Mrsa Plant

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Journal of Ethnopharmacology 116 (2008) 102–111

Antibacterial activity of plants used in traditional medicines of Ghana with particular reference to MRSA George A. Pesewu a,b , Ronald R. Cutler a,∗ , David P. Humber a a

b

School of Health and Bioscience, University of East London, Stratford, London E15 4LZ, UK Centre for Scientific Research into Plant Medicine (CSRPM), P.O. Box 73, Mampong-Akwapim, Ghana Received 20 June 2006; received in revised form 7 November 2007; accepted 8 November 2007 Available online 17 November 2007

Abstract Ethnopharmacological relevance: : In an ethno botanical survey carried out in the Akwapim-North district of the Republic of Ghana, 25 plant species, used in traditional medicine to treat skin disease and/or to treat antimicrobial (viral, bacterial or protozoan) infections were identified. Aim of Study: : To investigate the antimicrobial activity of traditional Ghanaian medicines with special interest in anti-methicillin-resistant Staphylococcus aureus (MRSA) activity. Materials and methods: : Chloroform, ethanol and aqueous extracts (including use of a Stomacher) of these plants were prepared and agar-well diffusion tests, MIC’s and MBC’s were used to investigate antimicrobial activity. Results: Extracts of 13 plant species inhibited the growth of one or more of the following bacteria: MRSA, methicillin-sensitive Staphylococcus aureus (MSSA), Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris. Extracts from 11 of these 13 plant species also inhibited the growth of three or more of 14 additional clinical isolates of MRSA. Aqueous extracts of Alchornea cordifolia were active against all 21 bacterial strains tested and showed the highest levels of antibacterial activity overall with MIC’s against MRSA in the range of 1.6–3.1 mg ml−1 and MBC’s in the range of 6.3–12.5 mg ml−1 . Conclusions: : The presence of antibacterial activity in extracts of Elaeophorbia drupifera, Rauwolfia vomitoria and the leaves of Solanum verbascifolium, plants traditionally used to treat skin infections, are reported for the first time. Extracts from Alchornea cordifolia, also used to treat wounds, had the widest spectrum of antibacterial activity. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Antibacterial; MRSA; Traditional medicines; Ghana

1. Introduction Many of the drugs currently used to treat bacterial and other infections were first isolated from natural sources including ethnomedicinal plants (Coe and Anderson, 1996). Such plants may provide new sources of therapeutic agents against multiply drug resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). MRSA is a major cause of nosocomial infection in UK hospitals and throughout the world. MRSA infections account for one fifth of all hospital-acquired infections, costing the UK National Health Service approximately £1 billion per year (Cepeda et al., 2005). The problem has been aggravated by the rapid spread and high incidence of MRSA in intensive-care

∗

Corresponding author. Tel.: +44 208 223 4162. E-mail address: [email protected] (R.R. Cutler).

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.11.005

units (Cepeda et al., 2005). The continuing rise in MRSA infection rates and its spread worldwide has led to calls for action to control infection and develop novel anti-MRSA agents (Cutler and Wilson, 2004; Hancock, 2007) and vaccines. Several ethnomedicinal plant species of Ghana have been identified and their usage documented; (Abbiw, 1990; Anonymous, 1992; Dokosi, 1998; Mshana et al., 2000; Agbovie et al., 2002; Anonymous, 2004). They have been used as antibacterial, antifungal, antiviral and antiprotozoan agents and for the general treatment of skin diseases, dermatitis, burns, diarrhoea, fever (pyrexia) of unknown origin, wounds, cuts, sores, coughs and localized skin swellings. In the present investigation, plants used in folk medicine in the Akwapim-North district of Ghana to treat bacterial and other skin conditions were identified and extracts were tested for antibacterial activity. Particular attention was given to activity against MRSA.

G.A. Pesewu et al. / Journal of Ethnopharmacology 116 (2008) 102–111

2. Materials and methods 2.1. Ethnobotanical survey and collection of specimens A survey was carried out in the Akwapim-North district of Ghana, a dry equatorial region where most of the natural vegetation has been cleared for the cultivation of food crops. It covers an area of 544 km2 and has a population of about 105,000 that includes the Cherepong, Larteh, and Akwamus ethnic groups. Interviews were conducted between July and September 2002 and between November and December 2003 with 81 herbalists and traditional healers in towns and villages. These villages included Adwaso, Dawu, Kunutiase, Kwamoso, Mamfe, New Mangoase, Obosomase, Okrakwadwo, Old Asuoyaa and Tutu. There were three objectives to the survey; to prepare an inventory of plants used to treat skin infections; to establish the origins, history and overall usage of these plants and to clarify the local procedures used to prepare and administer these medicines. Interviews were conducted according to the procedures recommended by Otshundi et al. (2000). A degree of consensus in the listing of medicinal plants attributable to a cultural group is important (Agelet and Valles, 2003) therefore only species with fidelity values (percentage of interviewees citing the same plant) of two or more were selected for this study. Specimens of medicinal plants were collected from natural populations. Voucher herbarium specimens were allocated to the Ghana Herbarium (Department of Botany, University of Ghana, Accra), the Ethnobotanical Herbarium, Centre for Scientific Research into Plant Medicine (CSRPM), Mampong-Akwapim, Ghana) and the School of Health and Bioscience (University of East London, UK). Samples for laboratory investigation were air-dried in the shade at room temperature (25–28 ◦ C), for at least 2 weeks. They were then dried at 45 ◦ C in a convection oven for 2 h to completely remove residual moisture, before milling into fine powder. These procedures correspond to those used by the herbalists consulted. The powders were sealed in air-tight bags to prepare them for storage and transport. 2.2. Plant extracts Crude extracts of the medicinal plants were obtained by both successive extraction methods and by the use a ‘Stomacher’ Lab-Blender (A.I. Seward, UK). Extracts were prepared from the plant organs specified by the herbalists (Table 1). Successive extractions were carried out using the procedures used at the CSRPM when screening herbal medicines from local practitioners. For each extraction, three different solvents (chloroform, ethanol then water) were used in succession. Twenty-five grams of dry milled plant powder was placed in a beaker (250 ml), 100 ml chloroform was added and the mixture left to soak overnight. This mixture was then vigorously stirred for 10 min and allowed to settle for 5 min. The supernatant liquid was passed through filter paper (Whatman No. 1) to remove solid plant material and the solvent evaporated from the filtrate in vacuo at 34 ◦ C in a rotary evaporator. Finally, the residue was dried to a constant weight in a hot air oven. This residual plant material was further extracted, first with 80% ethanol (100 ml)

103

and then with distilled water (100 ml) using the same procedure as was used for chloroform. The dry weight of each extract was expressed as a percentage of the dry weight of the original powdered tissue. Other workers have used successive extraction methods with good success for obtaining antimicrobial extracts (Camporese et al., 2003). In parallel to the above extraction methods, aqueous extracts were also processed using a novel stomacher (A.J Seward and Co, UK) based method. Twenty-five grams of powdered tissue was placed in a stomacher plastic bag, 100 ml of sterile distilled water was added and the mixture placed in the stomacher sample chamber. After processing for 5 min the solutions were passed through filter paper (Whatman No. 1) and the filtrate freeze dried. 2.3. Sources of bacterial cultures Plant extracts were tested against Escherichia coli (ATCC 25922), MSSA (methicillin susceptible Staphylococcus aureus – NCTC 6571), MRSA (UELSHB – University of East London School of Health and Bioscience, 102 and 103), Proteus vulgaris (UELSHB 241), Pseudomonas aeruginosa (ATCC 27853) and Streptococcus pyogenes (UELSHB 333). UELSHB strains are kept at the School of Health and Bioscience, University of East London. Tests were also carried out on 14 clinical isolates of MRSA obtained from the Royal London Hospital courtesy of Dr. P. Wilson. 2.4. Tests of antibacterial activity by the agar-well diffusion method The antibacterial activities of powdered plant extracts were compared with controls. Tests were based on BSAC (British Society for Antimicrobial Chemotherapy) procedures (Andrews, 2005) modified by Cutler and Wilson (Cutler and Wilson, 2004). Wells, 6 mm in diameter were punched in the agar medium and powdered plant extracts dissolved in sterile distilled water (100 (l of 50 mg ml−1 solutions) were added to each well. As with the test solutions, 100 (l of a purified aqueous extract of allicin at 0.5 mg ml−1 (Cutler and Wilson, 2004; Allicin International, Rye, UK) was used in wells as an herbal antimicrobial control. Standard paper discs (Oxoid Ltd., Basingstoke, UK), containing gentamicin (10 (g) or vancomycin (30 (g) were used as antibiotic controls. Plates were incubated for 24 h at 37 ◦ C and the diameter of the inhibition zone around the test wells or around the control discs were measured to assess antibacterial activity. All tests were replicated three times. A pooled estimate of the diameters of the zones of inhibition was calculated based on the sum of squares of the deviations of all replicates from their respective means. 2.5. Quantitative antimicrobial evaluation The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for the active plant extracts were determined using standard National Committee for Clinical Laboratory Standards methods (NCCLS, 1998). Concentrations of plant extracts were tested in the range

104

Table 1 Medicinal plants of the Akwapim North district of Ghana identified in the survey and used in bacteriological testsa Fcb

Species

Voucher number

Diseases treated

Plant part

Preparation

Administration

Apocynaceae

Alstonia boonei De Wild.

PA2002/5

Stem bark

Boil

Oral/topical

2

Rauwolfia vomitoria Afzel.

PA2003/8

Root

Latex/decoction

Topical

4

PA2002/9

Root

Boil

Oral

6

Balanitaceae Boraginaceae

Cryptolepis sanguinolenta (Lindl.) Schltr. Parquetina nigrescens (Afzel.) Bullock Balanites aegyptica (L.). Del.? Heliotropium indicum L.

Cleansing of suppurating wounds, open fractures Parasitic skin diseases, yaws Malaria

PA2002/8 PA2002/3 PA2003/1

Root Fruit Leaf

Poultice Alcoholic extract Juice

Topical insertion Topical Topical

2 3 2

Caesalpiniaceae

Cassia alata L.

PA2002/1

Leaf

Juice/infusion

Topical

14

Cassia podocarpa Guill. &. Perr.

PA2002/15

Leaf

Infusion

Topical

2

Cassia occidentalis (Linn.) Link. Crateva religiosa G. Forst

PA2003/2 PA2003/7

Leaf Stem bark

Infusion Boil

Topical Topical

2 2

Chenopodium ambrasioides L. Combretum micranthium G.Don. Terminalia avicennoides Guill.&.Perr. Vernonia amygdalina Delile Momordica charantia L. Alchornea cordifolia (Schum. & Thonn.) Mull. Arg. Elaeophorbia drupifera Thonn.

PA2002/4 PA2002/14 PA2003/10 PA2002/13 PA2003/4 PA2002/2

Leaf Root Stem bark Leaf Shoots Leaf

Infusion Boil Boil Boil Boil Juice

Topical Topical Topical Oral/topical Oral/topical Topical

5 3 2 5 2 4

Leaf

Boil

Topical

5

Ricinus communis Linn. Ocimum viride Willd. Hoslundia opposita Vahl. Persea americana Mill. Psidium guajava L. Gardenia ternifolia Schumach. & Thonn.

PA2002/12 PA2002/7 PA2002/11 PA2003/6 PA2003/9 PA2003/3

Seed Leaf Leaf Leaf Leaf Leaf

Oil Juice/poultice Boil Infusion Infusion Infusion

Topical Topical Oral/topical Topical Topical Topical

2 6 2 5 3 2

Mitracarpus villosus Cham. & Schlecht.

PA2002/6

Leaf

Infusion

Topical

3

Solanum verbascifolium L.

PA2003/5

Candidiasis Herpes zoster Erysipelas, thrush, Herpes zoster Herpes zoster, eczema, mycosis Wounds, sore dressing, skin ulcers Guinea worm, wounds Leprosy, swollen parts of the body Wound, skin infections Wounds, Guinea worm Boils Dermatitis Measles Dermatitis, Herpes zoster, ringworm, wounds Skin infections, Guinea worm Dermatitis, keratoderma Trichomoniasis Dermatitis Skin ulcers Measles, Herpes zoster Ulcers, syphilis, body itching Dermatitis; wound; leprosy Dermatitis

Leaf

Poultice

Topical

2

Asclepiadaceae

Capparaceae Chenopodiaceae Combretaceae Asteraceae Cucurbitaceae Euphorbiaceae

Labiatae Lauraceae Myrtaceae Rubiaceae

Solanaceae a b

PA2002/10

Only plants with fidelity values of 2 or more were used in bacteriological tests. Fc, fidelity level (number of respondents citing the same plant).

G.A. Pesewu et al. / Journal of Ethnopharmacology 116 (2008) 102–111

Family

G.A. Pesewu et al. / Journal of Ethnopharmacology 116 (2008) 102–111

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Table 2 Mean diameter of zones in agar plates where bacterial growth was inhibited by plant extracts placed in wells of 6 mm diameter Plant species

Mode of extraction

Mean diameters (mm)a of inhibition zones in 7 bacterial isolatesb MRSA I

MRSA II

MSSA

S. p.

E. c.

P. a.

P. v

Alchornea cordifolia

Chloroform Ethanol Water Blender

10 16 30 31

10 20 31 32

12 21 32 34

12 18 18

13 15 15

10 13 13

20 21 24

Chloroform Ethanol Water Blender

11 16 16

14 18 18

16 20 20

10 14 14

10 12 12

15 18 18

Chloroform Ethanol Water Blender

20 10 10

22 10 12

25 14 16

18 10 10

11 8 8

12 14

Cryptolepis sanguinolenta

Chloroform Ethanol Water Blender

14 24 18 22

14 26 22 25

22 28 24 28

12 20 16 18

8 13 10 10

10 22 20 22

Elaeophorbia drupifera

Chloroform Ethanol Water Blender

19 15 15

20 17 17

24 20 20

18 12 12

Chloroform Ethanol Water Blender

10 12 12

10 14 14

12 18 18

8 10 10

Chloroform Ethanol Water Blender

25 20 20

25 22 22

28 26 26

Cassia alata

Cassia occidentalis

Gardenia ternifolia

Mitracarpus villosus

Ocimum viride

Persea americana

Psidium guajava

Rauwolfia vomitoria

Solanum verbascifolium

Vernonia amygdalina

Controls: Allicin 100 ␮l of 0.5 mg ml−1 Gentamicin (10 ␮g) Vancomycin (30 ␮g)

Chloroform Ethanol Water Blender

12

Chloroform Ethanol Water Blender

26 22 23

28 25 26

28 26 30

21 16 18

10 10

8 8

20 20

Chloroform Ethanol Water Blender

16 20 18 22

18 22 21 24

20 25 24 27

20 10 15

15 10 10

10 8 8

18 10 10

10

12

16

20 20

20 22

21 24

Chloroform Ethanol Water Blender Chloroform Ethanol Water Blender Chloroform Ethanol Water Blender

8

8

12

10 10

10 10

14 14

8 8

10 10

Water extract Paper disc Paper disc

32 25 20

35 25 22

35 25 24

22 20 21

10 16 NTc

12 12 14 23 NT

20 18 NT

Extracts were obtained as serial extractions in chloroform, ethanol and water, or in aqueous solution in a ‘Stomacher’ blender. Blanks indicate no detectable inhibition of bacterial growth. a Values each represent the means of three replicates; the pooled S.D. of all means = 0.5 mm (all used 100 ␮l of 50 mg ml−1 solution). b Bacterial isolates: MRSA I, UELSHB 102; MRSA II, UELSHB 103; MSSA, NCTC 6571; S. p., Streptococcus pyogenes UELSHB 333.; E. c., Escherichia coli ATCC 25922; P. a., Pseudomonas aeruginosa ATCC 27853; P. v., Proteus vulgaris UELSHB 241. c Not tested.

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G.A. Pesewu et al. / Journal of Ethnopharmacology 116 (2008) 102–111

0.048–50 mg ml−1 except for allicin which was tested in the range 0.024–25 mg ml−1 . 3. Results 3.1. Ethnobotanical survey The study identified 25 plant species with fidelity levels of two or more used by herbalists to treat appropriate skin infections and conditions (Table 1). Herbalists used the leaves of plants as sources of extracts in 60% of species, the roots in 16%, the stem-bark in 12% and the seeds, fruits or shoots in 4% each. Local herbal practitioners used a number of methods to prepare these medicines some materials were prepared using more than one method. These methods were, infusions or decoctions of fresh or dried material (18 species), juice squeezed from leaves (5 species), poultices prepared from pulped plant tissues (3 species), alcoholic extraction (1 species), oil extraction (1 species) or latex (1 species). Medicines from Cassia alata, Ocimum viride and Rauwolfia vomitoria are prepared by more than one method. All of the prescriptions are used topically and 16% used both orally and topically (Table 1). The majority (76%) of the plant species were collected from the wild but 24%, mainly fast growing species, are cultivated for easy access.

3.2. Plant extracts Amongst those plant species with antibacterial activity (listed in Table 2) the yields of materials extracted using different methods varied. The ranges of percentage dry weights isolated from extracts were between 0.6% and 4% using chloroform, between 3.2% and 16% using ethanol and between 2.6% and 16.4% using water. Chloroform extracts produced the lowest yield of material and with the exception of Alchornea cordifolia and Cassia occidentalis; yields were generally lower from water than from ethanol. The yields obtained with the ‘Stomacher’ blender were in the range 4.4% and 16.0% equal to or slightly greater than those obtained in water extractions. The stomacher method was also quicker to use, taking minutes as opposed to overnight extraction. 3.3. Antibacterial activity The antibacterial activities of the variously dissolved extracts varied according to the species of bacteria tested. In 13 of the 25 plants tested at least one extract produced a zone of inhibition greater than 10 mm against at least one species. In the remaining 12 plants, no antibacterial activity was detected (Table 2). Zones of inhibition produced from aqueous and ethanolic extracts

Table 3 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of extracts, obtained with the ‘Stomacher’ blender, of selected plant species against seven bacterial isolates Plant species

MIC/MBC (mg ml−1 )

MRSA Ia

MRSA II

MSSA

S. p.

E. c.

P. a.

P. v.

Alchornea cordifolia

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

3.1 12.5 >50 >50 >50 >50 25 50 >50 >50 >50 >50 12.5 50 6.3 25 12.5 50 25 >50 >50 >50

1.6 6.3 25 >50 >50 >50 25 25 >50 >50 >50 >50 6.3 25 3.1 25 12.5 50 6.3 >50 >50 >50

0.4 0.8 6.3 50 50 25 3.1 12.5 6.3 >50 12.5 50 6.3 6.3 1.6 6.3 3.1 12.5 3.1 6.3 >50 >50

6.3 25 50 >50

6.3 50 50 >50 >50 >50 50 >50

12.5 50

1.6 6.3 12.5 50 50 >50 3.1 12.5

>50 >50 >50 >50

>50 >50 >50 >50

12.5 50 50 >50 >50 >50 12.5 >50

0.1 0.2 0.2 0.8 0.8 3.1

0.1 0.2 0.2 0.8 0.8 1.6

0.1 0.1 0.2 0.8 0.4 1.6

0.1 0.2 0.4 3.1 0.8 3.1

0.2 >0.2 0.4 3.1 NT NT

0.1 0.2 0.8 3.1 NT NT

Cassia alata Cassia occidentalis Cryptolepis sanguinolenta Elaeophorbia drupifera Gardenia ternifolia Mitracarpus villosus Persea americana Psidium guajava Solanum verbascifolium Vernonia amygdalina Controls: Allicin Gentamicin Vancomycin

MIC MBC MICa MBCa MICa MBCa

50 50 >50 >50 50 >50

12.5 50 25 50

>50 >50

>50 >50

>50 >50 0.2 >0.2 0.8 3.1 NTc NT

Blanks indicate no inhibition. a Values are in mg l−1 ; b Abbreviations: MRSA I, UELSHB 102; MRSA II, UELSHB 103; MSSA, NCTC 6571; S. p., Streptococcus pyogenes UELSHB 333; E. c., Escherichia coli ATCC 25922; P. a., Pseudomonas aeruginosa ATCC 27853; P. v., Proteus vulgaris UELSHB 241; c NT, not tested.

G.A. Pesewu et al. / Journal of Ethnopharmacology 116 (2008) 102–111

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Table 4 Mean diameter of zones where growth of MRSA isolates was inhibited by plant extracts Plant species

Alchornea cordifolia Cassia alata Cassia occidentalis Cryptolepis sanguinolenta Elaeophorbia drupifera Gardenia ternifolia Mitracarpus villosus Persea americana Psidium guajava Solanum verbascifolium Vernonia amygdalina Controls: Allicin 100 ␮l of 0.5 mg ml−1 Gentamicin 10 ␮g Vancomycin 30 ␮g

Mean diameters (mm)a of inhibition zones for 15 MRSA isolatesb A1

A2

A3

A4

A5

B1

B2

B3

C1

C2

D

E

F

G

102

26 10 12 24 10 12

25

30

28

20

15

28

30 14

27

28

22

10

26

10

10

20

14 21 17 20 12

15

20 22

12 18 10 20

29 15 22 18 20 21 10 12

8

30 15 14 30 12 20 22 19 24 10 10

21

20

27 20 10 23

16 20 10

29 15 8 22 10 12 15 18 21 10 8

31 16 18 22 14 20 20 24 23 20 10

32 25 20

32 26 20

28 24 21

30 24 20

25 22 20

31 21 18

32 25 20

30 26 17

22 8

12 24 16

32 25 20

20 11

34 25 21

30 26 21

24 18 16

32 25 20

44 40 30

32 24 20

26

18 20

10 18

Values each represent the means of three replicates; the pooled S.D. of all means = 0.3 mm. (All used 100 ␮l of 50 mg ml−1 solution). MRSA isolates and their origins: A: 1, 2, 3, 4, 5 – nose; B: 1, 2, 3 – leg ulcer; C: 1, 2 – wound swab; D: stoma; E: penis; F: vagina; G: eye; 102: UELSHB 102 (control). a

b

and were generally similar but antibacterial activity was found only in the ethanolic extracts of Ocimum viride and Rauwolfia vomitoria and only in the aqueous extracts of Vernonia amygdalina. In chloroform extracts antibacterial activity was found in only four plant species and the inhibition zones were generally smaller than those from ethanolic or aqueous extracts. The aqueous extracts of Alchornea cordifolia, Persea americana and Psidium guajava inhibited the growth of all bacterial species tested. Those of Cassia alata, Cassia occidentalis, Cryptolepis sanguinolenta, Elaeophorbia drupifera, Gardenia ternifolia, Mitracarpus villosus, Solanum verbascifolium and Vernonia amygdalina inhibited the growth of fewer bacteria but were active against all three isolates of Staphylococcus aureus. The most active extract of these was the ethanol extract from Cryptolepis sanguinolenta, average zone size 19 mm (range, no zone to 28 mm). No inhibitory zone was found against Pseudomonas aeruginosa. Only three species showed any activity against Pseudomonas aeruginosa, Alchornea cordifolia aqueous extracts were most active, giving a zone size of 13 mm, this compared well with the allicin control (14 mm). The diameters of the inhibition zones of the aqueous extracts of Alchornea cordifolia were similar to, or greater than those of all other plant species against corresponding bacteria average 23 mm (range 13–32 mm) (Table 2). In addition, the blender aqueous extracts of Alchornea cordifolia showed the greatest activity, average zone size 24 mm (range 13–34 mm) especially against Staphylococcus aureus. These results were also reflected in lower MICs (range, 0.4–3.1 mg ml−1 ) and MBCs (range, 0.8–12.5 mg ml−1 ) than those of the other plant species (Table 3). Blender extracts, from the 11 plant species showing antibacterial activity in aqueous extracts (Table 2) were tested against a further 14 clinical isolates of MRSA plus UELSHB 102 (Table 4). The extract of Alchornea cordifolia inhibited the growth of all 15 MRSA isolates and the diameters of the inhi-

bition zones (average 26 mm, range 15–31 mm) were similar to, or greater than, those of the other plant species against corresponding bacteria. Compared with the activity of Alchornea cordifolia extracts, MRSA were less inhibited by the extracts from the other species tested (Table 4). 4. Discussion Chloroform extracts generally showed lower activity than ethanolic or aqueous extracts (Table 2) and they inhibited a smaller range of bacteria. There was no other indication of variation in the ranges of antibacterial activity in the different extracts, which might suggest that the solvents had extracted different antimicrobial substances from the same plant. Although the activity of ethanolic extracts was generally greater than those of aqueous extracts, the use of an aqueous based ‘Stomacher’ Lab-Blender technique was preferred because it is quicker to use, and more efficient than the alternative methods. Samples were enclosed in a disposable plastic Stomacher bag and there is no direct contact between the sample and the device. The blender requires no cleaning between operations, two or more sample bags can be processed together and the extraction time (5 min) is shorter than for the various alternative methods used here or reviewed by Ong (2004). Antibacterial activity has previously been reported (Table 5) in 10 of the 13 plant species listed in Table 2. In the present study, the range of bacteria against which activity is attributed, is amplified and in addition aqueous extracts, often excluded in previous studies, were tested. We also tested the activities of extracts against clinical isolates of MRSA and in many cases highlighted the potential for these extracts to be used against such multiply antibiotic resistant organisms. Voss and Doebbeling (1995) pointed out that MRSA is a problem for the world and some hospitals in Africa have reported an increase in MRSA

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Table 5 Published data on antibacterial activity of plant extracts of species listed in Table 2 Plant species

Bacteria against which activity has been demonstrated

Authors

Alchornea cordifolia

Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae Bacillus spp., Lactobacillus casei, Stapylococcus spp., Streptococcus spp., agrobacterium tumefaciens, Citrobacter freundii, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Proteus spp., Pseudomonas aeruginosa, Salmonella spp., Serria marcescens, Trichomonas vaginalis Staphylococcus aureus, Bacillus subtilis, Escherichia coli, K. aerogenes, Proteus vulgaris, Ps. aerogenes Campylobacter coli, C. jejuni, Staphylococcus aureus, Streptococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurum, Shigella dysenteriae, Vibro cholerae, Mycobacterium fortuitum Campylobacter spp, Staphylococcus aureus Staphylococcus aureus, Bacillus subtilis, Streptococcus faecalis, Escherichia coli Staphylococcus aureus, Streptococcus faecalis, Pseudomonas aeruginosa, Proteus vulgaris, Bacillus subtilis, B. cerus, Escherichia coli, Enterococcus faecalis Streptococcus mutans Staphylococcus aureus, Mycobacterium phlei

Ajao et al. (1985), Okeke et al. (1999), Tona et al. (1999), Ajali (2000), Ebi (2001) Khan et al. (2001), Somchit et al. (2003)

Cassia alata

Cassia occidentalis Cryptolepis sanguinolenta

Gardenia ternifolia Mitracarpus villosus Ocimum viride

Persea americana Psidium guajava Vernonia amygdalina

Escherichia coli, Salmonella typhi, Shigella spp., Bacillus spp., Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris, Serria marcescens, Streptococcus spp., MSSA, MRSA.

isolation from 2% in 1985 to 50% in 1987. It is the view of ourselves and others (Hancock, 2007), that novel sources of agents active against MRSA, and other drug resistant micro-organisms, should be actively sought. This is the reason we highlighted the activity of these extracts against MRSA in this study. In many cases our findings agreed with, or showed greater antimicrobial activity, to that of earlier workers. In some cases however our findings showed less activity. Such variations were possibly due to the different extraction methods employed, the sources of plant materials and/or the different strains of bacteria tested. The fact that different extraction methods can affect antibacterial activity has been reported in earlier studies (Eloff, 1998). Aqueous extracts from Psidium gujava were particularly active against all 7 test strains with zones sizes of 24–27 mm for Staphylococcus aureus. The activity of aqueous extracts and ethanol extracts were similar the exception being the relatively higher activity of the ethanol extract against Proteus vulgaris (18 mm zone compared to 10 mm from aqueous). Abdelrahim and co-workers demonstrated that methanol extracts from the bark of P. gujava were more active than aqueous extracts against Escherichia coli. Their extracts were particularly rich in tannins. Gram positive organisms, including Staphylococcus aureus were not as susceptible. In keeping with the findings of Abdelrahim et al. ( 2002), we found ethanol extracts more active against Gram negative strains than aqueous extracts. The extracts from Cassia occidentalis were active against six of the seven original test strains; the greatest activity was from the ethanol extracts. From an ethanolic extract of roots of Cassia occidentalis Chukwujekwu et al. (2006) isolated the

Perez and Anesini (1994), Samy and Ignacimuthu (2000) Paulo et al. (1994), Silva et al. (1996), Cimanga et al. (1996), Cimanga et al. (1997), Gibbons et al. (2003), Sawer et al. (2005)

Silva et al. (1996) Irobi and Daramola (1994), Okunade et al. (1999), Bisignano et al. (2000) Orafidiya et al. (2001), Ngassoum et al. (2003)

Ofek et al. (2003) Jairaj et al. (1996), Nascimento et al. (2000), Abdelrahim et al. (2002) Akinpelu (1999), Iwalokun et al. (2003)

anthraquinone, emodin. They showed this agent was bacteriostatic against Gram positive organisms at very low levels (0.0078 mg ml−1 ). Our crude ethanolic extracts from Cassia occidentalis leaves also produced better activity against Gram positives than aqueous extracts. We additionally found that the aqueous extracts from the leaves were not only bacteriostatic but also bactericidal against MSSA (at 25 mg ml−1 ) this gives the possibility that at higher concentrations, Cassia-emodin could be bactericidal. This could be expected as emodin from other plants (e.g. Aloe-emodin) has been reported as bactericidal, affecting N-acetyl tranferase activity in some bacteria, Helicobacter pylori (Wang et al., 1998; Ferro et al., 2003). In contrast to the generally broad-spectrum activity from the plant extracts highlighted above, our extracts from the leaves of Solanum verbascifolium only produced activity against the three Gram positives. Glycosidal alkaloids from the fruits of Solanum plants have been associated with antibacterial activity in the past (McKee, 1959; Beaman-Mbaya and Muhammed, 1976; Fukuhara and Kubo, 1991). Solanum crystals from berry juice have strong activity against Gram positive bacteria and in addition are potent antifungal agents. Our aqueous extract from the leaves of Solanum verbascifolium showed particular activity against MSSA and MRSA and was bactericidal against MSSA (6.3 mg ml−1 ). There is some suggestion however, that although steroidal alkaloids have been associated in the past with antibacterial activity, that the antibacterial agents in the berry juice crystals are different and may be a phosphorylated purine and not steroidal alkaloids (Beaman-Mbaya and Muhammed, 1976).

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The antibacterial activities of extracts from Elaeophorbia drupifera, Rauwolfia vomitoria and from the leaves of Solanum verbascifolium are reported here for the first time. Activity against MRSA was also reported for the first time for many of these plant extracts. Overall the aqueous (Stomacher) extracts from Alchornea cordifolia, Psidium guajava and Persea americana were the most active antimicrobials found in this study. Tona and co-workers previously reported that extracts made from the root bark of Alchornea cordifolia demonstrated antibacterial, antiamoebic and antispasmodic action and as such had the potential to act as an anti-diarrheic agents (Tona et al., 1998; Tona et al., 1999). In our hands, aqueous (Stomacher) extracts from leaves were more active against MRSA and MSSA than against common intestinal bacterial pathogens. Against these organisms the extract was mainly bacteriostatic. We showed that an aqueous extract from Alchornea cordifolia was active against all MRSA strains (Table 4) and bactericidal against Staphylococcus aureus (Table 3). Although the MICs and MBCs of Alchornea cordifolia extracts were higher than those of the controls; purified allicin (Cutler and Wilson, 2004), gentamicin and vancomycin, one has to remember that the Alchornea cordifolia extract was a crude agent compared to these controls. When the active antibacterial agent of Alchornea cordifolia is isolated and purified its relative activity may increase. Ebi (2001) reported the inhibition of bacterial growth by leaf extracts of Alchornea cordifolia. Ebi found chloroform soluble extracts were active against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli but demonstrated greater anti-staphylococcal and anti-pseudomonal activity in the fractions that were insoluble in chloroform. Okeke et al. (1999) also reported that leaf extracts from Alchornea cordifolia were active against Gram positives. The activity they reported was similar but slightly lower than ours with MICs ranging from 5 to 20 mg ml−1 . We have also shown that Alchornea cordifolia extracts are not only inhibitory but can be bactericidal. Staphylococci were found to be most susceptible with MICs below 3.1 mg ml−1 and MBCs ranging for 0.8–12 mg ml−1 (concentrations that are 2–4 times the MIC). As to current knowledge on the toxicology of aqueous extracts of Alchornea cordifolia, a recent paper in a veterinary journal identified Alchornea cordifolia as one of five plants of the Euphorbiaceae family suspected of being poisonous to grazing animals. They reported that giving a crude aqueous extract orally to rats caused significant, potentially toxic, changes in blood chemistry. They also suggested that the tannins in the extract were responsible for these changes (Adedapo et al., 2007). In contrast to this, tannins from the same plant have been proposed as a possible adjuvant to penicillin in treating Staphylococcal skin infections. It is thought that they reduce the ability of the organism to coagulate plasma and form antibiotic resistant bacterial biofilms in wounds (Akiyama et al., 2001). Other workers have also reported from tests on mice, that the crude ethanol extract can be effective in treating Staphylococcus aureus infections at oral doses between 4 and 32 times lower than the lethal dose (Igbeneghu et al., 2007). Given the interesting potential of this agent against MRSA, further work is planned to separate out the various phytochemicals in our aqueous extract and test them for antimicrobial and toxicological activity. Current

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evidence suggests the crude aqueous extract may be toxic if taken orally however its use as a topical agent may provide a potential way forward, as was suggested above by Akiyama and co-workers. In contrast to the active agents discussed above, some of our extracts showed relatively low antibacterial activity but our findings compared well to the work of other workers, although different extraction methods and plant organs were sometimes used. Examples of these were extracts from Vernonia amygdalina and Cassia alata. Vernonia amygdalina is a vegetable regularly consumed in large amounts by Nigerians and other West Africans. Rotimi et al. (1988) found aqueous extracts of Vernonia amygdalina were very active against oral anaerobic bacteria. Currently aqueous extracts of Vernonia amygdalina are also under investigation as antitumour agents. Studies into its cytotoxicity using MTT assays showed no cytotoxic effects in the concentration range of 3–25 mg ml−1 . This was an improvement to the known toxic effects from chloroform extracts. These contain vernodaline, vernolide, and vernomygdine three agents reported to be toxic to 50% of cells at approximately 2000 mg ml−1 (Izevbigie, 2003). We found chloroform extracts from Vernonia amygdalina had very low antibacterial activity (<12 mm zones) but our aqueous extracts produced slightly better activity (<14 mm zones) against six of the original seven test strains. Iwalokun et al. (2003) found that their aqueous extracts of Vernonia amygdalina also low activity with zones of inhibition of approximately 16 mm against MRSA and MIC’s of 22–26 mg ml−1 . Our V. amygdaline extracts failed to produce such activity against MRSA and had MIC’s of >50mg ml−1 . By comparison, when the purified sesquiterpine lactones from Vernonia amygdaline; vernolide and vernodalol were tested, Erasto et al. (2006) reported MIC’s as low as 0.5 mg ml−1 against Staphylococcus aureus. These purified compounds were not tested against MRSA. Cassia alata extracts have been reported to produce low levels of activity against a wide range of micro-organisms (Khan et al., 2001; Somchit et al., 2003). The greatest activity came from petrol extracts of flowers with zones of 20 mm produced against Staphylococcus epidermidis (Khan et al., 2001). Leaf extracts produced no more than 16 mm zones of inhibition, aqueous extracts were not tested. Somchit et al. (2002) found water and ethanol extracts from barks produced a maximum zone of 16 mm against Staphylococcus aureus and Escherichia coli. These results were comparable to those found in our study; in fact our aqueous extracts produced equal activity to the petrol extracts cited above. The present study has shown that medicinal plants, collected following ethnobotanically directed interviews in Ghana; possess antibacterial properties that support their value in herbal medicine for the treatment of diseases and aliments. These studies highlight the importance in using an ethnomedicinal approach to identifying plants from which new therapeutic agents can be obtained. We specifically targeted topical antiinfectives in this study but, since the diagnosis of skin infections without the laboratory backup to determine the true cause of the disease is difficult, agents were selected with a general history of

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being used to treat skin complaints, as well as those recognized as broad-spectrum antimicrobial agents. The one exception to this was Cryptolepis sanguinolenta, this plant is used as a systemic antimicrobial against malaria. In our study, we also found that the extract was active against MRSA. Although there is no ethno botanical background for the use of Cryptolepis sanguinolenta extracts as an anti-infective, other workers have previously reported that Cryptolepis sanguinolenta alkaloids had antibacterial activity (Paulo et al., 1994; Cimanga et al., 1996) and the work of Sawer et al. (2005) further demonstrated that the alkaloid cryptolepine in particular had anti-staphylococcal activity (against MSSA). We have additionally demonstrated that Cryptolepis sanguinolenta extracts were active against a wide range of MRSA and using culture methods, confirmed the electron microscopy findings of Sawer that Cryptolepis sanguinolenta extracts are bactericidal. In their paper cryptolepine was bactericidal at four times the MIC; this is similar to our findings for MSSA. Another point of interest is that the extract showed little activity against the Gram negative organisms, Escherichia coli and Pseudomonas aeruginosa and was primarily active as a Gram positive antimicrobial. One potential problem with using cryptolepine extracts is the cytotoxicity. Earlier Cimanga et al. (1996) reported cryptolepine to be “quite” cytotoxic. This was also confirmed by Ansah and co-workers (Ansah and Gooderham, 2002; Ansah et al., 2005). They compared aqueous Cryptolepis sanguinolenta extracts with purified cryptolepine. They found that the aqueous extract and the pure cryptolepine were both cytotoxic and weak mammalian mutagens. However, they also suggested that because of the poor genotoxicity of the compounds and their potent cytotoxic action that they could be possible anticancer agents. This reported cytotoxicity may however be a problem with its use as an antibacterial agent. The plant extracts demonstrating the highest levels of antibacterial activity were from Alchornea cordifolia, Persea americana and Psidium guajava. These all had an ethno botanical history for treating skin complaints. Psidium guajava had previously been reported to be antibacterial and active against Staphylococcus aureus (Qadan et al., 2005). We have for the first time demonstrated its activity against MRSA. Persea americana extracts have only been reported as active against Streptococcus mutans, reducing is adherence capabilities (Ofek et al., 2003). We have now shown extracts additionally possess antibacterial and more specifically anti-MRSA activity. 5. Conclusions Multiple drug resistance in bacterial pathogens is a continuing problem throughout the world. There is an established need to develop new antimicrobial agents to combat these pathogens. In our study, we found that 11 out of 13 crude plant extracts with a history of ethnobotanical use in Ghana for the treatment of wounds, contain antibacterial compounds that we have demonstrated have a potential use against MRSA and other nosocomial pathogens. Further work is ongoing to identify the exact nature of these antimicrobials.

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