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Indian Journal of Experimental Biology Vol. 4 1, August 2003. pp. 885-889

Antifertility effect of Tinospora cordifolia (Willd.) stem extract in male rats

R S Gupta & Aruna Sharma

Reproduction Ph ys iology Secti on. Department of Zoology. Universi ty of Rajasthan. Jaipur 302004. India.

Received I J DC/libel' 2002 : rel'i.l'ed 23 Apr i l , 2003

Oral admi nistrat ion of 70% methanoli c ex tract of T. cordi/()Iia stem to malc rats at the dosc level of 100 mglrat/day for 60 days did not cause body we ight loss but decreased the weight of testes. ep ididymis, seminal vesicle and ventra l prostate in a signfic iant manner. Sperm motilit y as we ll as spe rm densi ty were reduced signi fica ntl y which resu l ted in reduction of male fertilit y by 100%. T he stem ex trac t brought about an interference wi th spermatogenesis. The round spermati ds were decreased by 73 .1 2%. However, the popu lati on of pre leptotene and pachy tene spennatoey tes were decreased by 47.60% and 52.85% respectively, foll owed by secondary spermatocy tes (48. 10%). Leyd ig cell nuclear area and mature Leyd ig cell numbers were significantly reduced when compared wit h controls. Serum testos terone level s showed signi ficant reduction after TiJlo.l'pom extract feed ing. Semini ferous tubule diameter. Leydig cell nuclea r area as we ll as cross sectional surface area of Serto li ce lls were reduced significantly when compared to controls. Biochemica l parameters i .e. protei n. siali c ac id, glycogen con tents of testes decreased signi ficantl y. Seminal vesicular fructose also depleted whereas, tes ticu lar cholesterol was elevated signi ficantl y followed by a reduction in tes tosterone levels. T hese resul ts suggested an tifert ility effects of the stem ex tract of T. cordifolia in male rats.

Key words: Antifertilit y ef fecl. Spermatogenes is. Test icu lar androgen, Ti Jl oSI )Ora cordi{olia

The deve lopment o f new fe rtility regul ating drug fro m medi cina l plants is an att racti ve prepos itio n. Tinospora cordifolia (Willd .) (family: Menispermaceae) is a traditi onal pl ant co mmo nly know n as " Neem­giloy". The roots o f T. cord!fo lia are used fo r alloxen diabetes 1 Pharmacological screening of crude methenolic ex tract o f T. corclifolia stem showed immuno-modul atory and anti -potenti ating acti viti es2

.

Some species have a lso been used fo r treatment of type-II di abetesJ

. The present study was under taken to evaluate the antife rtility e ffects o f T. cord/fo lia stem ex tract.

Mattrial and Methods EXlracl preparation - T. cordifolia plants were

collected from Ja ipur region. Shade dri ed powdered stem was ex tracted with 70% methano l in a sox hal et apparatus to obtain a solid vi scous brown mass, th at is "crude ex tract" . On chemical screening o f T. cord(folia stem two new compounds-c1erodane furano-diterpane (C2oH220 sh4 and c1erodane deri vati ve c le rodane diterpenoid5 have been obtained .

Animals and treatment - M ale a lbino rats o f Wistar-stra in weighing 150 to 200 g were used fo r ex perimentation.Animals were housed in pl asti c cages with proper aeration and temperature, mainta ined o n standard rat feed (Hindustan Lever Ltd) and wate r ad hbilium. The animals were di vided in to two groups of 10 animal s each.

Group I Rats received vehic le (di still ed water, 0. 5 mllrat/day) fo r 60 days .

Group II Rats treated with crude ex tract ( IOOmg/rat/day) for 60 days

Crude ex tract was di ssolved in 0. 5 ml di stilled wate r and admini ste red ora ll y. The mating exposure tests on contro l and treated gro ups were perfo rmed on day 55 using the method of WH06. The presence of sperms in the vag in al smear was the ev idence o f mating. On day 6 1 i.e. 24 hI' a ft er the last dose admini strati on animals were sac ri ficed using ether anaesthes ia. Blood was co ll ected by cardi ac puncture and serum was separated . Reproducti ve organs were di ssected out.

Hislopatholog ical preparation --:- Ti ssue were fixed in Bouin 's fluid , paraffin sectio ns were made and stained with hematoxy lin and eosin .

Sperm lIlotility and sperm density-Sperm motilit y and sperm density were assessed in cauda epididy mi s by the method of Prasad el a17

.

Tissue biochel1lislry-The parts o f tes tes. epididymi s, 3emi na l ves icle and ventral prostate fro m each rat were kept at -20°C until assayed fo r prote inS, sia lic ac id9

, glycogen lo. fructose ll and cho lestero l t 2

.

Testicular cell dynamics- The evaluati on of cell popul atio n dynami cs was based on the ca lcul ati ons made for each ce ll type per cross section of seminife rous tubul e. Va ri ous ce ll components were

886 IND IAN J EX P BIOL, A GUST 2003

quantitatively analyzed using spheri cal tubules iJ

Interstitial ce ll type such as fibroblast and mature degenerating Leyd ig cell s were es timated applying a di fferential count and stati sti ca ll y va ried by the binomi al di stributi on 14.

Horlllonal assay- Serum testos terone leve ls were assayed from frozen sampl es using radio immuno assay method (read il y available)15 The sensitivity of the assay was 10 pg/ml and intra assay error was 4.5%.

Stati.Hical ollalysis-The mean and standard error of mea n (SEM) were ca lculated by using Student's t­test 16.

Result Weight resjJonse- The orall y tested T. cordifolia

stem extract did not cause any significant change in the body we ight of treated rats. However, the weight of epid idymi s. sem in al ves icle and prostate gland

were decreased signifi cantly (P < 0.00 I ).The weights of testes and epididymis were not signifi ca ntl y different from controls (Table I).

Spe rll1 dynamics and f ertility test- The sperm motility in cauda epididymi s was decreased and severe impairment of sperm density in testes and cauda epididymi s were observed afte r trea tment. The fertility test showed 100% negative fert ility in T. cordi/olio treated rats (Table 2).

Biochelllical findings- A marked reducti on in protein and siali c ac id contents of testes and sex accessory organs were observed. However. tes ti cul ar glycogen decreased significantl y and fructose in seminal ves icle was al so lowered. The tes ti cu lar chol esterol was signifi cantl y elevated (P < 0.0 I) (Table 3).

Testicular histopathology and cell population dynalllics - Admi ni strati on of crude extract caused an effec ti ve inhibition of spermatoge nesis in male

Table I - Effect o f T cordijiJlia stem ext ract on body weight and o rga n we ights

I Va lues a re mean ± SE of s ix dete rminations ]

Organ weight s Body wt (g)

Fina l mglI 00 g body wI. ~

Testes Epid idy mi s Semin a l ves ic le Ventral prostate

Cont ro l 240 ± l AO 1345±4.70 529.5 ± 11 .20 605.7 ± 9.50 308.5 ± 2.02

TCordi{olia stem ex tract 100 mg/ rat! tby oral for 60 days 172.5±7.50 11 55. 15±95. 15 n' 427 .97±42.97n, 302A9±0.27** 183.97±9.53**

*p < (J.O I : ** p < 0.00 1 vs Control. (ns: on signifi cant )

Tabl e 2 - S pe rm dy na mics a lld Testos te rone leve l of T cordifolia trea ted m:J le rats

I Values are mean ± SE of s ix determinat ions I

Sperm motility (%) S~enn densit~ (milli on/ml ) Fe rtility tes t Testosterone Cauda epididy mides T es tes Cauda e pididy mides (ng/ml )

Cont ro l

r Cordifolia stem extrac t 100 mg/ rat! day o ra l fo r 60 days

** P < 0.00 I vs Contro l

72.00 ± 0.8 1

32.92 ± 1.94**

4.62 ± 0. 11 42.50 ± 0.92

2A5 ± 0.05 ** 11.1 3 ± 0.36**

Table 3 - Tissue biochemi stry of T cordijolia treated male rats

I Values are mean ± SE o r six determinations I

Protein (mg/g) Sialic acid (mg/g)

Cauda Vent ra l Seminal Cauda Ventral Seminal Testes Testes

epid idymides prostate ves icle epididymides prostate ves icle

Co ntl'll I 227 .80 268.00 162 .00 186.00 4.60 5.30 5.20 4.20 +4.56 ± 3.6 ±0. 18 ± 3.40 ±0. 16 ±0. 15 ±0.23 ±0.09

TCordijo/ill slcm 194 .30 '* 220.71 * 144. 19** 165.52 ** 3.91" 3.92** 3.64" 3.R2** ex tract 100 mgl ratl day

± 1.33 ± 2.96 ± 2.22 ±0.16 ± 0.04 ±0.04 ± 0.02 ± 0.02 oral for 60 days

* P<O.O I ;** P < O.OO I vsControl

100% (+) \ e 4.25 ± 0.03

100 % (-)ve 2.28 ± 0.02**

Glycogen Fruc tose

(mg/g) (l11g/g ) Cholesterol

Seminal ( l11g/g) tes ti s Testes

vesicle 4.85 4.52 7.30 ±0.6 ±0. 19 ± 0.30

2.76** 3.54** 12.50*

±0.02 ±0.04 ± 0.50

~-

t

GUPTA & SHARMA: ANTIFERTILITY EFFECT OF TlNOSPORA CORDIFOLIA STEM 887

rats. The production of round spermatids was decreased by 73. 12%. The number of pre-leptotene, pachytene spermatocytes and secondary spermatocytes were decreased by 47 .60%, 52.85% and 48. 10% respecti vel y. The mature Leydi g cell number was sign ifica ntl y (P <O.OOI ) depleted. The tota l number of Sertoli cell s and seminiferous tubule diameter were also reduced as compared to control group (Table 4) (Figs I & 2).

Hormonal assa), - Serum testosterone level of Til1ospora cordifolia stem extract treated animals was decreased significantly in comparision to contro l group (Table 2).

Discussion In the present ~tu dy, admini strati on of T. cordiIolia

stem ex tract to rats caused decrease in weight of testi s, epididymides, seminal ves icle and ventral prostate which may be due to low plasma level of testosteronel 7

, which IS found to be decreased fo ll owing T. cordifolia extract feedi ng to rats. Similar results we re revealed l8 after feed ing leaf ex tract of S/C'p liania hem andfolia. The decrease in the weight of accessory sex organs indicates the atrophy of glandul ar ti ssue and also reduction in secretory ab ilit y thus refl ecting the decrease level of testosteronel 9

.

The reduced testicu lar weight and shrunkun seminiferous tubul ar dimensions indicate wide spread damage2o.

The reduction in sperm motility in cauda epididymi s is of importance with regard to fertilization2i

. In many of the plant based contracept ives, inhibition of male fertility after admini strat ion of natural substance has been ascribed

,,' t ~ , .

/ ;. ,.

Fig. I - Microphotograph o r testes of cont ro l ral showing a ll lhe successive stages o f spermatogenesis. Leydi g ce ll s are present in in tertubul ar space. Lumen cont aining spermatozoa. X 200 HE

Fig. 2 - Microphotograph o r testes or rats treated with T. cordi/o ­lia stem ex tract 100 mglra t/day showing inhi bi ti on or spermato­genesis, note the abse ncc of secondary spennat ocy tes and sper­matids. Lumen is devoid or spermatozoa . X 200 HE

Table 4 - Testicu lar ce ll dynamics of T. cordi/olia treated mal e rat s

[Va lues are mean ± SE or s ix determin ati ons I

Teslicu lar ce ll counts/Cross sec tion Leydig ce ll dirrerenti al Seminiferous

Leydig count (%)

tubular ce ll

Secondary diam .

nuclear Spennato- Sertoli Pre-

Pachytene spemalo- Round Fibro-Mature

De- ciiam . goni t.lln cell le ptolene cy les spermatids blasl generated""",=- (I-lrr0-=----'

Conl ro l 6.87 2.8 1 19.C)5 29 .29 48.10 35. 10 43.30 99.70 57.00 268.00 11.1 0

± 002 ±0.02 ±0. 19 ±0.73 ± 0.60 ± 1.07 ± 3.60 ±0.26 :!: 2.8 ± 9.00 ± 0.02 T. Cordi/olia Slem ex t raCl 2.36** 1.65 * 10.45 ** 13.1;0** 12.34** 9.43** 63.94** 74.33** 62 .6 1 192.90** 4.93* 100 mg/ rat/ day oral ± 0 .28 ± 0. 13 ± 1.08 ± 0.68 ± 3.00 ± 2.68 :!: 5.37 ± 7.79 ±6.00 ± 0.22 ± 1.76 ror 60 days Percent

- 65.59 - 41.00 - 47 .60 - 52.85 - 74.32 - 92 .35 - 47.66 - 25.44 - 28 .02 - 55 .52 deviat ion

* P < 0.0 1; ** P < 0.00 1 vs Control

888 I DIAN J EXP BIOl, AUGUST 200]

d '1 ' d d . )) Jl D to ecrease sperm mot I Ity an enslty--·--. ecrease in sperm moti I i ty suggests alterati on of sperm maturation in the epid idymis24. Adminstratiori of T. cordifolia extract resulted in decrease sperm count which might be due to parti al arrest of spermatogenesis as it was al so confirmed by histopathologica l findings. A decrease in sperm reserve may be a reasonable cau se for reduction in the weight of epididymis. The significant decrease in tes tosterone level in the treated animals supports thi s viewc5. reduced androgen level may decrease sperm density in tes tes. The results indicate an inhibitory effect of the test substance on the tes ti cular sperm producti on and epididymal sperm maturation which consequentl y resulted in the gradual decline ill fe rtility rate, since decrease sp 'rm count has positi ve correlation with infertilitih .~7

The reduction in number of spermatogenic cell s llIay be due to non ava ilabilit y of testosterone, as spermatogenes is is acti vated by testosterone whi ch is synthesized by Leydig ce ll s and acts on sertoli and peritubular cell sc ~ . Sertoli cell s also facilitate the germ cell maturation but these cell s are highl y su spectible

' 9 d I to ex traneous damage- . Low counts an structura changes in sertoli ce lls consequentl y diminished secretory function i.e. ABP (Androgen Binding Protein) resulted in depleti on in number of spermatids along w ith secondary spennatocytes, since ABP is required for mai ntai ni ng i ntrat ubu lar androgen concentration 30 .

The impairment of Leydig ce ll functi on was ev inced by its reduced nuclear area and lower number of IYtature Leydig cell s. The number o f mature Leydig ce ll has direct bearing on spermatogenesi s. Deformation o f Leydig cell further indicates the ineffici ency of these cells to synthesize testosterone' l .

It IS evident th at decreased tes ti cular and epid idymal protein content cou ld be correlated with absence o f spermatozoa in the lumen 3~ . Structural integrity of acrosomal membrane is dependent upon sialic acid and due to al teration in its content the metabolism, motility and ferti li zing ability o f sperms also may be effected33 It al so acts as 'lubricant' to facilitiate the downward movement of sperms and

f · . ~ reduce ncllon among spermatozoa' .

Glycogen acts as an energy producing source in ti ssues and constant supply of carbohydrate (glucose) is essential for gonadal maturation and for proper function of testes, as it maintains the ti ssue integrity A TP production and protei n synthes is in the rat test es35

. Sign i ficant decrease in glycogen content after

the treatment possib ly cou ld be due 10 inhibition o f glycolysis during spermatogenesis.16 as the doses o f Tillospora cordifolia stem extract inhibited glycolysis in spermatozoa which wou ld ultimately influence fertilizing capacity o f male rats.

T he process of fructose formati on in semi nal vesic le is initiated and controlled by testi cular androgens37

. Hormonal defici ency causes a decrease or even disappearance of seminal fructose and a compensatory trea tment with androgens restores the ability of the accessory glands to produce this slIgar3

.

The results, thu s suggest s that oral admini strati on of crude methanolic ex tract o f T. cordifo /ia stem can lead to a infertile state in male rats due to interference in th e tes ti cular androgen leve ls altering the process o f spermatogenes is.

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