Oxytocic Activity of Leaf Extract of Spondias mombin.

Abstract

The oxytocic property of leaf extract of Spondias mombin. Linn. (Anacardiaceae) was evaluated in relation to its folkloric use by traditional birth attendants. The abortifacient effect of the methanol leaf extract (ME) obtained by cold maceration for 48 h, was evaluated in vivo. in pregnant mice and rats at different trimesters of pregnancy. The effect of ME on isolated gravid and non-gravid rat uteri preparations as well as on spontaneous contractile frequency and amplitude were investigated in vitro.. The results showed that ME caused abortion in rats only in the third trimester within 24 h of administration. An effective abortifacient dose (ED₅₀) of 105.33 mg/kg was established in pregnant mice. In vitro., the extract significantly (P < 0.05) increased the amplitude and frequency of spontaneous contraction of isolated uteri preparations, which were sustained for longer periods. The uterine-stimulant effects of acetylcholine, oxytocin, ergometrine, and PGF_2α. were all potentiated by ME in a dose-dependent manner. The in vivo. abortifacient activity was well corroborated by the uterotonic activity of ME in vitro.. Acute toxicity tests in mice revealed an intraperitoneal LD₅₀ of 282.44 mg/kg. Phytochemical tests showed the presence of saponins, flavonoids, tannins, glycosides, resins, protein, and triterpenes. These findings justify the use of S. mombin. by traditional birth attendants in labor induction, augmentation, and as postpartum astringent.

Keywords:

• Abortifacient
• Spondias mombin
• oxytocic
• uterotonic

Introduction

Although orthodox medicines are now available for many ailments and disease conditions, the use of herbal remedies is still encouraged due to affordability, accessibility, and acceptability. Plants and herbs belonging to various families have been employed by traditional birth attendants and native healers to induce labor, achieve relatively painless delivery, terminate unwanted pregnancy, and evacuate retained placenta in humans, sheep, and goats (Akah, 1994). Preliminary reports on some of these plants are documented (Lipton, 1963; Bouguet, 1979; Ojewole & Elujoba, 1982; Aguwa, 1987; Kamanyi et al., 1992; Akah, 1994). Spondias mombin. L. (Anacardiacea) is one such plant with a folkloric reputation for use as a oxytocic agent, particularly for the expulsion of retained placenta in women and animals when normal delivery of such is delayed or difficult and as an astringent in postpartum medication. The plant is also medicinally useful for its antidiarrheal (Iwu, 1983), antimicrobial, and astringent (Ajao et al., 1985; Caceres et al., 1993; Corthout et al., 1994) properties and in the treatment of superficial and internal wounds (Villegas et al., 1997). In the southeastern part of Nigeria, the plant is popularly known as “Ichikara” and commonly used by traditional birth attendants for its obstetric value.

S. mombin., also popularly known as “Hogplum,” is a deciduous tree widely cultivated for its yellow, pleasantly tasting acidic fruits and as a live fence. The leaf has a common stalk 12–24 inches long, 5–8 pairs of opposite leaflets, and an odd terminal one. The leaflets are very unsymmetrical acuminate, often with a blunt tip (Dalziel & Hutchintson, 1958; Keay et al., 1964).

The high success rate claimed in the use of the plant in obstetrics raises hope for the possibility of discovering a novel oxytocic agent or a lead compound. In pursuance of this, the leaf extract was screened in vivo. in pregnant rats and in vitro. on isolated uterine strips to establish a scientific basis for its use in herbal obstetrics therapy and possibly uncover the mechanisms underlying the pharmacological activities.

Materials and Methods

Plant material

Fresh leaves of S. mombin. L. were collected in the month of June 2001 from the wild plant growing in Nsukka, Nigeria. The identity of the plant was authenticated by Mr. J. M. C. Ekekwe of the Department of Botany, University of Nigeria, Nsukka, Enugu State, Nigeria. A voucher specimen is preserved in the Department of Pharmacognosy Herbarium for reference. The leaves were cleaned, air-dried, and then reduced to fine powder using a hand blender.

Extraction of plant material

The leaf powder (500 g) was extracted with methanol by cold maceration for 72 h (Nairn, 1990) with intermittent agitation. A fresh batch of leaf powder (500 g) was successively extracted with n.-hexane, ethyl acetate, and methanol in that order of increasing solvent polarity. Concentration of the extract and fractions under reduced pressure afforded 46.85 g of the methanol extract (ME), 31.5 g of the hexane fraction (HF), 13.0 g of ethyl acetate fraction (EF), and 22.5 g of methanol fraction (MF). The methanol extract (ME) was subjected to phytochemical analysis using the procedures outlined by Harbourne (1984).

Pharmacological tests

Animals

Pregnant female albino rats (130–240 g) and mice (29–40 g), non-pregnant female albino rats (120–180 g), and adult albino mice of both sexes were employed in the study. The animals were obtained from the laboratory animal facility of the Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria. Animals were housed in cages within the facility and maintained on standard pellets and drinking water ad libitum..

Preliminary uterotonic activity screening of extract and fractions

Preliminary assessment of in vitro. uterotonic activity of the extract and fractions on gravid and multigravida rat uteri preparations revealed a potency of activity of the order of magnitude: ME > MF > EF > HF. Based on this, the ME was subjected to further studies without fractionation.

Acute toxicity/lethality (LD₅₀) test of the ME

The acute toxicity and lethality (LD₅₀) of the methanol extract was estimated in mice by the intraperitoneal route using the method described by Lorke (1983). Briefly, 10, 100, and 1000 mg/kg of ME were administered in the first stage of the test to three groups of animals (n = 3). Treated animals were observed for number of deaths within 24 h. Based on the results obtained from the first stage, 200, 400, 800, and 1600 mg/kg doses were administered to four groups of animals (n = 1) and the number of deaths within 24 h recorded. The LD₅₀ was calculated as the geometric mean of the least lethal and the highest non-lethal dose.

Estimation of mean effective abortion dose (ED₅₀) of ME in pregnant mice

Preliminary experiments were carried out in pregnant mice to establish the effective abortifacient dose (ED₅₀) of the extract (Miller & Tainter, 1944) for subsequent in vivo. studies. Mice in the third trimester of pregnancy were randomized into three groups (n = 5). Each group received one of three dose levels (50, 100, and 150 mg/kg) administered intraperitoneally. The animals were observed for 24 h for signs of abortion assessed as vaginal bleeding, fetal discharge, and or placental expulsion. The ED₅₀ was determined from a plot of the percent response (as Probit) versus log dose.

Effect of ME on pregnant rats

Virgin rats (8–11 weeks old) were randomized into four groups (n = 5) and brought into estrus by cage confining with a male rat for 5 days. Thereafter, they were separated singly and placed with males of proven fertility and breeding potential until a dislodged sperm plug was observed on either the vaginal orifice or on plain white paper placed beneath the cage. Pregnancies were dated from the second day of such observation (Inglish, 1980). The first trimester group was used within the first 8 days of pregnancy; the second trimester group was used between the eighth and fifteenth day of pregnancy, while the third trimester group was used between the sixteenth and twenty-first day of pregnancy, when the rats were visibly heavy.

The methanol extract (100 mg/kg) was administered intraperitoneally. Control animals received 5 ml/kg normal saline. The animals were observed for 24 h for vaginal bleeding and fetal and/or placental expulsion. Animals that did not show signs of abortion after 24 h were kept and observed for the length of gestation. Moribund animals were sacrificed and opened up, and gross observation of the fetuses and uterus were made to ascertain the possibility of intrauterine death. The rats had free access to clean water and feed during and after the experiment.

In Vitro. pharmacological studies

Effect of ME on gravid and non-gravid uteri preparations

The effect of ME on isolated rat uterine smooth muscle preparations was studied using standard procedures (Turner, 1965). Gravid uteri in which the fetuses varied from recent implantation to near term were used. The non-gravid uteri were stimulated into oestrus by priming the animals with subcutaneous stilbesterol (100 ug/ml) 24 h prior to use. The animals were killed by a blow on the head and exanguinated. The abdomen was cut open and the two uterine horn dissected out, trimmed of fats and mesenteric materials, and prepared into 2 cm strips. A strip was suspended in a 30 ml organ bath containing De-Jalon's solution continuously aerated with a mixture of 95% O₂ and 5% CO₂ and maintained at 32°C. The tissue was allowed to equilibrate for 45 min on 1 g resting tension and flushed every 10 min.

After equilibration, responses of the tissue to the extract and standard drugs were recorded on a 2-channel recorder (Gemini 7070, Ugo Basile, Italy). The effect of the extract (0.1–1.6 mg/ml) on sub-maximal responses of acetylcholine (8 µg/ml), oxytocin (0.08 IU/ml), PGF_2α.(0.04 µg/ml), ergometrine (0.2 µg/ml), and histamine (32 µg/ml) was recorded. The sub-maximal dose of the standard drugs acting alone served as the control. The effect of some antagonists on the uterine-stimulant activity of the extract was tested with atropine (0.5 µg/ml), pirenzepine (16.67 µg/ml), and propanolol (5 µg/ml). Replicate determinations were obtained for each treatment and mean responses recorded.

Effect of ME on spontaneous rhythmicity of isolated rat uterus

Nonpregnant multigravida female albino rats pretreated with stilbesterol (100 µg/kg, s.c.) were used. The tissue was prepared and set up as described above. The isotonic developed tension (dT) and spontaneous contractile frequency (cF) were recorded at 15 min intervals for 120 min. The dT and cF were recorded in the presence and absence of ME and a calcium channel blocker-nifedipine (0.2 µg/ml).

The dT was measured as a function of amplitude of oscillation, converted to force values by a prior calibration of isotonic transducer. The (cF) was counted as number of contraction cycles in 15 min. The values of dT and cF were expressed as percentage change from the initial values; dTi and cFi were recorded during the first 30 min of equilibration before the addition of any treatment. Replicate determinations were made and mean values recorded. Percentage change in developed tension was calculated using the relation:

Similarly, the percentage change in contractile frequency was calculated using:

Statistical analysis

Data was analyzed by Student's t.-test. Differences between means were accepted as significant at P < 0.05.

Results

Extraction and phytochemistry

The extraction process yielded 9.37% (w/w) of ME, 6.3% (w/w) of HF, 2.6% (w/w) of EF, and 4.5% (w/w) of MF. Phytochemical analysis of the ME gave positive reactions to tannins, saponins, flavonoids, proteins, glycosides, resins, triterpenes, and steroids.

Acute toxicity/lethality (LD₅₀) test and mean effective abortifacient dose (ED₅₀)

The i.p. LD₅₀ of the ME in mice was estimated as 282.84 mg/kg, while the ED₅₀ was 105.53 mg/kg (Table 1).

Table 1.. Estimation of effective abortive dose (ED₅₀) of methanol extract (ME) in pregnant mice.

Dose (mg/kg)	Log dose	Abortion	Percentage abortion	Probit
50	1.699	0/5	0.0	0.2778
100	2.000	2/5	40.0	0.32011
150	2.176	4/5	80.0	0.83639

Regression equation: Probit = 1.6172 (Log dose) − 2.7722; 0.5 = 1.6172 (Log ED₅₀) − 2.7722; ED₅₀ = 105.53 mg/kg.

Effect of ME on pregnant rats

There was no abortion in the first and second trimester groups. However, the extract caused intrauterine death of fetuses in two of the rats in the first trimester and in one of the rats in each of second and third trimester groups. Two rats died within one week of induction of abortion in each of first and second trimester groups (Table 2).

Table 2.. Effect of methanol extract (ME) on pregnant rats.

Treatment	Trimester	Abortion	Abortion (%)	Intrauterine death	Death after one week
ME	1	0/5	0	2/5	2/5
Control		0/5	0	0/5	0/5
ME	2	0/5	0	1/5	2/5
Control		0/5	0	0/5	0/5
ME	3	4/5	80	1/5	0/5
Control		0/5	0	0/5	0/5

n = 5; ME = methanol extract.

Effect of extract on isolated rat uterus preparations

The methanol extract showed a delayed but consistent enhancement of amplitude of contraction and spontaneous contractile frequency of isolated rat uterus preparations at a dose of 0.1 mg/ml (Fig. 1). The uterotonic effect of the extract was more pronounced on gravida and multigravida than on virgin and quiescent uterus preparations. Also, the contractile effect of acetylcholine, oxytocin, egometrine, and PGF_2α. on the rat uterus was significantly (P < 0.05) potentiated by the extract. The uterotonic effect of the extract was abolished by the calcium channel blocker nifedipine (0.2 µg/ml) while atropine (0.5 µg/ml), pirenzepine (16.67 µg/ml) and propranolol (5 µg/ml) exhibited no inhibitory effect on the uterine contractility (Table 3).

Table 3.. Effect of methanol extract (ME) on submaximal responses of agonists on isolated uterus preparation.

	Height of response (mm)
Treatment	Control	0.1 µg/ml	0.2 µg/ml	0.4 µg/ml	0.8 µg/ml	1.6 µg/ml
Ach + ME	47.33 ± 1.45	50.33 ± 0.88	60.00 ± 1.16	65.00 ± 0.58	66.00 ± 1.16	74.67 ± 1.45
Oxytocin + ME	32.33 ± 1.86	29.67 ± 0.88	35.33 ± 0.88	36.00 ± 1.00	36.00 ± 0.58	39.00 ± 0.58
Ergometrine + ME	49.00 ± 2.52	51.33 ± 0.88	58.67 ± 0.88	60.33 ± 0.33	60.33 ± 0.88	59.00 ± 0.58
PGF2α. + ME	56.33 ± 0.88	56.33 ± 0.33	61.33 ± 0.33	67.67 ± 0.33	78.67 ± 0.67	83.33 ± 0.33
Histamine + ME	−14.00 ± 1.53	−12.33 ± 0.33	−13.00 ± 0.58	−15.33 ± 0.33	−11.33 ± 0.33	−10.33 ± 0.88

n = 3; ME = methanol extract.

Control values represent the contractile effect of the agonists: ach (8 µg/ml), oxytocin (0.08 IU/ml), ergometrine (0.2 µg/ml), PG_2α. (0.04 µg/ml), and histamine (32 µg/ml) without the extract.

†Relaxation of uterus.

*Significant at P ≤ 0.05 (compared to treatment with standard drug alone).

Figure 1 Effect of methanol extract on developed tension and contractile frequency of isolated rat uterus preparations.

Discussion

There are occasions when uterine stimulants can be used advantageously by experienced obstetricians to manage dysfunctional labor (Campbell & Lees, 2000). Labor induction, augumentation of labor, postpartum uterine atony, hemorrhage, and therapeutic abortion are key indications for the use of oxytocics (Derek, 1986). In traditional medicine practice, child delivery always involves the use of potent medicinal herbs to accelerate the process of parturition. Evaluation of the effect of leaf extract of S. mombin. on pregnant animals from conception to full term revealed a potent arbotifacient property that increased with gestational age. Administration of the extract earlier in the pregnancy did not provoke fetal expulsion nor did it reveal any abortive tendency. However, a pronounced degree of fetal expulsion occurred in the third trimester similar to what is obtained with other endogenous oxytocics, such as oxytocin and prostaglandins (Schrey, 1988; Gravas, 1996).

Although the retreat from maintenance of pregnancy and uterotonin induction of parturition have been suggested as possible theorems of initiation of parturition (Lopez, 2003; Woodcock et al., 2004), these suppositions are known to rely on careful regulation of the activity of myometrial smooth muscle cell contraction (Webb, 2003). Regulation of myometrial contractility is complex and involves changes in electrophysiological membrane function and hormone-receptor interaction (Huszar & Robert, 1982). The contractile state of myometrial cells is directly regulated by estrogen and progesterone receptors as well as by a variety of cell surface receptors (Lopez, 2003).

The extract potentiated the contractile effect of acetylcholine, oxytocin, ergometrine, and PGF_2α. on the uterus preparations, while the relaxant effect of histamine was inhibited. It is not certain if these effects were due to a direct interaction with the various receptors. However, the use of specific antagonists such as atropine, pirenzepine, and propranolol did not alter the effect of the extract on spontaneous contraction of isolated uterus, suggesting that the extract may not be interacting with muscarinic and β.-adrenergic receptors, particularly where the sensitivity of uterine muscarinic receptors has been shown to diminish in pregnancy (Nakanishi & Wood, 1971). In the same vein, we may not categorically state that the extract interacts with uterine 5-HT receptors where ergometrine has been shown to be a partial agonist (Hollingsworth et al., 1988) since we did not use any specific antagonist of ergometrine. Oxytocin and prostaglandin receptors as well as some of the cell surface receptors that mediate the contraction of the uterus are associated with G-protein-mediated activation of phospholipase C, which increases [Ca²⁺]i and myometrial cell contraction (Lopez, 2003).

Agents that act on myometrial smooth muscle cells to increase cytosolic concentration of calcium obviously promote contraction. In the present study, the abolition of the uterotonic effect of the extract by the dihydropyridine L-type calcium channel blocker, nifedipine, suggests a possible involvement of calcium mobilization in the activity of the extract. In the rat uterus, Ca²⁺ entry to the cytosol occurs almost entirely through the L-type Ca²⁺ channels (Wray et al., 2001), and extract of this plant has been shown to promote the entry of extracellular calcium into the cytosol through this type of channel (Uchendu & Nwankwo, 2005).

Acute toxicity tests revealed a relatively low LD₅₀ value. Estimation of the ED₅₀ also gave a low value. These results suggest a risk of acute intoxication and the possibility of a narrow therapeutic index. Intrauterine death recorded after induction of abortion indicates that systemic and fetal toxicity may not be ruled out as possible additional mechanisms of the abortifacient property of the extract. This factor may be responsible for the reproductive abnormalities caused in mice by this plant (Sierra & Buchelli, 1986). Fetal toxicity may equally result from compromised placental perfusion induced by contraction of uterine smooth muscle (Campbell & Lees, 2000) caused by the extract.

In the present study, solvent-guided extraction of the plant material was undertaken in an attempt to relate the activity of the extract to specific constituents. Several secondary metabolites, such as saponins, flavonoids, tannins, glycosides, resins, triterpenoids, and steroids, have been identified in the leaf extract of this plant. However, the results of pharmacological tests showed that the extract may not owe its activity to a single constituent since the magnitude of uterotonic effect decreased in graded solvent extracts of the plant leaves. The presence of whole constituents of the leaf may be a requirement for uterotonic activity.

In addition to these constituents, the isolation of anacardic acid derivatives (Coates et al., 1994) and a series of 6-alkenyl-salicylic acids (Corthout et al., 1994) have been reported. The role played by these constituents in the oxytocic activity of this plant is not known. However, some of the constituents identified in the plant, such as triterpenes, sterols, glycosides, and saponins, are known smooth muscle stimulants (Lipton, 1963) and may exert a direct musculotropic action (Offiah & Anyanwu, 1989) in a complementary manner.

Conclusion

The leaves of S. mombin. possess oxytocic activity that may derive from calcium ion mobilization into the cytosol and direct musculotropic activity of the constituents. The presence of whole constituents of the leaf may be a requirement for uterotonic activity. The results of both in vivo. and in vitro. tests are well corroborated and lend scientific credence to the use of the plant by traditional birth attendants. However, the possibilities of fetal toxicity and abortion may preclude use for other indications during pregnancy.

Acknowledgment

The authors thankfully acknowledge the financial support of Sylvester Nworu by the S. O. Awokoya Foundation for Science Education.