Phytochemical Characterization and Evaluation of the Antibacterial Activity of Leaf Extracts of Mitragyna inermis (Willd.) O. Ktze on the in Vitro Growth of Clinical Strains Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa Involved in Gastro Enteritis ()
1. Introduction
Traditional medicine has experienced renewed interest in recent years and occupied an important place in the health policy of several developing countries. According to the World Health Organization (WHO), nearly 80% of African populations use traditional medicine for primary health care [1] . These populations, mainly rural, use traditional medicine as an alternative to problems of accessibility to care, medications and the high costs of health services. The reasons linked to the phenomena of multi-resistance of pathogens to active substances [2] [3] [4] and the manifestation of severe side effects, even toxic in certain cases, encourage the use of traditional medicine [5] [6] . Certain molecules such as metronidazole, widely prescribed in the case of intestinal disorders, cause headaches and nausea. It is believed to be carcinogenic and teratogenic [7] . Non-steroidal anti-inflammatory drugs manifest side effects such as lesions, gastrointestinal irritation and renal toxicity while the use of opiates is reserved for severe pain because of their adverse side effects (respiratory depression, dependence, sweating) [8] . The consequences of the development of pharmaceutical industry have led researchers to undertake work relating to the pharmacological actions of medicinal plants [9] . It is in this context that we got interested in Mitragyna inermis used in traditional medicine to treat several pathologies. Indeed, M. inermis is a species widely used by healers, but differently depending on geocultural access [9] . The barks are often prescribed for gravida-puerperal conditions, stomach aches, dysentery, bilharzia, diabetes and urinary pain [10] . It is an antimalarial, an antispasmodic, a cardiotonic and anticholesteremic. The most used parts are the leaf and the bark of the trunk, removed by scraping the epidermis and subterm, which would be, after drying and reduction into powder, a healing agent for large wounds [10] .
2. Material and Methods
2.1. Material
2.1.1. Plant Material
The plant material consists of powdered leaves of Mitragyna inermis (Willd.) O. Ktze. Harvested in Boyo commune of Kouto, Bagoué region (northern Ivory Coast) and identified at the National Floristic Center of the Felix Houphouët Boigny University of Abidjan.
2.1.2. Reagents and Extraction Solvents
The developers and reagents used for phytochemical screening in tubes and by Thin Layer Chromatography (TLC) are: Sulfuric vanillin, Dragendorff (KOH) at 5% (AlCl3) at 1%, (FeCl3) at 2%. Distilled water and 96% ethanol were used as extraction solvents.
2.2. Methods
2.2.1. Harvest of the Plant
The leaves of Mitragyna inermis were harvested, then washed and then dried in ambient air at laboratory temperature. The dry leaves were ground using an electric grinder until a fine powder was obtained.
2.2.2. Preparation of Total Extracts
The extractions by maceration (aqueous and ethanolic) were carried out according to the method described by [11] and the decoction was made according to [12] .
2.2.3. Preparation of Selective Extracts
A volume of 20 mL of each total extract was exhausted by successive fractionations with (3 × 10 mL) hexane (C6H14), dichloromethane (CH2Cl2) and ethyl acetate (AcOEt). The different selective organic fractions were concentrated in an oven then stored in the refrigerator at 4°C. These fractions were used for phytochemical screening [13] .
2.2.4. Calculation of Extraction Yield
The total extracts (aqueous, decocted and ethanolic) were weighed in order to calculate the yield (Yield) of each extraction according to the formula below and then reduced into percentage.
2.2.5. Phytochemical Characterization of Extracts
The screening of secondary metabolites of the extracts was carried out on the one hand by means of coloring tests in test tubes according to the analytical techniques described by [14] , [15] [16] and on the other hand on TLC plate following the methods described by [17] .
2.2.6. Evaluation of Antibacterial Activity
1) Preparation of culture medium
The preparation of TBX, EPT Baird Parker, Cetrimide agar, Nutritive Agar and Müller-Hinton culture media was carried out following the manufacturer's instructions.
2) Verification of the purity of bacterial species
Under sterile conditions, a characteristic colony isolated on the specific culture medium is taken using a platinum loop then spread in streaks on a dish containing the nutrient agar. The box is then incubated at 37˚C for 24 hours.
3) Preparation of the inoculum
Using a Pasteur pipette, a colony aged 18 to 24 hours is taken and placed in a test tube containing 10 mL of sterile physiological water. The mixture is compared to the Mac Farland 0.5 control. This solution constitutes the 100% stock solution.
4) Inoculum counting
The enumeration of the inoculum is carried out by a 10th dilution from stock solution 100. The different decimal dilutions obtained as well as the stock solution 100 are inoculated using a calibrated loop of 2 μL by radial streaks on an agar Mueller Hinton antimicrobial free. This box constitutes box A. It is incubated at 37˚C for 24 hours.
5) Preparation of the concentration range of extracts
The concentration range was prepared according to [18] .
The concentration range of the plant extract from 64 mg/mL to 4 mg/mL was prepared in screw test tubes using the double dilution method. Thus, five (5) test tubes marked T1 to T5 were used. The first tube (T1) contains 10 mL of distilled water to which 0.6 g of plant extract has been added. The second tube (T2) contains 5 mL of distilled water as do all the other tubes (T3 to T5). The contents of the tube marked T1 are shaken vigorously mechanically. When the mixture becomes homogeneous, half of the contents of this tube (5 mL) is transferred to tube T2. The latter is also stirred in the same way as before and half of the content is transferred to tube T3 and so on just as at T5. Half of the content of the last tube is discarded into a jar provided for this purpose. The range thus prepared was sterilized in an autoclave at 121°C for 15 min.
6) Seeding the concentration range of extracts
Seeding the concentration range is carried out by adding 1 mL of the content of each tube of the concentration range to 1 mL of the inoculum (100% stock solution), around the Bunsen burner flame.
The Growth Control (TC) tube will contain 2 mL of the inoculum.
The Sterility Control (TS) tube will contain 2 mL of sterile culture medium.
7) Sensitivity test
The sensitivity of the strains to plant extracts was carried out by the diffusion technique in agar medium. Mueller Hinton medium was inoculated by swabbing. Using a sterile cookie cutter, wells of approximately 6 mm in diameter are made in the 4 mm thick agar. Each well received 80 µL of the substance to be tested at concentrations 16 and 8 mg/mL. After 15 min of diffusion at laboratory temperature, the Petri dishes are incubated at 37˚C for 18 to 24 h. The presence or absence of an inhibition zone was observed. The results were interpreted according to [19] .
8) Determination of the antibacterial activity ratio (MIC and CMB)
Antibacterial parameters were carried out according to [18] .
Minimum Inhibitory Concentration (MIC)
The MIC is determined from the spiked concentration range by observing the test tubes with the naked eye. Cloudy media indicates the growth of the culture and clear media is evidence that growth has been inhibited. The MIC corresponds to the smallest concentration inoculated without growth visible to the naked eye.
Minimum Bactericidal Concentration (MBC)
The MBC is determined by subculturing all experimental tubes in the seeded concentration range without growth visible to the naked eye. This subculturing constitutes box B. The CMB is defined by comparison of box A and box B. It corresponds to the smallest concentration of box B whose number of colonies is less than or equal to the number of colonies of the dilution 10-4 from box A.
2.2.7. Statistical Analysis of the Results
The results were analyzed using Statistica software version 7.1 and Microsoft Excel 2010. All data were expressed as mean ± standard deviation. The significance of the differences between the treated groups was evaluated by the Fischer test carried out at the 5% threshold. Differences were considered significant for p < 0.05.
3. Results and Discussion
3.1. Extraction Yield
The extraction yield, mass and color of the different extracts of M. inermis are presented in Table 1. The ethanolic extract obtained the highest extraction yield with 75.86% while the aqueous extract recorded the lowest extraction yield (61.8%). Ethanol would be the solvent that extracts the most compounds from M. inermis leaves compared to water.
3.2. Selective Extracts
Fractionation of the total extracts made it possible to obtain three selective extracts from each total extract. Leaching the extracts with hexane gave MAHex (hexane fraction of the total aqueous extract of M. inermis), MDHex (hexane fraction of the decocted total extract of M. inermis) and MEHex (hexane fraction of the total ethanolic extract of M. inermis). These extracts were used for the detection of sterols and terpenes. As for dichloromethane and ethyl acetate, their leaching made it possible to successfully obtain MADCM MDDCM and MEDCM and MAAcetatic MDAcetatic and MEAcetatic. These different extracts were used for the detection of secondary metabolites of interest other than sterols and terpenes.
3.3. Phytochemical Screening by TLC
3.3.1. Screening after Staining in Test Tubes
Table 2 presents the results of the phytochemical screening of the total extracts of M. inermis leaves. These results indicate the presence of polyphenolic compounds such as total polyphenols, flavonoids, tannins, saponins, sterols and terpenes, quinones and alkaloids. These compounds are more concentrated in the decocted extract compared to the aqueous and ethanolic extract. The ethanolic extract contains fewer polyphenolic compounds than the aqueous and decocted extract. It does not contain saponins and quinones. The results of this tube screening were reinforced by the thin layer chromatography method. The complexity of tube observation linked to the coloring of plant extracts sometimes reveals false positive or negative results. Detection by thin layer chromatography makes it possible to elucidate the results in test tubes. Thus, Table 3 indicates the results of the detection of tannins and phenolic acids. The gray color indicates the presence of tannins while the green color indicates the presence of phenolic acid. In view of these results, the MAAcetatic extract contains a single spot of tannins at Rf (0.95) and four spots of phenolic acids. As for the MEAcetatic extract, it contains two molecules of tannins with Rf (0.72 and 0.97) and three molecules of phenolic acid with Rf (00; 0.06 and 0.27). The MDAcetatic extract contains as many tannins as phenolic acid. MDAcetatic extract contains more tannins compared to other extracts. Mitragyna inermis leaf extracts contain more phenolic acids than tannins. The detection of alkaloids was also carried out in the visible. All the extracts showed spots of orange color on a yellow background testifying to the presence of alkaloids (Table 4). The detection of flavonoids was carried out in the visible. All extracts showed yellow-colored spots except the MEDCM extract. This extract recorded spots of green colors testifying to the presence of flavonoid at RF = 0.81 and 0.95 (Table 5). Regarding the detection of coumarins, Table 6 presents the results obtained. All the plates were found to be yellow in the visible, thus indicating the presence of coumarin. The observation of this same plate under UV λ = 366 nm, presented a blue coloring characteristic of coumarins with the MADCM extract at RF = 0.95 and with the MDDCM extract at RF = 0.25; 0.76 and 0.86.
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Table 1. Extraction yield of the different extracts of Mitragyna inermis.
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Table 2. Phytochemical characterization of Mitragyna inermis leaves.
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Table 3. Detection of tannins and phenolic acids, in the H2Cl2/AcOEt/ CH3COOH (1:3.5:1) (V/V/V) developer from acetate-ethyl extracts (AcOEt).
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Table 4. Detection of alkaloids in the CH2Cl2/AcOEt/C6H14 (2:2:1) (V: V: V) developer from dichloromethane extracts (CH2Cl2).
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Table 5. Detection of flavonoids in the developing CH2Cl2/AcOEt/C6H14 (2:2:1) (V/V/V) from dichloromethane extracts (CH2Cl2).
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Table 6. Detection of coumarins in the CH2Cl2/AcOEt/C6H14 (2:2:1) (V/V/V) developer from dichloromethane extracts (CH2Cl2).
3.3.2. Sensitivity of Germs with Regard to the Extracts
The results of the bacterial sensitivity test to the extracts are grouped in Table 7. The largest inhibition diameters were obtained at the concentration of 16 mg/mL. The germs of E. coli and S. aureus were sensitive to all extracts of Mitragyna inermis, however, P. aeruginosa was sensitive to the ethanolic extract but resistant to aqueous and decocted extracts since the inhibition diameters were less than 8 mm. At the highest concentration (16 mg/ml) and by comparison of the inhibition diameters between the germs at the level of each extract, there is no significant difference between the inhibition diameters of the aqueous extract at the level of E. coli and S. aureus. A similar observation was made between the inhibition diameters of the ethanolic extract at the level of P. aeruginosa and S. aureus. However, a comparison of the inhibition diameters of the extracts at the level of each germ gave more or less different results. There is a significant difference between the inhibition diameters of the aqueous, decocted and ethanolic extracts at the level of the germs of S. aureus and P. aeruginosa. A significant difference was also noted in the inhibition diameters of the ethanolic extract and the two other extracts at the level of E. coli. The most sensitive germ to Mitragyna inermis extracts was E. coli and P. aeruginosa was the least sensitive. All germs (E. coli, S. aureus and P. aeruginosa) were sensitive to the ethanolic extract with the largest inhibition diameters (17.87 ± 0.58 mm, 15.77 ± 1.44 mm and 15.07 ± 0.62 mm) respectively at the concentration of 16 mg/mL. These results were confirmed by the determination of the antibacterial parameters (Table 8, Table 9, Table 10). The ethanol extract showed a bactericidal effect on S. aureus and bacteriostatic effect on E. coli. As for the other extracts, they had a bactericidal effect on E. coli only. Their effects on other germs have not been determined based on the concentration range of this study. The ethanolic extract was therefore the most active extract.
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Table 7. Inhibition diameters of the leaves extracts of Mitragyna inermis.
Mean ± standard deviation of 3 trials.
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Table 8. Antibacterial parameters of the decocted extract.
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Table 9. Antibacterial parameters of the ethanolic macerated extract.
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Table 10. Antibacterial parameters of the aqueous macerated extract.
4. Discussion
The use of traditional medicine has known renewed interest in recent years and occupied a prominent place in the health policy of several developing countries. The present study showed that ethanol is the solvent that best extracts the active ingredients from the plant studied with an extraction yield of 75.86 ± 0.20%. These results are consistent with those of [20] . However, different results were obtained with water using leafy branches of Mitragyna inermis [21] . This difference may be due to the extraction method, the extraction conditions, and the chemical composition of the part of the plant studied. Phytochemical tube and thin layer chromatography screening revealed that the three extracts of Mitragyna inermis are rich in tannins, flavonoids, alkaloids, coumarin and phenolic acid. Similar results were obtained by [9] . These authors showed that polyphenols, flavonoids and catechic tannins are present in the methanolic, infused and decocted extracts but absent in the chloroform extract of Mitragyna inermis leaves. Saponosides are present only in the decoction while sterols, triterpenes and alkaloids were present in all extracts [9] .
Thin layer chromatography revealed the presence of several types of molecules from the family of tannins, flavonoids, coumarins, alkaloids, phenolic acid by the presence of several spots at various Rfs. These results help to justify that the TLC method highlights a large number of bioactive compounds present in plant extracts [13] [22] . This phytochemical profile is also comparable to that reported for Acacia nilotica extracts [23] . Tannins, flavonoids, sterols and triterpenes which are most involved in antibacterial activities [23] were highlighted in the extracts of the present study. The antibacterial analysis of Mitragyna inermis extracts showed that all the germs studied were sensitive to the ethanolic extract of Mitragyna inermis with inhibition diameters ranging from 15.07 ± 0.62 to 17.87 ± 0.58 to the concentration of 16 mg/mL. The aqueous macerated and the decoction obtained similar inhibition diameters but lower than those of the ethanolic extract. This difference may be linked to the extraction method and/or the extraction solvent used [24] [25] . The results obtained with the aqueous macerated and the decoction are substantially identical both in terms of the sensitivity of the germs and the antibacterial parameters. This indicates that the extraction temperature did not degrade the phytochemical composition of this plant. Similar results were recorded with total and freeze-dried extracts of the same plant [26] . These authors showed that Staphylococcus aureus MRSA germs were sensitive to total and hyophilized extracts of Mitragyna inermis. The difference in sensitivity observed could be due to the difference in structure of the germs. The CMB/MIC activity report showed that all Mitragyna inermis extracts exhibit bactericidal or bacteriostatic activity on the growth of E. coli and S. aureus. The antibacterial activity of the extracts may be due to the presence of flavonoids, tannins, polyphenols and coumarins. These phytochemicals act by either modifying the bacterial cell membrane, acting as an antimetabolite, or inhibiting nucleic acid, protein, and/or cell wall synthesis [27] .
5. Conclusion
This study established a scientific basis for the traditional use of Mitragyna inermis, a plant widely used in traditional medicine in the north of the Ivory Coast, in the treatment of gastroenteritis and gastric ulcers. It has allowed the characterisation of the main phytochemical groups of the extracts using two different methods to evaluate the antibacterial effects of the aqueous decocted, aqueous macerated and ethanolic macerated leaves. The findings revealed that ethanol extracts the active ingredients from M. inermis leaves better than water. Phytochemical characterization showed that M. inermis extracts contain several phytochemical compounds such as flavonoids, tannins, polyphenols, alkaloids, coumarins and sterols and terpenes. These compounds could be the basis of the antibacterial activity observed. These results could justify the use of M. inermis leaves by local populations as an alternative to primary health care, especially in cases of gastroenteritis.