Antibacterial and antispasmodic activities of a dichloromethane fraction of an ethanol extract of stem bark of Piliostigma reticulatum.

OBJECTIVES: This study presents the antispasmodic and antibacterial properties of an ethanol extract and fractions the of stem bark of Piliostigma reticulatum.
MATERIALS AND METHODS: The antispasmodic effects of the extract and its fractions were performed on isolated rabbit duodenum. The antibacterial properties were determined as minimal inhibitory and bactericidal concentration of the extract and fractions of P. reticulatum on susceptible and resistant strains of Escherichia coli, Vibrio cholerae, Staphylococcus aureus, Shigella dysenteriae and Salmonella tiphymurium.
RESULTS: The ethanol extract of P. reticulatum and fractions (except for heptane) produced concentration-dependent relaxant effects on isolated duodenum preparations. The IC50 of the extract and dichloromethane, ethyl acetate, butanol and aqueous fractions are 0.88452, 0.2453, 0.2909, 0.3946 and 0.3231 mg/ml respectively. The extract was found to significantly antagonize acetylcholine-induced contraction. The susceptible strains E. coli and V. cholerae were the most inhibited by the dichloromethane fraction at 60 mg/mL, as shown by their diameter of inhibition of 13.2 ± 0.76 and 13.3 ± 0.67 mm respectively. Conversely, the dichloromethane fraction, the most active antibacterial fraction, did not inhibit the resistant strains S. dysenteriae and S. tiphymurium.
CONCLUSION: The results showed that P. reticulatum stem bark possesses spasmolytic and antibacterial properties and this may contribute to its traditional medicinal use for the treatment of diarrhea.

Despite important progresses made in controlling major infectious diseases, infectious diarrhea remains one of the major causes of death of children under the age of 5 years in developing countries.[12] The search for new antimicrobial agents is necessary, due to the appearance of microbial resistance and occurrence of fatal opportunistic infections.[3] Chemotherapy is the main approach in the treatment of bacterial infections but in clinical treatment antibiotics encounter a major problem of resistance and this results in treatment failure.[24] Moreover these antibiotics are expensive and often produce several severe side-effects. One way to prevent antibiotic resistance of pathogenic species is by using new compounds that are not based on existing synthetic antimicrobial agents, but on natural drugs from medicinal plants.[567] Medicinal plants contribute to a significant proportion of pharmaceutical products in current used medicines derived from plants.[89] Thus around the world medicinal plants containing many compounds with antibacterial activity against Gram-positive and Gram-negative bacteria have been reported.[2810]

Piliostigma reticulatum which is the object of our study is traditionally used in Côte d’Ivoire for the treatment of diarrhoea.[1112] In our previous study, we showed that an ethanolic extract of the stem bark of P. reticulatum significantly reduced the gastrointestinal transit, the number, volume and weight of faeces of rats.[12] Again following this folkloric medicinal use, Babajide et al.[13] isolated piliostigmol (6-C-methyl-2-p-hydroxyphenyloxychromonol), from P. reticulatum, which inhibited Escherichia coli with an MIC of 2.57 μg/ml.

Aside bacterial infections, hyper contractility can also produce diarrhea. Treatment of spastic motility disorders continues to be challenging. Therapeutic options remain limited due in part to our lack of understanding of the pathophysiology and significance of these disorders.[14] Antispasmodics are drugs used to relieve or prevent smooth muscle spasms. By reducing the intestinal hypercontractility of smooth muscles, these drugs allow the gastrointestinal muscle to return to their proper tone, therefore reducing many of abdominal pains and symptoms.[15] Hence, antispasmodics are frequently prescribed for a number of gastrointestinal diseases, including irritable bowel syndrome, a condition which affects 10-25% of the general population.[1617] The main antispasmodic drugs include antimuscarinic compounds (e.g. the alkaloids derived from the plant belladonna and their synthetic derivatives) and calcium channel blockers (e.g. otilonium and pinaverium).[1718] Since the use of these drugs may be associated with the appearance of unwanted side effects (for example, dry mouth and urinary retention for antimuscarinic drugs, headache, nausea, vomiting and constipation for calcium blockers), the search for safer, plant-derived antispasmodics becomes a challenging option.

Some medicinal plant species are used in traditional medicine for the treatment of gastrointestinal disorders including diarrhoea, indigestion and constipation and these effects have been validated through pharmacological investigations.[19202122] Evaluation of antispasmodic activity on isolated animal organs have demonstrated that some of these medicinal plant species possess significant and interesting spasmolytic[232425] with demonstrable mechanisms underlying their antispasmodic activity.[26272829] Extensive investigations of active antispasmodic extracts have led to the isolation of active compounds belonging to different phytochemical groups.[63031]

Our previous studies had demonstrated that P. reticulum was endowed with anti-diarrheal activity.[12] Therefore, this study aimed at investigating the spasmolytic activity of an extract of P. reticulum and isolated fractions to lend some information on the possible mechanism for its reported anti-diarrheal properties. Furthermore, the antibacterial activity of the active fraction was evaluated against susceptible and resistant bacteria.

Materials and Methods

Plant collection

Stem barks of P. reticulatum (DC.) Horscht (Ceasalpiniaceae) were collected in Abidjan (South region of Côte d’Ivoire) in October 2007. The plant was identified and authenticated by late Pr Ake-Assi Laurent, National Centre of Floristic of the University of Cocody-Abidjan, where a voucher specimen (N°18033) was deposited.

Preparation of extract and fractions

Stem barks of P. reticulatum were washed with distilled water, cleaned, cut into smaller pieces and kept at room temperature for 2 weeks. Then, they were ground into a fine powder. The powder (100 g) was extracted with 2 L of a solution of ethanol (96%)/water (80:20) for 24 h under constant stirring (this operation was repeated twice). The extract was filtered twice through cotton wool, then through Whattman filter paper (N°1). The filtrate was evaporated to dryness using a rotavapor (Buchi R110/NKE6540/2) at 45°C, and dried under reduced pressure. Percentage yield was found to be 13.6%. The dried extract is subsequently referred to as extract of PRE. The five liquid fractions (heptane, dichloromethane, ethyl acetate, butanol and water) were obtained by successive liquid–liquid fractionation to exhaustion from the crude ethanol extract as describe by Harborn[32] and Samsam-Shariat.[33]

Animals

Rabbits (weighing 1.5-1.8 kg) of both sexes were obtained from UFR Biosciences (University of Cocody-Abidjan, Côte d’Ivoire). They housed in stainless steel cages (34 cm × 47 cm × 18 cm) with soft wood shavings as bedding, fed with normal commercial pellet diet (Ivograin®, Abidjan, Côte d’Ivoire) and given water ad libitum. They were allowed to acclimate to standard laboratory temperature conditions (temperature 24-28°C, relative humidity 60-70%, and 12 h light-dark cycle) for 1 week before the experiments. They were deprived of food for at least 18 h prior to experiments but allowed free access to drinking water. The equipment usage, handling and sacrificing of the animals were performed in accordance with the European Council legislation 87/609/EEC for the protection of experimental animals.[34] The protocols for the study were approved by the Departmental Ethics Committee.

Microorganisms used

Clinical strains of E. coli, Vibrio cholerae, Staphylococcus aureus, Shigella dysenteriae and Salmonella tiphymurium coming from Public Health Laboratory of Abidjan (Côte d’Ivoire) were used. Twenty strains of which ten sensible and ten resistant for each microorganism have been used in our study.

In vitro study on isolated rabbit duodenum preparation

The experiments were carried out according to Magnus general technique.[35] The animals were sacrificed by decapitation and the duodenum was removed and cut into segments of two centimeter long. A segment of duodenum was suspended in an organ bath containing 150 mL of a Tyrode's solution with the following composition (mM): NaCl 136.89, KCl - 2.68, CaCl2 - 1.80, MgCl2 - 1.05, NaHCO3 - 11.90, NaHPO4 - 0.42 and glucose - 5.55, maintained at 37°C. The solution was aerated with a mixture of 95% O2 and 5% CO2 under a resting tension of 1 g. The preparations were connected to a transducer coupled to a paper graph. The suspended duodenum was allowed to equilibrate for 30 min. The bath was washed after testing each concentration of extract and fractions.

The inhibition of duodenum contraction was expressed as percentage of mean ± standard error of the mean from six experiments and was calculated using the following formula:

where A is the amplitude (cm) of the normal duodenum contraction and B the amplitude (cm) of the duodenum contraction induced by the extract.[36] To determine IC50 values, crude extracts and fractions were tested at various concentrations (from 0.13 to 1.32 mg/ml) in organ bath successively. The IC50 value of each sample was derived from the sigmoid dose–response curves.

Preparation of stock solution

A total of 600 mg of the dichloromethane fraction was dissolved in 10 ml of distilled water to prepare the stock solution (60 mg/ml). 1 ml of dimethyl sulfoxyde was added to the solution. 1 mg of gentamycin was dissolved in 1 mL to obtain 1 mg/ml.

Preparation of inoculum

Two or three colonies of bacteria were deducted with Pasteur pipette and introduced to a tube containing 10 ml of Mueller-Hinton (MH) solution. The mixture was incubated at 37°C during 3 to 4 h. After, 0.3 ml of this solution was deducted and diluted in 10 mL of distilled water. After homogenization to vortex the inoculum (105 germs/ml) was used to flood the agar solution of MH in petriplates. The excess of inoculum was inhaled from the agar solution and petriplates (14 cm) were dried during 15 min before the incubation at 37°C for 24 h.

Preparation of seed agar plates and antibacterial assay

Agar diffusion method (well diffusion method) was used for antibacterial activity as reported by Kavanagh;[37] Leven et al.[38] and Perez et al.[39] Wells were made in seeded agar and the test sample was then introduced directly into these wells. After incubation, the diameter of the clear zones around each well was measured and compared with the zone of inhibition of the standard antibiotic, which is the gentamycin.

Thus, nutrient agar media already prepared by dissolving 20 g of the medium in 1 L of distilled water with pH 7 and autoclaved, was allowed to cool down to 45°C and were seeded with 10 ml of prepared inoculum. Petriplates (14 cm of diameter) were prepared by pouring 75 ml of the seeded nutrient agar in each plate and were solidified. 6 wells (holes) per plate were made with sterile Pasteur pipette (6 mm of diameter). The quantity of fraction of 100 μl was putting in each well, and the petriplates were incubated at 37°C during 24 h. Simultaneously, gentamycin was used as positive control at a concentration of 1 mg/ml.

Determination of minimal inhibition concentration and minimal bactericidal concentration

The minimal inhibition concentration (MIC) is the smallest concentration of fraction or antibiotic which inhibits all growth of bacteria after 24 h of incubation. The method of dilution of Oussou et al.[40] was used. Thus the concentration of 60 mg/ml was diluted successively 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 in order to have respectively the concentration of C1 = 60 mg/ml, C2 = 30 mg/ml, C3 = 15 mg/ml, C4 = 7.5 mg/ml, C5 = 3.75 mg/ml, C6 = 1.87 mg/ml, C7 = 0.93 mg/ml and C8 = 0.47 mg/ml. These concentrations have been putting in contact with bacteria in tubes.

The determination of the minimal bactericidal concentration (MBC) is as described as follows. Twenty-four hours prior to the MBC determination, four tubes containing the inoculum, the distilled water and the fraction were used in making the dilutions of 100, 10−1, 10−2, 10−3 and 10−4 of inoculum corresponding respectively to 100%, 10%, 1%, 0.1% and 0.01% of surviving bacteria. A control of the bactericidal activity was made. After the reading of the MIC, a streak on a new agar is made from each tube without visible growth of bacteria. These were then incubated at 37°C and 24 h after which comparisons were made to the control of the bactericidal activity. The MBC will be the smallest concentration of the subculture showing a growth of bacteria ≤ 0.01%.[4142]

Phytochemical analysis of the fraction

The crude extract and the active fraction were screened for the presence of tannins, flavonoids, alkaloids, sterols, saponins, polyphenols, polyterpenes and anthraquinones. Detection of these constituents was performed according to Bekro et al.[43]

Data analysis

The IC50 (dose responsible for 50% of the maximal effect) and inhibitory effects of drugs were analyzed by using an iterative computer least squares method, GraphPad Prism for Windows version 5.0 (GraphPad Software, San Diego, CA, USA) with the following nonlinear regression (four-parameter logistic equation).

Where, X is the logarithm of concentration. Y is the response and starts at a and goes to b with a sigmoid shape.

The fitted midpoints (IC50 s) of the curves were compared statistically using F-test[4445] for the spamolytic effects. GraphPad Prism Version 5.0 for Windows (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses and IC50 determination. The Dunnett's Multiple Comparison Test of ANOVA one way was used for the determination of the antibacterial activity. P < 0.05 was considered statistically significant in all analysis. The graphs were plotted using Sigma Plot for Windows Version 11.0 (Systat Software Inc., Germany).

Results

Extraction

The amount of crude ethanol extract was 13.6%. From dried ethanol extract (starting with 10 g in 100 mL of distilled water), heptane (800 mg; 8%), dichloromethane (900 mg; 9%), ethyl acetate (1700 mg; 17%), butanol (3200 mg; 32%) and aqueous (2100 mg; 21%) fraction were obtained respectively.

Effects of the crude ethanolic extract and it constituent fractions on the isolated rabbit duodenum

Isolated duodenum suspended in Tyrode's solution under 1 g tension had a stable tension after 30 min. Results showed a concentration-dependent reduction of the spontaneous contraction by the ethanolic extract [Figure 1]. At the concentration of 0.13 mg/mL, the intestinal contraction is reduced by 15.0% ±4.3%. At concentrations of 0.26, 0.52, 0.79 and 1.04 mg/mL the contraction is significantly reduced by 61.0 ± 3.1; 68.0 ± 2.6 and 97.0% ±2.8% respectively. The spontaneous contraction was totally inhibited at the extract concentration of 1.32 mg/ml [Figure 1]. The IC50 was 0.8840 mg/ml [Table 1].

Figure 1

Dose-response effects of crude ethanolic extract (0.13-1.32 mg/mL) and its constituent fractions (0.13-0.52 mg/ml) on spontaneous contractile response of isolated rabbit duodenum preparation. Each point represents mean ± standard error of the mean (n = 6)

Table 1

Inhibitory concentration (IC50) of the crude ethanolic extract of Piliostigma reticulatum and its constituent fractions on the contractile responses of isolated rabbit duodenum

The heptane fraction had no effect on spontaneous contraction at any of the concentrations tested. The spontaneous contraction was normal and not perturbed [Figure 2].

Figure 2

Dose-response effects of heptane fraction (0.13-0.52 mg/mL) on the spontaneous contractile response of an isolated rabbit duodenum

The dichloromethane fraction induced a concentration-dependent reduction of spontaneous contraction of the duodenum [Figure 1]. At 0.13 mg/ml, the fraction slightly reduced the duodenum contraction by 15.0% ±1.3% and then significantly by 65.0% ±2.12%, 96.0% ±0.54% 100%, respectively at 0.26, 0.39 and 0.52 mg/ml [Figures 1 and 3]. The IC50 was 0.2453 mg/ml [Table 1].

Figure 3

Dose-response effects of dichloromethane fraction (0.13-0.52 mg/mL) on the spontaneous contractile response of an isolated rabbit duodenum

The ethyl acetate fraction also induced a concentration-dependent reduction of spontaneous contraction of the duodenum [Figure 1]. The contraction was reduced to 18.0% ±1.3% at 0.26 mg/mL and was significant at concentrations of 0.39 (61.0% ±2.21%) and 0.52 mg/mL (71.0% ±1.58%) [Figure 1]. The IC50 was 0.2909 mg/ml [Table 1].

The butanolic fraction also induced a concentration-dependent reduction of spontaneous contraction of the duodenum [Figure 1]. The duodenum contraction was decreased at 0.39 mg/mL (46.0% ±2.07%). This reduction of contraction was significant at 0.52 mg/ml (82.0% ±1.05%). The butanolic fraction needed a high concentration to reduce duodenum contraction [Figure 1]. The IC50 was 0.3946 mg/ml [Table 1].

The aqueous fraction also induced a concentration-dependent reduction of spontaneous contraction of the duodenum [Figure 1]. The fraction reduced duodenum contraction of 16.0% ±2.2% at 0.26 mg/ml. This reduction was significant at 0.39 mg/mL (72.0% ±3.1%) and 0.52 mg/mL (88.0% ±4.1%) [Figure 1]. The IC50 was 0.3231 mg/ml [Table 1].

Antibacterial activity

The dichloromethane fraction inhibited all bacteria except the resistant strains of S. dysenteriae and S. tiphymurium. The susceptible strains were inhibited by dichloromethane fraction of Piliostigma reticulatum (DFPr) with a diameter most high in E. coli (13.3 ± 0.67 mm) and V. cholerae (13.2 ± 0.76 mm) at the concentration of 60 mg/mL. The gentamycin, a reference antibiotic, inhibited all bacteria [Table 2]. However, DFPr inhibited weakly S. aureus and S. tiphymurium respectively with 11.1 ± 0.62 and 11.2 mm ± 0.61 mm in susceptible strains [Figure 4]. Furthermore, gentamycin inhibited significantly all susceptible and resistant strains of bacteria compared with DFPr (P < 0.05).

Table 2

Diameters of inhibition, MIC and MBC of bacteria and gentamycin

Figure 4

Effects of the dichloromethane fraction of Piliostigma reticulatum on susceptible strains of bacteria: VC - Vibrio cholerae; SD - Shigella dysenteriae; ST - Salmonella tiphymurium; SA - Staphylococcus aureus and EC - Eschérichia coli (mg/L). Data are mean ± standard error of the mean (n = 10). nsP > 0.05, ***P < 0.001 compared to gentamycin treatments (Two-way ANOVA followed by a Bonferroni's post-hoc test)

The resistant strains of V. cholerae had the highest MIC with 15 mg/ml [Figure 5]. For the susceptible strains, the MIC was high in S. dysenteriae (15 mg/ml). These two bacteria are so resistant to the fraction comparatively to the other bacteria. The MBC in S. dysenteriae and S. tiphymurium were high with 15 mg/ml for susceptible strains. For resistant strains, V. cholerae had the highest MBC with 30 mg/ml [Table 2].

Figure 5

Effects of the dichloromethane fraction of Piliostigma reticulatum on resistant strains of bacteria: VC - Vibrio cholerae; SD - Shigella dysenteriae; ST - Salmonella tiphymurium; SA - Staphylococcus aureus and EC - Eschérichia coli (mg/L). Data are mean ± standard error of the mean (n = 10). ***P < 0.001 compared to gentamycin treatments (Two-way ANOVA followed by a Bonferroni's post-hoc test)

The quotients of MBC/MIC for each bacterium were lower to 4. This result means that our fraction possesses bactericidal power.

Phytochemical screening tests of the crude extract and the active fraction (dichloromethane fraction) for various constituents revealed the presence of major components such as tannins and flavonoids. Polyphenols and reducing sugars were present, and anthraquinones, alkaloids, coumarins, polyterpenes and sterols were absent.

Discussion

The present study sought to assess the spasmolytic activity of P. reticulatutum and also investigate to find the constituent fraction (s) responsible for the spasmolytic effect of the crude ethanol extract.

Our study showed that the crude ethanol extract significantly inhibited the duodenum contraction. A similar activity was also observed for all fractions except for the heptane fraction. The relative potency of the dichloromethane-, ethyl acetate-, butanol- and aqueous- fractions indicated that these fractions contain phytoconstituents which were responsible for the inhibitory action of P. reticulatum on the isolated rabbit duodenal contraction. Comparison of the IC50 values showed that dichloromethane-rich fraction (IC50 = 0.2453 mg/ml) is more potent than the ethyl acetate-rich fraction (IC50 = 0.2909 mg/ml), butanol-rich fraction (IC50 = 0.3946 mg/ml) and aqueous-rich fraction (IC50 = 0.3231 mg/ml). The descending order of potency, as illustrated as FDCM > FEAc > FBut > FW > FHept, may suggest that the dichloromethane fraction of the crude extract had the highest concentration of the phytochemical principle(s) responsible for the spasmolytic effects observed.

Diarrhea acts by various mechanisms. Often these mechanisms involve gastrointestinal disorders. Therefore, we can assume that the anti-diarrheal action of ethanol extract of P. reticulatum could be mediated by a mechanism involving the decrease of gastrointestinal mobility which results of inhibition of duodenum spontaneous contraction.

Our study sought to assess the antibacterial activity of P. reticulatutum and also search for the constituent fraction(s) responsible for the antibacterial effect of the dichloromethane fraction of ethanol extract of this plant. The susceptible strains were inhibited with a diameter most high in E. coli (13.3 ± 0.67 mm) and V. cholerea (13.2 ± 0.76 mm) at the concentration of 60 mg/ml. The resistant strains of V. cholerae had the highest MIC with 15 mg/ml. For the susceptible strains, the MIC had high in S. dysenteriae (15 mg/ml). And, the MBC in S. dysenteriae and S. tiphymurium were high with 15 mg/ml. The studies of dichloromethane fraction on microorganisms responsible of diarrhea have shown that it possesses antibacterial activity. Thus, all the susceptible strains have been inhibited. As for to resistant strains alone the Shigella and the Salmonella have resisted to plant fraction. A molecular, the piliostigmol isolated in P. reticulatum has shown an antibacterial activity in E. coli with a minimal inhibitory concentration of 2.57 μg/ml.[13] Also, others studies realized by Akinsinde and Olukoya,[46] have shown that aqueous extract of P. reticulatum leaves hadn’t inhibited the V. cholerae. The roots of P. reticulatum seemed not have effect on the Salmonella and E. coli.[47]

The phytochemical screening of stem bark of P. reticulatm showed that tannins and flavonoids are the major components while polyphenols and reducing sugars were minors. The result also showed the absence of sterols, polyterpenes, saponins, alkaloids, quinons, and coumarins. These components observed could be responsible of spasmolytic and the antibacterial activity of P. reticulatum.

Our results are in consonance with many studies in literature. It was reported that flavonoids,[2348] tannins, reducing sugars[49] ethyl acetate, butanol, aqueous extracts,[50] were responsible of spasmolytic properties of certain plants like Pynocycla spinosa, Morinda morindoides, Ficus sycomorus. The spasmolytic activity of flavonoids has been demonstrated and attributed to their ability to inhibit gastrointestinal mobility.[5152] In vitro experiments on animals have shown that flavonoids are able to inhibit the contractions induced by spasmogens.[5354] The spamolytic activity of P. reticulatum could therefore due be to the presence of flavonoids, tannins, polyphenols and reducing sugars contained in its crude ethanol extract. Further studies on the phytochemical distribution in the fractionated components of the crude ethanolic extract may be able to suggest the phytochemical principle(s) responsible for the observed effects of the extract.

The antibacterial activity of some tannins and flavonoids has been demonstrated by various researchers.[36555657585960] The antimicrobial activity of some isoflavonoids has been already studied.[61] For example, the C-méthyl flavonols of Piliostigma thonningii, plant species of similar chemical composition as P. reticulatum has also shown an antibacterial activity on E. coli and S. aureus at concentration of 20 mg/ml.[62] The antibacterial activity of polyphenols has been putting in highlight in Terminalia glaucescens by Bolou.[42]

In our previous study,[12] we demonstrated that P. reticulatum possessed antidiarrhoeal activity by inhibiting diarrheal feces and gastrointestinal mobility. This study can confirm the previous study by inhibiting intestinal contraction and bacteria responsible of diarrhea by a DFPr. The results could justify the use of P. reticulatum by rural populations to treat diarrhea.

Conclusion

Ethanolic extract of P. reticulatum possesses spasmolytic activity on isolated rabbit duodenum preparation and this activity lies in the dichloromethane, ethyl acetate, butanol and aqueous fractions of the extract and absent in the heptane fraction. Furthermore, our results show that the stem bark of P. reticulatum contains bioactive natural substances with antibacterial properties. These properties could be due to chemical constituents observed in crude extract and dichloromethane fraction such as tannins, flavonoids and polyphenols. Also these results justify the use of P. reticulatum in diarrhea management by traditional healers. However, further bioassay-guided fractionation studies are required to identify the active principle(s) and their mechanism of action. Moreover, the use of aerial parts of the plant could represent an alternative to the utilization of its roots, therefore limiting the biodiversity degradation.