Flavonoids from Maytenus buchananii as potential cholera chemotherapeutic agents

a Laboratory of Environmental and Applied Chemistry, Department of Chemistry, Faculty of Science, University of Dschang, PO Box 67, Dschang, Cameroon b Laboratory of Microbiology and Antimicrobial Substances, Department of Biochemistry, Faculty of Science, University of Dschang, PO Box 67, Dschang, Cameroon c Groupe Isolement et Structure, Institut de Chimie Moléculaire de Reims (ICMR), CNRS UMR 7312, Bat. 18 BP.1039, 51687 Reims, Cedex 2, France d Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkota 700 019, India


Introduction
Acute watery diarrhea accounts for 80% of the cases (death account for 50%) in the developing world (Tullock and Richards, 1993). Among the diarrheal diseases, cholera is a serious epidemic disease caused by the Gram-negative bacterium Vibrio cholerae (Nair et al., 1994). V. cholerae, serotypes O1 and O139 have the ability to produce an enterotoxin, cholera toxin that is a major determinant of virulence for cholera. Among the other virulence factors, ElTor hemolysin produced by V. cholerae is also reportedly a potent toxin with both enterotoxic and cytotoxic activities (Ichinose et al., 1987;Ramamurthy et al., 1993). Some drugs such as racecadotril and loperamide used to treat the secretary diarrhea, have side effects such as bronchopasm, vomiting and fever, and loperamide should not be administrated to children below 6 years of age, patients with constipation, and intestinal obstruction (Rogé et al., 1993;Salazar et al., 2000). Emergence of multiply drugresistant V. cholerae is a serious clinical problem in the treatment and containment of the disease, as reflected by the increase in the fatality rate from 1% to 5.3% after the emergence of drug-resistant strains in Guinea-Bissau during the 1996-1997 epidemic of cholera (Dalsgaard et al., 1999). The increasing incidence of drug-resistant pathogens has drawn the attention of the pharmaceutical and scientific communities towards studies on the potential antimicrobial activity of plantderived products, an untapped source of antimicrobial chemotypes, which are used in traditional medicine in different countries. Maytenus buchananii (Loes.) R. Wilczek belonging to the Celastraceae family is a worldwide distributed small evergreen tree 2-12 m high, with paired spines up to 2.8 mm long. It has been reported that plants of this genus are widely used in folk medicine as anti-tumor, anti-asthmatic, anti-ulcer, treatment of stomach problems, anti-inflammatory, analgesic and as antimicrobial (Ghazanfar, 1994;Muhammad et al., 2000;Orabi et al., 2001;Al Haidari, 2002). The biological activities associated with Maytenus species have been assigned to different classes of secondary metabolites such as triterpenes (Sannomiya et al., 1998;El Tahir et al., 2001;Lindsey et al., 2006;Martucciello et al., 2010), phenolic glucosides (Da Silva et al., 2008), alkaloids (Corsino et al., 1998a;Orabi et al., 2001) and flavonoids (Vilegas et al., 1999;Lindsey et al., 2006;Dias et al., 2007). Flavonoids, previously called bioflavonoids and included in aromatic compounds, are important constituents of plants. The basic structural feature of flavonoid compounds is the 2phenylbenzopyrane or flavane nucleus, consisting of two benzene rings linked through a heterocyclic pyrane ring. In total, there are 14 classes of flavonoids, differentiated on the basis of the chemical nature and position of substituents on the different rings. For centuries, preparations containing these compounds as the principal physiologically active constituents have been used to treat human diseases such as infections associated with bacteria and those related to oxidative stress. Many investigations revealed that flavonoids content contribute to the antimicrobial (Araujo et al., 2011;Garcia et al., 2012;Djouossi et al., 2015) and antioxidant (Pietta et al., 1998;Djouossi et al., 2015) activities of plants. In the course of our continuing search for secondary metabolites of biological importance from Cameroonian medicinal plants, we evaluate the antibacterial and antioxidant activities of extracts and flavonoids from the leaves of M. buchananii.

General
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. 1 H and 13 C NMR spectra were recorded on a Bruker Avance III 600 spectrometer equipped with a cryoplatform ( 1 H at 600 MHz and 13 C at 150 MHz). 2D NMR experiments were performed using standard Bruker microprograms (Xwin-NMR version 2.1 software). Chemical shifts (δ) are reported in parts per million (ppm) with the solvent signals as reference relative to TMS (δ = 0) as internal standard, while the coupling constants (J values) are given in Hertz (Hz). The IR spectra were recorded with a Shimadzu FT-IR-8400S spectrophotometer. UV spectra were determined as methanol solution with a Cary 50 UV/vis Spectrophotometer. HR-TOFESIMS experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK) with an electrospray source. The samples were introduced by direct infusion in a solution of MeOH at a rate of 5 μL/min. Column chromatography (CC) was performed on silica gel 60 (70-230 mesh, Merck) and gel permeation on Sephadex LH-20 while TLC was carried out on silica gel GF 254 pre-coated plates with detection accomplished by spraying with 50% H 2 SO 4 followed by heating at 100°C, or by visualizing with an UV lamp at 254 and 365 nm.

Plant material
The leaves of M. buchananii were collected at Dschang, Menoua Division, West Region of Cameroon, in March 2012. Authentication was done by Victor Nana, a botanist of the Cameroon National Herbarium, Yaoundé, where a voucher specimen (No. 12659/SFR/CAM) is deposited.

Extraction and isolation
The dried and powdered plant material (4 kg) was extracted two times (each for 24 h) with 15 L MeOH at room temperature. The filtrate obtained was concentrated under reduced pressure to yield a dark residue (540 g). This crude extract was fractionated with hexane, EtOAc and n-BuOH, yielding after evaporation to dryness 58, 141 and 65 g of hexane, EtOAc and n-BuOH fractions, respectively and 235 g of a brown gum.

Structural identification of the isolated compounds
Samples for NMR experiments were dissolved in CD 3 OD on a Bruker Avance DRX 600 Spectrometer (600 MHz for 1 H and 150 MHz for 13 C). Column chromatography was performed on silica gel 60 (70-230 mesh, Merck) and Sephadex LH-20. Fractions were monitored by TLC using Merck pre-coated silica gel sheets (60 F 254 ), and spots were visualized under UV light (254 and 365 nm) and by spraying with 50% H 2 SO 4 and heating at 100°C. 1D and 2D NMR experiments (COSY, TOCSY, ROESY, HSQC-Jmod, and HMBC) were performed using standard Bruker pulse programs (XW in NMR version 2.1). Quercetin

. Microorganisms
A total of six bacterial strains were tested for their susceptibility to compounds and these strains were taken from our laboratory collection (kindly provided by Dr. T. Ramamurthy, NICED, Kolkata). Among the clinical strains of V. cholerae used in this study, strains NB2 and SG24(1) belonged to O1 and O139 serotypes, respectively. These strains were able to produce cholera toxin and hemolysin (Thakurta et al., 2007;Bag et al., 2008). The other strains used in this study were V. cholerae non-O1, non-O139 (strains CO6 and PC2) (Bag et al., 2008); and Shigella flexneri (Acharyya et al., 2015). The V. cholerae non-O1 and non-O139 strains, were positive for hemolysin production but negative for cholera toxin production (Bag et al., 2008). The American Type Culture Collection (ATCC) strain, Staphylococcus aureus ATCC 25923, was used for quality control. The bacterial strains were maintained on agar slant at 4°C and subcultured on fresh appropriate agar plates 24 h prior to any antibacterial test. The Mueller Hinton Agar (MHA) was used for the activation of bacteria. The Mueller Hinton Broth (MHB) and nutrient agar (Hi-Media) were used for the MIC and MBC determinations respectively.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
MIC values were determined by a broth micro-dilution method as described earlier (Nyaa Tankeu et al., 2009) with slight modifications. Each test sample was dissolved in dimethylsulfoxide (DMSO) and the solution was then added to Mueller Hinton Broth (MHB) for bacteria to give a final concentration of 1024 μg/mL. This was serially diluted twofold to obtain a concentration range of 0.50-1024 μg/mL. Then, 100 μL of each concentration was added in each well (96-well microplate) containing 95 μL of MHB and 5 μL of inoculum for final concentrations varying from 0.25-512 μg/mL. The inoculum was standardized at 1.5 × 10 6 CFU/mL by adjusting the optical density to 0.1 at 600 nm using a JENWAY 6105 UV/vis spectrophotometer. The final concentration of DMSO in each well was b1% [preliminary analyses with 1% (v/v) DMSO did not inhibit the growth of the test organisms]. The negative control well consisted of 195 μL of MHB and 5 μL of the standard inoculum. The plates were covered with sterile lids, then agitated to mix the contents of the wells using a plate shaker and incubated at 35°C for 24 h. The assay was repeated three times. The MIC values of samples were determined by adding 50 μL of a 0.2 mg/mL piodonitrotetrazolium violet solution followed by incubation at 35°C for 30 min. Viable microorganisms reduced the yellow dye to a pink color. MIC values were defined as the lowest sample concentrations that prevented this change in color indicating a complete inhibition of microbial growth. For the determination of MBC values, a portion of liquid (5 μL) from each well that showed no growth of microorganism was plated on Mueller Hinton Agar and incubated at 35°C for 24 h. The lowest concentrations that yielded no growth after this subculturing were taken as the MBC values (Tamokou et al., 2011). Ciprofloxacin and ampicillin (Sigma-Aldrich, Steinheim, Germany) were used as positive controls.
2.5.3. The time-kill kinetic study (for antimicrobial drugs) against V. cholerae SG24 (1) Time-kill dynamic assay was performed using broth microdilution method as previously described (Avila et al., 1999) with minor modifications. Cultures of bacteria in MHB (1 × 10 6 CFU/mL) were incubated separately at 37°C for 0, 2, 4, 6, 10, and 24 h in the absence (control) and in the presence of the drug/extract at MIC and MBC of each sample. Compounds 2, 4, 6 and ciprofloxacin were used in the time-kill dynamic experiment. The final concentration of DMSO was 1%. A control sample was made using DMSO 1% and the inoculum. At each incubation time point, liquids (50 μL) were removed from the test solution for ten-fold serial dilution. Thereafter, a 100 μL liquid from each dilution was spread on the surface of the MHA plates and incubated at 37°C for 24 h, and the number of CFU/mL was counted. Experiments were carried out in triplicate. Time-kill curves were constructed by plotting the surviving log 10 of number of CFU/mL against time (hours).
2.6. Antioxidant assay 2.6.1. DPPH free radical scavenging assay The free radical scavenging activity of the MeOH extract as well as some of its isolated compounds was performed according to described methods (Djouossi et al., 2015). Briefly, the test samples, prior dissolved in DMSO (SIGMA) beforehand, were mixed with a 20 mg/L 2,2diphenyl-1-picryl-hydrazyl (DPPH) methanol solution, to give final concentrations of 1, 10, 20, 40, 80, 160, 320, 640 and 1280 μg/mL. After 30 min at room temperature, the absorbance values were measured at 517 nm and converted into percentage of antioxidant activity. L-Ascorbic acid was used as a standard control. The percentage of decoloration of DPPH (%) was calculated as follows: The radical scavenging percentages were plotted against the logarithmic values of the concentration of test samples and a linear regression curve was established in order to calculate the EC 50 (μg/mL), which is the amount of sample necessary to inhibit by 50% the absorbance of free radical DPPH. All the analyses were carried out in triplicate.

Statistical analysis
Data were analyzed by one-way analysis of variance followed by the Waller-Duncan Post-hoc test. The experimental results were expressed as the mean ± Standard Deviation (SD). Differences between groups were considered significant when p b 0.05. All analyses were performed using the Statistical Package for Social Sciences (SPSS, version 12.0) software.

Phytochemical analysis
The structures of the isolated compounds were established using spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments, COSY, TOCSY, HSQC, and HMBC and direct comparison with published information. The nine compounds isolated from the leaves of M. buchananii (Fig. 1) Kazuma et al., 2000). These compounds together with the extracts were tested for their antibacterial and antioxidant activities and the results are reported in Table 1 and Figs. 2---4.

Antibacterial activity
The antibacterial activity of the MeOH, n-BuOH, EtOAc, and hexane extracts as well as their isolated compounds were examined by microdilution susceptibility assay against six bacterial strains selected on the basis of their relevance as human pathogens. The experiments revealed that the extracts and isolated compounds exhibited variable MICs and significant antimicrobial activity, depending on the microbial strains ( Table 1). The MIC values of the extracts ranged from 32 to 512 μg/mL. No activity was noted with hexane extract on V. cholerae SG24 (1) and V. cholerae NB2 at concentrations up to 512 μg/mL while the most sensitive bacterial strains were found to be S. flexneri and S. aureus. EtOAc extract (MIC = 32-256 μg/mL) was the most active extract followed in decreasing order by n-BuOH (MIC = 64-256 μg/mL), MeOH (MIC = 128-512 μg/mL) and hexane (MIC = 512-N512 μg/mL) extracts. This observation suggests that the crude methanol extract contains several antibacterial principles with different polarities. Phytochemicals are routinely classified as antimicrobials on the basis of susceptibility tests that produce MIC in the range of 100 to 1000 μg/mL (Simões et al., 2009). Activity is considered to be significant if MIC values are below 100 μg/mL for crude extract and moderate when the MIC values vary from 100 to 625 μg/ml (Kuete, 2010). Therefore, the activities recorded with the n-BuOH fraction on S. flexneri and S. aureus and with EtOAc fraction on V. cholerae 2, S. flexneri and S. aureus can be considered as important.
The lowest MIC value of 16 μg/mL was recorded on S. aureus with compound 6 and on Escherichia coli with compound 8, whereas the lowest MBC value of 32 μg/mL was obtained on S. aureus with compounds 6 and 2 and on S. flexneri with compound 6. However, the highest MIC value of 512 μg/mL was recorded on V. cholerae SG24 (1) and V. cholerae CO6 with MeOH extract, and the highest MBC value of 512 μg/mL was obtained on V. cholerae SG24 (1) with the MeOH and EtOAc extracts and on V. cholerae CO6 and V. cholerae NB2 with the MeOH extract. A lower MBC/MIC (≤4) value signifies that a minimum amount of plant extracts/pure compounds is used to kill the microbial species, whereas, higher values signify the use of comparatively more amount of sample for the control of any microorganism (Djouossi et al., 2015).
The strains of V. cholerae NB2, PC2 (Thakurta et al., 2007;Bag et al., 2008) and S. flexneri (Acharyya et al., 2015) included in the present study were MDR clinical isolates and these were resistant to commonly used drugs such as ampicillin, streptomycin, tetracycline, nalidixic acid, furazolidone and co-trimoxazole. However, these bacterial strains were found to be sensitive to most of the tested samples, suggesting that their administration may represent an alternative treatment against V. cholerae, the causative agent of the dreadful disease cholera and S. flexneri, the causative agent of shigellosis. Taking into account the medical importance of the tested bacteria, this result can be considered as promising in the perspective of new antibacterial drug development. Although flavonoid compounds have been reported to possess antibacterial activity (Garcia et al., 2012;Djouossi et al., 2015), no study has been reported on the activity of these compounds against these types of MDR pathogenic strains.
With regard to the structure-activity relationship analysis, the eight flavonoids showed different degrees of antibacterial activity. Compounds 2 (MIC = 32 to 128 μg/mL) and 6 (MIC = 16 to 64 μg/mL) showed the largest antibacterial activities with the best MIC (16 μg/mL) recorded with compound 6 on S. aureus. These observations show that the sugar moieties and hydroxyl groups should be responsible for the difference in the observed activity. The mechanism of the active compounds (1-9) is still to be studied; nevertheless, their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. More lipophilic flavonoids may also disrupt microbial membranes (Cowan, 1999). For example, (−)-epigallocatechin gallate inhibit cytoplasmic membrane function, whereas the activity of quercetin has been at least partially attributed to the inhibition of DNA gyrase (Cowan, 1999;Fowler et al., 2011).

The time-kill kinetic study
The time-kill kinetic study for compounds 2, 4, and 6 against V. cholerae SG24 (1)

Antioxidant activity
Free-radical-scavenging activities of M. buchananii extracts and their isolated compounds were assessed by DPPH· and ABTS•+. The results were expressed as gallic acid equivalent antioxidant capacity of tested samples (Fig. 3) and as equivalent concentrations of test samples scavenging 50% of DPPH radical (Fig. 4). Both DPPH· and ABTS• + measure reductions of radical solutions in the presence of a hydrogen-donating antioxidant. The results of both assays found compounds 3 (EC 50 = 1.38 μg/mL; TEAC = 89.69 μg/mL), 5 (EC 50 = 1.56 μg/mL; TEAC = 90.93 μg/mL) and 6 (EC 50 = 1.42 μg/mL; TEAC = 89.76 μg/mL) to exhibit the most activity and compound 7 (EC 50 = 107.56 μg/mL; TEAC = 44.98 μg/mL) to exhibit the least activity. The results of the DPPH and ABTS free-radical-scavenging activities are not in the same order for the extracts. This difference in the activity may be due to the presence of potent molecule(s) in some extracts which is more capable of quenching one particular radical than another. The free-radical activity of the extracts can be explained by the presence of phenolic substances. Similarly, previous reports have shown phenolic compounds to contribute significantly to the antioxidant activity of medicinal plants (Lim et al., 2009;Zhao et al., 2010;Djouossi et al., 2015). Phenolic compounds such as flavonoids are known to be potential antioxidant due to their ability to scavenge free radicals and active oxygen species such as singlet oxygen, superoxide anion radical and hydroxyl radicals (Hall and Cuppett, 1997;Pietta et al., 1998).

Conclusion
Results obtained from this study may help to exploit the use of the M. buchananii leaf extracts and some of their flavonoid contents as pharmacological ingredients for promoting health, especially for cholera/ shigellosis and chronic diseases associated with oxidative stress.  (1) cells exposed to the compounds 2, 4, 6, and ciprofloxacin. Control: MHB medium with DMSO 1% + inoculums. Fig. 3. Gallic acid equivalent antioxidant capacity (TEAC; μg/mL) of tested samples. Bars represent the mean ± SD of three independent experiments carried out in triplicate. Letters a-i indicate significant differences between samples according to one way ANOVA and the Waller-Duncan test; p b 0.05. Fig. 4. Equivalent concentrations of test samples scavenging 50% of DPPH radical (EC 50 ). Bars represent the mean ± SD of three independent experiments carried out in triplicate. Letters a-h indicate significant differences between samples according to one way ANOVA and the Waller-Duncan test; p b 0.05.