Abstract
In vitro. antimicrobial activity of 32 extracts (ethanol, hexane, chloroform, and methanol) from eight different Libyan medicinal plants, namely Artemisia herba-alba. Asso (Compositae), Globularia alypum. L. (Globulariaceae), Helichrysum stoechas., DC. (Compositae), Peganum harmala. L. (Zygophyllaceae), Polygonum equisetiforme. Sibth. & Sm. (Polygonaceae), Pulicaria crispa. (Forssk.) Oliv. (Compositae), Rosmarinus officinalis. L. (Labiatae), and Thymus capitatus. Hoffmanns & Link. (Labiatae), was determined against seven bacteria and one yeast strain using the broth microdilution method. The results show that all plants tested possessed antimicrobial activity against at least one of the examined strains at a concentration ≤8 mg/ml. The extracts from H. stoechas., P. equisetiforme., P. crispa., R. officinalis., and T. capitatus. exhibited strongest activity against Gram-positive bacteria with minimum inhibitory concentrations (MICs) ranging from 0.5 to 8 mg/ml. Only a few extracts showed inhibitory activity against Gram-negative strains in this test, whereas those of the H. stoechas. and P. harmala. were among the strongest ones (MICs range 4–8 mg/ml). High anticandidal activity was observed for P. harmala., P. crispa., and T. capitatus., with MICs ranging from 0.25 to 1 mg/ml.
Introduction
The traditional medicinal methods continue to play a vital role in covering basic health needs in developing countries; medicinal plants used in folk medicine provide a still largely unexplored source for the development of potentially new anti-infective drugs. Despite a rich tradition of medicinal plants used for the treatment of various infectious diseases, inflammations, and injuries in many parts of Libya (Al-Kathe & Al-Ramah, Citation1997), there are only a few reports on their antibacterial or antifungal activities (Hussein Ayoub, Citation1989Citation1990; Hussain & Tobji, Citation1997). Therefore, it is of great interest to carry out an antimicrobial screening of these plants in order to validate their ethnopharmacological use and reveal their active constituents.
In this article, we report the results of in vitro. antimicrobial tests performed on 32 various plant extracts (ethanol, hexane, chloroform, and methanol) obtained from different parts of eight plant species that are commonly used in Libyan folk medicine for treating conditions likely to be associated with microorganisms against one yeast, five Gram-positive, and two Gram-negative bacteria.
Materials and Methods
Plant material
The plant species were selected based on their traditional uses reported in available literature (Boulos, Citation1983; Al-Yahya et al., Citation1984; Al-Kathe, Citation1995; Al-Kathe & Al-Ramah, Citation1997; Al-Kathe & Al-Maghrebi, Citation1999). summarizes the ethnobotanical data of the plants selected for the study.
Table 1.. Ethnobotanical data on medicinal plants.
The plant materials were collected from August 2003 to October 2004 from different regions of north, south, and east Libya. The taxonomic identification of plants was confirmed by a senior plant taxonomist, Dr. M. Sherif, at the Department of Botany, Faculty of Science, University of Garyounis, Benghazi, Libya. The voucher specimens are deposited in the Institute of Tropics and Subtropics Herbarium, Czech University of Life Sciences, Prague, Czech Republic.
Preparation of extracts
Each sample of dried and powdered plant material (40 g) was extracted successively using a Soxhlet apparatus with n.-hexane, chloroform, and methanol for 72 h. Crude extracts were also prepared by maceration of powdered plant material (15.0 g) with 80% ethanol (450 ml) at room temperature for 5 days. All solvents of analytical grade were purchased from Lach-Ner (Neratovice, CZ). The extracts were subsequently filtered and concentrated in vacuo. at 40°C. The evaporated residue was then resolved in 10% (v/v) solution of dimethyl sulfoxide (DMSO), obtained from Lach-Ner (Neratovice, CZ), and in Tris-buffered saline (TBS) of pH 7.6 (Sigma-Aldrich, Prague, CZ) to create a concentration of 32 mg/ml of stock solution. All samples were sterilized by filtration through a 0.2 µm membrane filter (Sartorius, Goettingen, DE) and stored at + 4°C until tested. The yields of dried residues are shown in .
Table 2.. Minimum inhibitory concentrations of extracts from some species of the Libyan medicinal plants (mg/ml)
Microbial cultures
The following American Type Culture Collection (ATCC) strains of bacteria were used: Bacillus cereus. ATCC 11778, Bacillus subtilis. ATCC 6633, Enterococcus faecalis. ATCC 29212, Escherichia coli. ATCC 25922, Pseudomonas aeruginosa. ATCC 27853, Staphylococcus aureus. ATCC 25923, and Staphylococcus epidermidis. ATCC 12228. The yeast strain used in this study was Candida albicans. ATCC 10231. All microbial strains purchased from Oxoid (Basingstoke, UK) were grown in Mueller-Hinton broth (Oxoid, Basingstoke, UK). The susceptibility of bacteria to ciprofloxacin and yeast to nystatin (Sigma-Aldrich, Prague, CZ) were checked as positive controls ().
Antimicrobial assay
In vitro. antimicrobial activity was determined by the broth microdilution method (Jorgensen et al., Citation1999) using 96-well microtitre plates. Two-fold dilutions (six) of each extract were prepared in Mueller-Hinton broth, starting from a concentration of 16 mg/ml. Each well was inoculated with 5 µl of bacterial suspension at a density of 107 CFU/ml. The microtiter plates were incubated at 37°C for 24 h (or 48 h for the yeast). The growth of microorganisms was observed as turbidity determined by the UV-VIS spectrophotometer Helios ε (Spectronic Unicam, Cambridge, UK) at 600 nm. For each dilution, the change of optical density was calculated by subtracting the absorbance of uninoculated sample from that of inoculated one. Minimum inhibitory concentrations (MICs) were identified as the lowest extract concentrations that resulted in 80% reduction in growth compared to that in the extract-free growth control. The solution of DMSO (5% v/v) in TBS was assayed as the negative control, simultaneously. All samples were tested in triplicate.
Results and Discussion
In , the plant part used, the percentage yield, and the obtained MIC values of the corresponding extracts are summarized. In accordance with previously published reports (Garcia et al., Citation2003Citation2006), only extracts with MIC values of 8 mg/ml or below were considered active.
The results show that all plants tested in this study exhibited antimicrobial activity, whereas 23 of the plant extracts inhibited growth of at least one of the examined strains at concentration ≤8 mg/ml. Extracts that exhibited strongest activity against Gram-positive bacteria were methanol extract from H. stoechas. (MICs range 1–4 mg/ml), chloroform extracts from T. capitatus. (MICs range 1–4 mg/ml), and R. officinalis. (MICs range 1–8 mg/ml) as well as ethanol extracts from P. crispa. (MICs range 1–4 mg/ml) and P. equisetiforme. (MICs range 2–4 mg/ml). Methanol extract of P. equisetiforme. inhibited growth of S. aureus. with MIC of 0.5 mg/ml, the lowest MIC value achieved for Gram-positive bacteria tested in this study.
In contrast, only a few extracts showed inhibitory activity against Gram-negative strains in this test, whereas methanol extract from H. stoechas. and chloroform extract from P. harmala. were among the strongest, with MICs ranging from 4–8 mg/ml. Methanol extract of G. alypum. and ethanol extract of R. officinalis. have also been found to be active against Gram-negative bacteria tested in this study, inhibiting both strains tested (MICs = 8 mg/ml). High anticandidal activity was observed for hexane extract from T. capitatus. (MIC = 0.25 mg/ml), followed by ethanol and hexane extracts from P. crispa. as well as by chloroform extract from P. harmala., with MICs not exceeding 1 mg/ml. The methanol extract from H. stoechas. exhibited the broadest spectrum of inhibitory activity against all microbial strains tested at concentrations of 8 mg/ml or below.
A potent antimicrobial activity of crude ethanol extract and its butanol fraction from whole plant of P. equisetiforme. (Ghazal et al., Citation1992), methanol and chloroform extracts from leaves (Erdogrul, Citation2002), as well as hexane and ethanol extracts from aerial part (Khafagi & Dewedar, Citation2000) of R. officinalis. have previously been described. Moreover, various compounds isolated from above-mentioned types of extracts of both these species, e.g., quercetin from P. equisetiforme. (Ghazal et al., Citation1992) or diterpenes and methoxyflavone from R. officinalis. (Oluwatuyi et al., Citation2004), showed microorganism-inhibiting properties in earlier studies. Our results demonstrating significant antimicrobial effects of P. equisetiforme. and R. officinalis. extracts are in correspondence with the above-mentioned reports.
The hexane, ethanol (Khafagi & Dewedar, Citation2000), and methanol (Yashphe et al., Citation1979) extracts from aerial part of A. herba-alba., together with ethanol extract from aerial part of T. capitatus. (Kandil et al., Citation1994), possessed antimicrobial activities in previous studies. Nevertheless, we detected no inhibitory action of A. herba-alba. hexane extract, the ethanol and methanol extracts exhibited promising antibacterial effect on certain strains tested in this study. In correspondence with the previous report, we also detected antibacterial effect of ethanol extract of T. capitatus., and, as a new observation, we have determined antimicrobial and anticandidal actions of chloroform and hexane extracts, respectively. With the exception of essential oil components such as carvone, piperitone (Saleh et al., Citation2006), or santolina alcohol (Yashphe et al., Citation1979) identified in A. herba-alba. and thymol or carvacrol found in T. capitatus. (Cosentino et al., Citation1999), there are no reports of antimicrobial activities of other classes of compounds isolated from both of these species.
Although crude chloroform extract of H. stoechas. aerial part (Rios et al., Citation1987) possessed significant antimicrobial activity in the previous study, we did not note any significant antimicrobial effect of this extract. On the other hand, methanol extract of this species showed significant inhibitory effect on all microorganisms tested in our study. With the exception of phloroglucinol acetophenone and α.-pyrone derivatives found in chloroform and dichloromethane extracts (Tomas-Barberan et al., Citation1990; Rios et al., Citation1991), there are no reports of isolation of other antimicrobially active components of H. stoechas..
Although some of our results demonstrating significant antimicrobial effects of A. herba-alba., H. stoechas., and T. capitatus. extracts are in correspondence with reports mentioned above, differences between activities of some extracts determined within our study and those published previously in the literature suggest further possibilities for exploration of antimicrobial properties of these three species. We also suppose that some of the previously identified antimicrobial compounds are responsible for antimicrobial effect of active extracts in this study; however, in accordance with our results, there are also certain indices that all antimicrobial principles responsible for activities of this species have not been fully explored yet.
In contrast to the previous report demonstrating very poor antibacterial activity of G. alypum. aqueous leaf extract (Oran & Raies, Citation2000), we have observed significant inhibitory effect of aerial part ethanol and methanol extracts against S. aureus. and S. epidermidis. (MICs ranging from 2 to 4 mg/ml), indicating their potential antistaphylococcal properties. Moreover, among the few extracts effective against Gram-negative bacteria, the methanol extract of G. alypum. exhibited notable inhibitory action on the growth of E. coli. and P. aeruginosa..
While aqueous extract of P. crispa. was recently found to be active against fish pathogenic bacteria Photobacterium damselae. subsp. piscicida. (Abutbul et al., Citation2005), this is the first report on its effect against food borne or potential human pathogens. Among the several compounds previously isolated from P. crispa., 2α.-hydroxyalantolactone has been tested for its in vitro. antimicrobial properties (Al-Yahya et al., Citation1984). Since only slight inhibitory activity of this compound against S. aureus. has been observed in the above-mentioned study, we believe that some other substance may be responsible for the marked antimicrobial activity of the plant determined in our test.
Although antimicrobial activity of P. harmala. seed extracts and their active principles is well reported (Al-Shamma et al., Citation1981; Prashanth & John, Citation1999), only a few authors examined aerial part of the plant with results of no detectable antibacterial activity and weak inhibitory effect on growth of Gram-positive bacteria for leaf ethanol (Awadh Ali et al., Citation2001) and aqueous whole plant extracts (Hussain & Tobji, Citation1997), respectively. A literature search did not reveal any published report on the antimicrobial effect of compounds isolated from aerial part of the plant. In correspondence with earlier reports, we observed only weak or moderate antibacterial activity of P. harmala. leaf extracts on growth of Gram-positive strains tested in this study. In contrast to both above-mentioned reports, we observed a certain degree of inhibitory action on Gram-negative bacteria and, as a new observation, we have detected significant inhibitory effect of chloroform extract against C. albicans., indicating its prospective antifungal properties.
In conclusion, the extracts from A. herba-alba., G. alypum., H. stoechas., P. harmala., P. equisetiforme., P. crispa., R. officinalis., and T. capitatus. possessed significant antimicrobial action against microorganisms tested in this study. Since, to the best of our knowledge, the main antimicrobial principles of G. alypum. and P. crispa. aerial parts and P. harmala. leaves have not been identified yet, bioassay-guided fractionation of these three plants is currently underway in our laboratories with a goal of establishing types of compounds responsible for their marked antimicrobial properties.
Acknowledgements
The authors acknowledge the financial support given by the Grant Agency of the Ministry of Education, Youth, and Sports (Grant MSM 6046070901) for this work. Special thanks go to Dr. M. Sherif, Department of Botany, Faculty of Science, University of Garyounis, Libya, for taxonomic identification of plant species.
References
- Abutbul S, Golan-Goldhirsh A, Barazani O, Ofir R, Zilberg D (2005): Screening of desert plants for use against bacterial pathogens in fish. Isr J Aquacult-Bamid 57: 71–80.
- Al-Kathe AA (1995): Usages of Some Plants in Libyan Folk-Medicine, Part 2., 2nd ed. Benghazi, Libyan National Library, pp. 3, 16, 86.
- Al-Kathe AA, Al-Maghrebi M (1999): Usages of Some Plants in Libyan Folk-Medicine, Part 3.. Beirut, Apophthegm House Press, p. 121.
- Al-Kathe AA, Al-Ramah SM (1997): Usages of Some Plants in Libyan Folk-Medicine, Part 1., 2nd ed. Beirut, Apophthegm House Press, pp. 59, 64, 72, 75, 80, 105, 121.
- Al-Shamma A, Drake S, Flynn DL, Mitscher LA, Park YH, Rao GS, Simpson A, Swayze JK, Veysoglu T, Wu ST (1981): Antimicrobial agents from higher plants. Antimicrobial agents from Peganum harmala. seeds. J Nat Prod 44: 745–747.
- Al-Yahya MA, Khafagy S, Shihata A, Kozlowski JF, Antoun MD, Cassady JM (1984): Phytochemical and biological screening of Saudi medicinal plants. Part 6. Isolation of 2-α.-hydroxy-alantolactone the antileukemic principle of Francoeuria crispa.. J Nat Prod 47: 1013–1017.
- Awadh Ali NA, Julich WD, Kusnick C, Lindequist U (2001): Screening of Yemeni medicinal plants for antibacterial and cytotoxic activities. J Ethnopharmacol 74: 173–179.
- Boulos L (1983): Medicinal Plants of North Africa. Algonac, Reference Publications, pp. 57, 67, 155.
- Cosentino S, Tuberoso CIG, Pisano B, Satta M, Mascia V, Arzedi E, Palmas F (1999): In vitro. antimicrobial activity and chemical composition of Sardinian Thymus. essential oils. Lett Appl Microbiol 29: 130–135.
- Erdogrul OT (2002): Antibacterial activities of some plant extracts used in folk medicine. Pharm Biol 40: 269–273.
- Garcia VMN, Gonzalez A, Fuentes M, Aviles M, Rios MY, Zepeda G, Rojas MG (2003): Antifungal activities of nine traditional Mexican medicinal plants. J Ethnopharmacol 87: 85–88.
- Garcia VMN, Rojas G, Zepeda LG, Aviles M, Fuentes M, Herrera A, Jimenez E (2006): Antifungal and antibacterial activity of four selected Mexican medicinal plants. Pharm Biol 44: 297–300.
- Ghazal SA, Abuzarqa M, Mahasneh AM (1992): Antimicrobial activity of Polygonum eqisetiforme. extracts and flavonoids. Phytother Res 6: 265–269.
- Hussain H, Tobji RS (1997): Antibacterial screening of some Libyan medicinal plants. Fitoterapia 68: 467–470.
- Hussein Ayoub SM (1989): Antimicrobial screening of Libyan medicinal plants. Planta Med 55: 650–651.
- Hussein Ayoub SM (1990): Antibacterial and antifungal activities of some Libyan aromatic plants. Planta Med 56: 644–645.
- Jorgensen JH, Turnidge JD, Washington JA (1999): Antibacterial susceptibility tests: Dilution and disk diffusion methods. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, eds., Manual of Clinical Microbiology, 7th ed. Washington, ASM Press, pp. 1526–1543.
- Kandil O, Radwan NM, Hassan AB, Amer AMM, El-Banna HA, Amer WMM (1994): Extracts and fractions of Thymus capitatus. exhibit antimicrobial activities. J Ethnopharmacol 44: 19–24.
- Khafagi IK, Dewedar A (2000): The efficiency of random versus ethno-directed research in the evaluation of Sinai medicinal plants for bioactive compounds. J Ethnopharmacol 71: 365–376.
- Oluwatuyi M, Kaatz GW, Gibbons S (2004): Antibacterial and resistance modifying activity of Rosmarinus officinalis.. Phytochemistry 65: 3249–3254.
- Oran SA, Raies A (2000): Antimicrobial activity of Globularia arabica. Jaub. and Spach and G. alypum. L. (Globulariaceae). Disar Pure Sci 27: 71–73.
- Prashanth D, John S (1999): Antibacterial activity of Peganum harmala.. Fitoterapia 70: 438–439.
- Rios JL, Recio MC, Villar A (1987): Antimicrobial activity of selected plants employed in the Spanish Mediterranean area. J Ethnopharmacol 21: 139–152.
- Rios JL, Recio MC, Villar A (1991): Isolation and identification of the antibacterial compounds from Helichrysum stoechas.. J Ethnopharmacol 33: 51–55.
- Saleh MA, Belal MH, El-Baroty G (2006): Fungicidal activity of Artemisia herba-alba. Asso (Asteraceae). J Environ Sci Heal B 41: 237–244.
- Tomas-Barberan F, Iniesta-Sanmartin E, Tomas-Lorente F, Rumbero A (1990): Antimicrobial phenolic compounds from three Spanish Helichrysum. species. Phytochemistry 29: 1093–1095.
- Yashphe J, Segal R, Breuer A, Erdreichnaftali G (1979): Antibacterial activity of Artemisia herba-alba.. J Pharm Sci 68: 924–925.