Journal of Ethnopharmacology 111 (2007) 657666

Antioxidant, antimicrobial and cytotoxic activities of selected medicinal plants from Yemen
Mohamed Al-Fatimi a , Martina Wurster c , Gudrun Schr oder b , Ulrike Lindequist c,
Faculty of Medicine and Health Sciences, Pharmacy Section, Department of Pharmacognosy, Aden University, Aden, Yemen b Friedrich L ofer Institute of Medical Microbiology, Ernst-Moritz-Arndt-University Greifswald, Germany c Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt-University Greifswald, 17487 Greifswald, Germany Received 4 September 2006; received in revised form 9 January 2007; accepted 13 January 2007 Available online 19 January 2007
a

Abstract Ninety crude extracts, including dichloromethane, methanol and aqueous extracts from 30 medicinal plants used in the Yemeni ethnomedicine to treat common infections, were screened in vitro for antimicrobial activities against three Gram-positive bacteria and two Gram-negative bacteria, Candida maltosa and ve opportunistic human fungal pathogens (two yeasts, three hyphomycetes). Most of the plants showed antibacterial activities. Extracts from Tamarindus indica owers and Ficus vasta fruits have been the most active. Of the 30 plants tested, 13 showed antifungal activity (40%) against one ore more human pathogenic fungi. The strongest inhibition was exhibited by Azima tetracantha (fruits), Sansevieria ehrenbergii (fruits) and Solanum incanum (fruits). Ten methanol extracts, especially those of Acacia asak barks and Solanum nigrum fruits, showed effective free radical scavenging activities in the DPPH assay. Remarkable cytotoxic activity against FL-cells was shown only for ve plants, among them Plicosepalus curviorus (stems). 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Medicinal plants; Yemen; Antimicrobial; Cytostatic; Antioxidant

1. Introduction The use of traditional medicine at the primary health care level is widespread in Yemen (Schopen, 1983; Badib, 1991; AlFatimi, 1999; Miller and Morris, 2004). But only a few species from Yemeni natural sources have been scientically investigated for their biological activity (Ali et al., 2001; Al-Fatimi et al., 2005a, 2005b, 2006; Mothana and Lindequist, 2005). Because of the resistance that pathogenic build against antibiotics, there is a great interest in the search for new antimicrobial drugs also from nature. Natural crude drug extracts and biologically active compounds isolated from plant species used in traditional medicine can be prolic resources for such new drugs. Therefore in the present study, medicinal plants were collected from different localities in Yemen to study their antimi-

crobial, antioxidant and cytotoxic properties according to their use in Yemeni traditional medicine and to investigate their in vitro activity in order to discover resources for new lead structures or to improve the traditional medicine. 2. Materials and methods 2.1. Plant material The plant material (Table 1) was collected from different localities of Yemen based on the information provided in the ethnobotanical survey conducted in the time from January 2002 to August 2003. The collected plants were identied in the Pharmacognosy Department Aden University, Aden, Yemen and at the Botanischer Garten and Botanisches Museum Berlin-Dahlem, Freie University, Berlin, Germany. Voucher specimens (Table 1/FT 001-FT 030) for each plant are deposited in the Department of Pharmacognosy, Aden University, Aden, Yemen.

Corresponding author. Tel.: +49 3834 864868; fax: +49 3834 864885. E-mail address: lindequi@uni-greifswald.de (U. Lindequist).

0378-8741/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.01.018

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M. Al-Fatimi et al. / Journal of Ethnopharmacology 111 (2007) 657666

Table 1 Ethnobotanical data of the investigated Yemeni plants Family/botanical name (voucher specimen no.) Amaranthaceae/Aerva javanica (Burm. l.) Juss. (FT 004) Aloaceae/Aloe vera L. Burm.f. (FT 005) Asclepiadaceae/Caralluma penicillata (Deers) N.E. Br. (FT 009) Asclepiadaceae/Leptadenia pyrotechnica (Forssk.) Decne. (FT 018) Asclepiadaceae/Orbea deersiana (Lavranos) Bruyns. (FT 012) Asclepiadaceae/Rhytidocaulon tortum (N.E. Br.) M.G. Gilbert (FT 023) Burseraceae/Commiphora foliacea Sprague (FT 012) Burseraceae/Commiphora kua (R.Br. ex Royle) Vollesen (FT 011) Caesalpiniaceae/Tamarindus indica L. (FT 029) Capparaceae/Cadaba farinosa Forssk. (MAB 007) Capparaceae/Cadaba glandulosa Forssk. (FT 008) Capparaceae/Maerua aff. oblongifolia vel macrantha (FT 019) Cucurbitaceae/Zehneria anomala C. Jeffrey (FT 030) Dracaenaceae/Sansevieria ehrenbergii Schweinf. ex Baker (FT 026) Euphorbiaceae/Euphorbia hadramautica Baker (FT 013) Fabaceae/Indigofera spinosa Forssk. (MAB 017) Labiatae/Ocimum forskolei Benth. (FT 020) Loranthaceae/Plicosepalus curviorus Tiegh. (FT 022) Malvaceae/Gossypium areysiamum De. (FT 015) Malvaceae/Gossypium barbadense L. (FT 016) Mimosaceae/Acacia asak Willd. (FT 001) Mimosaceae/Acacia nilotica (L.) Delile (FT 002) Mimosaceae/Acacia tortilis Hayne (FT 003) Moraceae/Ficus vasta Forssk. (FT 014) Salvadoraceae/Azima tetracantha Lam. (FT 006) Salvadoraceae/Salvadora persica L. (FT 025) Solanaceae/Solanum incanum L. (FT 027) Solanaceae/Solanum nigrum L. (FT 028) Part (tested) Fruits (Diaspores) Seeds Fruits Whole plant Whole plant Whole plant Stem cortex Leaves Local name ra Saber Uluth Marh Urza Quaran Rashaha Murr Site of collection Dhamar Tahama Taiz Schuqura Imkhudeera Alhudidah Alraqub Gischan Major traditional usesa Sedative (3) Malaria (3) Skin rash, scabies (3) Wound (3) Diabetes, wound (3) Wound, allergy (3) Wound, antiseptic Detergent for mouth and throat, bronchitis (2); face and skin antiseptic (1); cough (4) Skin antiseptic, insecticides (3) Urinary infection, haemorrhoids (3) Haemorrhoids (3) Asthma, skin diseases (3) Skin diseases, burns, wound (3) Warts, antiseptic (3) Bruises, eczema, wound (3) Kidney stones (3); cough, cold (4) Cosmetic, fever, skin infections (3) Cancer (3) Wound, dermatitis (3) Cosmetic (2); acne, rheumatism (3) Gastric ulcer, antiseptic, skin diseases (3) Expectorating, wounds, pharyngitis, bronchitis (2); diabetes (1) Stomach ache, digestive (3) Cough, expectorant (3) Rheumatism, cough (3) Dermatitis (3) Teeth antiseptic, toothache (1, 4); skin diseases (2) Skin antiseptic (1); diarrhea, expectorant, laxative (2); hemorrhages (3) Digestive (3); foodstuff, fever (4) Antiseptic, burns (3)

Flowers Leaves Leaves Gall Gall Fruits Whole plant Leaves Herb Stems Leaves Seeds Stem cortex Leaves Fruits Fruits Fruits Gall Fruits Fruits

Humar Gurduma Gurduma Gurduma Madh Seni Nafeez Hassar Dhumaran Saquar afra Uttub Talah Qarad Sumyrr Tawlaq Sor Rak Nuqum Qumqam

Hadramut Taiz Gaheen Gaheen Imkhudeera Lawdar Almahra Imaeen Imshaa Lawdar Imshaa Zingebar Aldhala Yafa Shabwah Radaa Lawdar Modyah Hadramut Lahaj

Vitaceae/Cissus rotundifolia Vahl (FT 010) Zygophyllaceae/Fagonia luntii Baker (FT 024)
a

Leaves Whole

Alfaq Hell

Hagah Lawdar

References for uses: (1) Al-Fatimi (1999); (2) Badib (1991); (3) Oral interview (2002, 2003); (4) Schopen (1983).

2.2. Extraction The plant materials were allowed to air dry and afterwards pulverized in grinder. Thirty grams of the pulverized materials were successively extracted with 300 ml of dichloromethane, 300 ml of methanol and 300 ml of water at room temperature for 8 h. The extracts were then concentrated under reduced pressure at 40 C, freezedried and stored in exsiccator until use. 2.3. Determination of antimicrobial activities 2.3.1. Microorganisms The following bacterial strains were employed in the screening: Staphylococcus aureus (ATCC 29213), Bacillus subtilis

(ATCC 6059), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Micrococcus avus (SBUG 16). As fungal strains Candida maltosa (SBUG 17), Candida albicans (ATCC 90028), Candida krusei (ATCC 90878), Aspergillus fumigatus (13550/99), Trichophyton mentagrophytes (05/2004), Absidia corymbifera (100798). The SBUG strains were given from the strain collection of the Institute of Microbiology (SBUG) and the strains 13550/99, 05/2004 and 100798 from the Friedrich L ofer Institute of Medical Microbiology of the Ernst-Moritz-Arndt-University Greifswald, Germany. 2.3.2. Antimicrobial assays Modied agar diffusion method (Bauer et al., 1966) was used to determine antibacterial and antifungal activities. The bacterial cell suspension was prepared from a 24 h culture and adjusted

M. Al-Fatimi et al. / Journal of Ethnopharmacology 111 (2007) 657666 Table 2 In vitro antibacterial activity of selected plants from Yemen Botanical name Extract and % yield Inhibition zones (mm) against S. a. Acacia asak D (2.8) M (18.5) W (16.2) D (2.9) M (17.2) W (14.8) D (2.6) M (11.3) W (15.4) D (1.8) M (10.5) W (9.7) D (1.3) M (12.8) W (11.8) D (1.9) M (18.7) W (7.9) D (3.4) M (17.2) W (9.5) D (3.1) M (16.8) W (1.3) D (2.7) M (9.5) W (8.6) D (3.2) M (14.5) W (11.2) D (1.7) M (17.1) W (11.8) D (2.8) M (12.5) W (16.3) D (1.5) M (16.7) W (9.1) D (1.7) M (15.9) W (12.4) D (2.3) M (18.7) W (12.2) D (1.8) M (16.8) W (18.8) D (9.9) M (17.1) W (6.6) D (2.6) M (14.3) W (9.4) 15 15 10 15 15 20 10 10 8 10 8 8 15 10 15 15 8 8 10 8 10 10 15 10 10 15 10 8 15 18 20 15 18 15 20 10 10 8 15 B. c. 10 20 10 20 15 20 8 8 8 10 10 15 20 8 8 10 15 8 10 10 8 8 10 12 15 8 25 15 15 10 15 8 15 M. f. 8 10 20 15 8 8 10 10 15 20 18 10 15 8 15 8 8 10 8 20 25 8 15 8 10 15 10 8 20 15 8 E. c. P. a. 15 10 8 15 15 10 10 20 8 8 10 18 8 10 8 10 15 18 15 10 10 10

659

Acacia nilotica

Acacia tortilis

Aerva javanica

Aloe vera

Azima tetracantha

Cadaba farinosa

Cadaba glandulosa

Caralluma penicillata

Cissus rotundifolius

Commiphora foliacea

Commiphora kua

Euphorbia hadramautica

Fagonia luntii

Ficus vasta

Gossypium areysiamum

Gossypium barbadense

Indigofera spinosa

660 Table 2 (Continued ) Botanical name

M. Al-Fatimi et al. / Journal of Ethnopharmacology 111 (2007) 657666

Extract and % yield

Inhibition zones (mm) against S. a. B. c. 8 10 15 8 8 10 8 20 8 15 10 8 20 10 8 10 10 10 10 15 20 20 15 8 28 N.T. M. f. 18 15 10 15 8 8 15 10 15 15 15 15 15 20 15 10 10 15 15 31 N.T. E. c. 10 8 10 8 15 18 20 N.T. 15 P. a. 10 10 15 8 10 10 8 15 10 10 15 10 20 20 N.T. 18

Leptadenia pyrotechnica

D (1.9) M (14.8) W (9.6) D (2.7) M (18.5) W (14.8) D (2.6) M (16.9) W (17.4) D (1.2) M (12.3) W (10.1) D (2.1) M (18.9) W (13.7) D (1.6) M (12.3) W (11.7) D (1.7) M (16.9) W (14.8) D (1.6) M (15.6) W (14.8) D (2.9) M (17.3) W (13.1) D (2.6) M (18.5) W (16.2) D (2.3) M (19.4) W (17.6) D (2.1) M (18.8) W (17.5) 10 g/disc 10 mg/disc

10 15 8 8 10 8 8 8 10 8 15 10 15 10 15 10 8 15 15 12 12 15 25 28 20 16 26 N.T.

Maerua oblongifolia

Ocimum forskolei

Orbea deersiana

Plicosepalus curviorus

Rhytidocaulon cf. tortum

Salvadora persica

Sansevieria aff.ehrenbergii

Solanum incanum

Solanum nigrum

Tamarindus indica

Zehneria anomala

Ampicillin Gentamicin

S. a.: Staphylococcus aureus ATCC 29213; B. c.: Bacillus subtilis ATCC 6059; E. c.: Escherichia coli ATCC 25922; P. a.: Pseudomonas aeruginosa ATCC 27853; M. f.: Micrococcus avus SBUG 16. Extracts: D, dichloromethane; M, methanol; W, water. Values are inhibition zone diameter (mm); (conc. 2 mg/disc); : no inhibition; N.T.: not tested; negative controls did not show any activity.

to an inoculation of 1 106 colony forming units per ml. Sterile nutrient agar (Immunpr aparate, Berlin, D, 26 g agar/l distilled water) was inoculated with bacterial cells (200 l of bacterial cell suspension in 20 ml medium) and poured into dishes to give a solid plate. Yeasts and hyphomycetes (1 105 colony forming units per ml) were inoculated into sterile Mueller-Hinton-agar (Becton Dickinson, Heidelberg) according to DIN E 58940-3 (DIN, 2004) for the agar disc-diffusion assay. Forty microliters of test material (equivalent to 2 mg of the dried extract), dissolved in the same solvent like for extraction, were applied on sterile paper discs (6 mm diameter, Schleicher and Schuell, D, ref. no. 321860). Ampicillin, gentamicin

and nystatin were used as positive control, and the solvents dichloromethane and methanol as negative control. The solvents were allowed to evaporate in a stream of air. The discs were deposited on the surface of inoculated agar plates. Plates were kept for 3 h in refrigerator to enable prediffusion of the substances into the agar. Plates with bacteria were incubated for 24 h at 37 C, plates with yeasts for 48 h at 36 C and plates with hyphomycetes for 72 h at 30 C. Inhibition zone diameters around each of the disc (diameter of inhibition zone plus diameter of the disc) were measured and recorded at the end of the incubation time. An average zone of inhibition was calculated for the three replicates.

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Minimal inhibitory concentrations (MICs) were determined by the agar diffusion technique as described by Rajbhandari and Sch opke (1999). The highest concentration of extract tested during the experiment was 1 mg/ml. The MIC corresponds to the lowest concentration of the test extract able to inhibit any visible microbial growth. 2.4. Cytotoxicity assay The cytotoxicity was measured by the neutral red uptake assay (Lindl and Bauer, 1989) using FL-cells, a human amniotic epithel cell line (Hilgenfeld et al., 1979). Only living cells are able to manage the active uptake of neutral red. FL-cells were cultivated in a 96-well microtiter plate (105 cell/ml EAGLEMEM, Sin, Berlin, D, 150 l/well) at 37 C in a humidied 5% carbon dioxide atmosphere. The EAGLE-MEM was completed by l-glutamin (0.10 g/l), Hepes (2.38 g/l), penicillin G (105 IE/l), streptomycin sulphate (0.10 g/l) and FCS (Gibco, 80.0 ml/l). After 24 h 50 l of the extract solution (extract dissolved in ethanol under stirring in ultrasonic bath for 5 min and then diluted with MEM: test mixture) or medium with equal amounts of ethanol (control) were added. After a further incubation for 72 h cells were washed three times with phosphate buffered saline solution. One hundred microliters of neutral red solution (SERVA, 0.3% in EGLE-MEM) were added per well. The cells were then incubated for 3 h at 37 C, followed by another three times washing with PBS. One hundred microliters of a solution of acetic acid (1%, v/v) and ethanol (50%, v/v) in distilled water were added. After shaking for 15 min the optical density was measured at 540 nm with an ELISA-Reader HT II (Anthos Labtec Instruments Salzburg, A). The mean of four measurements for each concentration was determined (n = 3). 2.5. Determination of antioxidant activity The free radical scavenging activity was measured by using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay. Qualitative determination was done as described in Sievers et al. (2000). Quantitative estimation was carried out according to the method of Brand et al. (1995). The reaction mixture contained 500 l of test extract and 125 l of DPPH in ethanol. Different concentrations of test samples (10, 50, 100, 500 and 1000 g/ml) were prepared while the concentration of DPPH was 1 mM in the reaction mixture. These reaction mixtures were taken in Eppendorf tubes and incubated at 37 C for 30 min, the absorbance was measured at 517 nm. Percent radical scavenging activity by sample treatment was determined by comparison with ethanol treated control group. Ascorbic acid was used as positive control. The DPPH radical concentration was calculated using the following equation: scavenging effect (%) = 100 A0 A1 A0

Table 3 Minimal inhibitory concentrations (MIC) of extracts from selected plant species from Yemen Botanical name Extract MIC (g/ml) S. a. Acacia asak D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W 1000 1000 NA 1000 500 500 500 500 1000 NA NA 1000 NA NA NA 500 10 250 500 NA NA 1000 NA NA NA NA 1000 1000 500 NA 1000 1000 500 NA 1000 NA NA 500 NA 125 125 500 NA 50 NA NA 500 20 1000 1000 NA NA 500 NA B. c. 1000 500 NA 1000 250 500 500 NA NA NA NA NA NA NA NA 1000 NA 1000 500 250 NA NA NA NA NA NA NA 1000 500 NA NA 1000 1000 NA NA NA NA 1000 NA 1000 500 NA NA 125 500 NA 500 NA 1000 500 NA NA 500 NA M. f. NA 1000 NA NA 1000 500 1000 NA NA NA NA 1000 NA NA 1000 500 20 NA 250 1000 NA NA 1000 NA 500 NA NA 1000 NA NA NA NA NA 250 125 NA NA NA NA NA NA 500 1000 1000 500 1000 NA NA NA 125 500 NA NA NA E. c. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA P. a. NA NA NA NA NA 1000 NA 1000 NA NA 1000 1000 NA NA 1000 NA 1000 250 NA NA NA NA NA NA NA NA 1000 NA 250 NA 1000 NA 1000 NA NA NA NA NA NA NA 1000 NA NA 500 500 500 1000 1000 NA NA 1000 NA NA NA

Acacia nilotica

Acacia tortilis

Aerva javanica

Aloe vera

Azima tetracantha

Cadaba farinosa

Cadaba glandulosa

Caralluma penicillata

Cissus rotundifolius

Commiphora foliacea

Commiphora kua

Euphorbia hadramautica

Fagonia luntii

Ficus vasta

Gossypium areysiamum

Gossypium barbadense

where A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample of the tested extracts.

Indigofera spinosa

662 Table 3 (Continued ) Botanical name Extract

M. Al-Fatimi et al. / Journal of Ethnopharmacology 111 (2007) 657666

MIC (g/ml) S. a. B. c. NA 1000 NA NA NA NA 500 NA NA NA 1000 NA 125 NA NA 500 NA NA NA 1000 NA NA 125 1000 1000 NA 1000 1000 1000 1000 500 125 125 NA 1000 NA N.T. M. f. NA NA NA NA NA NA 250 500 1000 500 NA NA 500 1000 500 NA NA NA 500 500 NA 500 500 125 NA 500 NA NA 1000 1000 NA NA 125 15 NA NA 0.25 E. c. NA NA NA NA 1000 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 1000 NA NA NA NA NA NA 1000 NA 500 NA NA 125 125 NA NA NA N.T. P. a. NA 1000 NA NA NA NA NA NA 1000 NA 500 NA 1000 1000 NA NA NA NA NA 500 NA NA 1000 NA NA NA 1000 NA 500 1000 NA 125 125 NA NA NA N.T.

Leptadenia pyrotechnica

D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W D M W

1000 500 NA NA NA NA 1000 NA NA NA 1000 NA 500 1000 NA 500 1000 NA NA 500 1000 NA 500 500 NA 1000 NA 1000 500 NA 125 25 125 NA 1000 NA 0.05

Maerua oblongifolia

Ocimum forskolei

Orbea deersiana

Plicosepalus curviorus

Rhytidocaulon cf. tortum

Salvadora persica

foliacea, Ficus vasta, Ocimum forskolei, Plicosepalus curviorus, Salvadora persica, Sansevieria aff.ehrenbergii, Solanum nigrum and Tamarindus indica. Gram-positive bacteria are more sensitive against the extracts than Gram-negative bacteria. In the agar diffusion assay 45 extracts were found to be active against Staphylococcus aureus, 51 extracts against Bacillus subtilis and 49 against Micrococcus avus. 31 extracts inhibited the growth of Pseudomonas aeruginosa and 2 that of Escherichia coli (Table 2). The lowest MIC values against Staphylococcus aureus showed the methanol extracts of Azima tetracantha (10 g/ml), Ficus vasta (50 g/ml) and Tamarindus indica (25 g/ml) and the water extract of Gossypium areysiamum (25 g/ml). Remarkable are also the low MIC values of the methanol extract of Azima tetracantha and of the dichlormethan extract of Zehneria anomala against Micrococcus avus (20 and 15 g/ml resp., Table 3). On the other hand ve plant species, namely Caralluma penicillata, Euphorbia hadramautica, Leptadenia pyrotechnica, Maerua oblongifolia, Rhytidocaulon cf. tortum, showed only very weak activities.
Table 4 In vitro antifungal activity of methanol extracts of tested Yemeni plant species Botanical name Inhibition zones (mm) against Cm Acacia asak Acacia nilotica Acacia tortilis Aerva javanica Aloe vera Azima tetracantha Cadaba farinosa Cadaba glandulosa Caralluma penicillata Cissus rotundifolius Commiphora foliacea Commiphora kua Euphorbia hadramautica Fagonia luntii Ficus vasta Gossypium areysiamum Gossypium barbadense Indigofera spinosa Leptadenia pyrotechnica Maerua oblongifolia Ocimum forskolei Orbea deersiana Plicosepalus curviorus Rhytidocaulon cf. tortum Salvadora persica Sansevieria ehrenbergii Solanum incanum Solanum nigrum Tamarindus indica Zehneria anomala Nystatin (100 g/disc) 8 8 25 15 15 8 25 Ca 17 8 17 24 26 Ck 11 19 18 18 22 Af 11 11 16 8 25 22 Ac 11 14 16 12 18 26 25 Tm 10 14 30 9 15 10 8 30 24 42 25

Sansevieria aff.ehrenbergii

Solanum incanum

Solanum nigrum

Tamarindus indica

Zehneria anomala

Ampicillin

S. a.: Staphylococcus aureus ATCC 29213; B. c.: Bacillus subtilis ATCC 6059; E. c: Escherichia coli ATCC 25922; P. a.: Pseudomonas aeruginosa ATCC 27853; M. f.: Micrococcus avus SBUG 16. Extracts: D, dichloromethane; M, methanol; W, water. NA, not active (MIC > 1000 g/ml); N.T., not tested.

3. Results The paper describes the antimicrobial, antioxidant and cytotoxic activity of 30 different plant species belonging to twenty families and used in Yemeni ethnomedicine. A summary of the ethnobotanical data of the medicinal plants studied is given in Table 1. The results of the antibacterial screening of a total of 90 extracts of 30 species against ve bacteria species are summarized in Table 2 (inhibition zones in the agar diffusion assay) and Table 3 (MIC values). Extracts of the following plants showed in the agar diffusion assay antibacterial activity against at least four bacterial strains: Acacia nilotica, Acacia tortilis, Commiphora

Fungi: Cm, Candida maltosa; Ca, Candida albicans; Ck, Candida krusei; Af, Aspergillus fumigatus; Ac, Absidia corymbifera; Tm, Trichophyton mentagrophytes.

M. Al-Fatimi et al. / Journal of Ethnopharmacology 111 (2007) 657666 Table 5 Free radical scavenging activities of the methanolic extracts of plant species, which were positive in the thin layer chromatography assay Extracts concentration Radical scavenging activity (%) 10 g/ml Acacia asak Acacia nilotica Acacia tortilis Aerva javanica Gossypium barbadense Indigofera spinosa Ocimum forskolei Plicosepalus curviorus Solanum nigrum Tamarindus indica Ascorbic acid 40.77 33.8 5 8.6 30.8 40.6 0.17 8.6 40.1 35.38 87 50 g/ml 89.7 77.1 11.9 13.6 34.66 44.9 1.7 16.7 93.6 59.39 96.9 100 g/ml 91.85 94.46 26.17 29.4 44.35 92.92 12.1 32 94 80.87 96.9 500 g/ml 92.6 94.62 92.06 91.6 94.07 93.08 53.8 90.8 94.5 94.98 97.0

663

1000 g/ml 92.77 94.62 92.6 91.8 95.21 92.92 90.26 92.8 95.3 93.86 97.2

Comparing the activities of the extracts prepared by different solvents methanol extracts exhibited the most interesting results. Therefore these extracts were tested also for other activities. The results of antifungal tests of methanol extracts are presented in Table 4. Thirteen extracts showed antifungal activity (40%). The most active plants against opportunistic human pathogens were Azima tetracantha, Sansevieria ehrenbergii and Solanum incanum. They showed broad spectrum antifungal activities. The strongest effect was observed with the extracts of Solanum incanum against Trichophyton mentogrophytes (inhibition zone 42 mm) and with the extracts of
Table 6 In vitro cytotoxicity of plant methanol extracts tested against FL-cells Extract Acacia asak Acacia nilotica Acacia tortilis Aerva javanica Aloe vera Azima tetracantha Cadaba farinosa Cadaba glandulosa Caralluma penicillata Cissus rotundifolius Commiphora foliacea Commiphora kua Euphorbia hadramautica Fagonia luntii Ficus vasta Gossypium areysiamum Gossypium barbadense Indigofera spinosa Leptadenia pyrotechnica Maerua oblongifolia Ocimum forskolei Orbea deersiana Plicosepalus curviorus Rhytidocaulon cf. tortum Salvadora persica Sansevieria ehrenbergii Solanum incanum Solanum nigrum Tamarindus indica Zehneria anomala IC50 % (g/ml) against FL-cells >1000 >1000 >1000 >1000 780 >1000 720 800 >1000 900 430 25 >1000 490 980 >1000 >1000 >1000 550 740 880 >1000 5 980 >1000 30 35 450 9 >1000

Azima tetracantha against Trichophyton mentogrophytes (inhibition zone 30 mm). Remarkable are also the specic effects of the methanol extract of Ocimum forskolei against Trichophyton mentogrophytes (inhibition zone diameter 30 mm). Seventeen plant species did not show any antifungal activity against the tested human pathogenic fungi. The methanol extracts of 10 plant species showed effective free radical scavenging in the DPPH assay. The methanol extracts of Solanum nigrum fruits and Acacia asak barks exhibited the highest antioxidant activity (Table 5). Among the 30 methanol extracts tested for cytotoxicity against FL-cells only ve extracts (Commiphora kua, Plicosepalus curviorus, Sansevieria ehrenbergii, Solanum incanum and Tamarindus indica) showed remarkable activities with IC50 values below 50 g/ml (Table 6). 4. Discussion and conclusions The results of our screening assays justify the use of the investigated plants in the Yemeni ethnomedicine. It is the rst report about biological tests with Acacia asak, Azima tetracantha, Cadaba farinose, Cissus rotundifolius, Commiphora foliacea, Gossypium areysiamum, Euphorbia hadramautica, Ficus vasta, Indigofera spinosa, Leptadenia pyrotechnica, Maerua oblongifolia, Ocimum forskolei, Orbea deersiana, Plicosepalus curviorus, Rhytidocaulon cf. tortum, Fagonia luntii, Sansevieria ehrenbergii and Zehneria anomala. Whereas other parts of Aloe vera, Tamarindus indica and Sansevieria ehrenbergii are well investigated, we describe the effects of the seeds of Aloe vera, the fruits of Sansevieria ehrenbergii and the owers of Tamarindus indica for the rst time. The existing knowledge about the other investigated plants is in the most cases very limited. Some authors describe antimicrobial effects of extracts of the roots of Acacia nilotica against Gram-positive and Gramnegative bacteria (Elnabi et al., 1992; Kambizi and Afolayan, 2001; Srinivasan et al., 2001). Tung et al. (2007) reported the antioxidant activities of other species of the genus Acacia, especially of ethanolic extracts from the bark of Acacia confuse. The antimicrobial activity and cytotoxicity of Aerva lanata was reported by Chowdhury et al. (2002). Some avonoid gly-

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cosides (Zadorozhny et al., 1986) and alkaloids (Zapesochnaya et al., 1991) could be found and are possibly responsible for the weak activities observed by us. Aloe vera is well known for its polysaccharides and anthraquinone derivatives. Besides, two new dihydrocoumarin derivatives with strong antioxidant activity could be isolated (Zhang et al., 2006). This activity could be shown also for some aloesin derivatives, e.g. isorabaichromone, feruloylaloesin and p-coumaroylaloesin (Yagi et al., 2002). In our investigation the seed extracts do not show strong bioactivities. For Azima tetracantha only some phytochemical results are known. Daulatabad et al. (1991) identied some fatty acids, namely ricinoleic acid and cyclopropenoid acid from the seed oil. Besides, the seeds contain a complex mixture of 26 avonoids, predominantly as glycosides and acyl-glycosides (Bennett et al., 2004). Isorhamnetin 3-O-rutinoside was identied in the leaves (Williams and Nagarajans, 1988). Rao and Rao (1978) reported the occurrence of triterpenoids and Rall et al. (1967) that of dimeric piperidine alkaloids (azimine, azcarpine and carpaine). Flavonoids as well as triterpenoids and alkaloids could be responsible for the observed antimicrobial activities. In Cadaba glandulosa avonol glycosides have been identied by Gohar (2002). In Cadaba farinosa Ahmad et al. (1990) found the sesquiterpene lacton cadabicilone. Oxypregnane glycosides, the penicillosides D-G, have been identied in the aerial parts of Caralluma penicillata (AbdelSattar et al., 2002). From Cissus quadrangularis tetracyclic triterpenoids (Bhutani et al., 1984; Guta and Verma, 1990) and stilbene derivatives (Adesanya et al., 1999) are known. The resins of Commiphora kua contain terpenes from bisabolen and dammaran type. Antifungal activities against plant pathogenic fungi could be shown (Dekebo et al., 2002; Manguro et al., 2003). Whether similar compounds are responsible for the observed antimicrobial and cytotoxic activities of the extracts of the leaves remains unclear. Whereas no reports about Ficus vasta are existing, Baumgartner et al. (1990) isolated two indolizidine alkaloids from the methanol extract of leaves of the related species Ficus septica. The chloroform extract of Gossypium hirsutum leaves was active against Staphylococcus aureus (Rojas et al., 2001). Bell et al. (1975) showed the antimicrobial activity of six terpenoids from Gossypium sp., namely gossypiumhemigossypol, 6-methoxyhemigossypol and 6-deoxyhemigossypol, gossypium-6-methoxygossypol and 6,6 -dimethoxygossypol. Possibly Gossypium areysiamum, tested here for the rst time, contains similar compounds. A pentacyclic triterpenoid named leptadenol was isolated from the hexane extract of Leptadenia pyrotechnica (Noor et al., 1992). The seeds of Leptadenia reticulata are rich in hyperoside and rutin (Subramanian and Nair, 1968) and the aerial part of Leptadenia arborea has been shown to contain pinoresinol, syringaresinol, leucanthemitol and E-ferulaldehyde (El-Hassan et al., 2003). From Leptadenia reticulata Srivastav et al. (1994) had isolated three pregnane glycosides. We did not found strong activities of Leptadenia pyrotechnica.

Besides the paper of Elegami et al. (2001) about the antimicrobial activities of some Plicosepalus species there are no further reports about this genus. The antimicrobial effects of bark and pulp extracts of miswak, Salvadora persica, were previously evaluated (Almas and Al-Bagieh, 1999). Abdelrahman et al. (2003) identied volatile compounds, oleic, linolic and stearic acids, esters of fatty acids and aromatic acids in crude Salvadora persica extracts, which have antimicrobial effects. Presently it cannot be decided whether these compounds are related to the effects observed by us. Like other Solanum species Solanum incanum and Solanum nigrum contain steroidal alkaloid glycosides. In Solanum incanum solamargin has been found. This compound shows cytotoxicity against human hepatoma cells (Kuo et al., 2000). Antitumor effects have also been described for Solanum nigrum (Son et al., 2003; Lee and Lim, 2006). The observed antibacterial and antifungal activities of Solanum species are well known and probably caused by the alkaloids (Mohamed et al., 1996; Kambizi and Afolayan, 2001). Besides, avonoids and chlorogenic acids have been documented for Solanum incanum (Lin et al., 2000). The antimicrobial activity of Tamarindus indica has been attributed to lupeol (Ali et al., 1998). The reported antioxidant activity of the leaves of this plant (Perez et al., 2003; Ramos et al., 2003) was similar to the activity of the owers observed by us. It should be caused by polyphenolic compounds which have been already isolated from the seeds (Luengthanaphol et al., 2004; Sudjaroen et al., 2005). The results of these screening investigations conrm the great potential of plants of the Yemeni ethnomedicine for production of bioactive compounds and are useful for rationalizing the use of medicinal plants in primary health care. The phytochemical characterization of the extracts, the identication of responsible bioactive compounds and quality standards are necessary. Acknowledgements The authors would like to thank Deutscher Akademischer Austauschdienst (DAAD) for a grant enabling the stay of Dr. Al-Fatimi at Ernst-Moritz-Arndt-University Greifswald that was used to carry out this research. Besides this we thank Mrs. Ruth Ball, University Greifswald, for technical assistance and Dr. Norbert Kilian, Botanischer Garten and Botanisches Museum Berlin-Dahlem Freie University, Berlin, for the assistance by the taxonomical identication of some plant species. References
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