Med. Sci., 32:152-65, 2003.
Bocke, S. J.; Boersma, M. G.; Alink, G. M.; Joop, J. A.; van
Loon, J. J.; van Huis, A.; Dicke, M. & Rietjens, I. M.
Safety evaluation of neem (Azadirachta indica) derived
pesticides. J. Ethnopharmacol., 94(1):25-41, 2004.
Roop, J. K.; Dhaliwal, P. K. & Garaya, S. S. Extracts of
Azadirachta indica and Melia azedarach seeds inhibit
folliculo-genesis in albino rats. Braz. J. Med. Biol. Res.,
38(6):943-7, 2005.
Bowen, R. Histology of the adenohypophysis. Endocrine
Index
Glossary,
2006.
Avalaible
in:
www.vivo.colostate.edu/hbooks/pathphys/endocrine.
Siddiqui, B. S.; Rasheed, M.; Ilyas, F.; Gulzar, T.; Toriq, R.
M. & Naim Ul Hassan, S. N. Analysis of insecticidal of
Azadirachta indica. A. Juss fractions. Z. Nature Forsh.,
59(1-2):104-12, 2004.
Chattophyay, R. R. Possible biochemical mode of antiinflammatory action of Azadirachta indica (A. Juss) in
rats. Indian J. Exp. Biol., 36(4):418-20, 1998.
dos Santos, T. M.; Costa, N. P.; Torres, A. L. & Junior A. L.
B. Effect of neem extract on the cotton. Pesq. Agropec.
Bras., 39(11):1071-6, 2004.
Garq, S.; Talwar, G. P. & Upadhyay, S. N.
Immunocontraceptive activity of guided fractionation
and characterization of active constituents of neem
(Azadirachta indica) seed extracts. J. Ethnopharmacol.,
60(3):235-46, 1998.
Gbotolorun, S. C.; Oremosu, A. A.; Noronha, C. C. &
Okanlawon, O. A. The effect of alcohol extract of neem
seed on ovulation, estrous cycle, and the fertility of adult
cyclic Sprague-Dawley rats. Nig. J. Health Biomed. Sci.,
3(2):116-9, 2004.
Subaprinya, R. & Nagini, S. Medicinal properties of neem
leaf; a review. Curr. Med. Anticancer Agents, 5(2):1469, 2005.
Talwar, G. P.; Shah, S.; Murkherfee, S. & Chabra, R. Induced
termination of pregnancy by purified extracts of
Azadirachta indica (neem): mechanisms involved. Am.
J. Reprod. Immunol., 37(6):485-91, 1997.
Yajurvedi, H. N.; Hooli, M. A. & Nadkarni, V. B. Effect of
gonadotropin releasing hormone (LH-RH) on the pars
distalis and testis of skipper frog, Rana cyanophlyctis
(Schn.). Cell. Mol. Life Sci., 4(8):1058-60, 1985.
Young, B. & Heath, J. W. The endocrine glands. In: Young,
B. & Heath, J. W. (eds.). Wheater’s functional histology:
a text and colour atlas. Edinburgh, Churchill Livingstone,
2004. pp.310-27.
Guyton, A. C. & Hall, J. E. Textbook of Medical Physiology.
10th ed. Philadelphia, S. W. Saunders Company, 2001.
Jin, P.; Pan, X. & Zhao, S. Toxicity and growth-regulating
activity of a neem seed kernel extract (azal-s) to the
larvae of Culex quinquefasciatus. Insect Sci., 2(1):64-9,
1995.
Khosla, P.; Bhanwra, S.; Singh, J.; Seth, S. & Srivastava, R.
K. A study of hypoglycaemic effects of Azadirachta indica (neem) in normal and alloxan diabetic rabbits.
Indian J. Physiol. Pharmacol., 44(1):69-74, 2000.
Koul, O.; Isman, M. B. & Kethar, C. M. Properties and uses
of neem, Azadirachta indica. Can. J. Bot., 68:1-11, 1990.
Correspondence to:
Dr. Moses B. Ekong,
Department of Anatomy
Faculty of Basic Medical Sciences
University of Uyo
Uyo,
NIGERIA
E-mail: [email protected]
Received: 25-11-2010
Accepted: 24-02-2011
Murray, R. K.; Granner, D. K.; Mayes, P. A. & Rodwell, V.
W. Harper’s Biochemistry. 25th ed. London, Appleton and
Lange, 2000.
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