Abstract
The ethanol extracts obtained from the leaves, rootstock, and the combined formulation of endemic Lamium tenuiflorum Fisch.& Mey (Lamiaceae) have been investigated for their antifungal activities against medical yeast Candida and Cryptococcus species by the visual broth macrodilution method. The MIC values ranged from 3.12 to 25 mg/mL. All the extracts exhibited a strong antifungal effect against the yeast cultures. The extracts exhibited greater antifungal effect against Candida species than Cryptococcus species. Our findings support the use of this plant in traditional medicine for fungal infections especially against candidiasis.
Introduction
In ethnomedicine, at least 80% of the population in developing countries uses plant materials as their source of primary healthcare (CitationFarnsworth et al., 1985). Plants are known to produce a variety of compounds to protect themselves against a variety of their own pathogens and therefore can be considered as a potential source of different classes of antimicrobial substances (CitationOlukoya et al., 1993).
Turkey has a rich flora and still unstudied for phytochemistry or bioactivity. However, the strong ethnopharmaceutical literature of Turkey provides us with valuable information on widely utilized plant material in folk medicine.
The genus Lamium L. (Lamiaceae) comprises almost 40 species spread throughout Europe, Asia, and Africa (CitationWillis, 1973). Some Lamium species have been reported to possess applications in traditional medicines worldwide for the treatment of trauma, fracture, putrescence, paralysis, leukorrhea, hypertension, and some women’s afflictions such as menorrhagia, uterine hemorrhage, vaginal and cervical inflammation, etc. (CitationBremness, 1995; CitationShuya et al., 2003).
There is evidence indicating various activities such as anti-inflammatory, antioxidant, free radical scavenging, and antiproliferative properties for Lamium plants (CitationTrouillas et al. 2003; CitationMatkowski & Piotrowska, 2006; CitationPaduch et al., 2007). In Turkey, 30 Lamium species are widely grown (CitationGuner et al., 2000). Lamium tenuiflorum Fisch. & Mey is endemic to Turkey (CitationDavis, 1982). Although there are many studies on the Lamium species, this particular plant has not been previously investigated. During our routine field excursions, it was determined that this plant is used to treat colds and bronchitis, externally for boils and abscesses, and for urinary tract infections. Therefore, this study was undertaken to determine the antifungal effects of some organic solvents and aqueous extracts obtained from this endemic plant against some Candida and Cryptococcus species.
Materials and methods
Plant material
The plant material was collected from Icel, Turkey in September, 2007. Voucher specimens of the plant were deposited in the Biology Department at Canakkale Onsekiz Mart University and identified by Ersin Karabacak from the same department.
Preparation of extracts
The plant parts were air-dried. Each dry powdered plant material (20 g) was soaked in ethanol (50% concentration) until complete saturation of the plant material. The extract was filtered using Whatman filter No. 1, and the filtrates were evaporated under reduced pressure and dried using a rotary evaporator at 55°C. Dried extracts were stored in labeled sterile screw-capped bottles at -20°C. The extract (in the form of sticky black substances) amounting to around 2 g was dissolved in DMSO (dimethyl sulfoxide) before testing. The combination of plant extracts (1:1 ratio) was used in this test (CitationSingh & Gulati, 1990). Candida species (C. albicans ATCC 10231, C. tropicalis ATCC 13803 and C. guilliermondii ATCC 6260) and Cryptococcus species (C. neoformans ATCC 90112 and C. laurentii 34142) as the test fungi were obtained from the Microbiology Research Laboratory in the Department of Biology, Uludag University, Turkey, and pure cultures were maintained on Sabouraud Dextrose Agar (SDA) plates and Sabouraud Dextrose Broth (SDB) in tubes.
Minimum inhibitory concentration (MIC) determination
MICs were performed by the visual broth macrodilution method (CitationDzoyem et al., 2007). Fungal suspensions were diluted into RPMI-1640 medium without bicarbonate (pH 7 with 0.165 morpholinepropanesulfonic acid) broth supplemented with glutamine, to a concentration of approximately 0.5 × 105 CFU/mL, verified by colony counts in SDA. A two-fold serial dilution of 0.2 mL each of extract was added to 1.8 mL of the RPMI-1640 medium. The concentrations were 0.390-200 mg/mL. Controls with medium without antifungal samples were used in the test. To compare the results with standard, ketaconazole was used. Tubes were then incubated at 35°C for 24-48 h. MIC was defined as the lowest concentration which did not yield visual growth. All experiments were performed in triplicate.
Results and discussion
The MIC values concerning in vitro antifungal activities of the extracts are presented in . The MIC results for the ethanol extract of leaf, rootstock and the combinations ranged from 6.25-25, 12.5-25, and 3.12-12.5 mg/mL, respectively, and showed that susceptibility of the extracts varied from one fungal strain to another. Comparing the obtained result with those of the antifungal agent ketaconazole used as a reference, it was noted that the combination of plant extracts (both leaves and rootstock) was stronger antifungal activity than the others. Candida albicans is more susceptible than other fungi, followed by Candida tropicalis, Cryptococcus neoformans, C. guilliermondii, and C. lautentii. In addition, extracts exhibited greater antifungal effect against Candida species than Cryptococcus species. In a previous study, ethanol was observed as the best solvent for extracting antimicrobial substances (CitationJonathan & Fasidi, 2003). The results in this study with ethanol are similar to those reported in the mentioned study. It is important to bear in mind that the concentration of extract used in the test may be correlated with a high activity of its chemical components.
Table 1. Minimum inhibitory concentration of the ethanol leaf and rootstock.
Fungi used in this study were chosen primarily on the basis of their importance as opportunistic pathogens of humans. According to findings from the National Nosocomial Infection Surveillance System (NNIS), 61% of reported nosocomial fungal infections were due to Candida albicans, followed by other Candida spp. and Cryptococcus spp. (CitationWalsh, 1992). Candida albicans, while naturally occurring in the intestinal flora, can cause oral thrush and systemic infections. Cryptococcus neoformans causes cryptococcosis, an opportunistic infection of the lungs, especially in AIDS patients.
The wide spectrum of Lamium album L. therapeutic activity is related to a variety of biologically active substances which could be found in that plant, including flavonoids, iridoids, phenolic and fatty acids, polysaccharides, triterpenes, saponins, phytoecdysteroids, amines, essential oil, tannins, and mucilage (CitationAlipieva et al., 2003; CitationBudzianowski & Skrzypczak, 1995; CitationDamtoft, 1992; CitationDamtoft & Jensen, 1995; CitationEigtved et al., 1974; CitationHegnauer, 1989; CitationSavchenko et al., 2001). However, there are no literature data available on the phytochemistry of L. tenuiflorum. The mentioned substances may be responsible for the antifungal activity in L. tenuiflorum.
Lamium album L., L. maculatum L., and L. purpureum L. have been reported to be used as tonics and for the treatment of constipation in Anatolia (CitationBaytop, 1999). In western Anatolia, Turkey, the whole plant of L. album and several other Lamium species are used to relieve pain in rheumatism and other arthritic ailments (CitationOzaydin et al., 2006). There is little information about antimicrobial activity of Lamium species. The essential oil of Lamium garganicum subsp. laevigatum Arcang. was reported to be active against some Gram-positive and Gram-negative bacteria (CitationRoussis et al., 1996). In another study, the MeOH, CH2Cl2, n-BuOH and H2O extracts of L. eriocephalum subsp. eriocephalum Bentham, L. garganicum subsp. laevigatum Arcang., L. garganicum subsp. pulchrum R.Mill, and L. purpureum var. purpureum L. were tested for their antimicrobial and free radical scavenging activities. The CH2Cl2 extracts of all plants showed highest antimicrobial activity. Among the tested extracts the CH2Cl2 extract of L. eriocephalum subsp. eriocephalum is considered as the most active one against all tested bacteria and fungi. In addition, the CH2Cl2 extracts of L. garganicum subsp. pulchrum and L. purpureum var. purpureum as well as the MeOH extract of L. garganicum subsp. pulchrum possessed the same antifungal activity as the CH2Cl2 extract of L. eriocephalum subsp. eriocephalum on Candida albicans (CitationYalcin et al., 2007). The results on antifungal effects obtained in this study are similar to those reported in the mentioned study.
A growth stimulatory activity of plant extracts was clearly observed in several cases. This effect is a known phenomenon (CitationGriffin, 1994). For example, in addition to nutrients like carbohydrates, other compounds and agents usually in low quantities can have growth enhancing activity. Sterols, aliphatic aldehydes, organic acids, and flavonoids are among the groups that can stimulate growth. The importance of these compounds is that they may antagonize the antifungal activity of other ingredients present in the extract. Depending on the concentration and other factors, net observed activity can be growth inhibition, stimulation or no activity (CitationSardari et al., 1998).
From our investigation, the results obtained confirm the therapeutic potency of Lamium tenuiflorum used in traditional medicine. In addition, the result supports the folkloric usage of the studied plant and suggests that the plant extract possesses certain constituents with antifungal properties that can be used as antimicrobial agents in new drugs for the therapy of infectious diseases caused by pathogens. The most active extracts need to be subjected to isolation of the therapeutic antimicrobials and further pharmacological evaluation.
Declaration of interest: The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper.
References
- Alipieva KI, Taskova RM, Evstatieva LN, Handjieva NV, Popov SS (2003). Benzoxazinoids and iridoid glucosides from four Lamium species. Phytochemistry 64: 1413–1417.
- Baytop T (1999). Therapy with Plants in Turkey (Past and Present), 2nd edn. Istanbul, Nobel Medical Books, pp. 520 [in Turkish].
- Bremness L (1995). The Complete Book of Herbs. London, Dorling Kindersley, p. 181.
- Budzianowski J, Skrzypczak L (1995). Phenylpropanoid esters from Lamium album flowers. Phytochemistry 38: 997–1001.
- Damtoft S (1992). Iridoid glucosides from Lamium album. Phytochemistry 31: 175–178.
- Damtoft S, Jensen, SR (1995). Hemialboside: A hemiterpene glucoside from Lamium album. Phytochemistry 39: 923–924.
- Davis PH (1982). Flora of Turkey and East Aegean Islands, Vol. 7. Edinburgh, Edinburgh University Press, pp. 947.
- Dzoyem JP, Tangmouo JG, Lontsi D, Etoa FX, Lohoue PJ (2007). In vitro antifungal activity of extract and plumbagin from the stem bark of Diospyros crassiflora Hiern (Ebenaceae). Phytother Res 21: 671–674.
- Eigtved P, Jensen SR, Nielsen B (1974). A novel iridoid glucoside isolated from Lamium album L. Acta Chem Scand B 28: 85–91.
- Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo Z (1985). Medicinal plants in therapy. B World Health Organ 63: 965–981.
- Griffin DH (1994). Fungal Physiology. New York, Wiley-Liss, pp. 130–157.
- Guner A, Ozhatay N, Ekim T, Baser KHC (2000). Flora of Turkey. Vol 11. Edinburgh University Press, Edinburgh, p. 656.
- Hegnauer R (1989). Chemotaxonomie der Pflanzen. Bd and Birkhauser, Basel, pp. 609–611.
- Jonathan SG, Fasidi IO (2003). Antimicrobial activities of two Nigerian edible macrofungi, Lycoperdon pusilum and L. giganteum. Afr J Biomed Res 6: 85–90.
- Matkowski A, Piotrowska, M (2006). Antioxidant and free radical scavenging activities of some medicinal plants from Lamiaceae. Fitoterapia 77: 346–353.
- Olukoya DK, Idika N, Odugbemi T (1993). Antibacterial activity of some medicinal plants from Nigeria. J Ethnopharmacol 39: 69–72.
- Ozaydin S, Dirmenci T, Tumen G, Baser KHC (2006) Plants used as analgesic in the folk medicine Turkey, In: Ertug F, ed., Proceedings of the IVth International Congress of Ethnobotany (ICEB2005), Izmir, Ege Publications, pp. 167–171.
- Paduch R, Wojciak-Kosior M, Matysik G (2007). Investigation of biological activity of Lamii albi flos extracts. J Ethnopharmacol 110: 69–75.
- Roussis V, Chinou I, Perdetzoglou D, Loukis, A (1996). Identification and bacteriostatic activity of the essential oil of Lamium garganicum L. ssp. laevigatum Arcangeli. J Essent Oil Res 8: 291–293.
- Sardari S, Amin G, Micetich RG, Daneshtalab M (1998). Phytopharmaceuticals. Part 1. antifungal activity of selected Iranian and Canadian plants. Pharm Biol 36:180–188.
- Savchenko T, Blackford M, Sarker SD, Dinan L (2001). Phytoecdysteroids from Lamium sp.: identification and distribution within plants. Biochem Syst Ecol 29: 891–900.
- Shuya C, Xingguo C, Zhide H (2003). Identification and determination of ecdysone and phenylpropanoid glucoside and flavonoids in Lamium maculatum by capillary zone electrophoresis. Biomed Chromatogr 17: 477–482.
- Singh H, Gulati IB (1990). Tribological behavior of some hydrocarbon compounds and their blends. Wear 139: 425–437.
- Trouillas P, Calliste CA, Allais DP, Simon A, Marfak A, Delge C, Duroux JL (2003). Antioxidant, anti-inflammatory and anti-proliferative properties of sixteen water plant extracts used in the Limousin countryside as herbal teas. Food Chem 80: 399–407.
- Walsh JJ (1992). Invasive fungal infections: problems and challenges for developing new antifungal compounds. In: Sutcliffe JA, Georgapapadakov NH, eds., Merging Targets in Antibacterial and Antifungal Chemotherapy. New York, Chapman and Hall, 349–373.
- Willis A (1973). A Dictionary of Flowering Plants and Ferns, VIII. Cambridge University Press, Cambridge, p. 624.
- Yalcin FN, Kaya D, Kilic E, Ozalp M, Ersoz T, Calis I (2007). Antimicrobial and free radical scavenging activities of some Lamium species from Turkey. Hacettepe Univ J Fac Pharm 27: 11–22.