sperm immobilization effect of Carica papaya seed extracts: an in vitro
K Lohiya, Lalit K Kothari1, B Manivannan, Pradyumna K Mishra,
Physiology Section, Department of Zoology, University of Rajasthan, Jaipur
- 302 004, India
Asian J Androl 2000 Jun; 2: 103-109
AbstractAim: To examine if the seed extracts of Carica papaya, which showed antispermatogenic/sperm immobilization properties in animal models, could cause human sperm immobilization in vitro. Methods: Chloroform extract, benzene chromatographic fraction of the chloroform extract, its methanol and ethyl acetate sub-fractions and the isolated compounds from the sub-fractions i.e., ECP 1 & 2 and MCP 1 & 2, of the seeds of Carica papaya were used at concentrations of 0.1%, 0.5%, 1% and 2%. Sperm motility was assessed immediately after addition of extracts and every 5 minutes thereafter for 30 minutes. Results: There were dose-dependent spermicidal effects showing an instant fall in the sperm motility to less than 20% at 2% concentration. Isolated compounds ECP 1 & 2 were more effective inducing a motility of less than 10%. Many of the spermatozoa became vibratory on the spot. Total inhibition of motility was observed within 20-25 min at all concentrations of all products. Scanning and transmission electron microscopy revealed deleterious changes in the plasma membrane of the head and mid-piece of spermatozoa. Sperm viability test and the number of abnormal spermatozoa after completion of incubation suggested that the spermatozoa were infertile. The effects were spermicidal but not spermiostatic as revealed by the sperm revival test. Conclusion: The results reveal spermicidal activity in vitro of the seed extracts of Carica papaya.
1 IntroductionSeveral plants which induce male antifertility, also possess spermicidal properties. Setty et al reported that out of 160 plant extracts tested for in vitro spermicidal activity at 2% concentration in rats and human spermatozoa, 30 plants showed spermicidal properties and 16 of them caused an instant human sperm immobilization. We have found earlier that out of various extracts of the seeds of Carica papaya tested for contraceptive efficacy, the chloroform extract and the partially purified benzene chromatographic fraction of the chloroform extract produced sperm motility inhibition in rats[3,4] and azoospermia in rabbits[5,6]. Methanol and ethyl acetate sub-fractions of the benzene chromatographic fraction also had similar effects in rats and rabbits and a sperm motility inhibitory action in langur monkeys (unpublished observations, Lohiya et al). The effects were systemic, resulting from oral administration of the seed extract/products and were free of toxicity. The chloroform extract of the seeds of Carica papaya has also been shown to possess in vitro spermicidal properties in rat, rabbit, and monkey spermatozoa. In the present study, the Carica papaya seed extracts which showed contraceptive efficacy in animal models were examined for their sperm immobilization effects in vitro, in human spermatozoa with a view to their potential as vaginal spermicidal contraceptives.
2 Materials and methods
seeds of Carica papaya (Caricaceae,Voucher No. RUBL 16590) of honey
dew variety, were examined using the extraction procedures described earlier.
Briegly, the seeds of Carica papaya were shade-dried, powdered
and soxhalated in chloroform at 58 for 123 hours. The soxhalated material
was concentrated under reduced pressure and further purified by silica
gel column chromatography using benzene, chloroform and ethyl acetate
as eluents. Following partial purification,
the benzene chromatographic fraction, which showed significant effect
on semen parameters[4,6], was subjected to methanol and ethyl
acetate sub-fractionation and the residues after evaporation of the solvents
were used for further purification by Thin Layer Chromatography (TLC).
Two compounds were obtained from each sub-fraction on TLC viz., MCP 1
& 2 and ECP 1 & 2, respectively. The compounds were scraped, extracted
with the corresponding solvents, evaporated to dryness under reduced pressure
and the residues were separated. The chloroform extract, the benzene chromatographic
fraction of the chloroform
acetate sub-fractions and the isolated compounds MCP 1 & 2
samples (n=15) from normal subjects after 48 hours of sexual abstinence
were subjected to routine semen analysis following liquefaction
at 37. Sperm counts above 50 million/mL with normal morphology, rapid
and linear and
progressive motility, and viability above 50% was considered for the in
vitro test. WHO's strict evaluation criteria were followed for assessing
sperm morphology. Spermatozoa free of seminal plasma were
obtained by centrifugation, washed once in BWW media and adjusted to the
final concentration of 50 million/mL.
to experiment, semen samples and the seed
were prewarmed to 37; 250 L semen sample containing 15 million
spermatozoa was added to 250 L of the extract preparation at various
concentrations. Sperm motility (a, b, c & d grades),
was assessed immediately after addition of the semen samples and thereafter
every 5 min for up to 30 min in a phase contrast microscope along with
placebo control. Following completion of the experiment, sperm viability
and morphological assessments were carried out by nigrosin-eosin and papanicolaou
staining methods, respectively.
completion of motility assessment, the spermatozoa were separated by centrifugation,
washed with phosphate buffer (0.1 mol/L; pH 7.2) and pelleted by centrifugation.
The pellets were immediately fixed in 2.5% glutaraldehyde in phosphate
buffer for 30 min and washed thrice in phosphate buffer. For scanning
electron microscopy (SEM), a thin film of spermatozoa was smeared on an
SEM stub with silver
paint, air dried, sputter coated with gold and observed under the scanning
electron microscope (Leo SEM 435 VP). For transmission electron microscopy
(TEM), the spermatozoa pellet was post-fixed in OsO4 for 30
min, washed in phosphate buffer, followed by distilled water, dehydrated
in acetone, embedded in low viscosity spur medium and polymerized at 60
for 48 h. The ultrathin sections were stained with uranyl acetate and
lead citrate and observed under the transmission electron microscope (Philips
Sperm revival test
completion of the experiment, the spermatozoa were washed twice with BWW
medium and incubated once again in the same medium free of extract/products
at 37 for 30 min to observe revival of sperm
motility, if any. Immotile spermatozoa showing vibratory movement
to progressive motility after incubation were considered revived.
motility from its initial level of 55.07%3.30% dropped immediately following
the addition of the chloroform extract, benzene chromatographic fraction
and its sub-fractions as well as the isolated compounds, ranged between
31.2%1.5%. The effects were dose-dependent and more pronounced effects
were observed with the compounds isolated from the ethyl acetate sub-fractions
(ECP 1 & 2). Although many of the spermatozoa showed only vibratory
movement after 5 min of addition of the drugs, particularly at 2% concentration,
total inhibition of motility was observed after 10 min in the chloroform
extract, benzene chromatographic
fraction of the chloroform extract and ECP 1 & 2 treated samples.
None of the spermatozoa showed motility, 25 min after addition of the
extract/fractions/compounds (Tables 1 & 2).
viability of spermatozoa, following completion of the experiment, fell significantly
(P<0.001; Range 22.8%0.6% to 45.4%1.2% as against 54.9%1.2%
in control) and the effects were more pronounced in the ECP 1 & 2
treated samples at 2%
concentration. Sperm abnormality showed a significant increase in its
pattern (P<0.001; Range 26.7%1.1% to 46.9%1.3% as against
control samples). Most abnormalities were found in MCP 1 &
2 treated samples at 2% concentration (Table 3).
1.Human sperm motility (%) following incubation with chloroform extract,
bezene chromatographic fraction and its sub-fractions of the chloroform
extract of the seeds of Carica papaya in vitro (meanSEM of
values are statistically significant to control (P<0.001)
2. Human sperm motility (%) following incubation with the partially purified
compounds of the sub-fractions of the bezene chromotographic fraction
of the seeds of Carica papaya in vitro (meanSEM of 15 samples)*.
values are statistically significant to control (P<0.001)
3. Sperm viability and abnormalities following incubation with chloroform
extract, bezene chromatographic fraction, its sub-fractions and isolated
compounds of the chloroform extracts of the seeds of Carica papaya
in vitro (meanSEM of 15 samples)*.
values are statistically significant to control (P<0.001).
Ultrastructure of Spermatozoa
Scanning Electron Microscopy (SEM)
SEM of normal human spermatozoa showed an oval head with distinct acrosomal
and post-acrosomal regions and well defined outer plasma membrane. The
mid-piece was long, slender, encircled with a distinct concentric mitochodrial
sheath. The tail region separated
from the mid-piece by a distinct groove, annulus (Figure
1). After completion of treatment in vitro with extract/fraction/compounds
of the seeds of Carica papaya at 2% concentration, the spermatozoa
depicted typical changes particularly at the level of mid-piece and acrosome.
Bent mid-piece was the
common observation. Membrane damage in the acrosome was also evident resulting
in acrosome ballooning (Figure 2).
1. SEM of the human spermatozoa from the placebo control samples.
The sperm appears with intact plasma membrane. 7,600.
Transmission Electron Microscopy (TEM)
human spermatozoa, prior to treatment, under TEM showed a triangular head
with distinct acrosomal envelope. The nucleus contained condensed chromatin
material. The neck region was short containing segmental
the head and mid-piece. The mid-piece contained well defined mitochondrial
sheath surrounding the axoneme (Figure
3. TEM of normal spermatozoa from control sample, showing intact acrosome
and mid-piece. 5,600.
completion of the treatment in vitro the head of the spermatozoa showed
deleterious changes. The typical observation was severe membrane damage
in the acrosome resulting in leach out of the acrosomal contents. The
nucleus appeared vacuolated and the mid-piece configuration was severely
damaged (Figure 4). The
morphological changes of the spermatozoa were uniform in all the extract/products
treated samples at 2% concentration.
4. TEM of the human spermatozoa following in vitro treatment (30 min
at 2%) with compound ECP 1 of Carica papaya showing membrane damage
in the mid-piece and acrosome. 7,600.
Sperm Revival Test
mechanism of action of many of the spermicidal compounds of plant origin
seems to be by surface action, disrupting the plasma membrane of the spermatozoa.
The currently used vaginal spermicide, nonoxynol-9 acts in a similar manner.
It produces disruption of lipids within the sperm membrane, particularly
on the acrosome and mid-piece, causing disruption and rapid loss of sperm
motility[9,10]. Praneem, a polyherbal cream
containing purified neem seed extract,
quinine hydrochloride and the reetha saponins extracted from the
pericarp of Sapindus mukorossi, at 10% concentration has high potency
the mucus and inhibit the penetration
of sperm. Inhibition of the sperm specific enzymes acrosin
and hyaluronidase, which play
an important role in the fertilization process
by plant derivatives, has also been reported. It
is believed that the flavonoids and their derivatives, flavonones and
flavonols, contain hyaluronidase inhibitory activity.
and Waller have screened a large number of plants for spermicidal
and reported that a majority of plant-derived spermicides are triterpene
saponins of several structural types, flavonoids and phenol compounds.
The saponins of Cyclomen persicum, Primula vulgaris and
Gypsophyla paniculata have been reported
to produce instant immobilization of human spermatozoa within 20
seconds. Acacic acid from the bark of Acacia concinna,
oleanolic acid and porceric acid from the roots of Albizza procera
and anagalligenone from the whole plant Anagallis arvensis also
produce instant sperm immobilization within one minute in human spermatozoa.
An instant spermicidal activity of pittoside A
& B, the sapogenins isolated from the plant Pittosporum nilghirense
the present study the chloroform extract, the benzene chromatographic
fraction of the chloroform extract and its methanol and ethyl acetate
sub-fractions and the isolated compounds,
ECP 1 & 2 and MCP 1 & 2, which exerted their systemic action
on the sperm parameters in our earlier studies, have also
shown a sperm immobilizing effect on human spermatozoa in vitro.
The effect is spermicidal and not spermiostatic as there is no revival
of motility after incubation with BWW medium free extracts. The isolated
compounds ECP 1 & 2 were found to be more
effective, lowering the motility to less than 10%, immediately after addition
of the compound at 2% concentration. SEM and TEM of spermatozoa after
completion of incubation
showed membrane damage in the head as well as mid-piece suggesting
that the mode of action appears similar to
of nonoxynol-9[9,10] and most other herbal spermicidal
agents. Although not specific, the membrane damage in the
sperm head could cause acrosin and hyaluronidase inhibition as well, as
reported for other herbal spermicides.
sperm immobilizing effect was dose-dependent. However, instant immobilization
of spermatozoa has not been observed in the present study, although
the compounds ECP 1 & 2 drastically affected the sperm motility
to less than 10% and most of
them showed slow progressive to
vibratory movement that may imply infertility in fertility trials.
A still higher concentration may induce instant immobilization as in CONSAP,
a herbal cream formulation
containing saponins of fruit pericarp of Sapindus mukorossi
where 2.5% concentration has been tested in woman volunteers.
possibly useful approach may be a combination of Carica papaya
products with other spermicides as in praneem, where the combination of
purified neem seed
extract, reetha saponins and quinine HCl dispensed in a water washable
cream base has been tried, for a better applicability.
Such a combination approach should, in addition to the
instant sperm immobilization action, contain an antimicrobial action
too, as in praneem which reportedly contains
antimicrobial action against the vaginal pathogens Chlamydia
trachomatis, Candida albicans and Gardeneralla vaginalis
tested in vitro in phase I clinical trial. Importantly,
extracts of Carica papaya are reported to possess antibacterial,
antiviral and antiyeast activities[16,17]. These extracts were
particularly sensitive to Candida albicans, tobacco mosaic virus,
ring spot virus and a number
of bacteria which includes Salmonella, Staphylococcus and Pseudomonas
species. This would be an added advantage for any Carica
papaya-based vaginal contraceptive.
These extracts were particularly advantageous over nonoxynol-9,
that it does not cause inflammatory reaction and irritation of vaginal
repeated use (unpublished observations, Lohiya et al). Further
in progress with a combination approach and its possible antimicrobial
action for a better applicability.
Farnsworth NR, Waller DP. Current status of plant products reported to
inhibit sperm. In: Zatuchni GI, editor. Research frontiers in fertility
regulation 1982; 2: 1-16.
NK Lohiya, Director, School of Life Sciences, Department of
Zoology, University of Rajasthan, Jaipur-302 004, India.