Tocolytic and Toxic Activity of Papaya Seed Extract on Isolated Rat Uterus j.lfs.2003.06.035

7/27/2019 Tocolytic and Toxic Activity of Papaya Seed Extract on Isolated Rat Uterus j.lfs.2003.06.035

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Tocolytic and toxic activity of papaya seed extract on isolated rat uterus

Adebowale Adebiyi, P. Ganesan Adaikan *, R.N.V. Prasad Department of Obstetrics and Gynaecology, National University of Singapore, National University Hospital,

5 Lower Kent Ridge Road, Singapore 119074, Singapore

Received 8 April 2003; accepted 3 June 2003

Abstract

Carica papaya L. seeds extracted with 80% ethanol (EEPS) caused concentration-dependent tocolysis of uterine strips isolated from gravid and non-gravid rats. Prostaglandin F 2 a and oxytocin-induced contractions of theisolated rat uterus were also inhibited in a concentration-dependent fashion by EEPS. Recoveries of the uterineactivity after EEPS-induced uterine quiescence were very weak. Higher concentration of EEPS caused prompt uterine quiescence, which was also significantly irreversible. Pre-incubation of the rat uterus in Ringer Lockesolution containing 10 mg/ml of EEPS for 1 hour prior to suspension in tissue baths led to significant depression of the spontaneous and KCl (60 mM)-induced uterine contractions relative to the solvent control ( P < 0.05). Crosssections of EEPS-pretreated non-gravid rat uterus (stained with hematoxyline and eosin) examined under light microscope revealed degeneration of the endometrium and myometrium with obvious cytoplasmic vacuolationindicating that EEPS could have direct toxic effect on the uterine tissues. Previous workers have reported benzylisothiocyanate (BITC) as the main bioactive and anthelmintic compound in different extracts of papaya seeds.Using electron impact ionization methods, the presence of BITC in EEPS was also shown in this study. Massspectra of both EEPS and standard BITC showed a base peak of benzyl/tropylium ion at m/z 91 (indicative of anaromatic compound) and the molecular ion peak of BITC (m/z 149). Our earlier studies have demonstrated BITC-induced functional and morphological derangement of isolated uterus. We thus conclude that at high concentration,

EEPS is capable of causing irreversible uterine tocolysis probably due to the damaging effect of BITC (its chief phytochemical) on the myometrium.D 2003 Elsevier Inc. All rights reserved.

Keywords: Papaya seeds; Uterine contractions; Benzyl isothiocyanate; Myometrium

0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2003.06.035

* Corresponding author. Tel.: +65-67724128; fax: +65-68723056. E-mail address: [email protected] (P. Ganesan Adaikan).

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Introduction

The seeds of Carica papaya L. (Caricaceae) have been used for decades in parts of Asia and SouthAmerica as vermifugal agent. In India, the seeds are administered with honey for expelling roundworms(Krishnakumari and Majumder, 1960) . In Panama, powdered papaya s eeds mixed with ho ney are takenorally followed by castor oil (as laxative) to get rid of intestinal worms (Gupta et al., 1979) . Results fromscientific studies showed that papaya seeds extracts are capable of killing worms like Toxascaris transfuga , Ascaris lumbricoides , Pheretima sp . and Caenorhabditis elegans in vitro and also deworm infectedanimals (Krishnakumari and Majumder, 1960; Dar et al., 1965; Lal et al., 1976; Kermanshai et al., 2001) .

Apart from their use as vermifugal agents, papaya seed preparations are reported to have been used infolk medicine to facilitate good menstru al flow and are thought to have abortifacient properties(Quisumbing, 1951; Rao and Jamir, 1982) .

Benzyl isothiocy anate (BITC), a chemopreventive phytochemical found in crucifero us vegetables

(Wattenberg, 1983) has also been shown to be present in different extracts of papaya seeds (Ettlinger andHodgkins, 1956; Tang, 1971; Tang et al., 1972; Sheu and Shyu, 1996; Kermanshai et al., 2001) . BITCexist in these plants as glucosinolate and is released by enzyme myrosinase-induced hydrolysis when thecellular/tissue integrity of the plants is disrupted by chopping during food preparation, seed crushing andother mechanical influences (Tookey et al., 1980) .

Our previous investigations have s hown the ability of BITC to cause functional aberrations of uterineactivity in vitro (Adebiyi et al., 2003) . Using uterine strips isolated from gravid and non-gravid Sprague-Dawley rats, the present study aimed to characterize the potential effect of papaya seed extracts onuterine functions.

Materials and Methods

Preparation of papaya seed extract

Fresh seeds of ripe Carica papaya L. fruits were collected locally, washed in distilled water and driedin an open space. 1.5 kg of the dried seeds was blended into powder with Knife Mill Grindomix GM 200(Retsch GmbH and Co., Dusseldorf, Cologne Germany) and extracted with 80% ethanol untilexhaustion. The mixture was filtered with Whatman No. 1 filter paper (Whatman International Ltd.,England). The filtrate was then concentrated at 40 j C by a rotavapor and the ethanol recovered (BuchiiLabortechnik AG, Switzerland). The resulting concentrate was freeze dried and gave a final yield of

6.2% w/w ethanolic extract of papaya seeds (EEPS) which was kept in the freezer (-10j

C) for subsequent use. EEPS was dissolved in normal saline or dimethyl sulfoxide (DMSO) accordingly.

Chemicals and tissue bathing medium

BITC (98% pure) was purchased from Sigma-Aldrich Chemical Company (St Louis MO, USA),oxytocin from American Pharmaceutical Partners Inc. (Los Angeles CA, USA) and prostaglandin F 2 afrom Upjohn (Kalamazoo MI, USA). Potassium chloride and DMSO were purchased from Merck KGaA; Darmstadt, Germany. Tissue bathing medium: Ringer Locke solution (mM): NaCl 154, KCl5.63, CaCl 2 2.16, MgCl 2 2.10, NaHCO 3 5.95, glucose 5.55.

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Animals

Virgin Sprague-Dawley female (180-220 g) and male (200-250 g) rats were housed in the animalholding unit of the Faculty of Medicine, National University of Singapore. The animals were maintainedunder a constant 12 hours light and dark cycle and an environmental temperature of 21-23 j C. Food andwater were available ad libitum . This study was conducted in accordance with institutional guidelinesand ethics.

Isolated gravid rat uterine tissues

Female rats were paired with males (1:1) until mating was confirmed by the presence of dislodgedvaginal plugs. The day the vaginal plugs were found was designated as day 0 of gestation. On day 17-20of gestation, the rats were euthanized by CO 2 asphyxiation. Myometrial tissues were excised in a

longitudinal direction from the gravid uterine horns and dissected into strips. The uterine strips(approximately 1.5 cm length) were mounted vertically under resting tension of 1 g in 4-channel (25ml) tissue baths connected to force transducers (Ugo Basile, Comerio VA, Italy). The tissue bathingmedium used was Ringer Locke solution maintained at pH of 7.4, temperature of 37 j C and gassed withcarbogen (95% O 2 and 5% CO 2 ).

Isolated non-gravid rat uterine tissues

Non-gravid rats were euthanized by CO 2 asphyxiation. The uterine body was excised above itsintersection with the cervix and at the junction of the uterine horns with the ovaries and then dissectedfrom the adherent fat and connective tissues. The two uterine horns were immediately dissected into four strips. Uterine strips (approximately 1.5 cm each) were mounted vertically under resting tension of 2 g in4-channel (25 ml) tissue baths connected to force transducers. The tissue-bathing medium used wasRinger Locke solution maintained at pH of 7.4, temperature of 37 j C and gassed with carbogen.

Concentration-response effect of EEPS on uterine contractions

The uterine strips were allowed to equilibrate in the bathing medium for at about 1 hour and thespontaneous rhythmic contractions were observed. Following the equilibration period, cumulativeconcentration-response effect of EEPS (0.05-3.2 mg/ml) on the spontaneous contractions of the uteruswas recorded by successive increase in EEPS concentration in the tissue baths with a contact time of 10

minutes for each concentration. After the final concentration of EEPS, the tissues were washed at least 4times and observed for recovery. The effect of a single but higher concentration of EEPS on the uteruswas also observed by adding 10 mg/ml of EEPS into the tissue baths and allowing it to act for 1 hour after which the tissues were washed 4 times and allowed to rest for at least 30 minutes. The viability of the uterine strips was later assessed by stimulating them with 60 mM of KCl.

In another set of experiments, strips from non-gravid rat uterus were pre-contracted with 5 Ag/ml of prostaglandin F 2 a (PGF 2 a ) and 10 mU/ml of oxytocin for 10 minutes after which cumulativeconcentration-response effect of EEPS (0.05-3.2 mg/ml) on the drug-induced contractions of the uteruswas observed by successive increase in EEPS concentration in the tissue baths with a contact time of 10minutes for each concentration. After the final concentration of EEPS, the same concentration of PGF 2 a

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and oxytocin were added to the tissue baths. The tissues were subsequently washed and observed for recovery. The control uterine strips in these experiments were treated with corresponding volume of normal saline or DMSO (50% concentration) used in the preparation of EEPS solution.

Effect of EEPS pre-treatment on uterine morphology and function

Uterine strips from the whole uterus of non-gravid rats were incubated in 10 ml of Ringer Lockesolution containing 10 mg/ml of EEPS. The controls were incubated in 10 ml of Ringer Locke solutioncontaining equivalent volume of DMSO (50% concentration) used for the preparation of EEPS. Theincubation was carried out at 37 j C, gassed with carbogen and shaken (400 RPM) for 1 hour in HeidolphUnimax 1010 rotary shaker (Heidolph Instruments GmbH and Co., Schwabach Germany). After 1 hour of incubation, the uterine strips were suspended in 4-channel (25 ml) tissue baths containing Ringer Lockesolution and washed at least 4 times. Spontaneous contractions of the uterine strips were monitored for 1

hour after which they were stimulated with 60 mM of KCl. After the recordings of the spontaneous andKCl induced-contractions, the uterine strips were removed from the tissue baths, and quickly frozen inJung tissue freezing medium (Leica Instruments, Nussloch Germany) using liquid nitrogen. 5 Am-thick cross-sections of the uterus were cut in Leica CM3050 S cryostat (Leica Microsystems AG, Wetzlar Germany) at -20 j C and mounted on polysine microscopic slides (Erie Scientific Co., Portsmouth NH,USA). The sections were stained in Harris hematoxyline and eosin following routine staining procedure.Sections were observed under light microscope for histological changes in the uterus.

Mass Spectrometry

The mass spectra analyses were done at the Department of Chemistry, National University of Singapore. Mass spectra analyses of EEPS and BITC (98% pure) by electron-impact ionization werecarried out on VG Micromass 7035 high-resolution double focusing mass spectrometer (Micromass UK Ltd, Manchester UK) equipped with Maspec Data System (Mass Spectrometry Services Ltd, Manchester UK) . The source temperature and ion current used was 200 j C and 70 eV respectively. Mass spectradata were reported in units of mass to charge (m/z) and percentage relative abundance.

Data analysis

Responses of the isolated uterus to the test substances were isometrically recorded on Mac Lab dataacquisition system running chart v3.6 software (AD Instruments, Castle Hill, New South Wales Australia).

The force of uterine contractions was represented as the maximum tension (g) above the baseline duringthe period of contact with the test substances. The results were expressed as the mean F SEM. Wherenecessary, students t test was applied and a P value of 0.05 or less was considered significant.

Results

Isolated uterus of gravid and non-gravid rats suspended in Ringer Locke solution exhibited regular spontaneous rhythmic contractions within 1 hour of the equilibration period. In contrast to the solvent control, EEPS (0.05-3.2 mg/ml) caused concentration-dependent inhibition of the spontaneous contrac-



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tions of the uterus (Fig. 1) . Following the rest period of at least 30 minutes, after washing off EEPS, theforce of spontaneous contractions of the uterus was significantly lesser ( P < 0.01) than the initial activity(Table 1) .

Table 1The force of contractions before and after EEPS-induced uterine tocolysis 1

Initial force of contractions (g tension)

Force of contractions (g)after EEP-induced quiescence

Spontaneous contractions of non-gravid rats 2.57 F 0.53 *0.74 F 0.46Spontaneous contractions of gravid rats 3.82 F 0.20 *1.80 F 0.32PGF 2 a -induced contractions of non-gravid rats 2.46 F 0.33 *0.32 F 0.16Oxytocin-induced contractions of non-gravid rats 2.33 F 0.53 *0.45 F 0.15

1 Following the rest period of at least 30 minutes, after washing off EEPS, the uterine strips were observed for the recoveryof the spontaneous, PGF 2 a and oxytocin-induced contractions. ( n = 6; * P V 0.01).

Fig. 1. Concentration-response effect of EEPS on uterine strips isolated from: (A) non-gravid and (B) gravid rats compared toequivalent volume of the solvent control (saline). Each bar represents mean F SEM (n = 6; * P


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10 mg/ml of EEPS caused prompt quiescence of the isolated uterus (Fig. 2) . Subsequent to the rest time (minimum of 30 minutes) after washing off EEPS, 60 mM of KCl failed to contract the non-graviduterine strips. The response of the strips from gravid rat uterus to 60 mM of KCl after EEPS-induceduterine quiescence was however 22 F 9% relative to the control.

PGF 2 a (5 Ag/ml) and oxytocin (10 mU/ml)-induced contractions of the rat uterus showed regular tonic and phasic contractile patterns. In contrast to the solvent control, cumulative concentration of EEPS (0.05 3.2 mg/ml) also caused concentr ation-dependent inhibition of PGF 2 a and oxytocin-induced contractions of the rat uterus (Fig. 3). Addition of the same concentration of PGF 2 a andoxytocin did not reverse the tocolytic effect of EEPS. Following the rest period of at least 30minutes (after washing off EEPS), the force of PGF 2 a and oxytocin-induced contractions of theuterus wa s significantly lesser ( P V 0.01) than the initial PGF 2 a and oxytocin-induced contractions(Table 1) .

Pre-incubation of the rat uterus in Ringer Locke solution containing 10 mg/ml of EEPS before

suspension in tissue baths led to pronounced depression of the force of spo ntaneous and KCl (60mM)-induced non-gravid rat uterine contractions relative to the control (Fig. 4). Spontaneouscontractions were totally abolished in most of the EEPS-pretreated uterine ti ssues. 60 mM of KClalso failed to contract quiescent uterus caused by EEPS pre-treatment (Fig. 5). Under light microscope, the uterus of non-gravid rats pretreated with DMSO (solvent control) showed normalmyometrium (comprising of dense bundles of smooth muscle cells) and simple endometrial epithelial

Fig. 2. Sample tracings showing the effects of DMSO (solvent control) on uterine strips isolated from (A) non-gravid and (B)gravid rats and the prompt tocolytic effects of EEPS (10 mg/ml) on uterine strips isolated from (C) non-gravid and (D) gravidrats.



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Fig. 4. Chart illustrating the effect of EEPS (10 mg/ml) pre-treatment on the spontaneous and KCl (60 mM)-inducedcontractions of uterine strips isolated from non-gravid rats compared to corresponding volume of the solvent control (DMSO).Each bar represents mean F SEM (n = 6; * P


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cells. However, EEPS-treated uterus revealed structural degenerations. The cy toplasm of the EEPS-

treated myometrium and endometrium showed marked vacuolation (Fig. 6). The electron impact spectra of both standard BITC and EEPS produced a base peak of tropylium/benzyl ion at m/z 91.

Fig. 6. Photomicrographs (magnification: 100 ) of sections of non-gravid rat uterus treated with: (A) DMSO (solvent control)and (B) 10 mg/ml of EEPS for 1 hour. The control and treated uterine strips were taken from the same rat and the samples were processed as described in the methods. E: endometrium; M: myometrium.

Fig. 5. Sample tracings showing effect of: (A), DMSO (solvent control) and (B), EEPS (10 mg/ml) pre-treatment on thespontaneous and KCl (60 mM)-induced contractions of uterine strips isolated from non-gravid rats.



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The next significant ion in the spectrum of both BITC and EEPS was the molecular ion peak of BITC (m/z 149). Other assortments of minor peaks were also present in both spectra (Fig. 7) .

Discussion

Under suitable physiological conditions, isolated mammalian uteri are capable of exhibitingspontaneous rhythmic contractions and are able to respond to oxytocic agents (e.g. oxytocin, ergome-trine and prostaglandin F 2 a ) in a similar fashion to their in vivo activity.

EEPS (0.053.2 mg/ml) caused concentration-dependent tocolysis of the uterus. Recoveries of theuterine activity after EEPS-induced quiescence were very weak. In addition, a single but higher

concentration EEPS (10 mg/ml) caused prompt quiescence of the isolated uterus, which was alsosignificantly irreversible. Pre-incubation of isolated rat uterus in 10 mg/ml of EEPS prior to recordingsof the spontaneous contractions of the uterine strips in tissue baths led to depression of the uterineactivity, despite washing the tissues many times. In most of the uterine tissues, spontaneous contractionswere totally abolished. Failure of 60 mM of KCl to reverse EEPS-induced uterine quiescence indicatesthat the tissues were less than viable. EEPS-induced uterine tocolysis could therefore be a result of concentration-dependent damage of the uterine tissues weakening the contractile capability of themyometrium. The cytoplasm of the myometrium consists of dense longitudinally aligned contractilefilaments containing the contractile proteins, actin and myosin (Romanini, 1994) . Morphologicalintegrity of the myometrium is thus essential for normal uterine contractions. As observed in the

Fig. 7. Representative electron impact ionization mass spectra of: (A) BITC and (B) EEPS.



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present study, sections of EEPS-treated uterus examined under light microscope showed structuraldisintegrity characterized by obvious cytoplasmic vacuolation of the myometrium and endometrium incontrast to the solvent control. Hence, EEPS-induced myometrial degeneration could be responsible for the tocolytic effect of EEPS.

BITC occurs naturally as glucosinolate, a secondary plant product of the cruciferous vegetable s. It isalso found in plants like Lepidium meyenii W, Tropaelum majus L and Lepidium sativum L. (Kjaer,1960; Gil and Macleod, 1980; P iacente et al., 2002) . One of the earliest reports on the presence of BITCin papaya seeds was made by Ettlinger and Hodgkins (1956) . Papaya seeds macerated in wa ter andorganic solvent yielded BITC (Ettlinger and Hodgkins, 1956) . Indeed, further investigations by Gmelinand Kjaer (1970) indicated that like Carica papaya L, other species of the Caricaceae family showed theoccurrence of benzyl glucosinolate (the parent compound of BITC). Recent report also suggests that BITC and not cyanogenic glucoside (which is present in minute quantity in papaya seed ex tracts) is themajor bioactive compound in papaya seeds and is responsible for its anthelmintic activity (Kermanshai

et al., 2001) . BITC is an aromatic compound with a molecular weight of 149. In the present study, theelectron impact spectra of both standard BITC and EEPS produced a base peak of benzyl/tropylium ionat m/z 91 which is indica tive of an aromatic compound with an alkyl side-chain attached to the ring(Bongiorno et al., 2000) . The molecular ion peak of BITC (m/z 149) was found to be the other significant ion present in the spectra of EEPS. Other minor assortments of peaks in both spectra couldhave arisen from complex ion fragmentation. The results of mass spectra analysis of EEPS in the present investigation supports earlier, well-established reports on the occurrence of BITC as the chief phytochemical in different extracts of papaya seeds.

BITC is capable of causing morphological and functional aberrations of certain cells/tissues in vitroand in vivo. For examples, BITC caused necrosis and cytomorphological cha nges characterized bysurface blebbing and swelling of vesicular structures of rat hepatocytes in vitro (Temmink et al., 1986;Bruggema n et al., 1986) . 20100 AM of BITC caused cell death via modification of mitochondrialfunctions (Nakamura et al., 2002) . Oral administration of BITC was also shown to induce significant histological changes in the ductus choledochus, liver and mesenteric lymph nodes of male rats(Lewerenz et al., 1992) . Pentane extract of papaya seeds and BITC damage d and caused irrever sibleinhibition of dog carotid arteries precontracted with phenylephrine and KCl (Wilson et al., 2002) . Our earlier studies showed that BITC is able to cause irreversible and concentrationdependent alteration of the spontaneous and druginduced uterine activity (Adebiyi et al., 2003) . Similar degenerative changesobserved in EEPStreated uterus were also seen in uterine tissues pretreated with BITC (Adebiyi et al.,2003) . Sample tracings demonstrating the irreversible inhibitory effect of BITC (100 AM) on the force of spontaneous uterine contractions is shown in (Fig. 8). It thus appears that the irreversible uterine

tocolysis caused by EEPS could due to BITCinduced myometrial damage. Should papaya seeds alter uterine activity in vivo, normal/druginduced uterine functions could be impaired.Furthermore, male albino rats given 100 mg/kg of crude papaya seeds (orally) showed degeneration

of the germinal epithelium, Leydig cells and the presence of vacuoles in the tubules (Udoh and Kehinde,1999) . Adult male rats pre-treated with papaya seeds extract exhibited significant decrease in thecontractions of their epididymal tubules in contrast to the control (Verma and Chinoy, 2002) . Fractions of papaya seed extracts also caused deleterious changes in the plasma membrane of the head and mid-pieceof human sperms leading to total inhibition of in vitro motility within 2025 minutes (Lohiya et al.,2000) . These effects may be due to the degenerative activity of BITC. The present and earlier studies byus and other workers thus suggest that the extracts of papaya seeds are capable of causing functional



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aberrations of different mammalian tissues/organs and systems probably due to the toxic effects of BITC,its chief phytochemical.

In Ayurverdic system of traditional medicine, 0.51 g of powdered papaya seeds is prescribed for medicinal use (Kapoor, 1990) . In Cuba, up to 4.5 g of papaya seeds are recommended per day asvermifugal agent (Roig y Mesa, 1974) . The concentration of BITC was shown to be as high as 2000 5000 ppm in macerated dry papaya seed preparations (Ettlinger and Hodgkins, 1956) and this isdependent on the environment/origin and maturity of the papaya fruits from which the seeds wereharvested (Tang, 1971; Tang et al., 1972) . The exact amount of BITC delivered to human systems via papaya seed consumption could therefore vary and not known. Like any other traditional medicine preparation, appraisal of the safety of papaya seed preparations is indeed necessary to protect the populace from the potential hazards of its use.

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Fig. 8. Sample tracings showing the effects of DMSO (solvent control) and 100 AM of BITC on the spontaneous contractions of uterine strips isolated from non-gravid rats. Strips from non-gravid rats were equilibrated in Ringer-Locke solution for 1 hour before the addition of BITC (100 AM) or equivalent volume of the solvent control (DMSO; 10% concentration) into the tissue baths. The tissues were washed and observed for recovery after 1 hour of rest.



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Tocolytic and Toxic Activity of Papaya Seed Extract on Isolated Rat Uterus j.lfs.2003.06.035

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