Anti-steroidogenic activity of methanolic extract of...

Indian Journal of Experimental Bio logy Vol. 4 1. June 2003. pp. 64 1-644

Anti-steroidogenic activity of methanolic extract of Cuscuta refl exa Roxb. stem and Corchorus olitorius Linn. seed in mouse ovary

M Gupta, U K MaZLl Illder, 0 K Pal' & S Bhattacharya

Department o f Pharmaceutica l Technology. Jadav pur Univers ity. Kolkata 700 032. Ind ia

' Seemanta Institute o f Pharmaceuti cal Sc iences, Jharpok hari a, May urbhanj . O ri ssa 757 086, Ind ia

Received II May 2002; revised 24 March 2003

Methanolic extrac t (M E) o f both Cre.f7e.w stem and Co/i/orills seed arres ted the normal oestrus cycle of adu lt fe male mouse and signi ficant ly dec reased the weight of ovari es and uterus. The cho lesterol and ascorbic ac id contents in ovaries

were s igni fica ntl y increased in the trea ted mi ce. Two key enzy mes, 1.l ·\-3~-hyd roxys tero i d dehydrogenase and g lucose-6-phosphate dehydrogenase, were decreased s igni fica ntl y in M E or both C re.f7exa stem and Co/i/orius seed afte r 17 days of trea tme nt. Hi gh leve l of substrates and low leve l of enzy mes indicate the inhibiti on o f steroidogenesis in treated mice and may be due to the presence o f f1 avonoids.

Key words : CUSCLIIa re/lexa seed ext rac t. C IISCil Ia o/i lOrious seed extrac t, G lucose-6-phosphate dehydrogenase,

1.l5-3 ~- hydroxys tero i d dehydrogenase, Mouse ovary, S teroidogenesis.

Cuscula reflexa Roxb. (Famil y: Convo lvulaceae, Swarnalata in Bengali , Aillarve l in Hind i) is a golden ye llow dodder - li ke paras ite. The plant is common th ro ughout Indi a, and is widely di stributed in plains of West Benga l. Various parts o f thi s pl ant were used by tribes in ailments like fits , me lancho ly and in saniti. It is also useful ex te rna lly aga inst itch and interna lly in fevers, ' rete ntion of wind ' and ' indurati o n of the liver,2-4. On pre liminary anal ys is, C. reflexa ste m, has been found to contain large quantity o f f1a vonoidss.6

and its diffe rent ex trac ts on pre liminary in ves tigati o n have been fo und to possess antife rtility effect? Corchorous olitorius Linn ., (Family Tiliaceae, Jute) is an annual he rb with s lender stems. It is cultivated in many parts of Indi a. The seeds are used as purgative and leaves are used as demul scent, diure ti c, febrifu ge (i nfu sion) and in chronic cyst iti s and dysuri a8

.

C. olilO rius seed is a traditio nal triba l medicine for birth contro l9. However, no deta iled s tudy has been undertaken on antifertility ac tivity o f C. reflexa ste m and C. oliforius seeds. In the present co mmunicatio n, we have assessed the in vivo anti -ste ro idogenic activity o f methano lic extract (ME) o f C. reflexa ste m and C. olitorius seeds by observing the changes in oestrus cycle, weight of the o vari es and uterus and biochemical parameters in mice. The changes in

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cho leste ro l and asco rbic ac id conte nt in ovaries were measured as choleste rol is the raw materi al fo r estrogen synthes is tO and ascorbi c ac id leve l se rves as a index for dete rmining the normal o vari an acti viti t.

The e ffect o f C. reflexa stem and C. olilOrius seed

extract on the activities o f L1s ,3~-hydroxys tero id dehydrogenase (5 HSD) and glucose-6-phosphate dehydrogenase (G-6- PDH), the two key enzy mes in vo lved Il1 ovan an steroidogenesis,. were also measured .

Preparation of extract- The ste ms of Cuscuta reflexa Roxb . and the seeds of Corch.orus oliLOrills Linn ., co llected locally were authenticated by the Di vi s io n of Pharmacognosy, Department o f Ph armaceutical Technology, Jadavpur University, Ko lka ta. The voucher specimens have been preserved in o ur laboratory. Shade-dried, powdered pl ant materi al was soxhle t ex trac ted firs t with petroleum e ther (40°-60°C) and then with methano l. The meth anolic extract was evaporated to dryness . The trace amo unt o f methano l which may be present within the solid mass of methano lic ex tract was remo ved by washing with ethy l alco ho l. For ph armacological tes ting, M E of C. refl exa stem and C. olitorius seed were disso lved in propylene g lyco l (PG).

Animal experiments- Adult female albino mice of Swiss stra in (22±2 g body weight) were acc limati zed

to laboratory conditi o ns (25°-30°C, 75-85% RH ,

642 INDIAN J EXP BIOL, J UNE 2003

12: 12 hr L:D) for o ne week and g iven pellet di et (Hindu stan Lever) and water ad libitulII. Experiments were performed under the guidance of The Ethical Committee, Jadavpur University , Ko lkata. The LDso va lues of ME of C.reflexa stem and C.olitorius seed are 435 and J 91 mg/kg body weight respectivelyl 2. Mi ce show ing normal oestrus cyc le for a period of 2 weeks were divided into 8 groups of 6 mice each and were g iven the fo ll owing treatment. Groups J and 2 served as normal sa line control (5 mllkg, 0 .9% NaCI w/v, ip) and vehicle control (5 mllkg, propy lene g lyco l, ip) respec tively . Groups 3, 4 and 5 were treated with M E of C.reflexa di ssolved in propylene glyco l at the doses of (25, 50 and 75 mg/kg, ip) respective ly and groups 6, 7 and 8 were treated with ME of C.olitorius disso lved in pro pylene glycol at the doses of (J 5, 20 and 25 mg/kg, ip) respectively. The doses were administered on alte rnate days for 17 days. Body wei ght was noted and oestrus cycle was examined everyday in the morn ing and in the evening by microscopical examination (x 100) of vagina l smear using methylene blue as staining solution.

On the 181h day, after 18 hr of fasting mice were sacrificed by cervical dis location. Normal saline and PG treated groups were sacrificed in the same oestrus phase of the ME treated groups (d ioestrus phase). Uterus was di ssected out, freed from fatty materials, wei ghed and kept on ice for further process ing.

Biochemical estimation-Cholesterol in the ova­ries was estimated by the method of Kingsley and Roscoe 'J . The absorbance was o bserved at 680 nm

and total cholesterol was quanti fied from the standard curve. Ascorbic acid in the ovarian tissue was meas­ured by the reduction of dichlorophenolindophenol (DCPIP) 14. The opt ical dens ity of the colour formed was measured at 520 nm aga inst DCPJP reagent as blank . For the es timation of L1s ,3 ~-hydroxystero id dehydrogenase (HSD) ovaries were homogeni zed with 1 ml normal saline and I ml of 0 . 1 M phosph ate buffer (pH 7.4) and centrifuged at 5000 g for 20 min. The supernatant was co ll ected and the enzyme was assayed by the method of Rab in et al ls. The protein content of ti ssue determined and the specific acti vity of HSD activity was expressed as U/mg of protein. G lucose-6-phosphate dehydrogenase (G6PD) in the ovarian tissue was meas ured by the method of Lohr et al ' 6

. Glucose-6-phosphate was used as the substrate and the formation of NADPH was mo nitored at 340 nm aga inst reagent blank for 10 min . The specific ac­tivity of G-6-PDH was calcul ated in terms of U/mg of protein . Prote in content of ovaries was estimated with Folin pheno l reagent l7.

Statistical analysis - Results are expressed as mean ± SE. Stati stical analys is was done by Student 's , t-test and the difference was considered statistically significant at P < 0.05 .

Results are summari sed in Tables 1 and 2. Vehicle contro l mice showed regular oestrus cycle.

Normal cyclical changes of the vaginal smear were examined in mice of groups 3, 4, 5, 6, 7 and 8 throughout the treatment period. After admini stratio n of 41h dose in low , medi um and high dose level of ME

Table 1- Effeci of crude eX lract o r C re.f7exa stem and Coli/orius seed on the we ights of ova ry and uterus in mature fema le mice

[Values are mean ± SE from 6 an imals in each group. Fi gu res in parentheses arc % increase (+) or decrease (-) over control]

Treatment Initi al body weight Final body weight Weight of ovaries Weight of uterus

(g) (g) (mg) (mg) Group

Saline 17.9 ± 2.5 27.6 ±1.9 9.5 ± 1.1 45. I ±O.9

(5 ml /kg, ip) Vehic le (PG)

18. 1 ± 1.9 27 .8± IA 9.1 ±0.9 46.0 ± 1.3

(5 ml /kg, ip) (+0.72) (-4.21) (+2.00) 2

ME of Creflexa 28.0 ± 1.0 5.8±0.7 30.0± 1.1 (25 mg/kg, ip)

17.8 ±1.2 (+ IA5 ) (-38 .95) (-33A8) 3

ME of Cre.f7exa 26. 1 ± 1.2 5.7 ±0.8 26.0± IA (50 Il1g/kg, ip)

17.9 ± 1.3 (-5 A 3) (-40.00) (-42.35)

4

ME of C reflexa 2S. I±1.8 5.6±0.5 27.3 ± 1.6 (75 mg/kg, ip)

18.0 ±1.1 (+ I.SI) (-41.05) (-39A7 ) 5

ME of Coli/orills 25 .7 ± 1.5 5.7±0.9 32.5 ± 1.3 (15 Il1g/kg, ip)

IS.7±1.5 (-6.8S) (- 40.00) (- 27 .94) 6

ME of Coli/orius 27.9 ±I A 5A±0.S 33 .4 ± 1.0 (20 Il1g/kg, i p)

18.5± IA (+ 1.09) (- 43.16) (-25.94) 7

ME of Coli/orills 28 .2 ± 0.9 5.2 ± OA 3 1.9± 1.2 (25 Il1g/kg, i p)

IS .7 ±1.6 (+2.17) (-45.26) (-29.27) S

*P <0.05 when Compared with control (Student 's / test)

NOTES 643

Table 2 - Effect of crude ex tract of C re.flexa stem and CoiilOrius seed on conte nt of ascorbic ac id, cholesterol and the act ivities of G-6-PDH and HSD in mouse ovary after 17 days of treatment

[Va lues are mean ± SE from 6 an imals in eac h group. Fi gures in parentheses are % increase (+)01' decrease (-) ove r cont rol]

Group

2

3

4

S

6

7

8

Treatm e nt

Sal ine (S ml /kg, ip) Vehi cle (PG) (S mllkg, ip)

ME of Crejlexa (2S mg/kg, ip)

ME of C reflexa (SO mg/kg, ip)

ME of Crejlexa (7S mg/kg, ip)

ME of Colitorius ( IS mg/kg, ip)

ME of Coiitorius (20 mg/kg, ip)

ME of Colitorills (2S mg/kg, ip)

Ascorbic ac id ( ~ g/mg ovary)

88.1 ± 4.3

90. 1±7.9 (+2.3)

130.3 ± 10.S (+47.9)

ISO.2± 12.8 (+ 70.S)

160.0± 11.2 (+81.6)

106.8 ± 7.3 (+2 1.2)

II S.2±6A (+308)

130.8 ± SA (+ 48S)

* P<O.OS when com pared with control (Student 's ttest)

of both C. rejlexa stem and C. oliwrius seed, the es­trus cycle was arrested at the dioestru s stage.

The wet weight of ovari es and ute rus was reduced significantl y (P<O.OS) whereas there was no significant change in the body weight of the animal (Table 1). The crude extrac t of C. rejlexa stem and C. olitorius seed significantly elevated the level of total cholesterol asco rbic ac id contents respectively in mouse ovaries as compared to the vehi cle control (Tab le 2). The activi ti es of G-6-PDH and HSD were inhibited signifi cantl y (P<O.OS) by crude ex tract of both C. rejlexa stem and C.olitorius seed.

Sequential changes of the vaginal smear in different phases of the oestrus cycle are c losely associated with simultaneous secretary patte rns of gonadal steroids l8. Ovarian hypofunctio n and anoestrus vagi nal smears appear to be due to the absence or decrease of c ircul ating gonadotropins 19.

Both the ex tracts reduced the wet weight of ovaries and arrested the oestrus cyc le at d ioestrous stage where minimum activity of stero id hormones has been reported2o

.22

. These di sturbances in the reproductive cycle and the decrease in the we igh t of the ovary and uterus in the present in vest igatio n a re re lated with the diminution of ovarian steroidogenesis. Thi s was associated with an e levat ion tn the level of cholesterol , whi ch serves as a precursor for the synthes is of steroid hormo nes in ova ri es~· l o, suggesting thereby that cholesterol was not utili sed23

.

The ovari an dysfunction was ev ident in the increase

Cholesterol G-6-PDH HSD

( ~lg/mg ovary) (Specific ac ti vity) (spec ific act ivity) (U/mg of protein) (U/mg of prote in )

S2.0±3.6 4.0±0. 1 1.0 ±0.04

363 ±I .R 4.1 ±0.26 0.9±0.06 (-30.2) (+ 2.S) (- 10.0)

17SA± 10.6 2.2± 0.O6 0.7 ±O.OS (+ 237 .3) (-4S.0) (-300)

240. 1 ±6.0 1. 8 ±0.03 0.6±0.02 (+361.7) (-SS.O) (- 40.0)

270.2± 12.9 IA±0.02 O.S ±0.02 (+4 19.6) (- 6S.0) (- SO.0)

209.7±9.2 1.6 ±0.04 0.7 ±0.03 (+303 .3) (-60.0) (-30.0)

2S 1.2± 10.7 1.3 ±0.02 O.S ±0.O2 (+383. 1) (-67.S) (- SO.O)

3 14.S± 12.8 1.2± 0.03 OA ± 0.03 (+S04.8) (-70 .0) (-60.0)

in ascorbi c ac id level after treatment with crude ex trac t of C. rejlexa stem and C. olitorius seed24

. To substanti ate these fac ts , the activities of G-6-PDH and HSD, the two key enzymes invo lved in steroidogenesis, were determined25

.26. ME of C. rejlexa and C. olitorius inhibited the activity of both enzymes to a significant extent and thus indi cating ant i­steroidogenic activity of the extracts27

.

Preliminary phytochemical tests indicate the pres­ence of f1avonoids in the ME of C. rejlexa stem 5.6.

Since various f1avonoids have been reported28.29 to

possess antiferti lity activity, the anti-steroidogeni c property of the ME of C. rejlexa ste m may be due to the presence of such compounds.

References Agarwal R R & Dull S, Chemical Exam inat ion of CUSCII /({

re/lexa Roxb. Part \. The constituents, J Indiall Cizem Soc, I ( 193S) 384.

2 Kirtikar K R & Basu B D, In dian medicillal plallls, Vol 8, 3rd ed n, ed ited by K S Mhaskar, E Blaller, J F Caius (Sri Satguru Publications, Delhi ) 2001 , 240 I.

3 Chopra R N, Nayer S L & Chopra 1 C, Glossary of Indian I/I edicin.al plan ts (Publi cations and In fo rmation Directorate, CS IR , New Delhi ) 1992, 8S .

4 Sastri B N, Raw material s, in The Wealth of India, Vol 2 (D irector, CS IR, Delhi ) 19S0, 326, 406.

S Rostogi R P & Mehrotra B N, Compendilll/l of Indian medicinal plants, Vol I(CDR I, Lucknow) loQ3 , 137.

6 Yadav S B, Tripathy V, Si ngh R K ~ Pandey H P. Anti ox idant activity of Cuscu/a reflexa s\ems, Indian J Pharm Sci, 6 (2000) 477.

644 INDIAN J EXP 1310L, J UNE 2003

7 Rao V S N, Dasaradhan P & Kri shnaiah K S , Antifcrti lity e ffcc t o f somc indigcnous pl ants, Indian J Med Res, 70 ( 1979) 517.

8 Challcljec A & Pakrashi C, The Iremise oj' lndian medicinal planls, Vol 2 (Publica tio ns and In formation Direc torate , CS IR, New Delh i) 1992 , 165.

9 C hallc rjec T K, Herbal Opliol/S (Central Book Agency, Kolkata) 1995 , 84.

10 Rang H P. Dale M M & Rit ter J M, Phan/wcologv (Churc hill Livings to ne) 1999, 438.

II Marc us R & Cou lston A M, The vitamins, in Goodlllan & Gilllwlln's The pharlllacological basis of Ih erapeUlics, 9 th cdn , edi ted by A G Gilman , L S Goodman , T W Rail & F Murad (McGraw-Hili Hcalth Pro fess ions Division, New York) 1996, 1569.

12 Litchfi e ld J T, Jr & Wilcoxon F, A s imple method of assess ing dose response rel at ionsh ips, J Plwrlllacol Exp Ther, 96 ( 1949) 99 .

13 Kingsley G R & Roscoe R S, Determination of frec and to tal cholesterol by direct chloroform ex trac ti on, J Biol Chem. 180 ( 1949) 315.

14 Omaye S T , Turnbull J D & Soubcrlich H E, Selected methods fo r the determination of ascorbi c ac id in animal ce ll s. ti ssucs and fluids, in Melhods in enZYlllology. Vol 62. edited by D B McCormic k and L D Wri ght (Academic Press, New York) 1979,3.

IS Rabin B L, Leipsner G & Deane H W , A rap id sensitivc assay proccdu re for adrc na l steroid 3 0 L dehydrogenase activity , Endocrinology, 69 ( 196 1) 619.

16 Lohr G W & Wallc r H D, Glucosc-6-phosphate dehydrogenase, in : Methods (~j' ellZYIIIOlic analysis, Vol 2, editcd by H U Burgmeyer (Vcrlag Chemie, Florida) 1974, 636.

17 Lowry 0 H, Rosenbrough N J. Farr A L & Randal R J, Protein measurement w ith folin phe no l reagent , J Biol Chelll , 193 ( 1951) 265.

18 Gupta M. Bandyopadhya S, Mazu mder S K & Paul B, Ovarian steroidogencs is in rats fo llowing Ochratox in H Treatmc nt. Toxicol Appl Plwl'llwcol , 53 ( 1980) 5 IS .

19 Behrman H R & Armstrong D T, Cholcsterol esterase stimulation by lutei ni zing hormone in lu tein ised rat ovarics, Elldocrinology , 85 (1969) 474 .

20 Wi es t W G , Wil cox R B & Kirschbuam H T, Rat ovari an 20-a lpha-hydroxysteroid-dehydrogcnasc : Effects of es trogen and pituitary gonadotropins, Endocrillology, 73 ( 1963) 588.

2 1 Ramirez V D & Mc Ca nn S M, Fluctuations in plasma lute ini zing hormo nc concentrations during the estrus cycle of the rat , Elldocrinology, 74 (1964) 8 14.

22 Anderson R R & McShan W H, Lute ini zing hormo ne levels in pig, cow and rat blood plasma during estrus cyc le. Endocrillology, 78 (1966) 976 .

23 Tamooki B & Pincus G , Biogenesis of progcs tcrone in ovari an ti ssues, Elldocrinology, 69 ( 196 1) 527 .

24 Guillemin R & Saki z E, Quantitative sllldy of rcsponses to LH after hypophyscctomy in the ovarian ascorbic acid depletion test: Effect of prolactin, Endocrinology, 72 (1963) 8 13.

25 Armstrong D G, 3~-Hydroxy - ~5 steroid dehydrogenase activ ity in the rapidl y growing ovarian follicles of the domestic fowl (Gal/us dOll1esl icus) , J Endocrinol, 93 (1982) 415 .

26 Suzuki S, Endo Y, Tanaka S & Li zuka R, Indirect il1lmunonuorescence studies o n the steroid producing activity of hamster ova, Am JObst GYllecol, 148 (1984) 76.

27 Mazumder P K. Dasgupta S, Mukhopadhya R K, Mazumder U K & Gupta M, Anti -s te riodogenic activity of the pe trolcum ether extrac t and fraction 5 (fa lly ac ids) of carrot (DauCtls carora L) seeds in mouse ovary, J Etlil1opliarlllGcol, 57 ( 1997) 209.

28 Hiremath S R & Rao S H, Antifertility efficacy of the plant Striga IUlefl (scrophulariaceae), Contraception, 42 ( 1990) 467.

29 Khanna U & Choudhu ry R R, Ant iferti lity screening or pl ants, part I. Invcsti gation o f BUlea IllOlIosperllla (Lam) Kutze, Indian J Med Res, 56 ( 1968) 1575.