Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Méndez-Vilas (Ed.)

Antifungal plant extracts Marcel Pârvu1 and Alina E. Pârvu2 1

Department of Biology, Faculty of Biology and Geology, "Babes-Bolyai" University, 42 Republicii Street, 400015 ClujNapoca, Romania. 2 Department of Pathophysiology, Faculty of Medicine, "Iuliu Hatieganu" University of Medicine and Pharmacy, 3 Victor Babes Street, 400012 Cluj-Napoca, Romania. Corresponding author: [email protected], [email protected] Plant diseases are a threat for global food safety. Allium obliquum, Allium fistulosum, Allium ursinum, Aloe vera, Berberis vulgaris and Chelidonium majus plant extracts were obtained by the repercolation method and the antifungal activity was determined by the agar-dilution assay against Apergillus niger, Botrytis cinerea, Botrytis paeoniae, Fusarium oxysporum f. sp. gladioli, Fusarium oxysporum f.sp. tulipae, Heterosporium pruneti, Penicillium gladioli, Penicillium expansum, and Sclerotinia sclerotiorum. Phytochemical screening was performed for phenolic compounds, allicin and allin in Allium species, for aloine in Aloe vera, and for berberine in Berberis vulgaris. Ultrastructural changes induced by the plant extracts were visualized by electron microscopy. The antifungal effects of the studied plant extracts recommend them as candidates for the in vivo biological control. Key words: plant extract, phytopathogenic fungi, antifungal

1. Introduction The development of strategies to control fungal infections may be an effective means for therapeutic interventions. Plant fungicides based on synthetic chemicals cause severe and long-term environmental pollution, are highly and acutely toxic, and can even cause cancer in humans and wild animals. Also, pathogens may become resistant to many of these chemicals. Consequently, the aim of new antifungal strategies is to develop drugs that combine sustainability, high efficacy, restricted toxicity, safety for humans, animals, host plants and ecosystems with low production cost. Since fungicides of biological origin have been demonstrated to be specifically effective on target organisms and are also biodegradable, biological control has become popular worldwide [1-5]. Medicinal plants remain a rich source of novel therapeutic agents. Many plant species are still unevaluated chemically or biologically. Several studies regarding the action of plant extracts against some phytopathogenic fungi have been performed. The quality and quantity of the biologically active compounds from the plant extracts significantly depend on the species, the plant organ and harvest time [6-9]. Different plant extracts from Allium obliquum, A. ursinum, A. fistulosum, Aloë vera, Chelidonium majus, and Berberis vulgaris were obtained in the Mycology Laboratory of Babes-Bolyai University, Cluj-Napoca, Romania, by modified Squibb’s repercolation method [10]. The agar-dilution assay was used to determine the in vitro antifungal activity, expressed as minimum inhibitory concentration (MIC) or minimum fungicidal concentration (MFC), against some phytopathogenic fungi (Aspergillus niger isolated from A. cepa bulbs; Botrytis cinerea from Rosa flowers; Botrytis paeoniae from Paeonia officinalis flowers; Botrytis gladiolorum, Fusarium oxysporum f. sp. gladioli and Penicillium gladioli from Gladiolus  hybridus corms; Fusarium oxysporum f.sp. tulipae from tulip; Heterosporium pruneti from Iris germanica flower; Penicillium expansum from Malus domestica fruits, and Sclerotinia sclerotiorum from Daucus carota ssp. sativus). As the quality of most plant extracts relies on organosulfur and polyphenols compounds, phytochemical screening was performed by LC/MS/MS [8-13]. The morpho-functional integrity of fungal cell components is required in order to maintain their viability and germination capacity. It has been demonstrated that MIC or MFC of some plant extracts induced fungal ultrastructural changes that were visualized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) [14,15].

2. Antifungal effect of Allium extracts Allium species have been used in traditional medicine for many centuries. Generally, there are more than 300 Allium species, some of them having been recently described [16]. Most of the research regarding the phytotherapeutic properties of Allium species is performed on A. cepa and A. sativum plants. A. sativum plant extract has antihypertensive, antidiabetic, hepatoprotective, immunostimulating, antioxidant and antitumor activities, antiviral, antifungal, antihelmintic and antiparasitic effects [16-22]. The aqueous extracts from A. cepa and A. sativum have antifungal action against C. albicans and other Candida isolates, against Malassezia furfur isolates and dermatophyte species. Also, A. cepa plant extract has antifungal action against Tricophyton rubrum and T. mentagrophytes species [23-25].

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Another species with antimicrobial activity is A. ascalonicum. It has antifungal action against C. albicans, dermatophytes (Microsporum gypseum, Trichophyton mentagrophytes, and Epidermophyton floccosum), Syncephalastrum spp., A. niger, Penicillium spp., Paecilomyces spp., Scopulariopsis spp., Cladosporium spp., Alternaria spp., Drechslera spp. at MIC of 0.25 % [7]. Ethnobotanical information from Romania mentions 32 wild and cultivated species of Allium. A. obliquum is a very rare perennial edible plant which can be found in a single place in Romania, i.e. on the limestone rocks of Turda Gorges. As a wild species, it is also encountered, in South-East Russia, Siberia and Central Asia [26]. In Table 1, A. obliquum plant extract had species dependent antifungal activity against S. sclerotiorum, B. cinerea, F. oxysporum f. sp. gladioli, A. niger and P. expansum. It was mostly active against S. sclerotiorum and B. cinere and least active against F. oxysporum f. sp. gladioli, A. niger and P. expansum [9]. Table 1 Minimum inhibitory concentration (MIC) of Allium plant extracts tested against phytopathogenic fungi

Fungus species

MIC of A. obliquum µl/ml

MIC of A. fistulosum µl/ml

Apergillus niger

80

Botrytis cinerea

100

MIC of A. ursinum flower µl/ml 100

MIC of A. ursinum leaves µl/ml 120

60

80

60

80

-

80

70

100

Fusarium oxysporum f. sp. gladioli

70

-

-

-

Fusarium oxysporum f.sp. tulipae

-

-

140

160

80

-

-

-

-

100

90

120

50

80

60

80

Botrytis paeoniae

Penicillium expansum Penicillium gladioli Sclerotinia sclerotiorum

One of the cultivated species of Allium is A. fistulosum (Welsh onion). This is a perennial species that has alimentary importance and is originated from Eastern Asia. It is not known as a wild species and it is cultivated for its leaves which are consumed fresh all over the year. In Table 1, A. fistulosum plant extract had antifungal effects against A. niger, B. cinerea, B. paeoniae, P. gladioli and S. sclerotiorum [27]. A. ursinum (ramson, wild garlic) is a wild-growing species found in European and Northern Asia forests. In recent years, the potential health benefits of ramson have been attributed mainly to the sulfur-containing compounds [28]. Several biological activities of A. ursinum plants, such as antioxidative, cytostatic and antimicrobial have been reported [29-31]. In Table 1 A. ursinum flower and leaf extracts had antifungal effects against A. niger, B. cinerea, B. paeoniae, F. oxysporum f. sp. tulipae, P. gladioli, and S. sclerotiorum [32]. The inhibitory effect of A. ursinum flower extract was stronger than that of the leaf extract for all concentrations and for all tested fungi. Allium plants and extracts contain different chemical compounds. In A. cepa, A. sativum and A. ampeloprasum extracts, different biologically active substances, such as organosulphurous compounds like alliin and allicin, E/Zajoene, sterols, flavones and polyphenolcarboxylic acids have been found [23,24]. Alliin is the precursor of allicin, formed by the action of allinase enzyme. There is also a secondary substance resulting from alliin decomposition, called ajoene [33]. Allicin has antibacterial, antiviral, antitumour, anticoagulation, antihypertensive, antiparasitic and hepatoprotective effects. It is also efficient against many fungal species, such as Aspergillus flavus, A. niger, Candida albicans, Fusarium laceratum, Microsporum canis, Mucor racemosus, Penicillium spp., Rhizopus nigricans, Saccharomyces spp., Trichophyton granulosum, F. oxysporum, B. cinerea, B. paeoniae, P. gladioli, and S. sclerotiorum [24,27]. Besides these biologically active substances, a novel antifungal peptide, called allicepin, was isolated from A. cepa bulbs [33]. In Table 2, the analysis of allicin by liquid chromatography-coordination ion spray-mass spectrometry method (LCCIS-MS/MS) in five Allium species extracts, A. obliquum, A. senescens subsp. montanum, A. schoenoprasum subsp. schoenoprasum, A. fistulosum, A. ursinum (leaves), A. ursinum (flowers) found that the extracts prepared by heating at 60 ºC are richer in allicin than room temperature extracts, proving that extraction at higher temperatures favors the transformation of alliin to allicin [34]. A. obliquum extract alliin content correlates with antifungal effect [9]. In A. ursinum plant extracts, alliin, isoalliin, methiin, flavonoid glycosides, saponins, polyphenolic compounds, volatile oil and other secondary metabolites have been determined. Alliin dominates in the widely used “garlic-type”, which includes wild leek (A. obliquum) and sand leek (A. scorodoprasum). Alliin and isoalliin rarely co-dominate, being only found in the cultivated Chinese leek (A. tuberosum). A mix of almost equal amounts of methiin, alliin and isoalliin is present in A. ursinum [28,30,31,35].

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Table 2 The allicin content found in various Allium extracts [34].

Allium species A. obliquum A. senescens subsp. montanum A. schoenoprasum subsp. schoenoprasum A. fistulosum A. ursinum (leaves) A. ursinum (flowers)

Allicin (mg/ml in 600C extract) 2.819 0.919 3.968 0.122 0.028 1.946

Allicin (mg/ml in room temp. extracts) 5.579 5.880 3.410 4.481 -

In A. ursinum leaves and bulbs, the highest amount of volatile precursors was found in March and April, shortly before flowering time [28]. In order to compare A. ursinum flower and leaf extracts, the plants were harvested shortly after blooming. The stronger antifungal activity of the A. ursinum flower extract compared to the leaf extract may be attributed to the higher content of allicin [32]. Polyphenols are bioactive molecules widely distributed in plant species, with a great variety of structures, ranging from simple compounds to highly complex polymeric substances. Phenolic compounds have been reported to have very wellcharacterised multiple biological effects: antioxidant, antimutagenic, antibacterial, antiviral, anti-inflammatory and antithrombotic [36-39]. Plant extracts which are rich in polyphenols are of increasing interest in the food industry because they retard the oxidative degradation of lipids and improve the quality and nutritional value of food [35,39]. In five Allium species from Romania, (A. obliquum, A. senescens subsp. montanum, A. schoenoprasum subsp. schoenoprasum, A. fistulosum and A. ursinum) six phenolic acids, three flavonoid glycosides and four aglycones were identified using a high-performance liquid chromatographic (HPLC) method. Ferulic acid and p-coumaric acid were identified in all samples. Sinapic acid derivatives were present in A. obliquum, A. senescens subsp. montanum, A. schoenoprasum subsp. schoenoprasum and A. fistulosum, while in the leaves and flowers of A. ursinum this compound was not detected. The pattern of flavonoids indicates large differences between all the five species, which can be used as potential taxonomic markers in order to distinguish the plants: isoquercitrin was identified in A. obliquum, A. schoenoprasum subsp. schoenoprasum and A. fistulosum, rutin in A. senescens subsp. montanum and A. schoenoprasum subsp. schoenoprasum, whereas quercitrin was identified only in A. fistulosum. Luteolin and apigenin were determined only in A. obliquum, before and after acid hydrolysis. A. obliquum and A. senescens subsp. montanum contain glycosides of kaempferol and quercetol, and A. ursinum contains only kaempferol derivatives. Kaempferol and quercetol were present in both the nonhydrolysed and hydrolysed samples of A. schoenoprasum subsp. schoenoprasum and A. fistulosum. The richest species in quercetol derivatives was A. schoenoprasum subsp. schoenoprasum, and the richest one in kaempferol derivatives was A. ursinum. Kaempferol derivatives are present in larger amount than the quercetol derivatives in A. schoenoprasum subsp. schoenoprasum and A. fistulosum. We observed only quantitative differences between the two samples of A. ursinum: the flowers contain a larger amount of kaempferol derivatives, whereas the leaves contain a larger amount of p-coumaric acid and ferulic acid. Kaempferol derivatives were previously identified in the aerial parts of A. senescens subsp. montanum and A. ursinum [35,40,41].

A

B

Fig. 1 TEM micrograph of oblique section of Fusarium oxysporum f.sp. tulipae hyphae: A. control hyphae; B. hyphae treated with Allium fistulosum plant extract MIC; C. cytoplasm; CW. cell wall; ES. extracelluar sheath; G. glycogen; L. lipids; M. mitochondrion; N. nucleus; S. septum; V. vacuole [27].

It is known that the genus Fusarium is a soilborne, necrotrophic, plant pathogenic fungus with many species that cause serious plant diseases around the world. Fusarium oxysporum causes primarily vascular wilts on many crops, whereas numerous species, especially F. solani, cause root and stem rots and rots of seeds that are accompanied by the

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production of mycotoxins. F. oxysporum consists of more than 120 formae speciales according to the hosts they infect. Each of them can be subdivided into physiological races with characteristic patterns of virulence on different host varieties [42]. In Figure 1 and Figure 2 Fusarium oxysporum f.sp. tulipae hyphae treated with A. fistulosum plant extract showed ultrastructural changes: the external sheath was slightly modified and the cell wall had irregular shape on the outside, the organelles were partly and/or entirely destroyed, the cytoplasm was degenerated and electron dense material appeared in the hyphal cells. The precipitation of the cytoplasm and the destruction of the organelles and nucleus caused the loss of hyphae’s viability [27].

A

B

Fig. 2 TEM micrograph of a cross section of Fusarium oxysporum f.sp. tulipae hyphae: A. control hyphae; B. hyphae treated with Allium fistulosum plant extract in MIC; C. cytoplasm; CW. cell wall; ER. endoplasmic reticulum; ES. extracelluar sheath; L. lipids; P. plasmalemma [27].

3. Antifungal activity of Aloe vera leaf extract Aloe vera is a shrubby or arborescent, perennial, xerophytic, succulent, pea-green color plant. It grows mainly in the dry regions of Africa, Asia, Europe and America [43]. A. vera has been used in folk medicine as the remedy for a variety of conditions. It possesses many pharmacological activities, including antiinflammatory, immunostimulant and wound healing, antiulcer, antidiabetic and antitumor. Due to the multiple biological effects, A. vera was considered as a miracoulous plant. Its activities were attributed to the variety of its chemical components including anthraquinones, glycoproteins, polysaccharides, vitamins and enzymes [44-47]. Aloin and aloe-emodin are the major anthraquinones in aloe plants. Since aloin and aloe-emodin contain a polyphenolic structure, they may be responsible for the antiinflammatory effects of aloe [48]. A. vera contains six antiseptic agents: lupeol, salicylic acid, urea nitrogen, cinnamonic acid, phenols and sulfur. They all have inhibitory action on fungi, bacteria and viruses [43,49]. The total hydroalcoholic plant extract obtained from A. vera fresh leaves harvested from the greenhouses of “Alexandru Borza” Botanical Garden in Cluj-Napoca had antifungal activity against the mycelial growth of B. gladiolorum, F. oxysporum f.sp. gladioli, H. pruneti and P. gladioli. A quantity of 0.017705 mg aloin/ml Aloe vera plant extract was determined by HPLC [50].

4. Antifungal activity of Berberis vulgaris extract Berberis vulgaris (barberry) is a common garden bush, native to Europe and the British Isles, and naturalized in North America. It has played a prominent role in herbal healing for more than 2,500 years [51]. Important antifungal activity of Berberis spp. has been demonstrated against some fungal strains with hydroalcoholic extracts, aqueous extract, methanolic or crude extracts, and alkaloidal fractions [12,13]. Alcoholic extracts provide more complete extraction and include fewer polar compounds [42]. Alcoholic extracts provide more complete extraction and include fewer polar compounds [42]. The in vitro antifungal activity of berberine isolated from the same sources has also been investigated and it was found that berberine alkaloids are cationic antimicrobials. Twenty-two alkaloids of medicinal importance have been reported so far from the roots, stems, leaves and fruit of Berberis spp. [52,53]. The alkaloid content differs in Berberis from different areas, species and organs. The main alkaloid that has been isolated from the roots and bark of B. vulgaris is berberine, an isoquinoline alkaloid with antibacterial and antifungal properties. The hydroalcoholic B. vulgaris plant extract had stronger antifungal effect against S. sclerotiorum than berberine. These results can be explained by the complex composition of Berberis plant extracts which contain besides berberine some other alkaloids, such as berbamine, oxyacanthine, magnoflorine, berberubine, etc. [12,13].

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Botrytis cinerea is a necrotrophic opportunistic plant pathogenic fungus also known as “gray mould fungus” which causes serious pre- and postharvest diseases in more than 200 plant species, including agriculturally important crops and harvested commodities, such as grapes, tomatoes, strawberries, cucumbers, bulb flowers, cut flowers and ornamental plants [54]. The broad host range of B. cinerea results in great economic losses, not only during growth but also during storage and transportation of products [55]. Necrotrophs kill their host cells by secreting toxic compounds or lytic enzymes and also produce an array of pathogenicity factors that can subvert host defences [56]. Botrytis cinerea strains are highly genetically and physiologically variable and several strains developed resistance to most of the fungicides used to control them [54,57,58]. Previous studies showed that the B. cinerea conidia cell wall has two layers and appears dark because of melanin, which protects the spores against enzyme action and probably UV [59]. The surface of dry B. cinerea conidia and other Botrytis spp. has many short protuberances (200–250 nm), visible with SEM and TEM. Hydration and redrying caused these protuberances to disappear [60,61]. Examination by SEM showed in Figure 3 revealed that B. vulgaris bark extract, at its MIC, induced large-scale damage to B. cinerea conidia, because the surface protuberances from the control disappeared. On TEM micrographs, as shown in Figure 4, B. vulgaris bark extract caused a disruption of the B. cinerea conidial cell wall, the external layer was more electron dense, the plasmalemma and the cytoplasm of the B. cinerea conidia had shrunk and detached itself altogether from the cell wall, the organelles and nucleus were also partly destroyed. Berberine treatment caused similar changes of the B. cinerea conidia as did B. vulgaris bark extract [51].

A

B

Fig. 3 SEM micrograph of Botrytis cinerea conidium: A. control conidium showing randomly positioned surface protuberances; B. conidium treated with Berberis vulgaris MIC showing surface protuberance damage [51].

Other studies have described B. cinerea conidial ultrastructure. The morpho-functional integrity of fungal cell components is required to maintain their viability and germination capacity. The TEM micrographs of control conidia showed a regular cell wall, approximately 300–400 nm thick, with a two-layer structure, plasmalemma, cytoplasm matrix with nucleus, mitochondria and vacuoles. The cell wall external layer was thin and electron dense, and the inner one was thick, uniform and less electron dense. The plasmalemma tightly adhered to the cell wall. The cytoplasm matrix (cytosol) was uniformly distributed, and the nucleus was up to 2 µm in diameter and ovoid or spherical in shape. Among cell organelles, mitochondria were numerous, usually ovoid and medium electron dense. Vacuoles were similar in size to mitochondria [60,62]. The electron microscopy data revealed that B. vulgaris bark extract, at its MIC, acts by causing irreversible ultrastructural changes to the B. cinerea conidia. Importantly, the antifungal effect was rapid (one h incubation), which should make it difficult for the pathogen to develop resistance. Even when berberine MIC was bigger than that of B. vulgaris bark extract, the electron microscopy examination showed that it had similar effects on the B. cinerea conidia. Due to these results, it is likely that most of the morpho-functional changes induced by B. vulgaris bark extract are due to the berberine content [51].

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A

B

Fig. 4 TEM micrograph of Botrytis cinerea conidium cross section: A. control conidium; B. conidium treated with Berberis vulgaris plant extract MIC, showing irreversible ultrastructural changes; C. cross section of a B. cinerea conidium treated with berberine in MIC, showing irreversible ultrastructural changes; CW. cell wall; P. plasmalemma; PS. periplasmic space; C. cytoplasm; M. mitochondrion; N. nucleus; V. vacuole; L. lipids [51].

C

B. vulgaris plant extract caused irreversible ultrastructural changes in S. sclerotiorum sclerotia, too. In Figure 5, TEM micrographs showed that the precipitation of the entire cytoplasmic content and the destruction of organelles and nucleus led to the loss of viability and germination capacity of sclerotia, after 3 hours of treatment with plant extract in MFC [12]. By these changes B. vulgaris plant extract abolishes forever the barrier function of the cell wall and the possibility to activate the enzymes bound to the cell wall.

A

B

Fig. 5 TEM micrograph showing ultrastructural changes in Sclerotinia sclerotiorum sclerotia (internal zone): A. control; B. treated with Berberis vulgaris plant extract in minimum fungicidal concentration; C. cytoplasm; CW. cell wall; G. glucan; L. lipid bodies; N. nucleus; IS. interhyphal space [12].

5. Antifungal activity of Chelidonium majus extract C. majus is a common, poisonous herbaceous perennial from the pappy family known as celadine. Plant extracts and their purified compounds have antibacterial, antiviral and fungicidal effects both in vitro and in vivo. Their properties were attributed mainly to alkaloids, several flavonoids and phenolic acids. The main alkaloids from C. majus extracts are chelidonine, chelerythrine, sanguinarine, coptisine and berberine [13,63].

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The hydroalcoholic C. majus plant extract obtained from powder of dried aboveground plant organs collected from a private homegarden in Cluj-Napoca, Romania during the summer of 2007 had antifungal effect against B. cinerea. On the SEM micrographs of the B. cinerea conidia treated with MFC of C. majus extract the shape and size did not change but the surface protuberances disappeared. The TEM micrographs showed important irreversible ultrastructural changes: the cell wall had a slightly irregular outline, loosely distributed components and was highly permeable; the cell wall external layer was more electron dense; the plasmalemma was mostly destroyed and did not adhere to the cell wall; precipitation of the entire cytoplasm and destruction of organelles and nucleus were seen. Due to these effects, the morpho-functional relationship between the cell wall and the cytoplasm was destroyed and a less electron dense band was formed between the altered cytoplasm and the cell wall [13]. In conclusion, the results showed that antifungal effects of plant extracts depend on the pathogenic species, on the type of plant extract and on the content of biologically active compounds. The antifungal effects of the studied plant extracts recommend them as good candidates for the in vivo biological control of phytopathogenic fungi, limiting the abuse of chemical fungicides. Acknowledgements: These studies were financially supported by the Romanian Ministry of Education and Research from the CNCSIS grants 46/220/2006, 43/220/2007 and PNII–IDEI 2272/2008.

References [1] Barker KS, Rogers PD. Recent insights into the mechanisms of antifungal resistance. Curr. Infect. Dis. Rep. 2006;8:449-456. [2] Carrillo-Munoz AJ, Giusiano G, Ezkurra PA, Quindos G. Antifungal agents: mode of action in yeast cells. Rev. Esp. Quimioter. 2006;19:130-139. [3] Fatehi M, Saleh TM, Fatehi-Hassanabad Z, Farrokhfal K, Davodi SJ. A pharmacological study on Berberis vulgaris fruit extract. Ethnopharmacol. 2005;102:46-52. [4] Strange RN, Scott PR. Plant disease: a threat to global food security. Annu. Rev. Phytopathol. 2005;43:83-116. [5] Ienaşcu IMC, Lupea AX, Hădăruga D, Hădăruga N, Popescu IM. The antimicrobial activity and quantitative structure – biological activity relationships evaluation of some novel 2-hydroxybenzamide derivatives. Revista de Chimie. 2008;59:247250. [6] Abubakar EL-MM. Efficacy of crude extracts of garlic (Allium sativum Linn.) against nosocomial Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa. J. Med. Plants Res. 2009;3:179-185. [7] Mahmoudabadi AZ, Nasery MKG. Antifungal activity of shallot, Allium ascalonicum Linn. (Liliaceae), in vitro. J. Med. Plants Res. 2009;3(5):450-453. [8] Pârvu M, Roşca-Casian O, Puşcaş M, Groza G. Antifungal activiy of Allium fistulosum L. Contrib. Bot. 2009;44:125-129. [9] Pârvu M, Pârvu AE, Roşca-Casian O, Vlase L, Groza G. Antifungal activity of Allium obliquum. J. Med. Plants Res. 2010;4:138141. [10] Ionescu-Stoian P, Savopol E. Extracte farmaceutice vegetale. Ed. Medicală, Bucureşti; 1977. [11] Bhandari DK, Nath G, Ray AB, Tewari PV. Antimicrobial activity of crude extracts from Berberis asiatica stem bark. Pharmaceut. Biol. 2000;38:254-257. [12] Pârvu M, Roşca-Casian O, Crăciun C, Barbu-Tudoran L, Vlase L, Tămaş M, Danciu RM. Ultrastructural changes in Sclerotinia sclerotiorum sclerotia treated with Berberis vulgaris plant extract. IOBC/wprs Bull. 2007;30:149-152. [13] Pârvu M, Pârvu AE, Crăciun C, Barbu-Tudoran L, Tămaş M. Antifungal activities of Chelidonium majus extract on Botrytis cinerea in vitro and ultrastructural changes in its conidia. J. Phytopathol. 2008;156:550-552. [14] Vlase L, Radu L, Fodorea C, Leucuta S, Gocan S. Determination of phenolic compounds from Geranium sanguineum by HPLC. J Liq Chromatogr R T. 2005;19: 3109–3117. [15] Hayat MA. Principles and techniques of electron microscopy: biological applications. Cambridge University Press, London; 2000. [16] Vimal V, Devaki T. Hepatoprotective effect of allicin on tissue defense system in galactosamine/endotoxin challenged rats. J.Ethnopharmacol. 2004;90:151-154. [17] Al-Qattan KK, Alnaqeeb MA, Ali M. The antihypertensive effect of garlic (Allium sativum) in the rat two-kidney–one-clip Goldblatt model. J. Ethnopharmacol. 1999;66:217-222. [18] Ayaz E, Alpsoy HC. Garlic (Allium sativum) and traditional medicine. Türkiye Parazitol. Derg. 2007;31:145-149. [19] Motsei ML, Lindsey KL, van Staden J, Jäger AK. Screening of traditionally used South African plants for antifungal activity against Candida albicans. J. Ethnopharmacol. 2003;86:235-241. [20] Lemar KM, Passa O, Aon MA, Cortassa S, Müller CT, Plummer S, O’Rourke B, Lloyd D. Allyl alcohol and garlic (Allium sativum) extract produce oxidative stress in Candida albicans. Microbiology. 2005;151:3257-3265. [21] Thamburan S, Klaasen J, Mabusela WT, Cannon JF, Folk W, Johnson Q. Tulbaghia alliacea phytotherapy: a potential antiinfective remedy for candidiasis. Phytother Res. 2006;20:844-850. [22] Guarrera PM. Traditional antihelmintic, antiparasitic and repellent uses of plants in Central Italy. J. Ethnopharmacol. 1999;68:183-192. [23] Duke JA, Bogenschutz-Godwin MJ, duCellier J, Duke PAK. Handbook of Medicinal Herbs. CRC Press, Boca Raton, London, New York and Washington D.C.; 2002. [24] Josling P. Allicin The Heart of Garlic. NWI Publishing Callahan Florida; 2003. [25] Ghahfarokhi MS, Goodarzi M, Abyaneh MR, Al-Tiraihi T, Seyedipour G. Morphological evidences for onion-induced growth inhibition of Trichophyton rubrum and Trichophyton mentagrophytes. Fitoterapia. 2004;75:645-655.

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[26] Ciocârlan V. Flora ilustrată a României Pteridophyta et Spermatophyta. Ed. Ceres, Bucureşti; 2009. [27] Pârvu M, Barbu-Tudoran L, Roşca-Casian O, Vlase L, Tripon SC. Ultrastructural changes in Fusarium oxysporum f.sp. tulipae hyphae treated in vitro with Allium fistulosum plant extract. Annals of RSCB. 2010;2:65-72. [28] Schmitt B, Schulz H, Storsberg J, Keusgen M. Chemical characterization of Allium ursinum L. depending on harvesting time. J. Agric. Food Chem. 2005;53:7288–7294. [29] Stajner D, Popovic BM, Canadanovic-Brunet J, Stajner M. Antioxidant and scavenger activities of Allium ursinum. Fitoterapia. 2008;79(4):303-305. [30] Sobolewska D, Janeczko Z, Kisiel W, Podolak I, Galanty A, Trojanowska D. Steroidal glycoside from the underground parts of Allium ursinum L., and their cytostatic and antimicrobial activity. Acta Pol. Pharm.-Drug Res. 2006;3:219-223. [31] Ivanova A, Mikhova B, Najdenski H, Tsvetkova I, Kostova I. Chemical composition and antimicrobial activity of wild garlic Allium ursinum of Bulgarian origin. Nat. Prod. Commun. 2009;8:1059-62. [32] Pârvu M, Pârvu AE, Vlase L, Roşca-Casian O, Pârvu O. Antifungal properties of Allium ursinum L. ethanol extract. J. Med. Plants Res. 2011;5:2041-2046. [33] Wang HX, Ng TB. Isolation of allicepin, a novel antifungal peptide from onion (Allium cepa) bulbs. J. Pept. Sci. 2003;10:173177. [34] Vlase L, Pârvu M, Toiu A, Pârvu AE, Cobzac SC, Puşcaş M. Rapid and Simple Analysis of Allicin in Allium Species by LCCIS-MS/MS. Studia Univ.Babeş-Bolyai, Chemia. 2010;4:297-304. [35] Pârvu M, Toiu A, Vlase L, Pârvu AE. Determination of some polyphenolic compounds from Allium species by HPLC-UV-MS. Nat Prod Res. 2010;24:1318–1324. [36] Bravo L. Polyphenols: Chemistry, dietary sources, metabolism and nutritional significance. Nutr Rev. 1988;11:317–333. [37] Duthie GG, Duthie SJ, Kyle JA. Plant polyphenols in cancer and heart disease: Implications as nutritional antioxidants. Nutr Res Rev. 2000;1:79–106. [38] Pratt DE. Phenolic compounds in food and their effects on health II. Washington, DC: American Chemical Society; 1992. [39] Shashidi F, Wanasundara P., Hong C. Phenolic compounds in food and their effects on health I. Washington, DC: American Chemical Society; 1992. [40] Selyutina LY, Tankhaeva LM, Olennikov DN. Acylated kaempferol diglycoside from Allium senescens. Chemistry of Natural Compounds. 2008;44(6):769–770. [41] Carotenuto A, De Feo V, Fattorusso E, Lanzotti V, Magno S, Cicala C. The flavonoids of Allium ursinum. Phytochemistry. 1996;2:531–536. [42] Webster D, Taschereau P, Belland RJ, Sand C, Rennie RP. Antifungal activity of medicinal plant extracts; preliminary screening studies. J. Ethnopharmacol. 2008; 115:140-146. [43] Atherton P. Aloe vera revisited. Br J Phytother. 1998;4:76–83. [44] Ozsoy N, Candoken E, Akev N. Implications for degenerative disorders: antioxidative activity, total phenols, flavonoids, ascorbic acid, beta-carotene and beta-tocopherol in Aloe vera. Oxid Med Cell Longev. 2009;2(2):99-106. [45] Choi S, Chung M-H. A review on the relationship between Aloe vera components and their biologic effects. Semin Integr Med. 2003;1:53-62. [46] Eshun K, He Q. Aloe vera: A valuable ingredient for the food, pharmaceutical and cosmetic industries-a review. Crit Rev Food Sci Nutr. 2004;44:91-6. [47] Hu Y, Xu J, Hu Q. Evaluation of antioxidant potential of Aloe vera (Aloe barbadensis Miller) extracts. J Agric Food Chem. 2003;51:7788-91. [48] Park MY, Kwon HJ, Sung MK. Evaluation of aloin and aloe-emodin as anti-inflammatory agents in aloe by using murine macrophages. Biosci Biotechnol Biochem. 2009;73(4):828-32. [49] Surjushe A, Vasani R, Saple DG. Aloe vera: a short review. Indian J Dermatol. 2008;53(4):163-6. [50] Roşca-Casian O, Pârvu M, Vlase L, Tămaş, M. Antifungal activity of Aloë vera leaves. Fitoterapia. 2007;78:219-222. [51] Pârvu M, Pârvu AE, Crăciun C, Barbu-Tudoran L, Vlase L, Tămaş M, Roşca-Casian O, Tripon SC, Persecă O, Molnar AM. Changes in Botrytis cinerea conidia caused by Berberis vulgaris extract. Notulae Botanicae. 2010;38(3):15-20. [52] Agrios GN. Plant pathology. 5th ed. Elsevier Academic Press; 2005. [53] Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA, Lewis K. Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor. Proc. Natl. Acad. Sci. U.S.A. 2000;97:1433-1437. [54] Kars I, Krooshof G, Wagemakers CAM, Joosten R, Benen JAE, van Kan JAL. Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris. Plant J. 2005;43:213-225. [55] Elad Y. Biocontrol of foliar pathogens: mechanisms and application. Commun. Agric. Appl. Biol. Sci. 2003;68:17-24. [56] Makovitzki A, Viterbo A, Brotman Y, Chet I, Shai Y. Inhibition of fungal and bacterial plant pathogens in vitro and in planta with ultrashort cationic lipopeptides. Appl. Environ. Microbiol. 2007;73:6629-6636. [57] Silva E, Valdés J, Holmes D, Shmaryahu A, Valenzuela PD. Generation and analysis of expressed sequence tags from Botrytis cinerea. Biol. Res. 2006;39:67-76. [58] van Baarlen P, Legendre L, van Kan JAL. Plant defence compounds against Botrytis infection. In: Elad Y, Williamson B, Tudzynski P, Delen N (eds.). Botrytis: Biology, pathology and control. Netherlands: Kluwer Academic Publishers; 2004. [59] Epton HAS, Richmond DV. Formation, structure and germination of conidia. In: Coley-Smith JR, Verhoeff K, Jarvis WR (eds), The biology of Botrytis. London, UK: Academic Press; 1980:41–83. [60] Coley-Smith JR. Sclerotia and other structures in survival. In: Coley-Smith JR, Verhoeff K, Jarvis WR (eds.). The biology of Botrytis. London, UK: Academic Press; 1980:314-317. [61] Doss RP, Christian JK, Potter SW, Soeldner AH, Chastagner GA. The conidial surface of Botrytis cinerea and several other Botrytis species. Can J Bot. 1997;75:612–617. [62] Segmüller N, Kokkelink L, Giesbert S, Odinius D, van Kan J, Tudzynski P. NADPH oxidases are involved in differentiation and pathogenicity in Botrytis cinerea. Mol. Plant. Microbe Interact. 2008;2:808-819. [63] Saglam H, Arar G. Cytotoxic activity and quality control determinations on Chelidonium majus. Fitoterapia. 2003;74:127–129.

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