Abstract

Stereospermum kunthianum is a plant used in traditional medicine of Burkina Faso. Ethnobotanical survey showed that this plant have many virtues but the toxicity and the effect of the leaves part on uterine contraction are not yet documented. To fill this gap, we investigated the toxicity effect on mice and the pharmacological effect of Stereospermum kunthianum on the contraction of rat isolated uterine horns. The acute and subacute toxicities were studied firstly. Secondary, the uterine segments were isolated and exposed to different concentrations of the aqueous extract of Stereospermum kunthianum leaves, oxytocin and KCl. The results showed that Stereospermum kunthianum aqueous extract decreased the amplitude and frequency of contractions. IC₅₀ values were 1.46 and 2.03 respectively for amplitude and frequency. Our study revealed that the extract inhibited uterine contractions induced by oxytocin and potassium chloride (KCl). These data suggest that Stereospermum kunthianum active compound could be used for calming uterine contractions. The effect of Stereospermum kunthianum showed that it could be useful to fight against diseases that caused uterotonic actions. It may be useful to prevent preterm birth and pain caused by menstruation, as indicated by its traditional use.

1. Introduction

Stereospermum kunthianum, is a plant used in traditional medicine of Burkina Faso. This plant is known for many therapeutic properties. Indeed, ethnobotanical surveys have revealed that a decoction of the roots was acknowledged to be effective against diabetes ¹. The other parts of the plant that is the pods were used in the treatment of cough, ulcers, leprosy, skin eruptions and venereal diseases. The leaf infusion was employed for washing wounds, while the macerated leaves were used to treat asthenia and exhaustion and the bark served as a haemostatic (in treating wounds) ^{2, 3}. Some other uses included the stem decoction as recipe for bronchitis, pneumonia and cough; the roots and leaves were used in the treatment of venereal diseases, respiratory ailments and gastritis; while the aqueous stem bark extract was documented as protective against generalized seizures in pentylenetetrazole and electro-convulsive models in rodents ⁴.

In Burkina Faso we have noted that the plant in addition to treating skin diseases, diabetes, and respiratory diseases can also be used to prevent preterm birth ². Nevertheless, the effect of Stereospermum kunthianum on preterm birth prevention is not yet documented. In addition, the studies on the toxicity of this plant are generally based on the stem bark. The other parts have not been much studied; it is the case of the leaves.

Concerning the studies on phytochemicals, Stereospermum kunthianum have shown the presence of tannins, iridoids, mucilage, chromogen, steroids, naphthoquinones, and steroidal saponosides ^{2, 5}. The presence of certain compounds such as steroids may be responsible for inhibiting the contractile activity of the uterus. The aim of this study is to evaluate the effect of Stereospermum kunthianum leaves extract on the isolated Wistar rat uterus after preliminary confirmation of its safety.

2. Materials and Methods

The plant sample was collected from its natural habitat in the “Parc urbain Bangr-Wéogo” located in Ouagadougou the capital of Burkina Faso. The samples were collected during the dry season and identified by the Herbarium of the Biodiversity Centre of University Joseph KI-ZERBO, where the voucher specimen (ID N^o: 16960) was stored. The leaves of the plant were dried in shade at room temperature (30 ± 2°C) for 14 days. The leaves were powdered and macerated using 100 g of powder in one liter (1 L) of distilled water for 24 h. The maceration was done using a magnetic stirrer and the solution was filtered using a Whatman no. 2 filter paper and then concentrated in a rotary vacuum evaporator. The powder obtained was stored at -4°C and used for the different tests.

Male and female mice were used for acute and subacute toxicity studies. According to effect of Stereospermum kunthianum on rat uterus contractility, the animals were treated as describe by Dabiré et al. (2021). Non-pregnant female Wistar rats (200–250 g) were used in this study. The oestrous cycle stages were monitored daily using vaginal smears and the rats were sacrificed only in the metoestrus or dioestrus ⁷. The non-pregnant rats were fed with granules containing 29 % of proteins provided by the western regional office of the “Centre de Promotion de l’Aviculture Villageoise” (CPAVI) of Bobo-Dioulasso (Burkina Faso) and they were kept at 22 ± 2 °C, 60 ± 10 % humidity. The rats were submitted to a 12-h light/dark cycle with food and tap water ad libitum. All procedures involving animals strictly followed ethical considerations for scientific research in the University Joseph KI-ZERBO.

The method used was as describe by Kharchoufa et al. (2020) with some little modifications. The single-dose acute oral toxicity study was evaluated following the recommendations of OECD Guidelines (423) ⁹. Acute toxicity studies were carried out in NMRI (Naval Medical Research Institute) mice, weighing 25–35 g each one, using a single oral dose. Forty-two mice, divided into 7 groups, were designed for the study of acute toxicity. Each group of 6 mice (3 males and 3 females) received, respectively, a single oral dose of 100, 300, 500, 800, 1000 and 2000 mg/kg body weight of Stereospermum kunthianum extract, while the control group was treated with distilled water. The general behavior of mice and signs of toxicity were observed continuously for 1h after the oral treatment and then intermittently for 4h and thereafter over a period of 24 h. The mice were further observed once a day up to 72 hours (3days) for following treatment for behavioral changes and signs of toxicity and/or death and the latency of death.

Subacute toxicity studies on the extract were carried out according to the guidelines for testing of chemicals ⁹. Forty mice of either sex were randomly allotted into four groups of ten animals per group. Animals were administered 500, 1000 and 2000 mg/kg/body weight/day of the extract. The control group received distilled water (5 mL/kg). All administrations were via the oral route once daily for a total of 28 days. The animals were maintained under standard laboratory conditions including 12h light /dark cycles and had free access to food and tap water during the study period. Body weight was taken before first administration and weekly (7 days) after the extract administrations began. Overt toxic manifestations and mortality were monitored during the study. The animals were anesthetized intraperitoneally by ketamine (87 mg/mL) and xylazin (13 mg/mL) 4 hours after extract treatment on day 28. The animals were sacrificed and dissected and the liver, heart, kidneys and spleen were isolated and weighed. Each organ was examined macroscopically for colour changes and any discernible pathological lesions.

The method used was the same as described by Dabiré et al. (2021). The study was undertaken at University Joseph KI-ZERBO. The rats were anesthetized with urethane (15%; 1,5g/kg) by intraperitoneal route. The isolated uterine horns were rapidly removed and placed in a physiological solution composed of [mM]: 154 sodium chloride (NaCl), 5.4 potassium chloride (KCl), 2 calcium chloride (CaCl₂), 1.2 magnesium sulphate (MgSO₄), 1.8 potassium dihydrogen phosphate (KH₂PO₄), 22 sodium bicarbonate (NaHCO₃) and 8 glucose, pH adjusted to 7.4. They were freed of the adhering connective tissues and fat. The isolated uteri were segmented into 10 mm-long sections. The part of the horn involved in this experiment is equivalent to the 10 mm section described by Chen et al. ¹⁰. The isolated segment was ligated at both ends and the lower end was attached to a fixed hook. Then, the uterine segment was mounted vertically in a tissue organ bath containing 10 mL of the physiological solution (pH 7.4 and temperature 37°C) and connected to a force transducer (Model FT03, Grass Instruments, Quincy, MA, USA) using silk threads. The maximum volume of the organ bath was 20 mL. Electrical signals from the transducer were amplified using a ETH-400 bridge amplifier (CB Sciences, Dover, NH, USA) and converted to digital signals to be recorded by a MacLab/8e digitizer (AD Instruments, Castle Hill, NSW, Australia) using the Chart software (v 4.2 for windows, AD Instruments Pty Ltd, Australia). The resting tension applied to the uterine segments was 0.5 g. An equilibrium period of 45 minutes was required to allow spontaneous and homogeneous contractions of the uterus before testing the leaves extract or any other drug.

After the equilibrium period, the extract was added cumulatively in the single organ bath containing the physiological solution to successively obtain concentrations of 10^-3, 10^-2, 10^-1, 1 and 5 mg/mL each 10 minutes of time interval. This allowed measurement of the amplitudes and frequencies of the extract effects.

In order to investigate a probable interaction of the extract with KCl (60 mM, bath concentration) or oxytocin (10 mIU/mL, bath concentration) which was purchased from Ciron Drugs (India), these substances were used to pre-contract the uterine horns. KCl an oxytocin were allowed to react with the uterine horn segments for 15 min and used as control. The extract was then added cumulatively to obtain 10^-3, 10^-2, 10^-1, 1 and 5 mg/mL bath concentrations, as mentioned previously.

The data were analyzed with Graph Pad Prism 5.00 software. The contractile activity of the last 5 minutes in the control solution or positive control solutions (oxytocin, KCl) was calculated and considered to be 100%. The inhibitory effect of each substance was then calculated by taking into account the control and the data were represented as a concentration-response curves. The amplitudes and frequencies were the parameters used in the analyses of the observed effects. IC₅₀ values were determined and Student's t-tests were used for comparisons. The differences in data sets were considered significant (*) if the p value was < 0.05.

3. Results

During the 72-hour follow-up of the animals in acute toxicity, we did not count any dead mice. All the mice have survived up to the maximum dose of 2000 mg/kg bw. We did not have any mice in latency of death. The mice showed apparent good health until the follow-up time. Beyond the 500 mg/kg bw, some mice scratched their mouths as if they were cleaning them. Otherwise, the mice showed no apparent signs of toxicity at doses of 100, 300, 500, 800, 1000 and 2000 mg/kg bw.

The animals showed an increase in body weight (1-3 g) from the beginning to the end of the treatment. There was no significant (p>0.05) change in body weight between treated and control rats measured every 7 days. At the end of 28 days of treatment, all the body weights of treated rats were lower than those of control rats (Figure 1). All animals have survived during the 28 days of treatments and no latency of death was recorded. Likewise we did not record any visible sign of toxicity on the rats.

The relative weights of the organs (heart, spleen, liver, kidney and lung) increased in a near dose-dependent manner in a way that at 2000 mg/kg bw the relative weight of each organ was greater than the relative weight of the organ in the control group. There was a non-significant decrease in relative weight at 500 and 1000 mg/kg bw for the lungs and at 1000 mg/kg bw for the liver (Figure 2). Macroscopic observation of the different organs of the groups treated with the aqueous extract of Stereospermum kunthianum and those of the control groups did not show any change in color or any change in organ morphology that would indicate toxicity.

The aqueous extract of Stereospermum kunthianum leaves at the different concentrations used inhibited the basic contractions of rat uterine horns (Figure 3). The effect was marked by a decrease in the amplitude and frequency of basal uterine contractions. Marked amplitude and frequency reduction appears from 1 to 5 mg/mL. Total relaxation, which corresponded to 100 % of amplitudinal decrease, was achieved at 5 mg/mL. The IC₅₀ values for amplitude and frequency were found to be 1.46 and 2.03 mg/mL, respectively (Figure 3B). This inhibitory effect occurred immediately after addition of the extract to the isolated organ bath, and the effect was maintained throughout the presence of the extract in the organ bath (Figure 3A). Furthermore, the extract did not alter basic tone compared with the control, and contractions returned to normal after two 15-min organ washes per wash.

Oxytocin stimulated the basic uterine contractions. It caused an increase in the amplitude, frequency, and basic tone of the uterine horns (Figure 4A). In the presence of the aqueous extract of Stereospermum kunthianum leaves added 10 minutes after oxytocin (10 mUI/mL), amplitude and frequency were decreased slowly from 10^-3 to 1mg/mL (Figure 4B).

About the amplitudes these decreases were 82 ± 5, 68 ± 15, 57 ± 13, 29 ± 8 % respectively for the same concentrations of extract. A complete decrease of mean percentage was observed at the concentration of 10 mg/mL. The IC₅₀ values were 1.90 and 5.40 mg/mL respectively for frequency and amplitude (Figure 4C).

KCl (60 mM) induced contraction in rat uterine horns (Figure 5A). The addition of the extract decreased the amplitude of the contraction in a concentration-dependent manner (Figure 5). The mean percentage of decrease obtained at 5 mg/mL was approximately 136 ± 10 % compared to the control considered as 0% (Figure 5B).

4. Discussion

This study was achieved by using the leaves of Stereospermum kunthianum, which are generally not widely studied. The results of this study indicated that the aqueous extract of leaves administered at different doses (100, 300, 500, 800, 1000 and 2000 mg/kg bw) did not cause mortality or signs of toxicity. According to the Globally Harmonized Classification System (GHS) ⁹ scale, the extract could belong to the category 5 chemical class. This category 5 represents the lowest risk chemical class. Our results can be compared with that of Okpo and Ching, (2013) who showed that the aqueous extract of stem bark of Stereospermum kunthianum does not cause death or signs of toxicity in mice up to a dose of 8 g/kg bw. We can therefore say that the leaves as well as the stem bark of Stereospermum kunthianum are practically non-toxic in case of short-term exposure.

The results on subacute toxicity were in line with those on acute toxicity. Indeed, there was no mortality or latency of death or even signs of toxicity during the test. In addition, analysis of the change in weight of the animals shows that there was no significant change compared to the controls. This was also the case for the weight of the vital organs measured. Our results on the toxicity test corroborate those of Egua et al. (2021) who showed that aqueous methanolic stem bark extract is safe up to 400 mg/kg bw. A previous study on aqueous stem bark extract showed that this aqueous extract could be safe up to 1000 mg/kg bw ¹⁰. This study presented a more advanced toxicity analysis than ours. Indeed, in addition to organ weight analyses it presented blood biochemistry and histopathology analyses and most of the toxicity effects occurred at 1000 mg/kg bw. This could involve that the leaf extract we studied is either not very toxic compared to stem bark or further analyses on biochemistry and histopathology are needed to reveal some signs of toxicity.

On uterine contractions, the results of the present study showed that the aqueous extract of Stereospermum kunthianum caused a decrease in the mean force amplitude and frequency of contractions. Uterine contraction is due to the presence of smooth muscle cells that compose the myometrium ¹¹. In the non-pregnant uterus, the contractions are produced to facilitate the journey of sperms to the fallopian tubes and to help expel the shed inner lining of the uterus (endometrium) during menstruation ¹². In some women these contractions may cause mild to severe pain, clinically known as dysmenorrhea ¹³. Increased plasma levels of prostaglandins and vasopressin are the main causes of primary dysmenorrhea in women ^{13, 14}. During primary dysmenorrhea these hormones increase uterine contractions and install pain ¹⁵. Treatment solutions aim to reduce pain through anti-inflammatory effects or to reduce uterine contractions ^{16, 17}. The work of Ching et al. (2009) showed the ability of Stereospermum kunthianum to reduce pain through its anti-inflammatory effect. The present study demonstrates the ability of this plant to reduce contractions of the uterus. The two effects combined are likely to give this plant a great power sustained in the fight against dysmenorrhea. The effects of Stereospermum kunthianum can be compared to those of Cinnamon zeylancium that also reduce pain and decrease the force amplitude of contraction ¹⁹.

A possible mechanism of action was demonstrated by using oxytocin and KCl. The results indicate that the aqueous extract of Stereospermum kunthianum inhibits uterine contractions induced by KCl (60 mM) and oxytocin (10 mIU/mL). The mechanism of KCl and oxytocin on uterine contraction involves an influx of extracellular calcium. These substances are therefore used to approximate the potential mechanism of action of the calcium pathway ⁶. KCl activates L-type voltage-dependent calcium channels, resulting in massive calcium ion entry into the myometrial smooth muscle cell ²⁰. This calcium entry combines with calcium release from the sarcoplasmic reticulum to induce contraction when the calcium level reaches a threshold. Our result suggests that the aqueous extract of Stereospermum kunthianum could probably modify the depolarization of the membrane or block voltage-dependent calcium channels.

As for oxytocin, it is known that this substance acts on specific receptors by activating a G protein. The latter stimulates phospholipase C which leads to the release of inositol triphosphate (IP3) and diacylglycerol (DAG) in the myometrial smooth muscle cell. IP3 blocks the SLO 2.1 potassium channel and gives rise to a membrane current ²¹. The latter stimulates voltage-gated calcium channels by increasing calcium influx through L-type calcium channels. IP3 also stimulates the sarcoplasmic reticulum (SR) calcium channel, resulting in calcium release. The release of calcium from the SR and the influx of calcium cause contraction of uterine smooth muscle ^{22, 23}. Through this mechanism of action we can say that the active compound of the extract could be an oxytocin antagonist or interferes with one of the pathways that leads to the increase of intracellular calcium.

5. Conclusion

This work is aimed at determining the effects of the aqueous extract of Stereospermum kunthianum leaves and investigating its probable mechanism of action. Our data showed that the aqueous extract of Stereospermum kunthianum has a relaxant effect on rat uterine smooth muscle. The aqueous extract of Stereospermum kunthianum acts by modifying the contractions produced by oxytocin and KCl. So, this plant active compound could be important to reduce uterine contractions but further investigation is needed to deepen the mechanism action. Our results are in agreement with the use of Stereospermum kunthianum in traditional medicine to fight dysmenorrhea or to prevent preterm birth.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgements

We are grateful to Professors Youssoufou Ouédraogo, Raymond G. Belemtougri, Maurice Ouédraogo and Balé Bayala, who allowed us to carry out this work.

References