Journal of Biology and Today's World

Antibacterial Activity of α and β Amyrin Isolated from Morinda lucida against Some Multidrug Resistant Enterobacteriaceae

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Introduction

Enterobacteriaceae is a family of bacteria often associated with the gut [1]. Enterobacteriaceae are responsible for a large proportion of serious, life-threatening infections and resistance to multiple antibiotics in these organisms is an increasing global public health problem [2].

Morinda lucida Benth. (Rubiaceae) is a tropical West Africa rainforest tree also called Brimstone tree [3]. The local names of the plant in Yoruba, Igbo and Hausa are Oruwo, Eze-ogu, and Huka respectively [4,5]. In many countries different parts of the plants are used in different ways. The leaves effectively treat and improve all forms of infertility in women. Brimstone tree is locally used in the treatment of irregular menstruation, insomnia and jaundice, also in the treatment of infected wounds, abscesses and chancre [6].

The aim of this study was to isolate a compound from the most active fraction of M. lucida leaves and to test its activity against some multidrug resistant Enterobacteriaceae.

Materials and Methods

Collection and identification of plants materials

Morinda lucida leaves were obtained from Ibadan, Nigeria and identified at the Herbarium Section, Department of Botany, Faculty of Life Science, ABU, Zaria and assigned Voucher numbers: 01862 for M. lucida (Family: Rubiaceae).

Preparation of plant materials

The Morinda lucida leaves specimen freshly obtained were separately washed with water and dried in a shade at room temperature to 1 kg weight. The dried plant samples were then grinded using motar and pestle into coarse powdered form. The powdered samples were stored in an air-tight sterile brown bottle within a desiccator until required for use [7].

Preparation of crude extracts

The extraction and screening was carried out using the method described by Ogbadoyi, et al. and Sankeshwari, et al. [8,9]. One kilograms (1 kg) of the dried powdered samples of M . lucida leaves was extracted with 10 L of methanol for 72 hours. Extracts were filtered using muslin cloth and solvents removed through evaporation using water bath at 40°C. The dried extracts were finally transferred into sterile sample bottles for storage at refrigerated temperature until when required for use [10].

Determination of Minimum Inhibitory Concentration (MIC)

The Minimum Inhibitory Concentration (MIC) of the extract was determined as described by Adeshina, et al. and Coker and oaikhena with little modification [11,12]. This was carried out for some resistant Enterobacteriaceae isolates at varying concentrations of 200, 100, 50, 25, 12.5, 6.25 and 3.125 mg/ml. Two (2) gram of methanol extract of M. lucida leaves was dissolved in 10 ml of sterile distilled water to give the desired concentrations of extract in milligram per millilitre (mg/ml), which were used for the MIC and MBC test. These concentrations in sterile melted Mueller Hinton agar plates were prepared using double dilution method. The solidified leaf extract-agar admixture plates were inoculated with 20 μl of standardized 18 h culture test organism on sterile discs placed on the solidified extract-agar admixture. The inocula were allowed to diffuse into the test agar plates for 30 minutes. The test agar plates were then incubated at 37°C for 18 h and the lowest concentration of the extract in the test agar plates that showed no visible growth was considered as the MIC of the extract against the test organism. An agar plate containing Mueller Hinton agar only was seeded with the test organism to serve as control.

Fractionation of the extract and elucidation of chemical constituents

Thin Layer Chromatography (TLC) of plant extracts: TLC of the plant extract was carried out. Different solvent systems based on their polarity were used. Two (2) μl of plant extract was applied to the TLC chromatogram. Solvent systems used are Hexane and Ethyl Acetate, 9:1, 8:2, 7:3, etc. Individual Rf for each spot was measured. TLC spots were visualized under UV light and adequate TLC reagents were used.

Fractionation was carried out using column chromatography with the best solvent system from the TLC profiling. Chemical elucidation was carried out using FTIR, NMR (H, ¹³C, and 2D).

Column chromatography: The column chromatographic method as described by Ewansiha, et al. with little modification was used to separate the fractions of the methanol extracts [13]. The column was prepared by plugging a Pasteur pipette with a small amount of cotton using a simple dry-pack method and with a wood applicator stick it was tamped down lightly. One hundred milligram silica gel was added as the stationary phase. The column was next pre-eluted by the addition of solvent (hexane) and it was allowed to flow slowly down the column by gravity. The column was loaded with the sample by the dry method which involves dissolving 5 g of the extract mixed with silica gel in a solvent (methanol), allowed to air-dried, crushed with sterile pestle and in a crucible before addition. It was then eluted as necessary by introducing more solvent (starting from the less polar solvent hexane 100%, hexane/ethyl acetate; ethyl acetate 100%; ethyl acetate/methanol to more polar solvent methanol) through the column which also prevents the silica gel from going dry. The fractions were collected in bottles according to their colour development.

Thin Layer Chromatography (TLC) of the pulled fractions: The different fractions obtained were pulled together based on their TLC profile (fingerprint). Thin layer chromatography was used, in which the TLC plate was spotted with the different fractions obtained and developed with different solvent systems in a chromatographic tank.

Antimicrobial analysis of M. lucida fractions and the isolated compound: Antibacterial susceptibility was tested using the modified Agar well diffusion method as used by Adeshina, et al. and Coker and Oaikhena. The surface of sterile Mueller Hinton agar was flooded with 1 ml of 0.5 Mc Farland standardized inoculum and allowed to cover the entire surface, after which the excess was discarded into a container of disinfectant. The inoculated Mueller Hinton agar surface was allowed to dry, after which wells were bored on the surface of the inoculated agar plates using sterile stainless cork borer (10 mm).

After adding a drop each of molten Mueller Hinton agar to seal the bottom of the wells, 20 μl of the different fractions were transferred into the wells using micro pipette. The wells were sufficiently spaced to prevent the resulting zones of inhibition from overlapping. Preincubation diffusion time (45 minutes) was allowed, after which the petri-dishes were incubated at 37°C for 24 hrs.

The zones of inhibition surrounding the wells were measured in millimeters using a ruler [14]. Clear inhibition zones around the wells indicated the presence of antimicrobial activity. The mean of the resulting zones of inhibition were determined.

Results

Fractionation test result

The MIC of the M. lucida active extract against the tested bacteria was within the range of 25 to 100 mg/ml (Table 1). One hundred and twenty-six (126) fractions were obtained and were pulled together to 27 fractions (M1-M27) based on their TLC fingerprint profiles (Table 2). Fraction M25 showed better activity with a zone of inhibition of 15.3 mm, compared to the other fractions. Others following are M18 and M24 having 15.1 and 15.0 mm respectively (Table 3).

Table 1. Minimum inhibitory concentrations of M. lucida against the isolates.

Table 2. M. lucida pulled fractions.

Table 3. Zone of inhibition (mm) for the M. lucida pulled fractions I.

Figure 1A-C showed the Thin Layer Chromatography (TLC) profile of some of the pulled fractions. From the TLC, the isolated compound had an Rf value of 0.74 and it is pink in color after spraying and heating (colourless under UV) (Figure 1D). The compound was whitish crystal and completely soluble in chloroform.

Figure 1. TLC profile of some of the pulled fractions (A, B and C) and that of the isolated compound (D).

Compound elucidation

Figures 2-8 showed the FTIR spetrum, and 2D NMR (Proton, Carbon 13, HSQC, HMBC, COSY and DEPT) spectra of the isolated compound. The peaks observed at 3414.2, 2926.0, 2855.1 and 1688.5 are functional groups indications of Hydroxyl (OH), asymmetric Methyl (-CH3) stretch, symmetric Methyl (-CH3) stretch and Alkenyl (C=C) stretch respectively in the compound isolated (FTIR spectrum). The proton spectrum of M25A in CC13 (400 Hz) showed the presence of eight methyl singlets, two close and similar triplets for two carbinylic protons at δH 5.27 (t, J=3.2, H-12 for α-amyrin) and 5.24 (t, J=3.5, H-12 for β-amyrin). An oxymethine proton (H-3) at δH 3.22 (α-amyrin) and 3.20 (β-amyrin) was also observed and integrated for two protons approximately. The rest of the signals were for methyl and methylene as well as other methine protons but they were overlapped for the two compounds. The 13C spectrum showed the distinct carbon atoms for the compounds while the rest of the carbons were close together or overlapped. For alpha amyrin the C-12 signal was observed at δC 123.03 and the C-13 signal at 139.7 ppm while for beta amyrin, the C-12 signal was observed at 120.91 and the C-13 at 143.43 ppm. The oxygenated C-3 for alpha amyrin was at 79.99 and for beta amyrin at 79.20 ppm. Based on the chemical shifts reported for the compounds and compared to our experimental values (Table 4), M25A was identified as a mixture of α and β-amyrins (Figure 9).

Figure 2. M25A FTIR spectrum.

Figure 3. M25A NMR_Proton spectrum.

Figure 4. M25A NMR_Carbon13 spectrum.

Figure 5. M25A NMR_HSQC spectrum.

Figure 6. M25A NMR_HMBC spectrum.

Figure 7. M25A NMR_COSY spectrum.

Figure 8. M25A NMR_DEPT spectrum.

Table 4. Characterization of the isolated compound M25A.

Figure 9. Structure of the proposed isolated compounds (Alpha and Beta amyrin).

The isolated compound (alpha and beta amyrin) (0.0928 μg/ml) showed better activity on Klebsiella, Pragia, Serratia, Enterobacter, Providencia, species and E. coli with zones in the range of 15-18 mm (Table 5). The zones of inhibition of the isolated compound was compared with that of some antibiotics, whereby alpha and beta amyrin had better activity (Figure 10).

Table 5. Zone of inhibition (mm) for the isolated compound (M25A).

Figure 10. Average zone of inhibition (mm) for the M. lucida methanol extract most active of the pulled fractions and the isolated compound.

Discussion

The proton spectrum for the mixture in CDCl3 showed two close and similar triplets for two olefinic protons at δ_H 5.28 (t, J=3.5, H-12 for α-amyrin) and 5.25 (t, J=3.5, H-12 for β-amyrin). This is characteristic for triterpenes having a C-12 olefinic proton such as amyrins and other similar triterpenes such as oleananes, ursanes and amyrins having a C=C double bond between C-12 and C-13. The triplet multiplicity is due to coupling with the H-11 protons usually at 1.84 and 1.87 ppm. An oxymethine proton (H-3) at δ_H 3.23 (α-amyrin) and 3.20 (β-amyrin) was also observed and integrated for two protons approximately. The rest of the signals were for methyl and methylene as well as other methine protons but they were overlapped for the two compounds. The ¹³C spectrum showed the distinct carbon atoms for the compounds while the rest of the carbons were close together or overlapped. For alpha amyrin the C-12 signal was observed at δ_C 122.8 and the C-13 signal at 139.7 ppm while for beta amyrin, the C-12 signal was observed at 125.8 and the C-13 at 145.2 ppm. The oxygenated C-3 for alpha amyrin was at 79.17 and for beta amyrin at 79.21 ppm. The rest of the carbon signals were in good agreement with literature reports. Based on the chemical shifts reported for the compounds and compared to our experimental values (Table 5), the fraction was identified as a mixture of α and β-amyrins [15,16].

The isolated compound (alpha and Beta amyrin) in M. lucida methanol extract is from the triterpenes family. It is a pentacyclic triterpenoid [17]. The isolated compound (0.0928 μg/ml) showed better activity against Klebsiella spp., Pragia spp., Serratia spp., Enterobacter spp., Providencia species and E. coli. Diaz-Ruiz, et al. reported that α-amyrin and β-amyrin isolated from copal (Bursera spp) and B. crassifolia respectively inhibited the growth of some bacteria, including S. mutans, at concentrations ranging from 64 to 1088 μg/ml. β-Amyrin acetate isolated from Heliotropium marifolum showed potent activity against Penicillum chrysogenum, Escherichia coli and Klebsiella pneumoniae [18]. Johann, et al. reported that Amyrin formate and acetate derivatives were the most active compounds, inhibiting all the opportunistic Candida species when tested in concentrations from 30 to 250 mg/ml [19]. Other studies have demonstrated that the α/β-amyrin mixture also has antiinflammatory, gastroprotective, antiallergenic and antinociceptive activities [20].

Conclusion

Implication of the findings in this study presents α and β-amyrin from M. lucida as a potential agent for the treatment of some infectious diseases that cannot be treated with commonly used antibiotics. Therefore, this study justifies the use of M. lucida in traditional medicine practices and especially as alternative medicine.

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