Abstract

We used the agar dilution method to evaluate the antibacterial effect of Terminalia macroptera leaf (Tml) extract against nine reference and clinical Neisseria gonorrhoeae strains, including penicillin- and tetracycline-resistant and -susceptible strains. Tml possesses anti-N. gonorrhoeae activity against all of the strains and the minimum inhibitory concentrations (MIC) were between 100 and 200 μg ml−1. We then used a liquid–liquid partition method to divide the Tml extract into five fractions and determined the anti-N. gonorrhoeae activity of each of the fractions. All of the fractions showed antibacterial activity. The most active one was identified as the diethyl ether fraction and had MIC values of between 25 and 50 μg ml−1 against all of the strains.

Introduction

Neisseria gonorrhoeae infection is a major health problem in Africa. The frequency of this infection is high and the prevalence of antimicrobial resistance, especially to penicillin and tetracycline, is increasing [[,,[]. Penicillinase-producing N. gonorrhoeae (PPNG) and tetracycline-resistant N. gonorrhoeae (TRNG) have emerged and spread remarkably quickly in several countries [[,[].

Considering the unobtainable or prohibitively expensive treatment of gonorrhoea in most developing countries[7] and the increase in international travel, gonococcal infections are a significant threat in Africa. Medicinal plants, including Terminalia macroptera Guill. and Perr. (Combretaceae) root decoctions, are used to treat infectious diseases in many West African countries [[,[]. This drug is found at the main Bissau's market and is referred by all inquired traditional healers as an effective anti-infectious agent[10]. In previous work, this medicinal plant showed an interesting profile of activity against enteropathogenic bacteria, namely Campylobacter spp.[11]. Recent studies on plants used in Guinea-Bissau's traditional medicine showed that extracts from plants used against venereal diseases presented in vitro activity against N. gonorrhoeae strains with different susceptibilities to penicillin and tetracycline[12]. Ethanol extract of T. macroptera (Tmr) root was one of the most active, with a minimum inhibitory concentration (MIC) of between 100 μg ml−1 and 200 μg ml−1. The most active fractions of this extract are the diethyl ether fraction (Tmr-2) and water fractions (Tmr-3, Tmr-5). Ellagic acid, gallic acid, punicalagin, terchebulin, 3,3′-di-O-methylellagic acid, 3,4,3′,4′-tetra-O-methylellagic acid and terflavin A were isolated and identified in these fractions [[2,[3].

To compare the biological activity of T. macroptera leaves and roots we studied the susceptibility of N. gonorrhoeae to an ethanol extract of T. macroptera leaf (Tml) and to five liquid–liquid partition fractions: the n-hexane fraction (Tml-1), the diethyl ether fraction (Tml-2), the ethyl acetate fraction (Tml-3), the water filtered fraction (Tml-4) and the water precipitate fraction (Tml-5).

Materials and methods

Plant material

T. macroptera leaves were collected in the Contúboel region of Guinea-Bissau and characterised by Dr. Adélia Diniz, ‘Centro de Botânica Tropical–IICT’, Lisbon, Portugal. Voucher specimen number 662 is preserved in the LISC Herbarium, Lisbon, Portugal.

Extract preparation

After air-drying and grinding, the leaves (284.3 g) were exhaustively extracted with ethanol (80% v/v) at room temperature. The extract was concentrated under reduced pressure (<40°C). A portion (38.1 g) of the total (80.6 g) dried ethanol extract (Tml) was fractionated by sequential liquid–liquid partition with n-hexane (Tml-1) (1.8 g), diethyl ether (Tml-2) (3.7 g), ethyl acetate (Tml-3) (15.5 g) and water. After sedimentation, the water fraction was filtered to give a water-soluble fraction (Tml-4) (8.6 g) and insoluble material (Tmf-5) (8.5 g). The dried extract and fractions (about 100 mg) were then dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 000 μg ml−1. The resulting solutions were used in the antibacterial studies.

Determination of minimum inhibitory concentrations

The nine strains of N. gonorrhoeae used in this study were from the microbiological culture collection of the Antibiotic Resistance Unit of National Institute of Health at Lisbon (Table 1). Four of the bacteria tested were susceptible to all antibiotics used to treat gonorrhoea (NGs); three were characterised by plasmid-mediated resistance to penicillin by penicillinase production (PPNG) and two were resistant to both penicillin and tetracycline (PPNG/TRNG). A reference strain was included for each of these phenotypes.

1

Tested N. gonorrhoeae strains

Code Strain Phenotype MIC (μg ml−1
Pen. Tetra. 
1N N. gonorrhoeae INSA 257 PPNG 16 0.5 
2N N. gonorrhoeae INSA 232 PPNG 16 
3N N. gonorrhoeae INSA 249 NGs 0.25 0.5 
4N N. gonorrhoeae INSA 219 NGs 0.125 0.25 
5N N. gonorrhoeae INSA 227 NGs ≤0.063 0.25 
6N N. gonorrhoeae INSA 195 PPNG/TRNG 16 32 
7N N. gonorrhoeae ATCC 49226 NGs 0.25–1 0.25–1 
8N N. gonorrhoeae CRA/INSA 7567 PPNG 
9N N. gonorrhoeae Bilthoven 7391 PPNG/TRNG 32 
Code Strain Phenotype MIC (μg ml−1
Pen. Tetra. 
1N N. gonorrhoeae INSA 257 PPNG 16 0.5 
2N N. gonorrhoeae INSA 232 PPNG 16 
3N N. gonorrhoeae INSA 249 NGs 0.25 0.5 
4N N. gonorrhoeae INSA 219 NGs 0.125 0.25 
5N N. gonorrhoeae INSA 227 NGs ≤0.063 0.25 
6N N. gonorrhoeae INSA 195 PPNG/TRNG 16 32 
7N N. gonorrhoeae ATCC 49226 NGs 0.25–1 0.25–1 
8N N. gonorrhoeae CRA/INSA 7567 PPNG 
9N N. gonorrhoeae Bilthoven 7391 PPNG/TRNG 32 

MIC, minimum inhibitory concentration, according to the NCCLS guidelines; Pen., penicillin; Tetra., tetracycline; INSA, Instituto Nacional de Saúde; ATCC, American Type Culture Collection; CRA, Centro de Resistência aos Antibióticos; PPNG, N. gonorrhoeae with plasmid-mediated resistance to penicillin by penicillinase production; NGs, N. gonorrhoeae susceptible to different antibiotics used in gonorrhoea treatment, such as penicillin, tetracycline, spectinomycin, ceftriaxone and ciprofloxacin; PPNG/TRNG, N. gonorrhoeae with plasmid-mediated resistance to penicillin and tetracycline; 1N to 6N are clinical isolates; 7N to 9N are reference strains.

Freeze-dried cell cultures were revived by culture on chocolate agar (Oxoid, Basingstoke, UK) and incubated at 35°C in a 5% CO2 enriched atmosphere for 24–48 h.

The MIC for penicillin (Wyeth Lederle Portugal Farma Lda, Algés, Portugal), tetracycline (Laboratórios Atral S.A., Castanheira do Ribatejo, Portugal), and for the T. macroptera extract and fractions, against the N. gonorrhoeae strains, were determined by the agar dilution method, according to NCCLS proceedings[14]. The MIC was determined as being the lowest concentration of sample that resulted in complete inhibition of growth. Two controls were included for each extract or fraction: one plate in the absence of the extract solution and the other in the presence of the solvent (DMSO). All experiments were carried out in triplicate as previously described, to obtain consistent values[12].

Results and discussion

We found that Tml had antibacterial activity against all of the strains and that the MIC values were between 100 and 200 μg ml−1 (Table 2, Fig. 1). All of the fractions (Tml-1 to Tml-5) exhibited some activity against the different strains. The most active was the Tml-2 fraction, with MIC of between 50 and 25 μg ml−1 (Table 2, Fig. 1).

2

In vitro anti-N. gonorrhoeae activity of T. macroptera leaf extract (Tml) and fractions (Tml-1 to Tml-5)

N. gonorrhoeae strains MIC (μg ml−1)a 
Tml Tml-1 Tml-2 Tml-3 Tml-4 Tml-5 
1N 100 100 25 100 200 100 
2N 100 100 50 100 200 200 
3N 200 100 25 200 400 200 
4N 200 200 50 200 400 200 
5N 200 100 50 200 400 200 
6N 200 200 25 100 400 200 
7N 200 200 50 200 200 200 
8N 200 100 50 100 200 200 
9N 200 100 25 100 400 200 
N. gonorrhoeae strains MIC (μg ml−1)a 
Tml Tml-1 Tml-2 Tml-3 Tml-4 Tml-5 
1N 100 100 25 100 200 100 
2N 100 100 50 100 200 200 
3N 200 100 25 200 400 200 
4N 200 200 50 200 400 200 
5N 200 100 50 200 400 200 
6N 200 200 25 100 400 200 
7N 200 200 50 200 200 200 
8N 200 100 50 100 200 200 
9N 200 100 25 100 400 200 

Tml, T. macroptera leaf extract; Tml-1, Tml n-hexane fraction; Tml-2, Tml diethyl ether fraction; Tml-3, Tml ethyl acetate fraction; Tml-4, Tml water filtered fraction; Tml-5, Tml water precipitate fraction.

aMIC values correspond to the average of three experiments.

1

Comparison between the MIC values of T. macroptera leaf extract (Tml) and fractions (Tml-1 to Tml-5) and T. macroptera root extract (Tmr) and fractions (Tmr-1 to Tmr-5) against N. gonorrhoeae strains (1N to 9N). Tmr-1, Tmr n-hexane fraction; Tmr-2, Tmr diethyl ether fraction; Tmr-3, Tmr water total fraction; Tmr-4, Tmr water precipitated fraction; Tmr-5, Tmr soluble water fraction.

1

Comparison between the MIC values of T. macroptera leaf extract (Tml) and fractions (Tml-1 to Tml-5) and T. macroptera root extract (Tmr) and fractions (Tmr-1 to Tmr-5) against N. gonorrhoeae strains (1N to 9N). Tmr-1, Tmr n-hexane fraction; Tmr-2, Tmr diethyl ether fraction; Tmr-3, Tmr water total fraction; Tmr-4, Tmr water precipitated fraction; Tmr-5, Tmr soluble water fraction.

Tml had similar antibacterial activity to Tmr in the range of the tested concentrations (400–12.5 μg ml−1)[12] (Fig. 1). All of the strains were more susceptible to Tml-2 than to the Tmr fractions[12] (Fig. 1).

As previously demonstrated with the Tmr extract and fractions (Fig. 1)[12], these results suggest that the activities of the Tml extract and fractions are not dependent on the different resistance mechanisms expressed by the tested strains (Tables 1 and 2). No significant differences were observed between the activities of Tml extract and fractions against the clinical strains and the reference strains.

Compared to MICs of penicillin and tetracycline, the MIC values of T. macroptera extracts were greater. Our results indicate the presence of chemical compounds in T. macroptera with antibiotic activity against N. gonorrhoeae comparable to penicillin or tetracycline. In most plant extracts, the compounds responsible for the biological activity are present within a range of 1–0.001%[15].

Chebulagic acid, chebulinic acid, ellagic acid, gallic acid, punicalagin and isoorientin were identified in Tml (Fig. 2)[16]. It will be interesting to use different phytochemical methods to determine the active compounds in Tml-2 which contain ellagic and gallic derivatives.

2

Structure of the compounds identified in T. macroptera leaf extract[15]: chebulagic acid (1), chebulinic acid (2), ellagic acid (3), gallic acid (4), isoorientin (5) and punicalagin (6).

2

Structure of the compounds identified in T. macroptera leaf extract[15]: chebulagic acid (1), chebulinic acid (2), ellagic acid (3), gallic acid (4), isoorientin (5) and punicalagin (6).

Further research for new, active, anti-N. gonorrhoeae agents appears to be warranted in developing countries where effective antibiotic therapy is not available. Although T. macroptera roots are traditionally used to treat gonorrhoea, our results suggest that T. macroptera leaves can also be used. As the leaves are a renewable resource their use may help to protect this West African medicinal plant. In the future, it will be possible to develop, locally, pharmaceutical formulations for clinical trials.

Acknowledgements

Work in the CECF laboratory was supported by the ‘Programa Operacional Ciência, Tecnologia e Inovação (POCTI) do Quadro Comunitário de Apoio III’, which was co-funded by the FEDER and by national funding.

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