Author’s Accepted Manuscript

Investigation of uterotonic properties of Ananas

comosus extracts

Faezeh Monji, P Ganesan Adaikan, Lang Chu Lau,

Baharudin Bin Said, Yinhan Gong, Huey Min Tan,
Mahesh Choolani
www.elsevier.com/locate/jep

PII: S0378-8741(16)30465-2
DOI: http://dx.doi.org/10.1016/j.jep.2016.07.041
Reference: JEP10306
To appear in: Journal of Ethnopharmacology
Received date: 23 December 2015
Revised date: 27 June 2016
Accepted date: 14 July 2016
Cite this article as: Faezeh Monji, P Ganesan Adaikan, Lang Chu Lau, Baharudin
Bin Said, Yinhan Gong, Huey Min Tan and Mahesh Choolani, Investigation of
uterotonic properties of Ananas comosus extracts, Journal of
Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.07.041
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Investigation of uterotonic properties of Ananas comosus extracts

Faezeh Monji, P Ganesan Adaikan*, Lang Chu Lau, Baharudin Bin Said, Yinhan Gong, Huey
Min Tan, Mahesh Choolani

Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National
University of Singapore, NUHS Tower Block, Level 12, 1E Kent Ridge Road 119228,
Singapore

* Corresponding author at Department of Obstetrics and Gynaecology, Yong Loo Lin School
of Medicine, National University of Singapore, NUHS Tower Block, Level 12, 1E Kent
Ridge Road 119228, Singapore
Tel: +6566015453
Fax: +6567794753
Email address: p_ganesan_adaikan@nuhs.edu.sg

Abstract
Ethnopharmacological relevance: In folklore medicine Ananas comosus (pineapple) is
reputed to act as an abortifacient and in expectant women as a means of inducing labor.
Several reports have claimed abortifacient property of A. comosus fruit (ripe or unripe). Ripe
fruit has been used orally as traditional medicine in inducing abortion in Kerala state of India
while the juice of unripe fruit was used for abortion in Bangladesh. However, scientific
evidence supporting the efficacy of pineapple extracts in inducing uterine contractions is
clearly lacking.

Aim of the study: This study investigated the pharmacological effects of different fractions of
pineapple extract with a range of maturities to identify the most potent uterotonic fraction.

Materials and Methods: The ethanolic crude extracts of pineapple (edible part) were
prepared and fractionated through a series of liquid-liquid partitions. Fractions were
separately tested on isolated uterine muscle from pregnant SD rats and human pregnant
myometrium, which were cut into strips along the longitudinal axis of uterus. The strips were
 mounted vertically in organ baths (37oC) and exposed to cumulative addition of fractions
(0.1-10mg.ml-1), serotonin (0.05-5µM) and different inhibitors to delineate the mechanism of
action of the active ingredients of the extract.
Results: Aqueous fraction (F4) possesses uterine stimulant property which was blocked by
verapamil but unaffected by indomethacin, prazosin and atosiban. Notably, ketanserin
(10µM) diminished the maximal contractile response induced by both F4 and 5HT by 74.3%
and 92.1% respectively.
Conclusions: These results may indicate the presence of 5HT or 5HT-like compound(s) and
serotonergic pathways may contribute to the uterotonic activity of pineapple extract.
Key words: Ananas comosus; obstetrics; abortion; uterus; in vitro

1. Introduction

Natural products including medicinal plants, remained an essential source of new drugs and new
drug leads (Butler, 2004; Newman et al., 2003). Historically this category of medicines is one of
the bestselling drugs in allopathy (Balunas and Kinghorn, 2005).

Ananas comosus, popularly known as pineapple is a plant of bromeliaceae family native to tropic
and sub-tropic countries (Lim, 2012). In folklore medicine, Ananas comosus is reputed to act as
an abortifacient as a means of inducing labor to avoid medical intervention of post-dates
pregnancy

Several reports and studies have claimed abortifacient property of Ananas comosus fruit (ripe or
unripe) in many parts of the world including India (Yabesh et al., 2014), Bangladesh (Rahman,
2014) and Malaya (Maurya et al., 2004). In India, the juices of the unripe fruits and leaves of
Ananas comosus have long claimed to have abortifacient property (Manjunath, 1948).

Abortifacient effect appears to be exerted at the implantation site when juices from unripe
pineapple fruit was evaluated in rats (Garg et al., 1970). Extraction of air-dried whole leaves of
pineapple yielded 4 steroidal fractions (consisting of ergosterol peroxide, 5 stigmastene-3β, 7α-
diol, β-sitosterol, campesterol, stigmastanol and campestanol) and 2 minor aliphatic esters
(Pakrashi et al., 1975). Subsequent investigation confirmed that the sterols derived from Ananas
comosus leaves possessed abortifacient effect in mice by decreasing the number of implantation
site (Pakrashi and Basak, 1976). However, recent study in Nigeria could not replicate the
abortifacient efficacy of unripe pineapple juice in Wistar rat model (Yakubu et al., 2011). The
fruit juice of ripe pineapple also shows the ability to contract uterus of non-pregnant rat in vitro
(Nwankudu et al., 2014).

In zebrafish model, the ethanol and chloroform extract of A. comosus at different post
fertilization periods decreased in survival of the embryo specifically in the blastula stage
(Abhinava et al., 2014). These studies focused mainly on anti-implantation and not on the ability
to contract uterus as the mechanism of action.

The folk botanical literature reveals that pineapple is also used traditionally for women's
healthcare such as treating uterine fibroids and menorrhagia in Dominican and Chinese healing
systems (Ososki et al., 2002), facilitating childbirth and regulating postpartum bleeding in
Western Uganda (Kamatenesi‐Mugisha and Oryem‐Origa, 2007), stopping uterine hemorrhage
and vaginal infection in eastern Nicaragua (Coe, 2008) and also treating uterine cancer in
Singapore (Siew et al., 2014).

Phytochemical screening has identified diverse antioxidants such as polyphenols, vitamin C and
β-carotene in pineapple fruit (Kongsuwan et al., 2009). Analysis of the phenolic composition
revealed the presence of tyrosine, dimethyl hydroxyl furanone, glutathione, p-coumaric acid,
syringic aldehyde, ferulic acid, caffeic acid and sinapic acid (Fernandez de Simon et al., 1992;
Wen and Wrolstad, 2002). 5-Hydroxytryptamine (5HT) or serotonin as a phenolic bioactive
compound also had been reported to be present in pineapple fruit (Bruce, 1960; Feldman and
Lee, 1985; Wen and Wrolstad, 2002). Previous studies have documented the role of the serotonin
in uterine contractility in vitro in rat (Minosyan et al., 2007) and human myometrium (Cordeaux
et al., 2009). These studies suggested that the 5-HT2A receptor may be potential therapeutic target
receptors using different selective agonists and antagonists; however, the other subtypes of 5-HT
receptors in uterine muscle are not well characterized.

The other compounds known to be present in A. comosus are bromelain (Hale et al., 2005;
Taussig and Batkin, 1988) and melatonin (Hattori et al., 1995; Sae‐Teaw et al., 2013). In human
and rat uteri, stimulation of protease activated receptors (PAR-1 and PAR-2) increase uterine
contractility by mediating prostaglandin E2 (Maul et al., 2003). Melatonin regulates circadian
rhythms; it is expressed by pregnant human myometrium. In the myometrium, melatonin may be
involved in the endocrine control of parturition (Bosc, 1987; Maurya and Singh, 2010).
In view of the wide range of biological effects of abovementioned compounds and also the
potential of other unknown compounds of the pineapple on uterine activity, this research is
expected to provide new and important leads against threatened abortion and postpartum
hemorrhage. This notion stems from the fact that there is lack of potent, selective and non-toxic
drug to manage abnormalities in uterine muscle during pregnancy and parturition. Also there is a
lack of evidence in the existing literature on the effect and mechanism of pineapple on uterine-
stimulation, although pineapple consumption, specifically unripe portion was regarded as a
traditional antenatal taboo in many countries. In addition, there is also a need to clarify possible
side effects due to consumption of pineapple in pregnant women.

2. Materials and methods

2.1.Plant material collection

Ananas comosus with a range of maturities including 1, 1.5, 2 months and 100 days (fully ripe)
were collected from the Johor region located in the southern part of Peninsular Malaysia
(103°45′28″ E, 1°27′55″ N altitude 32 m) in August 2014. The plant was identified and a
voucher specimen was deposited in the SING herbarium (herbarium number: SING0215652).

2.2. Preparation of crude extracts

The trimmed edible part of 10 pineapples of each abovementioned maturities was cut into small
pieces and subsequently blended to liquid form. The blended pineapple was stored in air tight
glass bottles and kept inside -80Ċ freezer overnight. Lyophilization was carried out the next day
over a period of 4 days. The lyophilized pineapple was mixed in twice their volumes with 70%
ethanol, sonicated for 1 hour, kept in cold room for 24 hours and then filtered. The ethanolic
pineapple extracts were evaporated to dryness using a rotatory evaporator at 42±1°C.

2.3. Phytochemical screening

Crude extracts obtained from A. comosus with different maturities were analyzed for
phytochemicals as previously reported by Evans for the presence of alkaloids, flavonoids,
anthraquinones, saponins, carbohydrates, steroids, triterpenes and tannins (Evans, 2009).

2.4. Liquid-Liquid partition

The crude extracts were re-constituted in a composition of methanol and water (20:80, v/v) and
fractionated through a series of liquid-liquid partitions using hexane (F1), ethyl acetate (F2) and
1-butanol (F3) respectively. The aqueous fraction was named F4. The fractions were dried down
using rotary evaporator at 42°C and further blow dried using nitrogen gas at 42°C.

2.5. Determination of total phenolic contents

The total phenolic content of the crude extracts and fraction 4 (ripe or unripe) was determined by
the Folin- Ciocalteu reagent (Baba and Malik, 2015; Maurya and Singh, 2010). Gallic acid was
used as a standard. Briefly, 200 µL of crude extract and fraction 4 (3 mg/mL) were made up to 3
mL with distilled water, mixed with 0.5 mL of Folin–Ciocalteu reagent for 3 min, followed by
the addition of 2 mL of 20% (w/v) sodium carbonate. The mixture was allowed to stand for a
further 60 min in the dark, and absorbance was measured at 630 nm. The total phenolic content
was calculated from the calibration curve, and the results were expressed as mg of gallic acid
equivalent per gram dry weight.

2.6. Animals

All the experimental procedure were executed in accordance with the international guideline for
animal research under due approval from institutional animal care and committee, National
University of Singapore (R12-145). Pregnant SD rats (Day 20±1) are acquired for this study.
Animal were allowed free access to standard laboratory diet and tap water. To obtain the
estrogenized uterus, virgin female rats are pre-treated with 17-β-estradiol via intraperitoneal
injection (40ug/kg body weight) 24 hours before the study.

2.7. Human tissue

All myometrial samples were collected from the National University Hospital, Singapore. The
research protocol was approved by the National Healthcare Group Domain Specific Review
Board (DSRB-ID-2011-00211) and all participating women gave written informed consent.
Pregnant myometrium specimens were obtained from the lower segment during elective
caesarean section at term and immediately transferred to the laboratory in chilled Krebs-
Henseleit solution.

2.8. Experimental protocol

Rats are euthanized in CO2 chambers after which bilateral uterine horns are excised and promptly
placed in chilled Krebs buffer. After clearing of adherences, the uterine horns are cut into 2mm
wide × 10mm long along the longitudinal axis of uterus. Human myometrial strips of similar
width and length were prepared.

Rat uterine strips were mounted in 10 mL thermostatically controlled organ baths (37°C)
containing Krebs-bicarbonate and human myometrial strips were suspended in Krebs-Henseleit
solution. Each strip was mounted under an applied resting tension of 1 g and aerated
continuously with 95% O2 / 5% CO2 at pH 7.4 (Lau et al., 2001). The rat and human uterine
strips were allowed to equilibrate for 1h and 2h respectively. After the equilibration period, the
myometrial strips were contracted with 120mM KCl for normalization of data. After stabilization
of the response, KCl was removed from the bath. After 1hour, crude extract, fraction 4 (aqueous)
from pineapple with different maturities, 5-HT and vehicles were tested.

To characterize the mechanism of action of pineapple extracts, uterine strips were pretreated with
established inhibitors or blockers of various pathways or receptors. In some experiments,
different concentrations of the inhibitors were employed on the same strip to investigate the level
of blockage and confirm the effect of inhibitor on uterine activity.

2.8.1. Drug used

The following compounds were used in this study: serotonin (0.05-5µM), a neurotransmitter that
binds to 5-HT receptors leading to increased contractility; verapamil (1-10µM), a L-type calcium
channel blocker; indomethacin (10µM), a non-selective cyclooxygenase enzyme inhibitor;
prazosin (10µM), selective α1-adrenergic receptor antagonist; atosiban (3µM), an oxytocin
receptor antagonist and chemicals for measuring the total phenolic content including Folin-
Ciocalteu reagent, sodium carbonate and gallic acid were obtained from Sigma-Aldrich (St.
Louis, Missouri, U.S.A.); KCl (120mM), a stimulus which activate smooth muscle (Merck-
Kenilworth, U.S.A.) ; ketanserin (10µM), a selective antagonist for serotonin 5-HT2A and 5-HT2C
(RBI- Ashland, U.S.A.) and aqueous fraction (F4) (0.1-10 mg.ml-1) of pineapple extract with
different maturities. Most of the drugs were dissolved in distilled water. Sodium carbonate was
used to dissolve indomethacin. Serotonin was dissolved in HCl (1N) and then diluted in distilled
water.
The Krebs bicarbonate solution contained (in mM): sodium chloride 119, potassium chloride
4.7, calcium chloride 2.4, sodium bicarbonate 25, magnesium sulfate 1.2, potassium phosphate
1.2, and glucose 11.1.

Krebs-Henseleit solution used for human myometrium consisted of (in mM): sodium chloride
115, potassium chloride 4.7, calcium chloride 1.8, potassium phosphate 1.2, magnesium sulfate
1.2, glucose 8.5, sodium bicarbonate 22.1 and disodium EDTA 0.03. All chemicals were of
analytical grades and purchased from Merck (Kenilworth, U.S.A.)

2.9. Data analysis

Uterine activity was quantified as the “area under the curve” (AUC) calculated from the “integral
over the minimum” provided by Powerlab software Chart (version 8.0.5). Baseline AUC was
calculated from 10 and 30 minutes in rat and human uterine activity respectively before addition
of first concentration of agonists and fractions. The changes in uterine activity were measured in
the 10 and 30 minutes in rat and human myometrium respectively in the presence of cumulative
concentrations of agonists and fractions normalized after subtraction of the basal activity. Values
were expressed as a percentage of 120mM KCl contraction. All responses were compared with
concurrent time and vehicle controls. The cumulative concentration responses were analyzed by
nonlinear regression analysis using GraphPad Prism (version 4.03; GraphPad Software Inc., San
Diego, CA), and EC50 values were obtained. EC50 is the molar concentration of the agonist that
produced 50% of the maximal response achieved.

All data are expressed as means ± S.E. The n in the text refers to the number of independent
experiments performed. Means were compared using Student’s t test and ANOVA between two
and three or more groups respectively. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate
statistically significant difference between the control and tested groups.

3. Results

3.1. Percentage yield

The weights of freeze dried powder of edible part of 10 pineapples (of each maturity) were
measured to estimate the yield of crude extract and fraction 4 (Table 1).
Table 1
Yield of A. comosus extracts of different maturities
Net weight of dried Yield of crude
Age of A. comosus Yield of fraction 4
powder extract
1 month 18.5g 22% 13.2%
1.5 months 260g 25% 16.5%
2 months 469g 31% 23.8%
3.3 months 1030g 37% 28%

3.2. Phytochemical screening

Phytochemical analysis was carried out on the crude extract of different maturities and the
results, as shown in Table 2, indicated the presence of bioactive compounds in crude extracts of
A. comosus.

Table 2
Phytochemical constituents of ethanol extract of A. comosus of different maturities
Phytochemical Test Positive Interference Negative Interference
constituents
Alkaloids Wagners 1M,1.5M and 2M Ripe
Flavonoids Sodium All the maturities _
hydroxide
Anthraquinones Ajaiyeoba _ All the maturities
Saponins Frothing 2M 1M, 1.5M and Ripe
Carbohydrates Benedict 1.5M, 2M and Ripe 1M
Steroids Liberman _ All the maturities
Buchard
Triterpenes Liberman All the maturities _
Buchard
Tannin Ferric chloride _ All the maturities
1M-1 Month; 1.5M-1.5 Months; 2M-2 Months
3.3. Phenolic content

The total phenolic content of the crude extract and fraction 4 (ripe and unripe) calculated from
the calibration curve (R2 = 0.999) was expressed as mg gallic acid equivalents per gram (Table
3).

Table 3
Total phenolic content (TPC) of Ananas comosus extracts.
A. comosus extracts TPC (mg gallic acid equivalent per gram dry weight)
Ripe crude extract 3.07
Unripe crude extract 2.39
Ripe fraction 4 1.77
Unripe fraction 4 1.77

3.4. Evaluation of contractile effect of ripe crude extract on the late pregnant rat uterine activity

Isolated uterine tissue from late-pregnant rat showed spontaneous contractions. Crude extract
(0.1-10mg.ml-1) increased the frequency and amplitude of contractions and basal tone (Fig. 1a).
There is a clear trend of increasing uterine contraction following cumulative addition of ripe
crude extract on strips with spontaneous contraction in Fig.1b (pEC50=3.15 ± 0.24µg.ml-1).
Fig. 1. Stimulation of contractions in the late pregnant rat uterine strip by crude extract. (a),
Representative tracing of the late pregnant uterine response to cumulative addition of crude
extract (0.1-10 mg.ml-1). (b), Crude extract produced a concentration-dependent increase in
contractions in late pregnant rat uterus (pEC50=3.15 ± 0.24µg.ml-1).

3.5. Evaluation of contractile effect of different fractions on human and late pregnant rat uterine
activity

Among fraction 1 to 4 of ripe batch, only F4 caused a concentration-related contractile response

at all concentrations tested on rat and human uterine strips (Fig. 2a). The values of pEC50 of ripe
F4 in human (3.01±0.28) and rat (3.19±0.22) uterus were similar (p = 0.62, Student’s t test, n=6),
as was the maximal response (122 ± 18 and 102 ± 9%) of KCl induced tone after subtracting
basal activity, respectively (p = 0.35, Student’s t test, n=6) (Fig. 2b).
Fig. 2. Stimulation of contractions in the late pregnant rat uterus and pregnant human
myometrium by ripe F4. (a), Representative tracing of the uterine response to cumulative
addition of fraction 4 (0.1-10 mg.ml-1). (b), Fraction 4 produced a concentration-dependent
increase in contractions with similar potency (represented by EC50) in rat and human uterine
tissues (p = 0.62, Student’s t test, n=6), as well as the maximal response of KCl induced tone (p
= 0.35, Student’s t test, n=6).

3.6. Evaluation of effect of ketanserin on ripe F4 and serotonin induced contraction on late
pregnant rat uterine muscle

Ketanserin (10µM), a 5HT2a,c receptor antagonist, markedly diminished the maximal contractile
response induced by both ripe F4 and 5HT respectively by 74.3% (p = 0.001, Student’s t test,
n=6) and 92.1% (p < 0.0001, Student’s t test, n=5) in late pregnant rat uterine strips (Fig. 3a and
3b). Fig. 3c illustrates the suppressing effect of ketanserin on the contractile response of
serotonin and ripe F4 in uterine muscle of late pregnant rat.
Fig. 3. Stimulation of contractions in late pregnant rat uterus by 5-HT (0.05-5µM) and ripe F4
(0.1-10 mg.ml-1) in the presence of 5-HT2 receptor antagonist. (a) and (b), Ketanserin (10µM)
diminished the maximal contractile induced by both F4 and 5HT significantly (p = 0.001,
Student’s t test, n=6 and p < 0.0001, Student’s t test, n=5 respectively) in late pregnant rat. (c),
Representative tracing of the uterine response to the cumulative addition of fraction 4 (0.1-10
mg.ml-1) and serotonin (0.05-5µM) in the absence and the presence of ketanserin (10uM) as
compared to a time-controlled parallel strip (lower panel)

3.7. Effect of indomethacin, prazosin, atosiban and verapamil on ripe F4 and serotonin induced
contraction in late pregnant rat uterine activity

To delineate the mechanism of action of bioactive compounds in ripe F4, the effect of F4 and
serotonin as the control was studied on the strips pretreated with indomethacin, prazosin,
atosiban and verapamil. The contractile effect of ripe F4 and serotonin was blocked by
verapamil, a L-type calcium channel blocker (Fig. 4a and 4b) but unaffected by indomethacin,
atosiban and prazosin (Fig. 4c). Pre-incubation of the uterine tissues with verapamil 1µM and
10uM inhibited the maximal contractile response of ripe F4 by 77% (p = 0.01 one-way ANOVA,
n=4) and 100% (p = 0.001, one-way ANOVA, n= 4) respectively (Fig. 4a). Fig. 4b shows the
representative tracings of the blockade of F4 and 5-HT induced contractions in the absence and
the presence of verapamil 10µM.
Fig. 4. Stimulation of contractions in late pregnant rat uterus by ripe F4 (0.1-10 mg.ml-1) and
serotonin (0.05-5µM) in the absence and presence of verapamil. (a), Verapamil 1µM and 10uM
inhibited the maximal contractile response of F4 by 77% (p = 0.01 one-way ANOVA, n= 4) and
100% (p = 0.001, one-way ANOVA, n= 4) respectively. (b), Representative tracings of the
uterine response to cumulative addition of fraction 4 (0.1-10 mg.ml-1) and serotonin (0.05-5µM)
in the absence and the presence of verapamil (10uM). (c), Representative tracings of the uterine
response to fraction 4-induced contraction in the presence of indomethacin (10µM), atosiban
(3µM) and prazosin (10µM)

3.8. Evaluation of contractile effect of F4 from different maturities in late pregnant rat

It can be seen from Fig. 6a, that among a range of maturities of 1, 1.5, 2-months and 100 days
(fully ripe) batches, ripe pineapple showed contractile response in all concentrations tested. The
2 months batch showed a potent uterotonic effect only at the concentration of 10 mg.ml-1 (Fig.
6b). There were significant differences between the maximal contractile response of ripe batch
and two unripe batches that include 1 and 1.5-months batch (p = 0.012 and p = 0.029
respectively, one-way ANOVA, n=10), while ripe batch and 2-months batch showed similar
efficacy in contracting the uterus at high concentration in late pregnant rat (p > 0.05, one-way
ANOVA, n=10).
Fig. 5. Effect of ripening on F4 induced contractility. (a), Concentration-related (0.1-10 mg.ml-1)
stimulatory response in late pregnant rat uterus by ripe F4. Ripe batch was more efficacious
compared to two unripe batches of 1 and 1.5-months (p = 0.012 and p = 0.029 respectively, one-
way ANOVA, n=10), while ripe batch and 2-months batch showed similar efficacy at high
concentration in contracting the late pregnant rat uterus (p > 0.05, one-way ANOVA, n=10). (b),
Representative tracings of different maturities of F4-induced stimulatory response in late
pregnant rat uterus

4. Discussion

This study sought to determine uterotonic effect of edible part of A. comosus. Indeed, our
investigation clearly indicates that crude extract (0.1-10mg.ml-1) of A. comosus produced
contractile response in rat isolated uterine tissues. Crude extract at concentration more than
10mg.ml-1 didn’t produce higher contractile response. The contractile effect of crude extract was
further shown in aqueous fraction (F4) which mediated concentration-dependent contraction with
maximal contractile response attained at 10mg.ml-1 while the other fractions (F1 to F3) didn’t
produce significant stimulatory response.

Serotonin is one of the known compounds in A. comosus. Although, the effect of serotonin on
uterine motility varies in different species, 5-HT2 and 5-HT7 have been identified as major
serotonin receptor subtypes which are involved in stimulatory and inhibitory (Nakamura et al.,
2008) response on uterine motility. 5-HT2A receptors mediating uterine contraction have been
characterized in rat and human (Cordeaux et al., 2009; Minosyan et al., 2007). In this study the
blockade of F4 and serotonin-induced contractions by 74.3% and 92.1% respectively after
pretreatment with ketanserin suggested the existence of serotonin like compound in F4. The
failure of indomethacin, prazosin and atosiban in blocking F4 stimulatory response indicated that
the uterotonic activity of F4 does not involve prostanoids, alpha1 adrenoceptors and oxytocin
receptors. It can thus be inferred that serotonin receptors is the major contributor to F4 induced
contraction of the uterus. Although several reports had indicated the presence of serotonin in
different parts of pineapple including edible part (Bruce, 1960; Feldman and Lee, 1985; Wen and
Wrolstad, 2002), the partial blockage of F4 in this study may be explained by the fact that in
addition to serotonin, other compounds may also be involved in uterotonic effect.
The study of contractile response of F4 in the presence of verapamil revealed the importance of
cellular entry of Ca2+ through calcium channel in bioactivity of F4. The uterotonic effect of F4
and serotonin was completely blocked after pretreating with verapamil (10uM). Thus it is likely
that F4 might cause the contraction of the isolated rat uterus through the stimulation of
extracellular Ca2+ influx which is similar to intercellular signaling cascade of 5-HT2 receptors. 5-
HT receptors couple preferentially to Gq/11 and increase the hydrolysis of inositol phosphates
which elevate cytosolic [Ca+ +] and produce an excitatory response.

Despite several claims of abortifacient properties of A. comosus, there is no scientific evidence

on comparison of the effect of A. comosus at different maturities on uterine activity. It is
interesting to note that only F4 obtained from the ripe pineapple stimulated the uterine
contractions at all the concentrations tested. However, F4 of 2-months unripe batch showed
potent contractile response in high concentration with similar efficacy as ripe batch. It is
tempting to hypothesize that different compounds may mediate the stimulatory response in
unripe batch. Further investigations are needed to isolate the active compounds in unripe and ripe
pineapples as the exact compound(s) which are involved in stimulatory response are not fully
known.

5. Conclusion

The present study was designed to determine the contractile effect of A. comosus extract and
delineate the probable mechanism of action of observed contractile effect of aqueous fraction of
pineapple extract on uterine activity. Despite its exploratory nature, this study offers some
insight into folkloric beliefs of abortifacient effects of A. comosus. This study has identified
firstly the uterotonic effect of crude extract of A. comosus and secondly the main role of
serotonin like compound(s) in aqueous fraction (F4) induced contraction of the uterus. The other
major finding of this study was concentration-related stimulatory response of ripe pineapple as
well as potent uterotonic effect of 2-month unripe batch at high concentration. This provides a
framework for an exploration of bioactivity of phytoserotonin derivative(s) and other agents that
may exist in pineapple. Further studies would include chemical identification of isolated
phytoserotonin structure in addition to other bioactive compound(s).
Conflict of interest

The authors have declared that there is no conflict of interest.

Author contributions

All authors contributed extensively to the work presented in this paper

P Ganesan Adaikan: supervised the project, contributed to conception and design

Faezeh Monji: performed experiments, analysis and interpretation of data

Lang Chu Lau: designed and performed experiments

Yinhan Gong and Huey Min Tan: developed and optimized the method of extraction

Mahesh Choolani: contributed to provide human samples

Acknowledgements

Professor Brian W Dymock and Dr Pondy Murugappan Ramanujulu are gratefully

acknowledged for their advice and support for chemical analyses. The authors also wish to thank
Cecille Arquillo Laureano and Cynthia Pamela Zapata Tagarino for their valuable assistance and
Abrar Al-Mahmood Siddiquee for his skillful experimental support.

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