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Journal of App lie d Sciences Research , 6(4 ): 27 5-279, 2010
© 20 10 , INSIn et Publication
Comparative Effect of Aqueous Extract of P. Guajava Leaves and Ascorbic Acid on
Serum Sex Hormones Levels in Male and Female Rats
F.E. Ub oh , E .E . Edet, M.U. Eten g and E .U . Eyong
Department of B iochemistry, Faculty of Basic M edical Science C ollege of M edical Sciences University
of Calabar - C alabar
Ab stract: Psidium g ua java has a ric h e thnom ed icina l history, particu larly in the trea tment of va rio us
gastroin tes tinal d isord ers. In this s tudy, 2 00 mg/kg body weight of aqu eo us e xtrac t of P. gu ajava
leav es(AE PGL ) an d asco rbic acid (vitC) were administered orally to male and female rats daily, for 30
days, to assess their respectiv e effe ct on the male and female repro du ctive fu nctions, using serum levels
of fo llicle stimulating hormo ne(FSH), luteinizing horm one(LH ), estradiol, pro ge stero ne and testosterone.
Th e results of this stud y showed that trea tment o f rats with A EP GL and vitC , respec tiv ely, had
insign ificant e ffe ct (P $ 0 .05) on the seru m levels of FSH a nd LH in m ale a nd female rats, a nd a
significa nt increase (P # 0 .05) in the se rum estradiol and progesterone in females, and testoster one in
males . AEP GL increased serum estradio l and pro gesterone leve ls by 17 .6 and 34. 6 percents, resp ectively,
in females, and testostero ne by 38 .6 perc ent in males, while vitC increas ed serum estradio l and
progestero ne levels by 15.2 and 30 .0 perc ents, respectively, in females, and testo ste rone by 28.0 perc en t
in male s rats. From these r esults, it w as o bs erved tha t the effect o f AEP GL on the le vels of these
hormones was higher than that of vitC in male and fem ale rats, and that th e percentage incre ase in male
sex ho rm o ne s w as significantly higher than the perc entag e increase in fe ma le s ex hormone s. T hese
ob serva tio ns ind icated th at AE PGL possess a higher pote ncy in stim ulating the se cretion of sex horm on es
tha n vitC , a nd tha t the effect is more significant in male tha n female rats; hence, AEP GL may be
recommend ed for males with various reprod uc tive dysfunctions.
Key w or ds: P. gua java, asco rb ic acid , sex hormones .
IN TR O DUC TION
Ps id ium g ua java, guav a, is a member of the
Myrtaceae family, w ith ab ou t 133 genera a nd more
than 3,80 0 sp ecies. P. gu ajava is a large tropica l
everg reen shr ub or sma ll sh ad e tree that grows up to
10 - 15 m in height. It is native to and widely
distributed in Mexico and Central Ame rica. Ho we ver,
the plant is cultiv ate d today from the w est coast o f
Africa to the Pacific region , includin g India and Chin a,
with va rie tie s origina lly introd uced over the past 3 00
yea rs fro m th e United States. Ge nerally, guava p lant
has sp re ad widely thro ughout the tro pics b ec au se it
thr ive s in a va rie ty of s oils, propagates easily, a nd
bears fruit rela tiv ely quickly. T he tree is easily
identifie d by its d istinc tive thin , smo oth, co pp er-co lored
bark that flakes off, sho win g a greenish laye r ben eath.
Th e g uava berry is an im po rtant tro pical fruit that is
mo stly consumed fre sh. T he fruit co ntain s several sm all
see ds an d consists of a flesh y pericarp and se ed ca vity
with pulp [1 -3 ]. Th e gu ava fruits are either eaten fresh,
or mad e into drink s, ice cre am , and preser ves. Gu ava
fru it is still enjo yed as a sweet tr eat by ind igeno us
peop les thr ou ghout the rain fores t, and the leave s and
bark of th e guav a tr ee have a lon g histo ry of medicinal
uses that are still emp loyed today.
Ph otochem ical analyse s re vealed that more tha n 20
compounds can be isolate d from gua va lea f prod uc ts,
inc luding alk aloid s, anthocyanins, car oteno ids, essen tia l
oils, fatty acids, lectins, phenols, sap on ins, tannins,
triterpenes , and about 8 0 mg o f v ita min C ( ascorbic
acid) per 100 g of guava . The essential oil co nta ins
[4 -10 ]
alp ha pinene, caryop hyllene, cine ol, D-limonene ,
eu ge no l, and myrcene . T he majo r constitue nts of th e
volatile a cids inc lud e (E )-c inn am ic a cid and (Z)-3-
hexen oic acid. Carbohyd rate es ter s have also been
iso lated from the fruit . Howeve r, the m ain active
[5, 8]
constituent in the plan t is rep or ted to be quercetin. Th e
spasm olytic and antidiarrhe al effects are re po rted to be
as soc iated with its q ue rc etin-der iv ed fl avo noi d
glycosid es, wh ich sup port use of this ancient lea f
remedy in treating gastro intestinal disorders .
[3 ]
Scien tific investigations on the m ed icina l p roperties
of guava le af pro ducts date back to the 1940s. Som e
Corresponding Author: F.E. Uboh, Department of Biochemistry, Faculty of Basic Medical Science College of Medical
Sciences University of Calabar - Calabar.
E-mail: fridayuboh@yahoo.com
275
J. App. Sci. Res., 6(4): 275-279, 2010
in ves ti gations exa m ine d a n tia m ebic , a ntibi otic,
ant idi a rrh e ic, a nti h yp er gly c emic , a nti m uta g eni c ,
antisp asmo dic, and sedative effects, as we ll as
anticoug h a nd narcotic-like ac tiv ities of the p lan t.
Ho we ver, mo st sc ientific e vidence rela tes to the clinical
ef fi cac y o f g uav a in t rea ti ng ga stroi ntest in al
disord er s . Th e you ng leaves of the plant have been
[3 ,5]
used as a to nic to treat d ige stive co nd itions such a s
chole ra a nd diarr hea in Br az il and M exico. C ur rent
Mexican med icinal data doc um ent the treatm en t of
ac ute diarr hea, fla tulenc e, and gastric pain by using a
guava leaf w ater decoc tion fo r oral administra tio n 3
tim es daily. A dec oc ti on of yo ung le aves and sho ots is
presc rib ed as a feb rifuge and a sp as mo lytic. In Bolivia
and E gyp t, guava lea ves are used to treat cough and
pulmona ry dise ases. Y ou ng guava leaves are us ed a s an
anti-infla mm atory and hem osta tic agent, as well as an
agent fo r the tr eatmen t of co ug h in Bo livia, Egyp t,
India , an d C hina . Also, aqueous extracts fro m
[2,4,5,11,12]
P. gua java is rep orted to have antioxidant or ra dical-
sca venging activity. M o st of the activity is asso cia ted
with the polyph enols; however, the guava extracts also
contain antioxid an ts such a s a scorbic ac id and
carotenoid s .
[2,13,14]
Several plant extr acts are known to possess
stimulato ry activity on the reproductiv e function s. For
33 4 0 15
instan ce , Icarii n (C H O , molec ular weight: 676.6 7),
a flavonoid isola ted fro m the pla nt H erba epimedii ,
[15,16]
has b een used as a to nic d rug in traditiona l C hinese
medic ine (TC M ) for ce ntu ries to im prov e e rectile
fun ction . Also, it increase s the weights o f the
[17 ,1 8]
pituitary gland , testis and epididymis and the
tes tosteron e con ce ntration in immatu re male rats, and
has te sto ste ro ne mimetic prope rties . I n folk
[19-21]
medic ine, especially in some traditio na l African
local ities, decoctions fro m P. g ua java leaves are
co mmo nly emplo yed to treat a wide range of
repro du ctive disord er s. In the practice of local herb al
medic ine, P . gua java leaf extracts are pa rti cularly
believed to improve ere ction and impotency in ma les.
Ho we ver, th ere is paucity of d oc um en ted evide nce to
sup po rt th is practice.
Sex ho rmones are known to reg ula te the
repro du ctive functions and ch aracteristics in both male
and female orga nis ms . M easurem en t of serum sex
[22,23]
hormones profile is the refore ve ry useful in asses sin g
the reprod uc tive integrity in both an imals an d humans.
Ac cording to Jenner e t al. , seru m estradio l
[24 ]
me asurement is a va luable index in ev aluating a variety
of menstrua l dysfunc tio ns in females. T his indic ates
tha t suppre ssion of r ep roducti ve fun ctions may
evide nced in redu ce d seru m sex h ormo ne profile and
vic e versa. T his study a sse ssed the e ffect of P. g uaja va
lea f ex tracts and v ita min C o n se rum se x hormonal
profile in male and female rats, with the aim of
inv estiga tin g the vita mi n C- property, and the valid ity
or ot herwise of the use of the leaf extract in
repro ductive disorders in folk me dicine.
M AT ERIAL S AN D M E TH O DS
Identificatio n an d Preparation of Plan t M ateria ls:
Fresh leaves of P . gu ajav a w ere co lle cte d in Ma y 20 09
fro m lo cal garden at the University of Ca labar,
Ca la ba r, N igeria. T he sam ple of the plant specim en
was identifie d and authentica ted by a Botanist fr om the
bo tanical garden, and the Voucher specim en w as
depo sited in the herbari um o f the same Univer sity.T he
lea ves w ere sorted to eliminate a ny de ad ma tte r a nd
other unwanted particles. Th e leaves were air-dried for
2 weeks and then ground into fine po wd er using a n
ele ctric dr y m ill (M ou lin ex ). 200g of th e g round
po wder wa s soak ed in 1.0l of distilled water fo r 48
hours at ro om te mp erature. T he mixture wa s filtere d
into 500 ml c onical flask w ith Watman filte r pap er
(N o.1). The filtrate was dried at a temp eratu re of 30ºC
for 10 hours to produce a gel-like extract, which
weighed 20.5 g. Approp riate concen tration of the extra ct
was then sub sequ en tly mad e by dilu tio n with distilled
water into 20 0/mg /kg body weig ht and administered to
the anim als.
Handling and Treatme nt of Anim als: A tota l of 36
adult albino rats (18 males an d 18 fe ma les) we ighing
betwe en 150 -300g ob taine d fr om the dis ease free stock
of the anim al house, B io ch em istry Department, C o llege
of M edical Scie nces Univer sity of C ala ba r, Calabar
Nigeria, were u sed fo r the study. The rats were div ided
according to sex into six gro up s with six rats each, as
follows:
Gr ou p I. Mc (Ma le co nt ro l group rece iving distille d
water as plac ebo),
Gr ou p I. Fc (Female control group rec eiving distille d
water as plac ebo),
Gr ou p II. M vitC (M ale t est g roup receiv ing vitam in
C), Group II.Fv itC (Female test gr oup
receiving vitamin C),
Gr ou p III. MPg (M ale tes t group rece iving aq ue ous
extract of P. gua java le aves) ,
Gr ou p III. FP g (Fe ma le test group r eceiving aqu eo us
extract of P. gua java le aves) .
Th e rats were a cclimatize d in the exp erime ntal
anima l house for one wee k befor e the commencem ent
of the e xp erime nt. T he anim als, housed in sta inless
ste el cages un de r standard conditio ns ( am bient
tem peratur e, 28.0±2.0º C and hu mi dity,46%, with a 12
hr light/d ark cycle), wer e fed with the no rmal rat
pelle ts. A ll the rats in both test and control groups
276
J. App. Sci. Res., 6(4): 275-279, 2010
were allo wed fre e acce ss to food and water ad libitum ,
thr oughou t th e experimental period. Goo d hygiene wa s
maintain ed by co ns tan t cleanin g and rem oval of faeces
and spilled feed from cages daily.
The anim als in test groups II.M vitC, II.F vitC an d
III .M Pg, III.FP g rec eived 200 /mg/kg body we ight ora l
daily d os es of vitamin C a nd aqueo us e xtr act of P .
guajava leaves, respectiv ely, using o rogastric tubes and
syringes. This la ste d for a perio d of 30 da ys and the
experim ents were co nd uc ted be tween the hours of
09 .00 am and 10.0 0a m daily. Ra ts in the contro l
group s I.Mc, I .Fc we re adm iniste red, b y oral gavag e,
with 5ml of distille d water (p lacebo).
Synthetic vitamin C was ob ta ined from the Sigma
Ch emicals, Poole England an d u sed fo r the study. A
sto ck solution of vitam in C was prepared by dissolving
20 g of vitamin C powder in 500ml of distilled wa ter
ou t of which a dose of 200 mg/kg body weig ht was
admin istere d to anim als in 5m l of vehic le daily for 30
days. All the an im al experiments were carried out in
accordanc e with the g uid elines o f th e Institution’s
Animal Eth ical Comm ittee.
Co llection an d A nalysis of Blood: A ll the anim als
were anaesthetiz ed with ch loroform v ap ou r, twenty-fo ur
hours after last day of extract and vitam in C
admin istratio n, and dissected fo r blood c ollec tio n.
Bloo d samp les we re co llecte d by card iac punc ture into
a set of pla in sa mp le bottles, and allowed to clot. T he
clo tted blood sa mp les we re spun in a b ench top
ce ntr ifu ge (MSE, En gland ) to obtain sera. The serum
sam ples were sep arate d in to anothe r set o f plain sample
tub es. The separated se ru m samples we re sto re d in th e
re frigerator until req uir ed for the ho rm on al assay. All
assays w ere done within 24 ho urs of the sample
collection. The serum samp les wer e assa yed for FSH ,
LH , estradiol, progesterone and testostero ne u sin g
enzym es immu noassay meth ods. T h e re sp ecti ve
im mu noa ssay re ag ent kits we re o btai ne d from
Diagnos tic A utom ation Inc., 23961 C raftm an R oad,
Suite E/E, Cala ba sas, CA 9 13 02. Mic ro plate read er
(D ialab Instrum en ts Ltd.) was used in ta kin g the
absorba nce. Calc ulatio ns of th e co nc en tratio ns of
hormones were made ac co rd ing to the me tho d given in
the kits handbook.
Statistical Analyses: Th e results o btained from this
study were analyzed by o ne-wa y analysis of var iance
(ANO VA), fo llo we d by Student’s t-tes t to evaluate the
significa nce of the difference between the mean value
of the me asure d para me ters in the re spective test and
control g roup s. A significant chang e was consid ered
acceptable at P < 0.05 .
RE SUL TS A ND DISC US SION
Re sults: The results ob tained fr om this pr esent study
are summarized in Ta ble 1. Fro m th ese resu lts, it was
disco vered that tr eatment of ra ts m odel with aq ue ou s
extract of P. guajav a leaves an d vitamin C (che mical
tes t agents), resp ectively, caused no significant effect
(P $ 0.05) on the levels of serum FS H and LH in bo th
male an d female rats (T ab le 1). Howeve r, it was also
ob serve d th at trea tment of th e anim al model with the
respe ctive chemical test agents cau sed a sign ifican t
inc re as e (P # 0 .05) in the levels of ser um estradio l an d
progestero ne , in fem ales, and testo sterone in male,
when compared respe ctively with the serum FSH and
LH leve ls obtaine d for the re spective contro l gro up s
(T ab le 1).
In this study, it wa s interestin g to noticed th at
while treatme nt of rats with the aqueous ex tra ct of P.
guajava lea ve s in cre ase d ser um e st ra diol and
progestero ne levels by 17 .6 and 34.6 percents,
respe ctively, in fema les, a nd testosterone b y 38.6
percent in males; treatme nt of the animals with vitamin
C incr eased se rum estra diol a nd pro geste rone levels
only by 15 .2 a nd 30.0 percents, resp ec tiv ely , in
fem ales, and testosterone by 28.0 percent in males.
From these observ ations, it w as noted that the
effect of the aqueo us extract of P. guajava le aves on
the le vels of these se x hormones relea sed into
cir culation was higher than the effect of vita min C in
bo th m ale a nd female rats model. Moreover, the
percentag e increase in male serum sex hormones was
ob serve d to be signific an tly hig her th an the perc entag e
inc rease in fe male serum sex horm on es following
tre atment of the anim al model with aqueou s extrac t of
P. guajava leave s and vitamin C. Th ese ob servations
ind icated that the aqueous extract of P. gu ajava le av es
po ssess a hig her po tency in stimu lating th e se cretion of
sex ho rm on es than vitamin C, and th at the effec t se em s
to be more significant in male than fe ma le rats mo del.
Discu ssion : En do cr ine ch anges and d ec line in
endocrine fu nction invo lve tissu e respo nsive ness,
reduced se creto ry outp ut from perip heral glan ds and
alterations in the ce ntral mech anism c on tro lling the
te mpor al org a niz a ti o n o f h o rm ona l rel eas e .
[2 5 ]
Hypogonadism is a clinical co nd ition in which low
level of seru m sex hormones, including te sto stero ne in
ma le s, as well as estradiol and pro ge ste rone in females ,
is found in association with sp ec ific sign s and
sym ptom s. Th ese signs and symptom s may inclu de
diminished libid o and sense of v itality, erec tile
dysfunction , dysmeno rrhoea, reduce d musc le mass an d
bo ne density, dep ression an d anemia. B y re storing these
serum sex hormones leve l to th e normal range using
such agents as hormone supplement therapy, ma ny of
277
J. App. Sci. Res., 6(4): 275-279, 2010
Ta ble 1: Ser um sex h orm on es lev els of m ale and fe male r ats tre ate d wi th aq ue ous extr act of P. gu ajav a leave s an d vita min C
Gr oup FS H(m IU/m l) LH (mIU /ml) Pr oge ste ron e(ng /m l) Es tra dio l(n g/m l) Te sto ste ron e(ng /m l)
I.M c 2.4±0 .6 3 .8± 1. 1 - - 13 .2± 2. 3
II.M vi tC 3.0±1 .0 4 .0± 2. 3 - - 16 .9± 2. 4*
III.M P g 2.8±1. 3 4. 2±2 .1 - - 1 8.3±3.1 *
I.F c 2.6±0 .2 4 .3± 0. 2 28 .0± 1. 6 68 .3± 2. 7 -
II.F vitC 2 .9±0. 1 4.4±0 .2 3 6.4 ±1 .4 * 7 8. 7± 1.4 * -
III.F Pg 2.6±0 .4 4 .5± 1. 1 37 .7± 1. 7* 80 .3± 2. 6* -
Va lues a re p resen ted as m ean ± SE M, n = 6, *P < 0. 05 co m pa red wi th the cont rol. I.M c = M ale con tro l gro up r ece iving dis tille d wat er a s
pla ceb o, I.Fc = Fem ale con trol gro up re ce ivi ng dist illed water as plac ebo, II.M vitC = M ale te st group r eceiv ing vitam in C, II.Fv itC = Fem ale
tes t gr ou p rece ivin g v itam in C, III.M Pg = Ma le t est gro up r eceiv ing aqu eou s ex trac t o f P . g uaja va leav es, III.F Pg = F em ale test grou p rec eiv ing
aq ueo us ext rac t of P. guajav a lea ves .
these sym ptoms ca n be relieved . Reports indicate
[26,27]
tha t testos teron e supplem ent the ra py and Icariin, from
the plan t Herba epime dii, might prod uc e a wide range
of b en efits for m en with hypo go nadism th at includ e
impro ve me nt in lib ido, bo ne density, m uscle m as s, body
compositio n, mood, erythropoiesis and c ognition[1 7-
.
21,28,29]
Se x horm ones, pa rticularly estr ad io l and
progestero ne in females, and testosterone in male s, are
produce d p rimarily in th e gonads under the influenc e
of FSH and L H. The increa ses in th e conc entrations of
sex ho rm ones a re known to exe rt p osit ive feedback
influence at the le ve l of the pituitary gla nd , wher e th ey
regulate the se cretion of gona do tropins . In non-
[30 ,3 1]
pregn ant fema les w ith a nor ma l menstrua l cyc le, th e
pro ges te ron e lev el r ema ins r e la tiv e ly c ons ta n t
thr oughout the follicular phase of the men strual cy cle
and the inc rease s rapidly following the o vulation , wh ile
the estradio l secre tio n fo llo ws a cyclic b ip hasic p attern,
wit h the high est co nc entratio n found im mediate ly prior
to ovulation .
[32-34]
In this stud y, w e fo un d that tre atm en t of male and
fem ale rats with P. guajava leaf extr act and vitamin C
inc rease d the circula ting level of testo ste ro ne in males,
as well as estradiol and p ro gesterone in fema les.
How ever, in the present stu dy, there was no sig nific an t
differ ence in seru m levels of LH and FSH between the
rats in control gro up and those in the grou ps treated
wit h P. gu ajava leaf e xtrac t and vitamin C ,
respe ctively. From the resu lts o f this stud y, it see ms
tha t P . guajava le af e xtract and vitamin C have a
po sitive effect mainly o n the go nads, stimu lating the
sec retion of these gon ad al horm ones into circulation in
male an d fem ale rats. On the whole , this gonadal
hormones sec retion stim ulating effect was o bserv ed to
be higher in ra ts trea ted with P . guajav a leaf extract
tha n those trea ted w ith vitamin C . M oreo ve r, this e ffect
was observe d to be remarkab ly greate r in m ale tha n the
fem ale rats for bo th P. g ua java leaf extract a nd vita min
C treated groups. The observatio n made fro m the
resul ts of this stu dy supported the re po rt that P.
guajava lea f extracts conta in vitamin C, in add ition to
other chem ical agents .
[4- 10 ]
Th e results of this present study revealed that so me
of the ch em ical a gent(s) which a re contained in P.
guajava leaf extracts p ossess stimulato ry activity on the
repro ductive fun ctions in rats mod el, mainly the ma les.
Th is asser tio n hyp othesized an d documented, in support
of the practice of African folk medic ine , th at P.
guajava leaf ex tra cts may be strongly re co mme nded to
males, and mildly to female s with vario us deg rees of
repro ductive d ysfunctions. However, th e specific
chemical agent(s) resp onsib le fo r the enhanc ed e ffe ct of
P. g ua java l ea f extract over vitam in C and th e
mechanism(s) of gonadal ho rm on es stimulating effec t
are subjec ts for furth er investig ation.
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