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Research Article
 

Bactericidal Activity, Isolation and Identification of Most Active Compound from 20 Plants used in Traditional Mexican Medicine Against Multidrug-Resistant Bacteria



C. Rivas-Morales, V.M. Rivas-Galindo, J. Rodriguez-Rodriguez, S.A. Galindo-Rodriguez, C. Leos-Rivas and D.G. Garcia-Hernandez
 
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ABSTRACT

Background and Objective: Plants are used in Mexico as traditional medicine for the treatment of diverse illnesses such as stomach pain, fever, diarrhea, insomnia, flu and other respiratory diseases. Twenty were selected to determine their bactericidal activity. The aim of this study was the isolation of molecules from plants used in Mexican traditional medicine. Materials and Methods: Using chromatographic procedures, the responsible bactericidal molecules from rosemary was extracted and then identified by spectroscopic analysis IR, 1H NMR, 13C NMR, DEPT, HSQC and GC-MS. Measures of central tendency were determined by statistical analysis. Results: Ten of these plants showed bactericidal activity against multidrug-resistant bacteria. This biological activity was reported for Carya illinoensis against Pseudomonas aeruginosa, also for Equisetum robustum, Stevia rebaudiana and Castela texana against Methicillin resistant Staphylococcus aureus (MRSA). The methanolic extract of Rosmarinus officinalis (rosemary) showed important bactericidal activity against MRSA (ATCC BAA-44) and clinically isolated MRSA. Conclusion: Rosemary’s bactericidal molecules were isolated and then identified as a mixture of betulinic, oleanolic and ursolic acid (MIC = 725 μg mL–1).

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  How to cite this article:

C. Rivas-Morales, V.M. Rivas-Galindo, J. Rodriguez-Rodriguez, S.A. Galindo-Rodriguez, C. Leos-Rivas and D.G. Garcia-Hernandez, 2018. Bactericidal Activity, Isolation and Identification of Most Active Compound from 20 Plants used in Traditional Mexican Medicine Against Multidrug-Resistant Bacteria. International Journal of Pharmacology, 14: 203-214.

DOI: 10.3923/ijp.2018.203.214

URL: https://scialert.net/abstract/?doi=ijp.2018.203.214
 
Received: August 11, 2017; Accepted: November 27, 2017; Published: January 15, 2018


Copyright: © 2018. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

An important prerequisite for the success of primary health care is the availability and use of suitable drugs. Plants have always been a common source of drugs, either in their traditional preparations or as pure active principles1.

Current research in drug discovery from medicinal plants involves a multifaceted approach that combines botanical, phytochemical, biological and molecular techniques. Medicinal plant drug discovery continues providing new and important leads to various pharmacological targets. Although, research of medicinal plants is an important source of new drug leads, numerous challenges are encountered including the procurement of plant materials, the selection and implementation of appropriate high-throughput screening bioassays and the scale-up of active compounds2.

Antibiotic resistance is the organism’s acquired capacity to resist the effects of a chemotherapeutic agent to which it is usually sensitive3. This is due to bacterial chromosome mutations, plasmids or transposons which can transfer some resistance to many microorganism species faster than the new drugs developed for treatment4-6.

Antibiotic resistance is an important public health problem in the world mainly in underdeveloped countries where their inadequate use has caused the development of multidrug-resistant bacteria. Resistant strains are highly disseminated due to the inefficient infrastructure of the public health system and incorrect control of infection treatments. The group of antibiotics most often used is β-lactams, which includes penicillin, cephalosporins, cephamycins, carbapenems and monobactamics. These antibiotics have low toxicity levels and a broad sprectrum7,8.

The high incidence of infectious disease due to Enterobacteriaceae as well as the rise of multidrug-resistant strains are the main elements for current and future medical problems. Beta-lactam antibiotics have a structure similar to the binding sites of bacterial substratum, this similarity can deactivate some proteins required for cell wall peptidoglycan synthesis9.

The spread of drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases. The plants extracts have evoked interest as sources of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases10-13.

The aim of this study is the isolation of bioactive molecules from plants and their application in multidrug-resistant bacteria.

MATERIALS AND METHODS

This study was carried in Laboratorio de Química Analítica, Facultad de Ciencias Biológicas, UANL, México, with the collaboration of Tecnologico de Monterrey, México and Departamento de Química Analítica, Facultad de Medicina, UANL, México (January, 2015-December, 2016).

Plant material: Twenty plants used in traditional Mexican medicine (Table 1) were obtained commercially by PACALLI®.

Test organisms: Twelve strains were used (Table 2), 6 multi drug resistant bacteria (ATCC) and another six clinically isolated bacteria (CI) obtained from the Department of Microbiology of the Hospital Universitario Dr. José Eleuterio Gonzalez, Universidad Autónoma de Nuevo León, México.

Extraction: Dry and ground plant material (30 g) was extracted in a Soxhlet with 500 mL methanol (CTR Scientific, Monterrey, Mexico) to exhaust the extraction. Then, the solution was evaporated to dryness under reduced pressure using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) at 40°C. Later, its extraction percentage was calculated and the material was stored at room temperature in amber vials until its use14.

Antibacterial assay: Twenty methanol extracts were screened for antibacterial activity using the disk diffusion method, which is normally used as a preliminary test to select the most efficient extracts12. These were performed using the M26-A protocol15 with different culture media as a modification16 An 18 h culture at 37°C in 3 mL of C. Rivas broth was performed. The cultures were adjusted to approximately 106 CFU mL–1 by the McFarland turbidity method. One hundred microliters of these suspensions were spread over the plates containing C. Rivas agar (Patent number 9810892) using a sterile Driglasky spreader in order to achieve uniform microbial growth.

The methanol extracts were dissolved in methanol (CTR Scientific) at a final concentration of 50 mg mL–1. Under aseptic conditions, empty sterilized discs (Whatman No. 5, 6 mm diameter) were impregnated with 20 μL of the extracts and placed on the agar surface. The paper disc was moistened with methanol and placed on the seeded Petri dish as a negative vehicle control. Petri dishes were incubated at 37°C for 24 h. After incubation, the inhibition zone was measured with a rule.

MIC assay: The minimum inhibitory concentration (MIC) was determined based on the previous screening. Only the most active extract was tested according to the M07-10 document in a 96 well microplate17.

Table 1: Traditional medicine plants selected for this study

Table 2: Clinically important bacteria in this study
CI: Clinical isolate

These were tested from 2900-45.31 μg mL–1 concentrations. Inhibition of bacterial growth in a well containing the test extract was judged by comparison with the growth control well. The MICs were determined as the lowest concentration of extract inhibiting visible growth of each organism in the well.

Thin layer chromatography (TLC): The extract with the highest bactericidal activity was analyzed by TLC. The TLC plate (TLC silica gel 60, Fluka, 25×75 mm, without UV indicator) was developed in benzene/acetone (9:1), was sprayed with cobalt chloride/sulfuric acid (2, 19% v/v) and visualized by heating to calculate the retention factor (Rf). Ursolic acid standard used as a control reference due to its presence in the plant.

Bioautography test: The contact bioautography method was used for assaying antibacterial activity18. TLC plates were developed as mentioned previously. In order to sterilize the plates, they were allowed to dry inside the Petri dish in a laminar flow hood with UV light for 30 min. Then, C. Rivas agar was cut with a sterilized spatula and deposited on the TLC plate. Thereafter, 100 μL of a standardized bacterial solution was spread over the agar layer using a sterile Driglasky spreader in order to achieve uniform microbial growth. Plates were incubated at 37°C for 24 h and then the inhibitions zones on the agar layer were observed, which indicated the presence of antimicrobial compounds.

Table 3:
Column chromatography separation from methanolic rosemary extract
100 mL per fraction was recovered. The material was measured in miligrams

Flash chromatography: Flash chromatography was performed using an Isolera One 2.0.8 device (Biotage, Charlotte, NC, USA). The sample (173.2 mg) was charged in a SNAP Ultra 10 g cartridge and eluted automatically via Isolera Spektra (Biotage) software. Hexane and ethyl acetate were performed as eluents. The fractions obtained were monitored with lambda-all UV detection mode (254, 280 nm).

Silica gel chromatography: Silica gel 60G (Sigma Aldrich, 63-200 mesh) was packed into an open column (28 mm diameter and 330 mm length). The sample (3.5 g) was also adsorbed in silica. Then an eluent gradient system, which consisted of hexane/acetone/methanol was prepared (Table 3). One hundred milliliters of each system was collected as a fraction, then this was dried under reduced pressure using a rotary evaporator (Buchi Rotavapor, Buchi Labortechnik, Essen, Germany) at 40°C for antimicrobial activity tests.

Table 4: Antibacterial activity of extracts and halo inhibition growth (mm)
CI: Clinical isolate, ±Standard Deviation, n = 3, Not applicable, Halo was measured in mm, Negative data is not shown

Infrared spectroscopy: The infrared spectroscopic assay (IR) was performed in FT-IR Frontier spectrometer (Perkin Elmer, Tempe, AZ, USA). A 10 mg sample was dissolved in acetone and put into a universal attenuated total reflectance device (UATR). Measurements were made in triplicate taking 64 scans in the range of 400-4000 cm–1 and analyzed with Spectrum V10.4.00.0190 software (Perkin Elmer).

Nuclear magnetic resonance (NMR) spectroscopy: For NMR analysis, 10 mg of the isolated compound was dissolved in acetone-d6 with 0.3% of TMS as zero reference. The NMR spectra were recorded at 25°C using a Bruker Avance III HD 400 spectrometer (Bruker Corp., Billerica, MA) equipped with gradients and a 5 mm multinuclear probe at a base frequency of 100 MHz for 13C and 400 MHz for 1H. The spectrogram was analyzed via Topspin 3.0 software (Bruker Corp.).

Gas chromatography-mass spectrometry (GC-MS): Coupled gas chromatography-mass spectrometry (GC-MS) analysis was performed with an Agilent GC 6890 coupled to an MSD 5973N (Agilent Technologies Inc., Santa Clara, CA, USA), under the following instrumental conditions: HP-5 capillary column (30 m×0.25 mm×0.25 μm) with oven temperature programmed at 70°C for 2 min, then increased to 200 at 10°C min–1, after that 200-320 at 10°C min–1. Total run time: 37 min, injector temperature: 270°C, injector type: Split 1:20, detector temperature: 250°C, carrier gas: Helium (1 mL min–1). MS conditions: Acquisition mode: Scan (m/z 30-650).

Statistical analysis: Measures of central tendency were performed in triplicate and mean value was calculated using Social Sciences (SPSS) software (version 17.0 for Windows, SPSS Inc., Chicago IL) the results were expressed as mean±SD (Table 4).

Fig. 1:
Antimicrobial susceptibility test against S. aureus Rosenbach ATCC BAA-44 [S: Sensitive, R: Resistant], ampicillin (AM) = R, cephalothin (CF) = R, cefotaxime (CTX) = R, ceftazidime (CAZ) = R, cefuroxime (CXM) = R, dicloxacillin (DC) = R, erythromycin (E) = R, gentamicin (GE) = R, pefloxacin (PEF) = R, penicillin (PE) = R, trimethoprim+ sulfamethoxazole (SXT) = S, tetracycline (TE) = S

RESULTS

The extraction percentage of chamomile produced the highest percentage (58.82% p/p), while canary seed the lowest (3.42% p/p).

At first, the antimicrobial susceptibility to MRSA BAA-44 was tested, only trimethoprim/sulfamethoxazole and tetracycline were effective against this microorganism. Then bactericidal activity was performed (Fig. 1) and rosemary extract presented the greatest activity against both strains (ATCC and CI) so, the decision was to continue this study only with this extract. The activities of the other extracts are shown in Table 4.

Fig. 2(a-c):
(a) TLC rosemary extract revealed with CoCl2, (b) TLC rosemary extract revealed with UV light, (c) Bioautography assay against S. aureus Rosenbach ATCC BAA-44. The black arrow shows the Rf 0.42 which developed the inhibition area in build c

Fig. 3:
Thin layer chromatography from fractions obtained from rosemary methanolic extract by column chromatography. Lane 1: Complete methanolic extract (MeOH), lanes 2-8: Obtained fractions and lane 9: Ursolic acid as a reference control. Rf = near 0.42

In order to separate rosemary extract, we decided to perform a TLC in which one had an Rf = 0.42, so we decided to use this system for performing the bioautography assay.

Fig. 4:
Antimicrobial test from fractions 3-9 from rosemary methanolic extract against S. aureus Rosenbach ATCC BAA-44 in C. Rivas agar, n = 3, NA: Not applicable, SD: Standard deviation

Fig. 2 shows the results from the eluate system. The inhibition growth area was located on the Rf point.

Column chromatography produced 9 fractions described in Table 3. The highest percent recovery was corresponded to fraction 3, while the lowest corresponded to fraction 1. These fractions were performed again in TLC and the results are shown in Fig. 3. Fractions 3-9 were displaced near the same Rf.

Fractions 3-9 were tested for antibacterial properties. Fig. 4 shows the results. Fraction 3 was more active than the others. Once the TLC and antibacterial results were obtained, we decided to join some fractions according to these results. It was concluded joining fractions 4, 5 and 7-9. A sample of fractions 4 and 5 (630 mg) were dissolved in acetone and then cooled at -20°C for 72 h. The precipitate (white powder, 300 mg) was recovered by filtration.

Fig. 5:
Isolera one (Biotage) report. Mode: Lambda all. Two peaks were observed named isolated 1 and isolated 2 (1 and 2, respectively) through the flow time from automated flash chromatography

Fig. 6:
Thin layer chromatography collected fractions from rosemary methanolic extract by flash chromatography revealed with CoCl2. Lane 1: Isolated 1, Lane 2: Isolated 2, Lane 3: Methanolic extract as a control (MeOH)

From this precipitate, a sample (173.2 mg) was evaluated by flash chromatography. The Isolera Spektra software determines the flow for eluents. Figure 5 presents the report from the device. It was observed that two peaks in all detection modes that were programmed, materials collected before and after were discarded. Table 5 also shows recovered material. These peaks were named "isolated 1" and "isolated 2", respectively.

Fig. 7:
Antibacterial test from partitions to isolated 2 chloroform and methanol soluble partitions from fractions 4 and 5 chloroform, acetone and methanol soluble (1) Chloroform soluble compound 2, (2) Methanol soluble compound 2, (3) Methanol soluble fractions 4 and 5, (4) chloroform soluble fractions 4 and 5, (5) acetone soluble fractions 4 and 5

Once these fractions were recovered, they were tested with TLC and the antibacterial test was applied as shown in Fig. 6 and 7. The TLC from compound 2 presents a dark point just under the reference Rf. This is the reason to partition this compound into a soluble fraction in chloroform and methanol and test these partitions for antibacterial activity. The result showed that the methanol soluble fraction was responsible for its antibacterial activity. Likewise, the union of fractions 4-5 of rosemary extract was partitioned in chloroform, acetone and methanol and the acetone soluble partition had activity.

Table 5:
Collected material for automated flash chromatography from the joined fractions 4 and 5 from rosemary methanolic extract
Flash chromatography was performed on a ISOLERA ONE (Biotage) device

The MIC for isolated 2 against S. aureus Rosenbach ATTC BAA-44 was 725 μg mL–1.

The obtained fraction (isolated 2) had the following characteristics: IR are show in Fig. 8. 1H NMR (acetone-d6), 13C NMR are show in Fig. 9 and 10, respectively. Two-dimensional HSQC test it’s showed in Fig. 11. The GC-MS (Retention time = 32.67 min) are show in Fig. 12. According to interpretation and literature results, it was concluded that a mixture of betulinic acid, ursolic acid and oleanolic acid was isolated.

Fig. 8: IR spectrogram from methanol soluble partition to isolated 2 from rosemary methanolic extract

Fig. 9:
1H NMR from isolated 2 from rosemary methanolic extract. X represents the dissolvent signals. Acetone d-6 was used as a dissolvent

Fig. 10:
13C NMR from isolated 2 from rosemary methanolic extract. X represents the dissolvent signals. Acetone d-6 was used as a dissolvent

Fig. 11:
HSQC test. The double bond signals from the three different carbons and five hydrogens are shown in their coupling signals by red lines. Sample of isolated 2 from rosemary methanolic extract

DISCUSSION

The aim of this study was the isolation of molecules from plants used in Mexican traditional medicine. The search for molecules in plants as new antimicrobial agents, today brings the traditions of indigenous peoples and their knowledge to the treatment of diseases with the most inexpensive medicine accessible to all. In this study, 20 plants were selected that are used around the world as spices, flavors and as principal ingredients to make infusions.

Fig. 12: Fragmentation pattern (MS) from isolated 2 from rosemary methanolic extract

Fig. 13:
DEPT (Distortionless Enhancement by Polarisation Transfer) 135 and 90 analyses from isolated 2 from rosemary methanolic extract

Early research of plants extracts report some importance or clinical of strains that are the etiologic agents of opportunist diseases. Oluwatuyi et al.19 reported antibacterial activity against S. aureus Efflux (NorA) from chloroformic aerial parts of rosemary. Other plant extracts against multidrug resistant bacteria consisted of methanolics and essential oils20,21. Ten of the plants had antibacterial activity against reference ATCC and clinical isolate strains. The first antibacterial activities reported were for C. illinoensis (pecan nut) against P. aeruginosa ATCC-27853, E. robustum (horsetail), S. rebaudiana (Stevia) and C. texana (goat-bush) against S. aureus Rosenbach ATCC BAA-44 and C. citratus (lemon grass), horsetail, A. adstringens (cuachalalate) against A. baumannii. While R. officinalis (rosemary) showed major activity in both strains, the decision was to isolate the molecule(s) responsible for this property. Firstly, after column chromatography, the white amorphous powder that was separated had a coincident with Ghias Uddin et al.22, who isolated betulinic acid from a methanol extraction of Grewia optiva and Chung et al.23 from Callicarpa farinose, who tested betulinic acid against MRSA and MSSA strains. The IR data according to Kovac-Besovic et al.24 is very similar with an O-H stretching signal of 3484 cm–1 (3435 cm–1 observed), C = O stretching signal of 1686 cm–1 (1689 cm–1 observed), C-OH stretching signal of 1432 cm–1 (1457 cm–1 observed). These signals were referenced to an organic acid. In the search of references of organic acids in rosemary, three principal acids were found: Ursolic acid, oleanolic acid and betulinic acid, all of which were triterpenic compounds. Their differences were defined by the positions of their unsaturations and some substituents25,26 (Fig. 12). For the 13C NMR spectrogram, the signals were coupled as pentacyclic triterpene compounds due to their COOH signals (172-180 ppm)27. Fifty-nine signals were observable, the DEPT analyses (135 and 90) revealed 13 CH3, 19 CH2, 13 CH and 14 did not show carbons signals (Fig. 13). The HSQC revealed the 105.17 ppm (13C NMR, bottom signal) that was coupled with the 4.65 and 4.7 ppm (1H NMR) as a = CH2, the 122 ppm (13C NMR, up signal) was coupled with 5.23 ppm (1H) as a = CH and the 125 ppm (13C NMR, up signal) was coupled with 5.25 ppm (1H NMR) as a = CH, corresponding to carbons number 29 from betulinic acid, 12 from oleanolic acid and 12 from ursolic acid (Fig. 11) as according to Seebacher et al.28. For 1H NMR a comparison was made with the literature, Peng et al.29 and Adesanwo et al.30. Derivatization was performed with BSA (N,O-bis (trimethylsilyl) acetamide), which replaces the active proton in -OH, -COOH31,32, in the chemical structures of pentacyclic triterpenes of the ursane, oleane and lupane families, the COOH position is carbon 28 and the OH position is carbon 333, for this reason the silyl derivative contains two trialkylsilyl groups (146.296 g/mol). Ursolic, oleanolic and betulinic acid present a molecular weight of 456.70 g/mol as a consequence of the loss of two protons, with the addition of trialkylsilyl groups, the molecular weight from this derivative results in 600.982 g/mol34 (Fig. 12).

The pentacyclic triterpenes mixture showed an MIC = 725 μg mL–1, according to Wang et al.35, who reported an MIC>128 μg mL–1 for the same microorganism, this information is important since it can use an objective dose in the treatment of opportunist diseases caused by S. aureus Rosenbach, also, if combine another molecule that is present in the plants it can improve new therapy or use it as a coadjuvant to a synergistic molecule. In addition, the betulinic acid has these reported activities: Apoptotic and anticancer. Some derivatives are protease inhibitors (against HIV)36-39. Now this study provides a method for isolating this beneficial molecule.

CONCLUSION

In conclusion, the antimicrobial activity of methanolic extracts from 20 plants used in traditional Mexican medicine was tested against multidrug-resistant bacteria. Half of these showed biological activity. For the first time Carya illinoensis against Pseudomonas aeruginosa and also Equisetum robustum, Stevia rebaudiana and Castela texana against S. aureus Rosenbach was reported. A mixture conformed by betulinic, ursolic and oleanolic acids (MIC 725 μg mL–1) was isolated from Rosmarinus officinalis which showed major activity against S. aureus Rosenbach as an ATTC reference strain (BAA-44) as well as against clinical isolates.

SIGNIFICANCE STATEMENT

This study establishes the antibacterial activity of Carya illinoensis, Equisetum robustum, Stevia rebaudiana and Castela texana against multidrug-resistant bacteria. In addition, the isolation of a mixture compound by triterpenic acids from Rosmarinus officinalis, which has activity against ATCC reference and clinically isolated bacteria, identified by chromatography and spectroscopic methods is reported. This study help the researchers to discover critical areas of new sources of antibacterial molecules and alternative treatments for multidrug resistant bacteria which many researchers are not able to explore. Thus, a new theory on plant extracts and their molecules as antimicrobial agents may be reached.

ACKNOWLEDGMENT

This project was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT, México) under registration number 377431.

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