Assessment of in vitro. Antifungal Activities of Various Extracts of Indigenous Bahraini Medicinal Plants

ABSTRACT

The in vitro. antifungal activity of the aqueous, ethanol, chloroform, petroleum ether, and residue extracts from 10 indigenous Bahraini plants used in folk medicine for the treatment of various diseases is reported. Extract efficacy was evaluated using the agar well diffusion assay against four filamentous fungi and two yeasts monitored by standard antifungal disks. The results showed that all but, in particular, ethanol and chloroform of the examined plant extracts reveal variable degree of bioactivity against at least two of the tested microbes. The highest ethanol extract activity was exhibited by Cressa cretica. L. against Penicillium citrinum. Thom (32.2 mm) followed by Candida albicans. (C. P. Robin) Berkhout (25.7 mm). The diffusable metabolites of Heliotropium curassavicum. also demonstrated marked inhibitory effect against the same microorganisms. Chloroform extract of Emex spinosa. Campd. displayed an elevated potency against Alternaria alternata. (Fries) Keissler (27.9 mm) and Saccharomyces cerevisiae. (27.5 mm). Zone of inhibition against other fungi varied from 19.9 mm to 25.9 mm, indicating a novel class of metabolite. However, the highest growth inhibition was encountered with Fagonia indica. Burm f. against Penicillium citrinum. (29.3 mm). With the exception of chloroform extracts from cultivated soils, various extracts of plants randomly collected from saline-affected soils exhibited higher fungal radius inhibition than plants from cultivated soils. The significance of these results in relation to ethnobotanical data are discussed.

Keywords:

• Antifungal activity
• Bahraini
• Cressa cretica
• Emex spinosa
• fungi
• Heliotropium curassavicum
• medicinal plants

Introduction

Bahrain is a small semiarid island nation in the Arabian Gulf with rich plant resources. About 310 plant species are reported from various ecological habitats (El-Oqlah & Abbas, 1994). Of these, 80 species were documented as folk remedies for both internal and external use (Abbas & Al-Saleh, 2002). The people of Bahrain have very long-standing tradition in the trade and use of ethnomedicine due to the island's strategic location and several other sociocultural factors. The practice is still strong in the treatment of minor ailments with these plants including ulcers, pneumonia, stomach disorders, rheumatism, diabetes, renal problems, and bronchitis (Abbas et al., 1992). Researchers are increasingly turning their attention to folk medicine and antimicrobial compounds from plant species used in herbal medicine in Bahrain (Abbas et al., 1992; Al-Saleh et al., 19931997; Mahasneh et al., 1996; Taha & Hashem, 2000). Moreover, herbal medicine is improved as an alternative effective solution to health problems and cost of pharmaceutical products. However, little work has been done to match this ethnobotanical information with analytical research to identify active chemical compounds.

Plants used in traditional medicine can offer potential sources of new biologically active compounds, many of them with anticancer agents, anti-HIV agents, and antifungal activity. Examples of these compounds include flavanoids, saponins, phenolics, glucosinolates and cyanogenic glycosides (Bennett & Wallsgrove, 1994; Grayer & Harborne, 1994). Also, these plants can serve as a source of model compounds for synthetic or semisynthetic structure modification (Balandrin et al., 1993). Potential natural or synthetic substances with biocidal activity are considered candidates for developing new drugs for treatment of various chronic as well as infectious diseases.

In hot, humid countries like Bahrain, human infection by fungi, especially skin and vaginal and mucosal surfaces, constitute a serious problem. Furthermore, the number of reported cases of immunocompromised and HIV patients with opportunistic and superficial mycoses like cryptococcosis, candidiasis, and aspergillosis has increased in recent years (World Health Organization, 1998). The use of several antimycotic drugs available at present is limited by emergence of new resistant strains, toxicity, poor solubility, and low potency (Navarro-Garcia et al., 2003). Therefore, it is of prime importance to search for new, safe, and more effective antifungal agents. In this connection, indigenous medicinal plants continue to be a rich source of therapeutic drugs. The active principles of many drugs are usually found in plants as secondary metabolites. Nevertheless, relatively few studies are focused on developing antifungal compounds (Jones et al., 2000; Portillo et al., 2001; Quiroga et al., 2001; Navarro-Garcia et al., 2003) from medicinal plants compared to antibacterial substances (Al-Saleh et al., 1997; Mahasneh et al., 1996).

The aim of this screening was the selection of the most promising plant species for further bioactivity-guided fractionation of active antifungal compounds. We report results of in vitro. evaluation against fungi of various extracts of 10 selected indigenous traditional medicinal plants. No data are available on the use of these plants in folk remedies. Also, the therapeutic efficacy and antimycotic activity of these plants have not been evaluated previously. It is hoped that the data presented here will not only provide useful information but also encourage further interest and research on Bahraini medicinal plants.

Materials and Methods

Plant material

Ten plant species were collected in 2002 in their natural habitats from various regions of Bahrain. Dr. D. Al-Esawi, Department of Biological Sciences, Faculty of Science, University of Jordan, Amman, Jordan, identified the plant materials according to the checklist of El-Oqlah and Abbas (1994). Voucher specimens were deposited in the herbarium collection of the Department of Biology of the University of Bahrain. Acquisition code numbers are listed in Table 1.

Table 1. Ethnobotanical data of medicinal plants used in antifungal assays.

Botanical name	Family	Local name	Life form	Parts used	Extract yield (%)^a.	Collection code^b.	Collection site	Popular uses
Heliotropium curassavicum.	Boraginaceae	Kharees	Herb	Leaves & stem	1.6	D E Q1 S	Adhari	Emetic, snake bite, laxative, diuretic
Emex spinosa. Campd.	Polygonaceae	Hammaidh	Herb	Leaves & stem	1.12	D E Q 2 C	Hamad Town	Purgative, diuretic, cure dyspepsia and biliousness
Frankenia pulverulenta. L.	Frankeniaceae	Mollaih	Herb	Leaves & stem	1.2	D E Q 3 S	Mugsha	Mucilaginous, demulcent
Cressa cretica. L.	Convolvulaceae	Nedewah	Herb	Leaves & stem	2.6	D E Q 4 S	Jid Hafs	Aphrodisiac, expectorant, antibilious, aphrodisiac
Fagonia indica. Burm f.	Zygophyllaceae	Shuwaika	Shrub	Leaves & stem	2.73	D E Q 5 C	Jid Hafs	Astringent, prophylatic against small pox, febrifuge, snake bite.
Cenchrus echinatus. Torr.	Gramineae	Sabat	Grass	Leaves & stem	0.92	D E Q 6 C	Mugsha	Pectoral, diuretic
Convolvulus arvensis. L.	Convolvulaceae	Ollayg	Herb	Leaves & stem	2.91	D E Q 7 C	Mugsha	Purgative, cholagogic, antihemorrhagic, harnnorrhage
Pluchea ovalis. DC.	Composieae		Shrub	Whole plant	2.2	D E Q 8 C	Adhari	Carminative, astringent, antipyretic, diaphorytic
Capparis spinosa. L.	Capparaceae	Fakauha	Shrub	Leaves, stem & fruit	6.13	D E Q 9 S	Adhari	Tonic, expectorant, anthelmintic, diuretic, depurative, vasoconstrictive
Tamarix arabica. Bung.	Tamaricaceae	Ethl	Shrub	Leaves	1.54	D E Q 10 S	Hamad Town	Astringent, antidiarrheic, diuretic

^a.Dry residue of the extract in terms of dry starting material.

^b.Collection codes are preceded by DEQ for Drs. Dawood Al-Esawi and Qaher and S for saline or C for cultivated.

Preparation of the plant extracts

Plant parts, mostly leaves and stems, were shade-dried at room temperature (25–30°C) and later ground into a fine powder using a household blender and sieved with a 2-mm-diameter mesh. Pulverized material (50 g) was extracted sequentially with ethanol, petroleum ether, and chloroform as follows.

Pulverized air-dried materials (ca 50 g) were first extracted continually for 48 h in a Soxhlet extractor using 200 ml of 96% ethanol. The insoluble material was filtered off using Whatman filter paper no. 4, and the filtrate was dried (anhydrous MgSO₄) and concentrated by complete evaporation of the solvent in a vacuum rotary evaporator at 40°C (water bath temperature). Percentage yield of ethanol extract (EE) of each plant material was determined from the resulting residue (0.56–1.46 g). Each of the ethanol extracts, after setting aside a portion for the antifungal assay, were further extracted with petroleum ether (b.p. 60–80°C) three-times (3 × 10 ml). The supernatant solutions were separated by decantation, combined, dried, and the solvent evaporated in the rotary evaporator as above to give the petroleum ether extract. Petroleum ether, part of which was used in antifungal tests, was further extracted with three portions of chloroform (3 × 10 ml). The mixture was filtered, separated from droplets of water, and dried (anhydrous MgSO₄). Complete evaporation of chloroform afforded the chloroform extract. The material remaining after chloroform separation was considered as residue extract.

In vitro. antifungal test solutions of these extracts and controls were prepared by dissolving an equivalent of 100 mg in 1 ml of 5% dimethylsulfoxide (DMSO, Merck). The mixture resulted in a homogenous solution that was placed in small vials and stored at 5°C.

For the aqueous extract, 10 g of plant powder were soaked in 50 ml of distilled de-ionized water for 72 h with shaking over a water bath at 40°C. The mixture was left to stand for 3 h at room temperature and then the supernatant liquid was centrifuged at 1000 × g at room temperature. The filtrate was oven-dried at 40°C until complete dryness. The dried aqueous crude extract was weighed, and the concentration was adjusted to 100 mg/ml with distilled sterilized water.

Fungal cultures and growth conditions

Test fungi used in this study (Table 2) were chosen primarily on the basis of their importance to plants and humans as pathogens. The human pathogens were the yeast Candida albicans. (C. P. Robin) Berkhout (clinical isolate from American Mission Hospital, Bahrain) and Saccharomyces cerevisiae.. Stocks were maintained on Sabouraud's dextrose agar (SDA) (Oxoid, UK) slants at 4°C. The filamentous plant pathogens Alternaria. alternata. (Fries) Keissler, Penicillium citrinum. Thom, Aspergillus flavus. Link, Aspergillus niger. van Tieghem, and Fusarium oxysporum. Schlecht were locally isolated from diseased plant parts and identified by the first author. Stock cultures of these fungi were maintained on potato dextrose agar (PDA) (Oxoid) slants at 4°C prior to use for antifungal tests.

Table 2. In vitro. antifungal activity of crude aqueous and ethanol extracts of 10 Bahrain medicinal plants.

		Zone of inhibition diameter (mm)^b.
Plant species	Extract^a.	F. oxysporum.	A. niger.	A. flavus.	P. citrinum.	A. alternata.	C. albicans.	S. cerevisiae.
H. curassavicum.	EE	18.7 ± 0.7	15.8 ± 0.1	10.0 ± 0.6	25.5 ± 0.4	14.7 ± 0.9	21.3 ± 0.5	20.4 ± 0.4
	AE		8.7 ± 0.3		12.6 ± 0.1
E. spinosa.	EE		11.4 ± 0.0	8.2 ± 0.8	10.1 ± 0.2	12.2 ± 0.4	18.0 ± 0.0	16.6 ± 0.6
	AE							11.1 ± 0.1
F. Pulverulenta.	EE	11.3 ± 0.3	13.2 ± 0.3	13.3 ± 0.1	13.8 ± 0.4	13.9 ± 0.3		14.2 ± 0.7
	AE
C. cretica.	EE	12.5 ± 0.1	13.6 ± 0.6	12.7 ± 0.3	32.2 ± 0.6	22.1 ± 0.7	25.7 ± 0.6
	AE		10.0 ± 0.3		15.6 ± 0.9
F. indica.	EE			12.0 ± 0.0		11.1 ± 0.0	13.1 ± 0.1
	AE				9.3 ± 0.1			12.7 ± 0.3
C. echinatus.	EE	16.1 ± 0.0	12.1 ± 0.5	11.8 ± 0.5		10.1 ± 0.1
	AE				11.1 ± 0.5
C. arvensis.	EE			13.0 ± 0.5			10.0 ± 0.0
	AE
P. ovalis.	EE	14.6 ± 0.7	11.1 ± 0.6	12.4 ± 0.1	14.0 ± 0.0		18.9 ± 0.3	10.2 ± 0.3
	AE				9.1 ± 0.6
C. spinosa.	EE	12.7 ± 0.4		12.1 ± 0.3		9.6 ± 0.3	13.2 ± 0.6	11.2 ± 0.1
	AE
T. arabica.	EE	11.9 ± 0.1	12.1 ± 0.2	13.1 ± 0.8			13.7 ± 0.2
	AE					8.2 ± 0.6

^a.Extract: EE, ethanolic; AE, aqueous.

^b.Results are mean ± SD (mm) of three replications.

Test for antifungal activity

Antimycotic activity of aqueous, ethanol, petroleum ether, chloroform, and residue extracts of each plant species was evaluated by the agar well diffusion assay modified from Quiroga et al. (2001).

Overnight cultures of yeasts (Candida albicans.and S. cerevisiae.) were prepared by inoculating the organisms in a 250-ml Erlenmeyer flask containing 50 ml Sabouraud's broth medium. The culture was incubated at 30°C for 18 h. Inocula of filamentous fungi (Alternaria alternata., Aspergillus niger., Aspergillus flavus., Penicillium citrinum. and Fusarium oxysporum.) were prepared by growing these microbes on PDA in 250-ml Erlenmeyer flask for 8 days at 30°C in an alternate cycle of 12-h light/dark photoperiod. Spore suspension was raised by pouring 5 ml of distilled sterilized water into the flask, vortexing for 1 min, and sieving through 8 layers of cheesecloth. A final inoculum density of 10⁵/ml for yeast and 10⁶ spore/ml for the filamentous fungi was calibrated using a hemocytometer.

An aliquot of 50-µl inoculum of either yeasts or filamentous fungi was aseptically mixed with 20 ml melted SDA or PDA media cooled at 45°C. Media-spore suspension was gently mixed and poured aseptically into 9-cm-diameter plastic Petri dishes. Plates were allowed to stand for 1 h at room temperature and a small well (5 mm) was cut in the middle of each solidified medium using a sterilized cork borer. Each plant extract (100 mg/ml) was slowly loaded in the well using micropipette. The dishes were then preincubated at 4°C for 2 h to allow uniform diffusion of the extract into the agar. After preincubation, the plates were incubated aerobically in the dark in a humid incubator at 30°C for 48 h for yeasts and 72 h for filamentous fungi.

Code numbers to maintain objectivity blinded the identity of the plates. Appropriate treatments including wells loaded only with sterilized distilled water or DMSO were considered as negative controls. Additionally, for comparative purposes, disks containing standard antifungal disks like nystatin and miconazol nitrate (50 µg/ml) served as positive control. Each experiment was carried out in triplicate and repeated at least twice simultaneously. The results were recorded by measuring the zone of growth inhibition around the agar well and expressed in millimeter diameter. The mean of three readings per zone was noted and standard deviation of all replicates determined.

Results

Table 1 shows the ethnobotanical data, extract yields (%), and collection codes of 10 selected Bahraini medicinal plants. They are represented by nine families and were mostly in the form of herbs or shrubs. Plant parts consisted mostly of leaves and stems except for Capperis spinosa. for which fruits were also included in the assay. This plant resulted in the highest extract yield (6.13%) compared to other plants. Five plants, namely Heliotropium curassavicum., Frankenia pulverulenta. L.., Cressa cretica. L.., Capparis spinosa. L.., and T camarix arabica., Bung. were sampled from salt-affected soils, whereas the remaining plants were collected from cultivated locations. Most of these plants had been used in folk remedies in different forms for various afflictions (Abbas et al., 1992). Nonetheless, no information of their antifungal application and usage frequency to combat for infectious diseases whose etiologic origin appears to be microbial or anti-inflammatory has been previously reported. Thus, selection of plants for study for antifungal assay was based on availability of collection.

Plant bioactivities were tested against five filamentous fungi and two yeasts using the agar well diffusion method. The methanol and aqueous plant extracts are summarized in Table 2. The extent of activity of these extracts was quantitatively assessed by measuring the diameter of zone of inhibition around the well, which was ultimately expressed in millimeters. Comparisons of data with standard fungicidal disks were included to monitor the experimental conditions and to facilitate better evaluation of the results with other published reports.

From these results, it was obvious that all of the examined plant extracts demonstrated varying degrees of biological activity at least against one of the tested microbes. Ethanol extracts were superior to aqueous crude extracts and showed a broad spectrum of activities as shown in Figure 1. In particular, ethanol extract of H. curassavicum. was found to be active against all the fungal strains tested. Also, extracts from Emex spinosa., Frankenia pulverulenta., Cressa cretica., Pluchea ovalisoc., and Capparis spinosa. proved active against most of the fungi assayed. Overall, the highest inhibitory effect of ethanol solution was exhibited by Cressa cretica. against Penicillium citrinum. (32.2 mm) followed by Candida albicans. (25.7 mm) and Alternaria alternata. (22.1 mm). Furthermore, the ethanol crude solution of H. curassavicum. showed marked activity toward Penicillium. citrinum. (25.5 mm) and the yeasts Candida albicans. and S. cerevisiae. with an activity of 21.3 and 20.4 mm, respectively. Apart from Alternaria alternata., extracts from Pluchea ovalis. revealed elevated inhibitory effect against all microbes evaluated. Mainly, growth reduction was noted in opposition to Candida albicans. (18.9 mm) followed by Fusarium oxysporum. (14.6 mm) and Penicillium citrinum. (14 mm). However, the efficiency of all other ethanol extracts encountered was somewhat intermediate with a total average of 12.7 mm inhibition zone. The lowest antifungal activity was reported for Fagonia indica.and Convolvlus arvensis..

Figure 1 Mean fungal inhibition zone (mm ± SD) by each of the two plant habitat types for various solvents used against filamentous and yeasts fungi. EE, ethanolic; AE, aqueous; PE, petroleum ether; CE, chloroform; RE, residue extracts.

Among filamentous fungi, the highest susceptibility frequency toward all plant extracts was observed with Aspergillus flavus. (100%) followed by Alternaria alternata. (80%) and Penicillium citrinum. (70%). Among the yeasts, Candida albicans. was more sensitive to plant extract (80%) than S. cerevisiae. (60%).

Aqueous crude extracts showed, in general, poor bioactivity against the assayed microorganisms (Table 2). The inhibitory effect ranged from as low as 8.2 mm for T. arabica. against Alternaria alternata. to as high as 15.6 mm for Cressa cretica. against Penicillium citrinum..

The results of Table 2 show that ethanol extracts possess pronounced antimycotic properties and that some plants like Cressa cretica. and H. curassavicum. retain increased biological attributes. As a result of the above observations, the ethanol extract of some plants were further fractionated. The resulting petroleum ether, chloroform, and residue extracts from H. curassavicum., Emex spinosa., Frankenia pulverulenta., Fagonia indica., and Cenchros echinatus. were mycologically assayed as in Table 3.

Table 3. In vitro. antifungal activity of petroleum ether, chloroform, and residue extracts of five Bahraini medicinal plants.

		Zone of inhibition diameter (mm)^b.
Plant species	Extract^a.	F. oxysporum.	A. niger.	A. flavus.	P. citrinum.	A. alternata.	C. albicans.	S. cerevisiae.
H. curassavicum.	PE	13.7 ± 0.4	12.0 ± 0.0	15.2 ± 0.6		14.6 ± 0.6	15.5 ± 0.7
	CE	12.8 ± 0.9	9.7 ± 0.1	8.8 ± 0.3	19.9 ± 0.3	19.1 ± 0.4	18.3 ± 0.2	21.0 ± 0.0
	RE	13.1 ± 0.2	9.1 ± 0.4		17.2 ± 0.8	14.4 ± 0.9		11.6 ± 0.8
E. spinosa.	PE	13.2 ± 0.3	15.8 ± 0.3	16.2 ± 0.6	8.1 ± 0.6	14.0 ± 0.0	12.1 ± 0.1
	CE	21.4 ± 0.9	23.6 ± 0.6	20.2 ± 0.1	19.9 ± 0.9	27.9 ± 0.7	25.9 ± 0.6	27.5 ± 0.6
	RE	11.8 ± 0.6	10.1 ± 0.8		11.6 ± 0.5	13.2 ± 0.2	10.2 ± 0.3
F. pulverulenta.	PE	14.7 ± 0.1	9.1 ± 0.2	14.0 ± 0.3	15.0 ± 0.0	12.0 ± 0.0	10.0 ± 0.0	16.6 ± 0.4
	CE	11.1 ± 0.1		10.8 ± 0.2	10.6 ± 0.3	11.1 ± 0.2		13.6 ± 0.2
	RE	10.9 ± 4	11.7 ± 0.8
F. indica.	PE	11.7 ± 0.3	12.6 ± 0.6		11.6 ± 0.7	15.7 ± 0.8	12.2 ± 0.6	15.5 ± 0.6
	CE	19.2 ± 0.5	20.1 ± 0.9	15.5 ± 0.7	29.3 ± 0.3	10.6 ± 0.4	25.6 ± 0.8
	RE		12.0 ± 00	10.0 ± 0.0				13.7 ± 0.2
C. echinatus.	PE	8.8 ± 0.8	13.3 ± 0.3	12.6 ± 0.1	16.0 ± 0.1	12.9 ± 0.8	12.0 ± 0.1
	CE	17.1 ± 0.6		20.1 ± 0.3	16.8 ± 0.3	18.7 ± 0.6	19.9 ± 0.9	18.6 ± 0.3
	RE		14.4 ± 0.9				11.1 ± 0.4

^a.Extracts: PE, petroleum ether; CE, chloroform; RE, residue extracts.

^b.Results are mean ± SD (mm) of three replications.

The plant extracts reveal differential mycotic activity against all the microbes tested. All the solvent extracts showed a broad-spectrum activity at 100 mg/ml toward at least two of the fungi assayed. Chloroform extracts of H. curassavicum. and Emex spinosa. and the petroleum ether extracts of Frankenia pulverulenta. demonstrated high efficacy potential against all of the microbes examined. The highest extract potency was displayed by chloroform extract, whereas petroleum ether and residue extracts indicated rather similar pattern of inhibition.

Among all the fractionations, the broadest and prominent activity was detected in chloroform extract of Emex spinosa. against Alternaria alternata. (27.9 mm) and S. cerevisiae. (27.5 mm) (Table 3). Likewise, the activity against other fungi of the same extract varied from 19.9 to 25.9 mm for Penicillium citrinum. and Candida albicans., in that order. However, the highest zone of inhibition was exhibited against Penicillium citrinum. (29.3 mm) for Fagonia indica., followed by Candida albicans. (25.6 mm). The diffused chloroform extracts from the agar wells of H. curassavicum. and Cenchros echinatus. also revealed marked activity with a highest zone of 21 and 20 mm against S. cerevisiae. and Aspergillos flavus., respectively. Reduced antifungal potential against yeasts and filamentous fungi was encountered with Frankenia pulverulenta..

Petroleum ether crude fractions of three plants, namely Emex spinosa, Frankenia pulverulenta., and Cenchros echinatus., illustrated the highest comparable inhibitory level with an average of 16.2 mm against Aspergillus flavus, S. cerevisiae., and Penicillium citrinum.. A pronounced antifungal activity (average 15 mm) was also noted in the petroleum ether extract of H. curassavicum. against Aspergillus flavus. and Candida albicans. and in the extract of Fagonia indica. against Alternaria alternata. and S. cerevisiae.. Other filamentous fungi and yeasts were also susceptible at different levels toward plant fractions.

Residue extracts yielded a somewhat similar pattern of microbial reduced growth but showed narrow-spectrum efficiency. The lowest inhibitory activity was more than 9 mm in radius against Aspergillus niger.. The residue extracts of H. curassavicum. attained the highest growth reduction (17.2 mm) against Penicillium citrinum.. This plant and the diffusable extract of Cenchros echinatus. both exhibited analogous inhibition radius of 14.4 mm against Alterneria alternata. and Aspergillus niger.. The other residue fractions were active against at least two fungi assayed.

In Figure 2, the mean inhibitory zone diameter for extracts from each habitat (five saline and four cultivated soils) is presented for filamentous fungi and yeasts. For all microbial species, Penicillium citrinum. was the most susceptible microbe toward the overall diffusable metabolites of saline habitat (18.05 mm), followed by the yeasts Candida albicans. (16.8 mm) and S. cerevisiae. (15.5 mm). The latter yeasts also displayed an increased susceptibility level toward extracts from plants of cultivated soils.

Figure 2 Mean fungal inhibition zone (mm ± SD) by each of the two plant habitat types against filamentous and yeast fungi.

The efficacy of various solvents from each plant habitat type on mean antifungal activity is shown in Figure 2. Unlike chloroform extracts from cultivated soils, which revealed elevated mycoactivity, extracts of plants from saline-affected soils showed slightly increased activity than those from cultivated habitats. In general, the efficacy of solvents can be arranged in the order of magnitude chloroform > ethanol > petroleum ether > residue > aqueous.

Discussion

This study clearly shows that all but, in particular, chloroform and ethanol extracts possess substantial yields of ingredients active against the assayed microbes (Table Tables 2 and 3). The yeasts appeared to be quite susceptible to the metabolites diffused from the ethanol extracts of Cressa cretica. and H. currasavicum. as revealed by the zone radius of 25.7 and of 20.4 mm, respectively (2). Likewise, chloroform extracts of both Emex spinosa. and Fagenia indica. also proved active in restricting the growth of Candida albicans. (27.5 mm) while Fagonia indica. alone was active against S. cerevisiae. (27.5 mm). This observation is of particular interest because Candida albicans. is a ubiquitous pathogen common in pathogenesis of urinary tract infections, endocarditic, vulvovaginalis, and oral thrush (Greenspan & Greenspan, 1997). The yeast also causes serious systemic infection, including opportunistic infections in HIV patients.

For filamentous fungi, the ethanol diffusable substances of Cressa cretica. and H.. curassavicum. showed prominent biological activity against Penicillium citrinum. at a diameter of restriction of 32.2 and 25.5 mm, respectively (Table 3). In addition, the assayed microbes concealed measurable degree of susceptibility toward the chloroform extracts of Emex spinosa., and the uppermost attained activity was found against Alternaria alternata. (27.9 mm) followed by Aspergillas niger. (23.6 mm) (Table 3). Penicillium citrinum. was the most sensitive fungus to the diffusable metabolites of Fagonia indica. (29.3 mm). Members of Aspergillus., Altenaria., Penicillium., and Fusarium. are well-known for their production of toxins. (e.g. aflatoxins of Aspergillus flavus.). These secondary metabolites are potent carcinogens, hepatotoxins, teratogens, and immunosuppressor compounds (Quiroga et al., 2001). Species of Fusarium oxysporum. causes important plant diseases such as wilts, damping-of, root rots, and seed decay. Species of Fusarium. produce potent mycotoxins in food commodities and are commonly prevalent fungi affecting corn (Marassas, 1991). These fungi represent threats not only to plants but also to animals and humans consuming contaminated feed and food.

These findings suggest that the antifungal properties in these plants are most likely due to the presence of broad-spectrum biological compounds or general metabolic inhibitors. Indigenous plants sampled randomly from saline soils demonstrated somewhat higher inhibition level, except in opposition to Penicillium citrinum. and Candida albicans. (Fig. 2). It is probable that plants inhabiting salt-affected soils are more likely weakened by stress conditions, especially by salinity, compared to cultivated soil plants, the former being more prone to pathogen attack and thus as a defense mechanism produces antimicrobial secondary metabolites in response. Examples of such synthesized metabolites include tannins, phenolic compounds, and prolines (Rizq, 1986; Al-Saleh et al., 1993; Bennett & Wallsgrove, 1994). Similarly, plants are known to produce antifungal compounds in response to pathogen attack or other abiotic stress factors (Osbourne, 1998; Wojtaszek, 1997).

With the exception of one study (Mahasneh et al., 1996), no previous reports on the antifungal properties and chemical nature of the inhibitory compounds of the examined plant taxa could be found in the literature. In their study, Mahasneh et al. (1996) reported a mild inhibitory effect (average 11.5 mm) of various extracts of Capparis spinosa. against Fusarium oxysporum. and Candida albicans.. This inhibitory level is comparable to the reported activity in this study of the ethanol extract against the same microorganisms tested (Table 2).

Analysis of compounds in many plant families revealed the occurrence of common phytochemical constituents. In this regard, Pluchea ovalis. belongs to the Compositeae, and this family is well-known for its sesquiterpene lactones as a bioinhibitor (Bohlman, 1988). Many lactones are antibacterial agents, especially, against Gram-positive bacteria (Bruneton, 1995). Emex spinosa. was reported to contain anthraquinones as well as quercitrin and rutin flavanoids, coumarins and alkaloids (Rizq, 1986). Cressa cretica. was reported to contain quercetin glycosides, alkaloids, coumarins, and sterols (Rizq, 1986). Convolvules arvensis., known locally as ollaiq., contain saponins, sterols, and alkaloids. Fagonia indica. was reported to contain triterpenoids, saponins, alkaloids, flavanoids, and tannins (Rizq, 1986). Pluchea. spp. contains also quercetin glycosides. H. currassavicum. was reported to contain pyrrolizidine alkaloids, and Frankenia pulverulenta. was reported to contain flavanoids, coumarins, and tannins (Rizq, 1986). Certain compounds produced by these plants were reported to have antifungal activity. Anthraquinones were reported to have antifungal efficacy against Aspersillus niger. (Ali et al., 1999). whereas pyrrolizidine alkaloids were reported to have antimicrobial activity (Singh, 2002).

The understanding of the inhibitory mechanism would provide better direction toward the development and efficient production of new chemical classes of novel metabolites (Hostettman, 1998). This is the first report showing the antifungal activities of different Bahraini medicinal plants. The use of these plants in treatment of various infectious diseases, whose symptoms might be of fungal origin, highlights the importance of ethnobotanical approach for the future selection of plants in the discovery of new bioactive compounds. Further work, including structure-function relationship and bioactivity-guided fractionation to isolate and purify antifungal compounds in some of these plants, is now in progress.