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Original Article
2021
:2;
6
doi:
10.25259/JRHM_33_2020
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Utero-relaxing and anti-free-radical effects of hydro-ethanolic extracts of Desmodium ramosissimum in the wistar female rats

Department of, Animal Physiology and Pharmacology, University of Lomé, Lomé, Togo
Department of, Chemistry, University of Lomé, Lomé, Togo
Department of, Biochemistry and Nutrition, Faculty of Sciences, University of Lomé, Lomé, Togo
Corresponding author: Mamatchi Melila, Department of Biochemistry and Nutrition, Faculty of Sciences, University of Lomé Togo. mamatchimelila@gmail.com
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How to cite this article: Madjalani H, Metowogo K, Pakoussi T, Nabede A, Kanabiya E, Melila M, et al. Utero-relaxing and anti-free radical effects of hydro-ethanolic extracts of Desmodium ramosissimum in the wistar female rats. J Reprod Healthc Med 2021;2:6.

Abstract

Objectives:

In sub-Saharan Africa, the therapeutic use of medicinal plants is of arousing interest. Reproductive disorders are a public health problem like everywhere else in the world. The population, especially in rural areas, uses medicinal plants for their beneficial effects. In Africa, the population uses these medicinal plants for their healthcare, including solving problems related to childbirth where there is an increase in the number of women known as “traditional birth attendants.” The effects of its medicinal plants relate to the various reproductive processes in women, from pregnancy to induction of labor, elimination of placental retention, and management of postpartum hemorrhage. To contribute to the enhancement of medicinal plants traditionally used in reproductive healthcare in Togo and to make information on their pharmacological effects available to the population, this work focused on the evaluation utero-relaxing and anti-free radical effects of extracts of Desmodium ramosissimum in the female rats of the Wistar strain.

Material and Methods:

The effects of hydro-ethanolic extracts of leaves, stem barks, and roots of D.ramosissimum were then tested on shreds Myometer of the female rats mounted in an organ vat and the antioxidant activity of these extracts tested was then determined.

Results:

The results show that hydro-ethanolic extracts from leaves, stem bark, and roots of D.ramosissimum cause a reduction in the frequency of spontaneous contractions of the uterine muscle. This reduction shows that these extracts have relaxing effects on the contraction of this muscle. The leaves extract showed better antioxidant activity compared to other extracts.

Conclusion:

This antioxidant activity is probably due to the phenolic constituents and its metabolites would be partly responsible for the pharmacological activities of D.ramosissimum. D.ramosissimum could thus be used to reduce uterine contractions in order to avoid Threats of Premature Labor.

Keywords

Desmodium ramosissimum
Uterine smooth muscle
Utero-relaxing effect
Antioxidant activity

INTRODUCTION

In recent years, the use of traditional medicine has been booming throughout the world, particularly in rural areas, although it is more practiced in some cultures than in others.[1] According to the World Health Organization,[2] 80 % of the world’s population depend on traditional medicine for primary healthcare. This traditional medicine is based on the pharmacological and biochemical activity of some medicinal plants used for therapeutic purposes.[3] The therapeutic use of medicinal plants is applied in different pathologies[2] and reproductive disorders in women are not spared.[4] These disorders are in part the causes of maternal morbidity and mortality, especially in many African countries where the rate of childbirth-related mortality is high.[5] According to the 2010 RGPH4 report in Togo, maternal mortality at the national level is 437 maternal deaths/100,000 live births, a rate of 0.59%.[6] In Africa, the low income of the population, the high cost of medicines and the inaccessibility to health structures, encourage the use of medicinal plants. Among these plants is Desmodium ramosissimum which is characterized by its antioxidant, anti-inflammatory, and various therapeutic properties such as treatment of gastrointestinal infections, diarrhea, dental problems, and complications related to childbirth.[7] However, macerations, tinctures, decoctions, and infusions constitute the forms of preparations used in the traditional system.[8] Nevertheless, in many cases, the whole plant is used in this preparation of traditional medicines with the consequent destruction of biodiversity. This is indeed the case of the species D.ramosissimum which is often uprooted by users of medicinal plants. The pharmacological effects of these medicinal plants during parturition may be such as to modulate uterine contractions at work, resulting in either the stimulation or inhibition of Myometer contractions. Therefore, it appears necessary to investigate the pharmacological properties of these plants, including those that facilitate childbirth, to develop products adapted to human health and to make available Improved Traditional Medicines to the population and at a lower cost. Thus, the objective of this study is to assess the parturient activity of D.ramosissimum through its effect on spontaneous contractions of the uterine smooth muscle.

MATERIAL AND METHODS

Material

Plant material and preparation of the extract

The plant material consisted of D.ramosissimum plants collected in July 2019 in the prefecture of Agoè-Nyivé, 15 km from Lome (Togo). The plant has been identified and deposited under the number TOGO15658 in the herbarium of the Faculty of Science of the University of Lomé. The leaves, stem bark, and roots were then removed, dried, crushed, and stored in jars protected from light and humidity. Fifty grams (of vegetable powder from the various organs of D.ramosissimum were macerated in 500 ml of a mixture of water and ethanol 95° (50/50, v/v). The preparation was macerated at room temperature for 72 h, protected from light. After filtration through Whatman N° 1 filter paper, the macerates were evaporated to dryness under vacuum using a rotary evaporator of mark HEIDOLPH and then dried in an oven at 40°C for 24 h. The soluble dry extracts obtained were weighed and stored in tubes at a temperature of 4°C and protected from light until use.

Animal material

The experiments were carried out on female rats’ pubescent of Wistar strains, weighing between 110 g and 180 g, reared in the animal house of the Department of Animal Physiology of University of Lome. They were given free access to water and food and were subjected to a regular cycle of 12 h of light and 12 h of darkness.

Pharmacological tools and experimental device

Ethanol, DPPH, gallic acid, sulfuric acid, sodium hydrogenphosphate, ammonium molybdate, ascorbic acid, Krebs–Henseleit solution, and various laboratory reagents (methanoic solution of DPPH, Phosphomolybdate reagent) were used for this study. The recording of uterine contractions is made possible thanks to a device consisting of a 10 ml organ vat (SA-068, UGO BASILE), a sensor equipped with an amplifier and a transducer (BIOPAC Systems Inc., MODEL MP100A, Santa Barbara, California) coupled to a microcomputer controlled by the Acq Knowledge III MP100 software. The absorbance was read using the UV-Visible spectrophotometer (METASH, UV-5200 PC) fitted with data acquisition software such as MetaSpec Pro.

Methods

Preparation of the organ

The female rats were sacrificed under ether anesthesia. The uterine horny was quickly removed, cleaned of adhesions, and cut into sections 5 mm long and 3 mm wide. These shreds were then mounted in an organ vat containing Krebs-Henseleit physiological solution (in millimoles): NaCl 118; KCl 4.7; CaCl2 2.5; KH2PO4 2.5; MgSO4 1.2; NaHCO3 25; and glucose 11; pH 7.4. The organ was fixed longitudinally with a wire in the organ vat and subjected to a preload of 1 g. It is then continuously oxygenated and then maintained at a temperature of 37°C.

Effect of the extract on spontaneous contractions of the uterus

For each extract a uterus shred was mounted in the organ vat containing Krebs-Henseleit solution and left for 45 min. During this time, the shred was washed 3 times to restore balance. After equilibration and stabilization of spontaneous contractions, different concentrations of extract at 100 and at 200 mg/ml were added to the vat cumulatively with 10 min interval. For the initial concentration of 100 mg/ml, the extract volumes of 5; 10; 20 and 40 μl were added to the vat; which corresponds respectively to the final concentrations of 0.05; 0.1; 0.2 and 0.4 mg/ml into the vat. For the initial concentration of 200 mg/ml, the extract volumes of 40; 80; 160 and 320 μl were added to the vat; which corresponds respectively to the final concentrations of 0.8; 1.6; 3.2; and 6.4 mg/ml into the vat.[9]

Evaluation of the anti-free-radical activity of D. ramosissimum

DPPH free-radical scavenging test

The determination of the anti-free-radical activity by the DPPH test was carried out according to the method described by Molyneux.[10] A methanoic solution of DPPH˙ was prepared by dissolving 4 mg of this product in 100 ml of methanol. Then, to 250 μl of extract at the concentrations indicated was added 3 ml of the DPPH solution. The extracts of the different parts of D. ramosissimum as well as the reference (gallic acid) were tested at different concentrations varying from 0 μg/ml to 90 μg/ml, and then the absorbance’s were read at a wavelength of 517 nm after 30 min incubation in the dark. The results were expressed as percent inhibition (%), according to the following formula:

Percentage of anti free radical activity=Control absorbance Sample absorbanceControl absorbance×100   1

The IC50 values were determined graphically by linear regression.

Molybdate ion reduction test

The reducing power of the molybdenum ion (Mo6+) of the extracts was determined according to the phosphomolybdate reduction method described by Prieto et al.[11] One milliliter of extract of 30 mg/ml concentration was added to 9 ml of phosphomolybdate reagent. The solution was brought to a water bath at 95°C for 90 min, and then allowed to cool to room temperature. The absorbance was read at the wavelength of 695 nm using the UV-Visible spectrophotometer. Ascorbic acid was used as a standard. The equation of the calibration curve obtained from successive dilutions of ascorbic acid was used to determine the results in mg Ascorbic Acid Equivalent/g of dry extract.

Preliminary phytochemical screening of D. ramosissimum

The different chemical groups such as flavonoids, alkaloids, tannins, and saponins have been characterized with reference to the techniques described by the previous studies.[12,13]

Statistical analysis of data

Results were presented as the mean with the Standard Error of the Mean (M ± S.E.M). They were processed using Graph Pad Prism 8 software (Graph Pad Software Inc. California) which was also used to construct the curves and histograms. Analysis of variance followed by Dunnett’s test was used to compare the means of the data from the treated rats compared to those from the controls. The differences between the results were considered significant at the 5% level (P < 0.05).

RESULTS

Effect of the extract on spontaneous contractions of the uterus

Effects of the leaf extract on uterine smooth musculature

Figure 1a shows the effects of different concentrations of D.ramosissimum leaf extract on spontaneous myometer contractions. The result indicates that the extract has no effect on the amplitude of spontaneous contractions of uterine smooth muscle [Figure 1b]. However, at 0.2 and 0.4 mg/ml, the extract significantly (P < 0.05) reduced the frequency of spontaneous contractions of the uterine smooth musculature [Figure 1c]. The results show percentages of frequency reduction of 54.60 ± 7.80% and 44.29 ± 2.97%, respectively, at the concentrations of 0.2 and 0.4 mg/ml of extract compared to the control estimated at 100%. D.ramosissimum leaf extract, therefore, reduces the frequency of spontaneous Myometer contractions without affecting their amplitude.

Effects of stem bark extract on uterine smooth musculature

Figure 2a shows the effects of different concentrations of D.ramosissimum stem bark extract on spontaneous Myometer contractions. The 0.2 and 0.4 mg/ml extract produced non-significant (P > 0.05) decrease in the amplitude of spontaneous uterine smooth muscle contractions [Figure 2b]. However, from 0.1 to 0.4 mg/ml, the extract significantly (P < 0.05) reduced the frequency of spontaneous contractions of the uterine smooth musculature [Figure 2c]. A reduction in the frequency of Myometer contractions of 66.07 ± 8.93% was thus observed; 39.29 ± 10.72% and 19.65 ± 5.36%, respectively, at concentrations of 0.1; 0.2; and 0.4 mg/ml of extract compared to the control which is estimated at 100%. D.ramosissimum stem bark extract then reduces the frequency of spontaneous Myometer contractions without significantly affecting their amplitude.

Effects of root extract on uterine smooth musculature

Figure 3a shows the effects of different concentrations of D.ramosissimum root extract on spontaneous Myometer contractions. At low concentrations, the extract had no effect on the amplitude and frequency of spontaneous uterine smooth muscle contractions [Figures 3b and c]. Furthermore, at a concentration of 3.2 mg/ml of the extract, a reduction in spontaneous contractions of the Myometer [Figure 3d] was observed, as well as a non-significant decrease (P > 0.05) in amplitude [Figure 3e] and a significant decrease (P < 0.05) in the frequency of myometer contractions [Figure 3f]. The results show a percentage reduction of the frequency of 55 ± 5% at 3.2 mg/ml of extract compared to the control which is estimated at 100%. D.ramosissimum root extract also causes, as in the case of leaves and bark, a reduction in the frequency of spontaneous contractions of the uterine smooth musculature without affecting their amplitude.

Figure 1:: Effect of Desmodium ramosissimum leaf extract on spontaneous Myometer contractions. Reduction of spontaneous contractions of the uterine muscle from the concentration of 0.1 mg/ml extract (a). The arrows indicate the time of addition of the extract to the contents of the organ vat. Histograms showing the non-effect of the leaf extract on the amplitude of spontaneous Myometer contractions (b) and its effect on reducing the frequency of spontaneous Myometer contractions (c). Results are presented as the mean±S.E.M (n=4). Significantly different from the control: *P<0.05; **P<0.01 (Analysis of variance followed by Dunnett).

Antioxidant activity of D. ramosissimum

DPPH radical trapping test

The IC50 values for gallic acid and the hydro-ethanolic extracts of the organs of D.ramosissimum are shown in Figure 4. From the IC50 obtained, it is observed that the hydro-ethanolic extract of the leaves exhibits the lower IC50 (77.53 10-6 mg/ml), followed by that of roots (90.83 10-6 mg/ml) and stem bark (103.6 10-6 mg/ml). The estimate of this antioxidant activity shows that the leaf extract has remarkable reducing power for the DPPH radical scavenging test.

Molybdate ion reduction test

The IC50 values for ascorbic acid and the hydro-ethanolic extracts of the organs of D.ramosissimum are shown in Figure 5. From the IC50 obtained, it can be seen that the hydro-ethanolic extract of the leaves exhibits lower IC50 (2.021 mg/ml), followed by that of stem bark (8.643 mg/ml) and roots (14.126 mg/ml). The leaves, therefore, have greater antioxidant activity than the stem bark and roots. However, its antioxidant activity is low compared to ascorbic acid (1.201 mg/ml).

Preliminary phytochemical screening of D. ramosissimum

Table 1 presents the results of the qualitative phytochemical tests of D.ramosissimum organs. These tests revealed that the hydro-ethanolic extract of the leaves and bark of the stems of D.ramosissimum do not contain flavonoids, unlike the roots which do. On the other hand, these different organs contain tannins, saponosides, and alkaloids.

Figure 2:: Effect of Desmodium ramosissimum stem bark extract on spontaneous Myometer contractions. Reduction of spontaneous contractions of the uterine muscle from the concentration of 0.1 mg/ml extract (a). The arrows indicate the time of addition of the extract to the contents of the organ vat. Histograms showing the non-effect of bark extract on the amplitude of spontaneous Myometer contractions (b) and its effect on reducing the frequency of spontaneous Myometer contractions (c). Results are presented as the mean±S E M. (n=4) values. Significantly different from the control: *P<0.05; **P<0.01 (Analysis of variance followed by Dunnett).
Table 1:: Results of the phytochemical screening of Desmodium ramosissimum.
Chemical groups Flavonoids Saponosides Tannins Alkaloids
Leaves + + +
Stem bark + + +
Roots + + + +

+: Presence, –: Absence.

DISCUSSION

The use of medicinal plants in the treatment and management of pathologies is a perpetual practice of traditional medicine despite the spectacular advances in modern medicine.[14] Indeed, the parturition is a process that involves several factors such as hormonal factors, but also and above all, the dynamics of uterine smooth muscle.

Hydro-ethanolic extracts of leaves, stem bark, and roots of D.ramosissimum caused a reduction in the frequency of spontaneous contractions of the uterine muscle. This reduction in the frequency of spontaneous contractions of the Myometer shows that these extracts have relaxing effects on the contraction of uterine smooth muscle. The effect of hydro-ethanolic root extract on the frequency of uterine contractions is less pronounced than that of leaves and stem bark. This difference is due to the active ingredient which is probably more or less important in the roots than in other parts of the plant. The preliminary phytochemical screening showed the presence of tannins, saponosides, alkaloids, and flavonoids in the roots with an absence of flavonoids in the hydro-ethanolic extracts of the leaves and the bark of the stems. Hooper et al.[15] were able to identify the presence of C-glycosyl flavonoids, tannins, coumarins, and alkaloids in the roots of D.ramosissimum. Alli et al.,[16] them, have identified flavonoids, tannins, and phenols in the aqueous extracts of the leaves. This difference could, therefore, be explained by the type of solvent used. Caffeine, an alkaloid extracted from plants, inhibits the contractile activity of the Myometer,[17] as does yohimbine, another plant alkaloid.[9] In fact, alkaloids constitute a group of secondary metabolites contained in plants which have an inhibitory effect on spontaneous contractions of uterine smooth muscles.[18] At the same time, studies have shown that there are various subclasses of alkaloids which contain different chemical structures that contribute to the activity of the alkaloids as both stimulants and inhibitors of uterine contractions.[19] The reduction of the frequency of spontaneous contractions of the Myometer in the presence of various extracts of D.ramosissimum is probably due to the presence of alkaloids in all three organs. Tannins and flavonoids are also chemical compounds that affect the availability of intracellular calcium for uterine smooth muscle contraction.[20] The presence of tannins in the different extracts should allow an increase in spontaneous contractile activity of uterine smooth muscle. The more pronounced effect of hydro-ethanolic extracts from the leaves and stem bark on the frequency of uterine contractions could be explained by an imbalance in the stimulant/inhibitor balance in favor of the inhibitory compounds. On the other hand, the less pronounced effect of the hydro-ethanolic extract of the roots on the frequency of uterine contractions would be due to the reduction of this imbalance in the stimulant/inhibitor balance.

Figure 3:: Effect of Desmodium ramosissimum root extract on spontaneous contractions of the myometrium. The extract low concentrations have no effect on spontaneous contractions of the uterine muscle (a) but in high concentrations, it reduces these spontaneous contractions of the Myometer (d). The arrows indicate the time of addition of the extract to the contents of the organ vat. Histograms showing the non-effect of the root extract at low concentrations on the amplitude (b) and on the frequency (c) and at high concentrations on the amplitude (e) of spontaneous contractions of the Myometer, then the reduction in the frequency of spontaneous contractions of the Myometer in the presence of the extract (f). The results are presented as the mean±S E M. (n=4). Significantly different from the control: *P<0.05 (Analysis of variance followed by Dunnett).
Figure 4:: Gallic acid calibration line (a) and histograms showing the IC50 values determined by the DPPH test (b).
Figure 5:: Calibration line for ascorbic acid (a) and histograms showing the IC50 values determined by the molybdenum ion reduction test (b).

Bade et al.[8] pointed out in an ethnobotanical study that the leafy stem of D.ramosissimum is traditionally used to treat complications of delivery. Indeed, to have a good delivery four conditions are needed, including a correct uterine dynamic[21] which allows the expulsion of the placenta. The decrease in the frequency of uterine contractions, by the bark of the stem, the leaves and even the roots, shows that our results are opposed to those of the ethnobotanical investigations carried out in Benin by Bade et al.[8] Djoumbissie et al.[22] isolated from whole plant methanolic extract nine (9) secondary metabolites including b-sitosterol which increases contractility of uterine smooth muscle in rats.[23] These results, which confirm the ethnobotanical study by Bade et al.[8] and contradict our results would be explained by the type of extraction used in our study, namely, hydro-ethanolic extraction. This is because the extraction and isolation of b-sitosterol begins with maceration in methanol[24] and not in ethanol. In addition, the form in which the plant is traditionally used, in decoction and not in the hydro-ethanolic form and probably in combination with other medicinal plants, would explain the relaxing effect observed on the contraction of the uterine smooth musculature. The decoction would contain the compounds involved in the contraction of the uterine muscle. Our results show that the hydro-ethanolic extracts of the bark of the stem, leaves, and roots of D.ramosissimum can be used during threats of preterm birth to allow the uterine muscle to regain its relative quiescence.

Stress is a commonly cited factor in reproductive failure such as miscarriages, preterm births, and inhibition of fetal development.[25] A compound’s antioxidant activity is its ability to resist oxidation. Among the various methods used to assess antioxidant activity, we used the DPPH radical scavenging method and the molybdenum ion reduction method. The results obtained showed a lower IC50 for leaves 77.53 10-6 mg/ml) followed by that for roots (90.83 10-6 mg/ml) and bark (103.6 10-6 mg/ml) with the method of trapping the free-radical DPPH. The results observed with the molybdenum ion reduction method also showed that the leaf extract exhibited the best anti-free radical activity with an IC50 of 2.021 mg/ml, followed by those of the bark (8.643 mg/ml) and the roots. (14.126 mg/ml). By comparing the IC50 of the hydro-ethanolic extract of the leaves of D.ramosissimum compared to that of the reference antioxidants (Gallic acid and ascorbic acid), we notice that the capacity of the scavenging of the free-radical DPPH and the reduction of the molybdenum ion by this extract are relatively interesting and close to what it is with gallic acid (60.2 10-6 mg/ml) for DPPH and ascorbic acid (1.201 mg/ml) for the ion molybdenum. These anti-radical activities are probably due to the phenolic constituents.[26] Saker et al.[27] in their studies have shown high levels of oxidative stress markers during normal pregnancy. The perinatal period and childbirth are critical times for maintaining a balance between free-radical production and antioxidant protection for the fetus and newborn. Indeed, childbirth represents a significant oxidative stress for the fetus which passes from a hypoxic intrauterine space to a normo-oxic environment. Our results justify the traditional use of D.ramosissimum in the fight against oxidative stress to prevent miscarriages or premature deliveries.

CONCLUSION

Traditional medicine makes use of medicinal plants for the treatment of various pathologies including those which affect parturition. Hydro-ethanolic extracts from the leaves, stem bark, and roots of D.ramosissimum significantly reduce the frequency of spontaneous uterine contractions in female rats of the Wistar strain. This herb can be used in the fight against the threats of preterm labor. The present work revealed the existence of secondary metabolites such as flavonoids, alkaloids, saponosides, and tannins in the organs of D.ramosissimum used; which justifies its pharmacological properties. The results thus justified the traditional use of this plant. The leaves showed better antioxidant activity and a better result in moderating uterine contractions. This observation makes it possible to advise the use of the leaves instead of the whole plant, for the safeguarding of the biodiversity. This study being a preliminary study aiming at contributing to the valorization of the therapeutic use of D.ramosissimum merit to be thorough by other studies such as the fractionation of the extracts and the evaluation of the activity of these fractions on the uterine smooth musculature, the insulation and the identification of the chemical compounds contained in the various bodies of the plant by techniques of HPLC and NMR and the research of the mechanisms of action of the various bodies of this plant on the contraction of the smooth musculature of the uterus.

Declaration of patient consent

Patient’s consent not required as there are no patients in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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