Antibacterial Activity and Cytotoxicity of Selected Egyptian Medicinal Plants

• Abstract
• Introduction
• Material and Methods
• Plant material
• Extraction and isolation
• General experimental procedure
• Microbial strains and culture media
• Cell lines and treatment
• Chemicals
• MIC determination for antibacterial assay
• Resazurin cell growth inhibition assay
• Statistics
• Results and Discussion
• Acknowledgements
• Authors' contributions
• References

Abstract

Medicinal plants have been used as a source of remedies since ancient times in Egypt. The present study was designed to investigate the antibacterial activity and the cytotoxicity of the organic extracts from 16 selected medicinal plants of Egypt. The study was also extended to the isolation of the antiproliferative compound jaeschkeanadiol p-hydroxybenzoate (FH-25) from Ferula hermonis. The microbroth dilution was used to determine the minimal inhibitory concentration (MIC) of the samples against twelve bacterial strains belonging to four species, Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli, while a resazurin assay was used to assess the cytotoxicity of the extracts on the human pancreatic cancer cell line MiaPaCa-2, breast cancer cell line MCF-7, CCRF-CEM leukemia cells, and their multidrug resistant subline, CEM/ADR5000. The results of the MIC determination indicated that all the studied crude extracts were able to inhibit the growth of at least one of the tested bacterial species, the best activity being recorded with the crude extracts from F. hermonis and Vitis vinifera, which were active against 91.7 % and 83.3 % of the studied bacteria, respectively. The lowest MIC value of 128 µg/mL was recorded against P. stuartii ATCC 29916 and E. coli ATCC 10536 with the extract from V. vinifera and Commiphora molmol, respectively. In the cytotoxicity study, IC₅₀ values below 20 µg/mL were recorded for the crude extract of F. hermonis on all four studied cancer cell lines. FH-25 also showed good cytotoxicity against MCF-7 cells (IC₅₀: 2.47 µg/mL). Finally, the results of the present investigation provided supportive data for the possible use of the plant extracts investigated herein, mostly F. hermonis and V. vinifera in the treatment of bacterial infections and jaeschkeanadiol p-hydroxybenzoate in the control of cancer diseases.

Introduction

All over the world, people depended on herbs for the treatment of various ailments before the advent of modern medicine. In Egypt, many plants are used today in folk medicine and are sold at herbal vendors and shops [1]. The ancient Egyptians were familiar with many medicinal herbs and aware of their usefulness in the treatment of various diseases. They used the plant organs such as roots, rhizomes, flowers, leaves, fruits, seeds, and oils. They applied their medicaments in the form of powders, pills, suppositories, creams, pastes, and ointments [2], [3]. However, scientific evidence for the medicinal properties of such plants is not always demonstrated. We therefore undertook the present work to assess the antibacterial and cytotoxic properties of 16 selected medicinal plants from Egypt, namely Ferula hermonis Chirch el. (Apiaceae), Bidens pilosa L. (Asteraceae), Crategus sinaica B. (Rosaceae), Carduncellus eriocephalus B. (Asteraceae), Verbesina encelioides (Cav.) Benth. & Hook. f. ex A. Gray (Asteraceae), Carthamus tenuis L. (Asteraceae), Echinopus spinosissimus L. (Asteraceae), Haplophyllum tuberculatum (Forssk.) A. Juss. (Rutaceae), Commiphora molmol Jacq. (Burseraceae), Cynara cornigera (ssp. sibthorbiana) Lindl. (Asteraceae), Cynara scolymus L. (Asteraceae), Camellia sinensis (L.) Kuntze (Theaceae), Cichorium intybus L. (Asteraceae), Foeniculum vulgare var. azoricum (Apiaceae), Foeniculum vulgare (ssp. piperitum) (wild) (Apiaceae), and Vitis vinifera L. (Vitaceae). The study was then extended to the isolation of the antiproliferative compound from F. hermonis.

Material and Methods

Plant material

C. eriocephalus, V. encelioides, C. tenuis, C. spinosissimus, H. tuberculatum, C. cornigera (ssp. sibthorbiana), and Foeniculum vulgare (ssp. piperitum) were collected from Marsa Matroh, Egypt in April 2010; B. pilosa was collected from Wadi Houf, Helwan, Egypt in April 2010; C. sinaica Boiss was collected from St. Catherin (South Sinai, Egypt) in October 2010; C. scolymus, C. intybus, and F. vulgare var. azoricum were collected from the farm of NRC, Egypt; F. hermonis, C. molmol, C. sinesis, and V. vinifera were purchased from the local market. The plants were identified by Prof. Dr. Ibrahim El-Garf, Department of Botany, Faculty of Science, Cairo University, Cairo, Egypt and Prof. Dr. Salwa Kawashty, Department of Plant Systematics, National Research Centre, Cairo, Egypt. The voucher specimens ([Table 1]) have been deposited at the Herbarium of the National Research Centre (NRC), Cairo, Egypt.

Extraction and isolation

The dried and powdered sample (100 g) of each plant [except those from Ferula hermonis (1 kg) and Commiphora molmol, which were extracted with dichloromethane] was macerated in 80 % methanol. The combined extracts were concentrated under reduced pressure. Ninety grams (90 g) of the total F. hermonis extract was chromatographed over a silica gel column (100 cm × 3 cm), eluted with 1000 mL of hexane (I; 2.25 g), 5000 mL of hexane/EtOAc (1 : 1) (II; 53.3 g), 4000 mL of EtOAc (III; 23.12 g), then 2000 mL of methanol (FH-2; 4.42 g). Fraction I, II, and III were combined to give FH-1 (78.67 g). FH-1 fraction was subjected to silica gel column chromatography and eluted with a gradient polarity 10 % of hexane/EtOAc (from pure hexane to hexane/EtOAc and finally 100 % EtOAc). Ten subfractions were collected. Compound FH-25 was eluted with 30 % hexane/EtOAc (25 g).

General experimental procedure

An aluminum sheet precoated with silica gel 60 F ₂₅₄ (Merck) was used for thin layer chromatography (TLC). The spots were visualized using both ultraviolet light (254 and 366 nm) and 5 % H₂SO₄ spray reagent and 1 % vanillin followed by heating. ¹H (600 MHz) and ¹³C (150 MHz) NMR spectra were recorded on a JEOL JNM-ECA 600 spectrometer with tetramethylsilane as an internal standard. The melting point (m. p.) was determined using a Kofler microhot stage apparatus. Mass spectra were recorded with an API QSTAR pulsar mass spectrometer. The structure of the isolated compound was confirmed by comparing it with reference data from available literature.

Microbial strains and culture media

The studied microorganisms included reference strains of Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter arerogenes, and Escherichia coli obtained from the American Type Culture Collection. They were maintained on agar slants at 4 °C and subcultured on fresh appropriate agar plates 24 h prior to any antimicrobial test. Nutrient agar was used for the activation of bacteria. The Mueller Hinton broth (MHB) was used for MIC determination [4].

Cell lines and treatment

The human pancreatic cancer cell lines, MiaPaCa-2, breast cancer MCF-7 (poorly differentiated), CCRF-CEM leukemia cells and their multidrug resistant subline, CEM/ADR5000 (moderately differentiated), were obtained from the American Type Culture Collection. Leukemia cells were maintained in RPMI 1640 containing 100 units/mL penicillin and 100 mg/mL streptomycin and supplemented with heat-inactivated 10 % fetal bovine serum (FBS). MiaPaCa-2 and MCF-7cells were maintained in DMEM containing 100 units/mL penicillin, 100 mg/mL streptomycin, and 10 % FBS (Invitrogen). All cultured cells were maintained in a humidified environment at 37 °C with 5 % CO₂.

Chemicals

Chloramphenicol ≥ 98.0 % (Sigma-Aldrich) was used as a reference antibiotic (RA) against bacteria. p-Iodonitrotetrazolium chloride (INT; Sigma-Aldrich) was used as a microbial growth indicator [5], [6]. Doxorubicin-hydrochloride ≥ 98.0 % (Sigma-Aldrich) was used as a positive (cytotoxic) control.

MIC determination for antibacterial assay

The MIC determination of bacteria was conducted using a rapid INT colorimetric assay according to described methods [5], [6] with some modifications. Briefly, the test sample was dissolved in 10 % (v/v) DMSO/MHB to give a final concentration of 512 µg/mL and serially diluted twofold to obtain the desired concentration ranges. 100 µL of each concentration was added in a well (96-well microplate) containing 95 µL of MHB and 5 µL of inoculum (standardized at 1.5 × 10⁶ CFU/mL by adjusting the optical density to 0.1 at 600 nm SHIMADZU UV-120-01 spectrophotometer) [7]. The final concentration of DMSO in the well was less than 3 % (preliminary analyses with 3 % (v/v) DMSO did not alter the growth of the test organisms). The negative control well consisted of 195 µL of MHB and 5 µL of the standard inoculum [8]. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a plate shaker and incubated at 37 °C for 24 h. The assay was repeated three times in triplicate. The MIC of samples was detected following the addition (40 µL) of 0.2 mg/mL p-iodonitrotetrazolium chloride and incubation at 37 °C for 30 min [5], [6]. Viable microorganisms reduced the yellow dye to a pink color. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth [9].

Resazurin cell growth inhibition assay

Alamar Blue or Resazurin (Promega) reduction assay [10] was used to assess the cytotoxicity of the studied samples. The assay tests cellular viability and mitochondrial function. Briefly, adherent cells were grown in tissue culture flasks and then harvested by treating the flasks with 0.025 % trypsin and 0.25 mM EDTA for 5 min. Once detached, cells were washed, counted, and an aliquot (5 × 10³ cells) was placed in each well of a 96-well cell culture plate in a total volume of 100 µL. Cells were allowed to attach overnight and were then treated with samples. After 48 h, 20 µL resazurin 0.01 % w/v solution was added to each well, and the plates were incubated at 37 °C for 1–2 h. Fluorescence was measured on an automated 96-well Infinite M2000 Pro™ plate reader (Tecan) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. For leukemia cells, an aliquot of 5 × 10⁴ cells/mL (obtained from overnight suspension) was seeded in 96-well plates, and extracts were added immediately. After 24 h incubation, plates were treated with resazurin solution as mentioned above. Doxorubicin was used as the positive control. The concentration of DMSO was kept at or below 0.1 %. Each assay was done at least three times, with two replicates each. The viability was compared based on a comparison with untreated cells. IC₅₀ values were the concentration of sample required to inhibit 50 % of cell proliferation and were calculated from a calibration curve by linear regression [11], using Microsoft Excel.

Statistics

The one-way ANOVA at 95 % confidence level was used for statistical analysis.

Results and Discussion

The structure of the compound isolated from the fraction FH-1 of F. hermonis ([Fig. 1]) was established using spectroscopic analysis, especially NMR spectra in conjunction with COSY, HMQC, and HMBC. The compound was identified as jaeschkeanadiol p-hydroxybenzoate (ferutinin) [FH-25; m. p. 121–122 °C; m/z 358, RF: 0.5 with the solvent system Hexane-EtOAc 1 : 1)] [12], [13].

The results of the MIC determination ([Table 2]) indicated that all the studied crude extracts were selectively active. All extracts were able to inhibit the growth of at least one of the tested bacterial species, with F. hermonis and V. vinifera being the most active [F. hermonis, 11 of the 12 tested bacteria (91.7 %) and Vitis vinifera, 10/12 (83.3 %)]. It should be noted that both black and brown seeds of V. vinifera had similar activity spectra though the black seed extracts were more active against P. aeruginosa PA124, P. stuartii, and K. pneumoniae KP55. The lowest MIC value (128 µg/mL) was recorded against P. stuartii ATCC 29916 and E. coli ATCC 10536 with the extract of V. vinifera and Commiphora molmol, respectively. Phytochemicals are routinely classified as antimicrobials on the basis of susceptibility tests that produce an MIC in the range of 100 to 1000 mg/mL [14]. Activity is considered to be significant if MIC values are below 100 µg/mL for crude extracts and moderate when 100 < MIC < 625 µg/mL [15]. Therefore, the activity recorded with V. vinifera (on most of the tested bacteria) and F. hermonis [on 4/12 (33.3 %)] can be considered moderate. A less restrictive criterion has been described by Fabry et al. [16], which consider extracts having MIC values below 8 mg/mL to have noteworthy antimicrobial activity. Under these less stringent criteria, the overall activity recorded with several extracts, most notably F. hermonis and V. vinifera, could be considered important. However, the tested samples were less active than chloramphenicol, used as a reference antibiotic on all of the microbial strains. Also, the compound isolated from F. hermonis (FH-25) was not active on the tested bacteria contrary to the crude extract. Nevertheless, the activity of this compound against Mycobacterium smegmatis [17] and Staphylococcus aureus [18] was demonstrated. In the previous studies reported by Hilan et al. [19], the resin and oil extracts of F. hermonis were found to be highly effective against gram-negative and gram-positive bacteria. Also, a fraction of the essential oil from the rhizome exhibited good anti-infective effects, and its major component jaeschkeanadiol benzoate (FH-25) was found to have strong antifungal activity against Trichophyton mentagrophytes with an MIC of 0.25 µg/mL [20]. The crude extracts from V. vinifera varieties (Boğazkere and Oküzgözü) grown in eastern Anatolia (Elazig) were also found to possess antibacterial activities against gram-positive and gram-negative bacteria [21]. Therefore, the activity of the extract from the black seeds of V. vinifera reported herein corroborates the hypothesis that different parts of this plant can be sources of potential antimicrobial drugs. However, this must be confirmed with further pharmacological and toxicological studies. Also, investigations will be carried out in order to isolate the antibacterial compounds from both F. hermonis and V. vinifera.

It has been recommended that ethnopharmacological usages such as immune and skin disorders, inflammatory, infectious, parasitic, and viral diseases should be taken into account when selecting plants used to treat cancer, since these reflect disease states bearing relevance to cancer or cance-liker symptoms [22], [23]. Consequently, all the studied extracts were tested for their cytotoxicity against cancer cell lines. Leukemia CCRF-CEM and CEM/ADR5000 cell lines were used in the preliminary step to select samples to be tested on breast MCF-7 and pancreatic MiaPaCa-2 cells. The results summarized in [Fig. 2] revealed that only the extracts from Crategus sinaica, Carduncellus eriocephalus, Verbesina encelioides, and Carthamus tenuis on CCRF-CEM and those from Bidens pilosa, C. sinaica, C. tenuis, Haplophyllum tuberculatum, and Vitis vinifera (brown seeds) on CEM/ADR5000 cells did not prevent any cell growth. All other extracts were able to inhibit in various extents the proliferation of the CCRF-CEM as well as CEM/ADR5000 cells, but only the fraction from F. hermonis (FH-1) and the compound isolated from that fraction (FH-25) induced more than 50 % inhibition. The IC₅₀ values of these two samples were then determined on the four studied cancer cell lines ([Table 3]). In the US NCI plant screening program, a crude extract is generally considered to have in vitro cytotoxic activity if the IC₅₀ value following incubation between 48 and 72 h is less than 20 µg/mL. Boik [24] (2001) defined this cutoff point as 4 µg/mL for compounds. Herein, IC₅₀ values below 20 µg/mL were recorded with the crude extract from F. hermonis on the four studied cancer cell lines, and this extract can consequently be considered as a promising anticancer source. FH-25 also showed strong cytotoxic activity on MCF-7 cells with an IC₅₀ value of 2.47 µg/mL, confirming the activity of its mother fraction (FH-1). Nevertheless, the activity of FH-25 was still lower than that of doxorubicin on three of the four studied cancer cell lines ([Table 3]). Previous studies demonstrated the antiproliferative capacity of this compound against human colon cancer cells [25]. The present work therefore provides more data on the cytotoxicity of FH-25. It is important to note that the crude extract from F. hermonis showed less than 50 % growth inhibition of the leukemia cell lines, but FH-1 and FH-25 were significantly active, suggesting that active compounds could also be isolated from other active extracts with lower inhibitory activities.

Finally, the results of the present investigation provided baseline information for the possible use of the plant extracts investigated herein, especially F. hermonis and V. vinifera in the treatment of bacterial infections as well as FH-25 from F. hermonis in the control of cancer diseases.

Acknowledgements

The authors are thankful to Mr. P. K. Lunga of the University of Dschang (Cameroon) for language editing.

Authors' contributions

VK, BW, M-EFH, TAM, and AGF carried out the study; VK, AAS, and TE conceived the experiments and wrote the manuscript. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare that they have no competing interests.

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Prof. Dr. Thomas Efferth

Department of Pharmaceutical Biology
Institute of Pharmacy and Biochemistry
University of Mainz

Staudinger Weg 5

55128 Mainz

Germany

Phone: +49 6 13 13 92 57 51

Fax: +49 6 13 13 92 37 52

Email: efferth@uni-mainz.de

References

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  Search in Google Scholar
• 2 Shahat A A, Pieters L, Apers S, Nazeif N M, Abdel-Azim N S, Berghe D V, Vlietinck A J. Chemical and biological investigations on Zizyphus spina-christi L.  Phytother Res. 2001;  15 593-597
  Search in Google Scholar
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Prof. Dr. Thomas Efferth

Department of Pharmaceutical Biology
Institute of Pharmacy and Biochemistry
University of Mainz

Staudinger Weg 5

55128 Mainz

Germany

Phone: +49 6 13 13 92 57 51

Fax: +49 6 13 13 92 37 52

Email: efferth@uni-mainz.de