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Evaluation of the activity of 16 medicinal plants against Neisseria gonorrhoeae
International Journal of Antimicrobial Agents 33 (2009) 86–91
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
Evaluation of the activity of 16 medicinal plants against Neisseria gonorrhoeae P. Shokeen a , M. Bala b , V. Tandon a,∗ a b
Dr B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India Regional STD Teaching, Training and Research Centre, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India
a r t i c l e
i n f o
Article history: Received 17 May 2008 Accepted 17 July 2008 Keywords: Neisseria gonorrhoeae Antimicrobial susceptibility Medicinal plants
a b s t r a c t 50% Ethanolic extracts of various parts of 16 medicinal plants were evaluated for potential activity against clinical isolates and WHO strains of Neisseria gonorrhoeae, including multidrug-resistant (MDR) strains. Activity was calculated as percentage inhibition in comparison with penicillin and ciprofloxacin and strains were categorised as less sensitive, sensitive or highly sensitive to the extracts. The extracts caused differential inhibition of N. gonorrhoeae, with greater inhibition of the MDR strains. Among the extracts tested, 60% exhibited high activity whereas 20% showed moderate activity and 20% had little activity against N. gonorrhoeae. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Gonorrhoea affects more than 60 million people worldwide every year [1] and is a global health problem. Gonococci not only cause a high incidence of acute infections and complications but also play a major role in facilitating human immunodeficiency virus (HIV) acquisition and transmission [2]. Considering the expensive cost of treatment of gonorrhoea and the increase in international travel, gonococcal infections are a significant threat in developing countries. Although the frequency of asymptomatic infections, lack of innate or acquired immunity, and changes in human sexual behaviour have all contributed to the continued spread of gonococcal infections, the major contributing factor has been the development of antimicrobial resistance. Over the last several years, Neisseria gonorrhoeae strains have developed a high level of resistance to several antibiotics, including penicillin, tetracycline and quinolones [3,4]. Recently, isolates with reduced susceptibility to ceftriaxone as well as a substantial proportion (23.3%) of multiresistant strains have been reported from New Delhi, India [5]. Moreover, some of the newly investigated fluoroquinolones have reported problems of adverse drug reactions, some of which can be serious or fatal. Consequently, there is an urgent need for safe, alternative antigonococcal compounds that can be administered orally and have effective potency, allowing high therapeutic efficacy (>95.0% cure rate) preferably with a single-dose regimen. Plants produce a variety of secondary metabolites that have long been of interest to man. In recent years these are being used,
∗ Corresponding author. Tel.: +91 11 2766 6272/7151; fax: +91 11 2766 6248. E-mail addresses: [email protected], [email protected] (V. Tandon).
either directly as precursors or as lead compounds, in the pharmaceutical industry and it is expected that plant extracts showing target sites other than those used by antibiotics will be active against drug-resistant microbial pathogens. However, very little information is available on such activity of medicinal plants and, of the 400 000 plant species on Earth, only a small percentage has been systematically investigated for their antimicrobial activities. India is fortunate in possessing the world’s richest flora, with approximately 120 families of plants comprising 130 000 species. Additionally, there is a rich local ethnobotanical bibliography describing the species most frequently used by the population to cure various diseases. Although screening of Indian medicinal plants has revealed varying degrees of antimicrobial activity against pathogenic and opportunistic microorganisms [6], there is still a lack of experimental scientific studies confirming the possible antigonorrhoeal properties of a great number of these remedies. In a previous study, various extracts of Ocimum sanctum, Drynaria quercifolia and Annona squamosa showed considerable activity against N. gonorrhoeae [7], and bioassay-guided fractionation of the crude extract of O. sanctum led to the identification of eugenol as an active component [8]. Thus, it was considered worthwhile to screen a few of those medicinal plants that have been used in traditional medicine in one form or the other for activity against N. gonorrhoeae. 2. Materials and methods 2.1. Plant material The plant parts used were as follows: the roots of Adhatoda vasica; the stem bark of Albizzia lebbeck, Drynaria peregrina and Michelia champaca; the seeds of Alangium salviifolium; the fruits of
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P. Shokeen et al. / International Journal of Antimicrobial Agents 33 (2009) 86–91
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Table 1 Susceptibility of clinical isolates and WHO strains of Neisseria gonorrhoeae to various antibiotics WHO strain/clinical isolate
-Lactamase test
PEN
CFX
SPM
WHO A WHO B WHO C WHO G WHO K WHO L WHO O 1 2 3 4 5 6 7 8 9 10 11
−ve −ve −ve −ve −ve −ve +ve −ve +ve −ve −ve −ve −ve +ve −ve +ve −ve −ve
S LS LS LS R R R LS R LS LS LS LS R LS R S LS
S S S S LS LS S S S S S S S S S S S S
R S S S S S R S S S S S S S S S S S
TET CDS test
NCCLS method
Not TRNG Not TRNG Not TRNG TRNG Not TRNG Not TRNG Not TRNG Not TRNG TRNG Not TRNG Not TRNG TRNG Not TRNG Not TRNG Not TRNG Not TRNG TRNG Not TRNG
S S R TRNG R I R S TRNG S S TRNG S S LS S TRNG S
CIP
NAL
S S S LS R R S R R LS R R LS R R R R R
S S S R R R S R R R R R R R R R R R
PEN, penicillin; CFX, ceftriaxone; SPM, spectinomycin; TET, tetracycline; CIP, ciprofloxacin; NAL, nalidixic acid; CDS, calibrated dichotomous sensitivity; NCCLS, National Committee for Clinical Laboratory Standards; S, sensitive; R, resistant; LS, less sensitive; I, intermediate; TRNG, tetracycline-resistant N. gonorrhoeae.
Anethum sowa; the leaves of Azadirachta indica, Cedrela toona and Eugenia camaldulensis; the entire plant of Euphorbia hirta and Phyllanthus fraternus; the leaves, stem and stem bark of Plumeria rubra; the stem and roots of Ricinus communis; the leaves and stem bark of Salvadora persica; the tubers of Solanum tuberosum; and the bulbs of Urginea indica. Seeds of A. salviifolium and fruits of A. sowa were purchased from Abirami Botanical Corporation (Tuticorin, Tamil Nadu, India), whereas tubers of S. tuberosum were purchased from a local market. All other plant parts were collected from inside or places nearby the campus of the University of Delhi, Delhi, India, except bulbs of U. indica that were collected from Pushkar, Rajasthan, India. All plant materials were identified by taxonomist Prof. S.R. Yadav prior to their use.
1000 g of each extract were used instead of antibiotic disks. Autoclaved distilled water-impregnated disks were used as a control. To check the antimicrobial activity of plant extracts, samples were made in autoclaved distilled water and filtered through a 0.2-m filter. The activity of the plant extracts against N. gonorrhoeae was measured, indicated by clear zones of inhibition. Percentage inhibition of N. gonorrhoeae by the extracts was calculated by considering inhibition by each of penicillin (0.5 IU) and ciprofloxacin (1 g) to be 100% and then measuring the corresponding percentage inhibition by the extracts. Each experiment was performed in triplicate and the average value of inhibition and standard deviation were calculated.
2.2. Extraction of plant material
3.1. Antimicrobial susceptibility
As described previously [7], 100 g of each plant part was washed with distilled water, shade dried and extracted with 50% ethanol (E. Merck, Darmstadt, Germany).
WHO strains and clinical isolates showed varying susceptibilities to the different antibiotics. WHO strains K, L and O and Isolates 2, 5, 7, 9 and 10 were multidrug-resistant (MDR) as they were resistant to three or more antibiotics (Table 1).
2.3. Neisseria gonorrhoeae clinical isolates and World Health Organization (WHO) strains Seven WHO control strains (A, B, C, G, K, L and O) were used in the present study. In addition, 11 clinical isolates (Isolates 1–11) were used, which were isolated from patients with acute gonococcal urethritis and were identified using standard methods [7,9]. 2.4. Antimicrobial susceptibility testing Susceptibility testing of clinical isolates and WHO strains to penicillin (0.5 IU), ceftriaxone (0.5 g), spectinomycin (100 g), tetracycline (10 g), ciprofloxacin (1 g) and nalidixic acid (30 g) (Oxoid, Basingstoke, UK) was performed as described previously [7]. 2.5. Testing the activity of plant extracts Susceptibility of N. gonorrhoeae to the extracts was determined by the same method as used for testing antibiotics [7] except that Whatman filter paper disks (6 mm diameter) impregnated with
3. Results
3.2. Inhibition of N. gonorrhoeae by different extracts Autoclaved distilled water used as a control showed no inhibition of N. gonorrhoeae, whilst the 50% ethanolic extracts of different plant parts inhibited N. gonorrhoeae differentially. 3.2.1. Inhibition compared with penicillin All the WHO strains showed variable sensitivity to the extracts of different plant parts. WHO A was sensitive to penicillin but was less sensitive to the plant extracts. WHO strains B, C and G were less sensitive to penicillin. Of these three strains, WHO B was sensitive to extracts of A. salviifolium, A. indica and M. champaca and stem bark of S. persica, but was less sensitive to the remaining 16 extracts; WHO C was sensitive to 14 extracts and was less sensitive to A. vasica, A. lebbeck, D. peregrina, P. rubra (leaves and stem) and S. tuberosum; and WHO G was sensitive to 13 extracts whilst it was less sensitive to A. vasica, A. indica, C. toona, D. peregrina, S. tuberosum, U. indica and stem bark of P. rubra. The remaining three WHO stains (WHO K, L and O) were resistant to penicillin but were highly sensitive to all the plant extracts (Table 2).
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Table 2 Inhibition of various clinical isolates and WHO strains of Neisseria gonorrhoeae in comparison with penicillin Plant part
Resistanta
Less sensitiveb
Sensitivec
Highly sensitived
Adhatoda vasica roots Clinical isolates WHO strains
Nil Nil
1, 3, 4, 5, 6, 8, 10, 11 A, B, C, G
9 Nil
2, 7 K, L, O
Albizzia lebbeck stem bark Clinical isolates WHO strains
Nil Nil
1, 3, 5, 6, 8, 10, 11 A, B, C
4, 9 G
2, 7 K, L, O
Alangium salviifolium seeds Clinical isolates WHO strains
Nil Nil
4, 10, 11 A
1, 3, 5, 6, 8 B, C, G
2, 9, 7 K, L, O
Anethum sowa fruits Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B
1, 3, 4, 5, 6 C, G
2, 7, 9 K, L, O
Azadirachta indica leaves Clinical isolates WHO strains
Nil Nil
5, 6, 8, 10 A, G
1, 11 B, C
2, 3, 4, 7, 9 K, L, O
Cedrela toona leaves Clinical isolates WHO strains
Nil Nil
1, 5, 8, 10, 11 A, B, G
3, 4, 6 C
2, 7, 9 K, L, O
Drynaria peregrina stem bark Clinical isolates WHO strains
Nil Nil
1, 3, 4, 5, 6, 8, 10, 11 A, B, C, G
9 Nil
2, 7 K, L, O
Eugenia camaldulensis leaves Clinical isolates WHO strains
Nil Nil
8, 10 A, B
1, 3, 4, 5, 6, 11 C, G
2, 7, 9 K, L, O
Euphorbia hirta entire plant Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B
1, 3, 4, 5, 6 C, G
2, 7, 9 K, L, O
Michelia champaca stem bark Clinical isolates WHO strains
Nil Nil
8 A
1, 5, 6, 10, 11 B, C, G
2, 3, 4, 7, 9 K, L, O
Phyllanthus fraternus entire plant Clinical isolates WHO strains
Nil Nil
8, 10 A, B
1, 3, 5, 6, 11 C, G
2, 4, 7, 9 K, L, O
Plumeria rubra leaves Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B, C
1, 3, 5, 6 G
2, 4, 7, 9 K, L, O
P. rubra stem Clinical isolates WHO strains
Nil Nil
3, 6, 8, 10, 11 A, B, C
1, 4, 5, 9 G
2, 7 K, L, O
P. rubra stem bark Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B, G
1, 3, 4, 5, 6, 9 C
2, 7 K, L, O
Ricinus communis stem Clinical isolates WHO strains
Nil Nil
3, 6, 8, 10, 11 A, B
1, 4, 5 C, G
2, 4, 7, 9 K, L, O
R. communis roots Clinical isolates WHO strains
Nil Nil
1, 5, 8, 10 A, B
3, 4, 6, 9, 11 C, G
2, 7 K, L, O
Salvadora persica leaves Clinical isolates WHO strains
Nil Nil
8, 10 A, B
1, 3, 5, 6, 11 C, G
2, 4, 7, 9 K, L, O
S. persica stem bark Clinical isolates WHO strains
Nil Nil
8, 10 A
1, 3, 5, 6, 11 B, C, G
2, 4, 7, 9 K, L, O
Solanum tuberosum tubers Clinical isolates WHO strains
Nil Nil
1, 5, 6, 8, 10, 11 A, B, C, G
3, 4, 9 Nil
2, 7 K, L, O
Urginea indica bulbs Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B, G
1, 3, 5, 6 C
2, 4, 7, 9 K, L, O
a b c d
No inhibition. 1–50% inhibition. 51–99% inhibition. ≥100% inhibition.
P. Shokeen et al. / International Journal of Antimicrobial Agents 33 (2009) 86–91
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Table 3 Inhibition of various clinical isolates and WHO strains of Neisseria gonorrhoeae in comparison with ciprofloxacin Plant part
Resistanta
Less sensitiveb
Sensitivec
Highly sensitived
Adhatoda vasica roots Clinical isolates WHO strains
Nil Nil
1, 3, 4, 6, 7, 9, 11 A, B, C, G, O
5, 8 Nil
2, 10 K, L
Albizzia lebbeck stem bark Clinical isolates WHO strains
Nil Nil
1, 3, 6, 7, 11 A, B, C, G, O
4, 5, 8, 9 Nil
2, 10 K, L
Alangium salviifolium seeds Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 4, 6, 7, 8, 9, 11 Nil
2, 5, 10 K, L
Anethum sowa fruits Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 4, 6, 7, 8, 9, 11 Nil
2, 5, 10 K, L
Azadirachta indica leaves Clinical isolates WHO strains
Nil Nil
6 A, C, G, O
1, 5, 7, 8 B
2, 3, 4, 9, 10, 11 K, L
Cedrela toona leaves Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 4, 5, 6, 7, 8, 9, 11 Nil
2, 10 K, L
Drynaria peregrina stem bark Clinical isolates WHO strains
Nil Nil
1, 3, 6, 7, 9, 11 A, B, C, G, O
4, 5, 8 Nil
2, 10 K, L
Eugenia camaldulensis leaves Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8 Nil
2, 4, 5, 9, 10, 11 K, L
Euphorbia hirta entire plant Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 11 Nil
2, 4, 5, 9, 10 K, L
Michelia champaca stem bark Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 6, 7, 8, 9 Nil
2, 3, 4, 5, 10, 11 K, L
Phyllanthus fraternus entire plant Clinical isolates WHO strains
Nil Nil
7 A, B, C, G, O
1, 3, 6, 8 Nil
2, 4, 5, 9, 10, 11 K, L
Plumeria rubra leaves Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 9, 11 Nil
2, 4, 5, 10 K, L
P. rubra stem Clinical isolates WHO strains
Nil Nil
3, 6, 7 A, B, C, G, O
1, 4, 8, 9, 11 Nil
2, 5, 10 K, L
P. rubra stem bark Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 9, 11 Nil
2, 4, 5, 10 K, L
Ricinus communis stem Clinical isolates WHO strains
Nil Nil
3, 6, 7 A, B, C, G, O
1, 8, 9, 11 Nil
2, 4, 5, 10 K, L
R. communis roots Clinical isolates WHO strains
Nil Nil
1 A, B, C, G, O
3, 4, 5, 6, 7, 8, 9 Nil
2, 10, 11 K, L
Salvadora persica leaves Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 9 Nil
2, 4, 5, 10, 11 K, L
S. persica stem bark Clinical isolates WHO strains
Nil Nil
Nil A, C, G, O
1, 3, 6, 7, 8 B
2, 4, 5, 9, 10, 11 K, L
Solanum tuberosum tubers Clinical isolates WHO strains
Nil Nil
1, 6, 7, 9 A, B, C, G, O
3, 5, 8, 11
2, 4, 10 K, L
Urginea indica bulbs Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 9, 11 Nil
2, 4, 5, 10 K, L
a b c d
No inhibition. 1–50% inhibition. 51–99% inhibition. ≥100% inhibition.
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P. Shokeen et al. / International Journal of Antimicrobial Agents 33 (2009) 86–91
Isolates 2, 7 and 9 were resistant to penicillin but were highly sensitive to all plant extracts, except Isolate 9, which was sensitive to A. vasica, A. lebbeck, D. peregrina, P. rubra stem and stem bark, R. communis roots and S. tuberosum. Isolate 10 was sensitive to penicillin as well as to stem bark of M. champaca, although it was less sensitive to all the other extracts. Isolates 1, 3–6, 8 and 11 were less sensitive to penicillin as well as to some extracts, whilst they were sensitive to the other extracts (Table 2). 3.2.2. Inhibition compared with ciprofloxacin WHO strains A, B, C and O were sensitive to ciprofloxacin and were less sensitive to all the plant extracts, except WHO B, which was sensitive to the leaves of A. indica and stem bark of S. persica. WHO G was less sensitive to ciprofloxacin as well as to all plant extracts. In contrast, WHO K and L were resistant to ciprofloxacin but were highly sensitive to all plant extracts (Table 3). Isolates 3 and 6 were less sensitive to ciprofloxacin, but Isolate 3 was highly sensitive to the extracts of leaves of A. indica and stem bark of M. champaca, sensitive to 13 extracts and less sensitive to the extracts of A. vasica, A. lebbeck, D. peregrina, P. rubra stem and R. communis stem. Similarly, Isolate 6 was sensitive to 13 extracts and less sensitive to the remaining 7. All of the other isolates were resistant to ciprofloxacin and showed different susceptibilities to different extracts. Isolates 2 and 10 were highly sensitive to all the extracts. Isolate 4 was highly sensitive to 12 extracts, sensitive to 7 extracts and less sensitive only to the roots of A. vasica. Isolate 1 was sensitive to 15 extracts, but was less sensitive to A. vasica, A. lebbeck, D. peregrina, R. communis roots and S. tuberosum. Isolate 5 was highly sensitive to 13 extracts and sensitive to A. vasica, A. lebbeck, A. indica, C. toona, D. peregrina, R. communis roots and S. tuberosum. Isolate 7 was sensitive to 13 extracts and less sensitive to A. vasica, A. lebbeck, D. peregrina, P. fraternus, stem of P. rubra and R. communis, and S. tuberosum. Isolate 8 was sensitive to all plant extracts. Isolate 9 was highly sensitive to A. indica, E. camaldulensis, E. hirta, P. fraternus and S. persica stem bark, sensitive to 12 extracts and less sensitive to A. vasica, D. peregrina and S. tuberosum. Isolate 11 was highly sensitive to 7 extracts and sensitive to 10 extracts, although it was less sensitive to A. vasica, A. lebbeck and D. peregrina (Table 3). Thus, the extracts of A. vasica, A. lebbeck, D. peregrina and S. tuberosum showed little activity, as most of the strains were less sensitive to these extracts; extracts of C. toona, stem of P. rubra and roots and stem of R. communis showed moderate activity as strains were less sensitive, sensitive or highly sensitive to these extracts. The remaining 12 extracts exhibited high activity against N. gonorrhoeae, as most of the strains were either highly sensitive or sensitive to these extracts (Tables 2 and 3). 4. Discussion The worldwide increase in resistance of N. gonorrhoeae to different antibiotics necessitates the search for alternative remedies for the treatment of gonorrhoea. Ethnopharmacology and natural product drug discovery remains a significant hope in the current target-rich, lead-poor scenario [10]. All the plant parts used in the present study have been used traditionally in various forms, especially for the treatment of various diseases including those caused by microorganisms, and have a very high margin of safety. For example, all parts of A. indica, especially the leaves, have been used traditionally for the treatment of inflammation, infections, fever, skin diseases and dental disorders. The aqueous extract of the leaf was not toxic to mice up to oral doses of 1000 mg/kg, and methanolic leaf extracts showed an
oral LD50 of about 13 g/kg in acute toxicity studies in mice [11]. In the present study, extract of A. indica leaves exhibited high activity against N. gonorrhoeae. The roots of R. communis had moderate activity in the present study and were found to have no toxicity even at a dose of 10 g/kg body weight when given via the oral route in rats (Shokeen et al., Manuscript Number- FCT-D-07-00743, accepted in Food and Chemical Toxicology). Euphorbia hirta is used in cough, asthma, colic, dysentery and genitourinary diseases and its aqueous extract did not induce any toxic effect when administered intraperitoneally and orally even with high doses (≥100 mg of dried plant/kg) in mice [12]. This plant showed good activity in the present study. The approximate LD50 of D. peregrina extract in mice was found to be 2570 ± 81 mg/kg by the oral route [13]. In a similar earlier study by Caceres et al. [14], tincture of plants used popularly in Guatemala for the treatment of gonorrhoea was tested for in vitro activity against five clinical strains of N. gonorrhoeae, of the 46 plants investigated, 13 (28.3%) showed evident inhibition zones, 7 (15.2%) showed little activity and 26 (56.5%) showed no activity. In the present study, of the 20 plant extracts tested, 4 extracts (20%) had comparatively less activity, 4 extracts (20%) exhibited intermediate activity, whereas 12 extracts (60%) were found to be highly active against N. gonorrhoeae. This is the first report of its kind on the screening of these plants for activity against N. gonorrhoeae. It is worthwhile noting that in the present study WHO strains K, L and O were MDR strains. Moreover, WHO K and L were highly sensitive to all plant extracts compared with penicillin and ciprofloxacin, whereas WHO O was highly sensitive to all the extracts only compared with penicillin. A similar pattern of greater inhibition of MDR clinical isolates was also observed. Such an observation has been made previously by us with different extracts of O. sanctum, D. quercifolia and A. squamosa [7] as well as purified fractions and the active component of O. sanctum [8]. A similar observation has been made by Selvakumar et al. [15] in studies with Mycobacterium tuberculosis, where trifluoroperazine was found to be more bactericidal to drug-resistant isolates than to sensitive isolates. The decreased growth rate of drug-resistant isolates compared with drug-sensitive isolates was found to be one of the reasons of the observed phenomenon. Also in the present study a change in the cell wall permeability of the organism may account for the observed interesting results. The sensitivity of N. gonorrhoeae (including MDR strains) to the plant extracts motivates us to isolate the active components from the highly active plants and to study their mechanism of action. Acknowledgments PS is indebted to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for a Senior Research Fellowship. The authors thank Dr J. Tapsall, Neisseria Reference Laboratory, Prince of Wales Hospital, Sydney, Australia, for supplying low-concentration antibiotic disks and World Health Organization strains, and to Prof. S.R. Yadav, Shivaji University, Kolhapur, Maharashtra, India, for providing bulbs of U. indica. They also acknowledge Mrs Leelamma Peter, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India, for excellent technical assistance. Funding: PS received a Senior Research Fellowship from the CSIR, New Delhi, India. Competing interests: None declared. Ethical approval: Not required. References [1] Ison CA, Dillon JAR, Tapsall JW. The epidemiology of global antibiotic resistance among Neisseria gonorrhoeae and Haemophilus ducreyi. Lancet 1998;351:8–11.
P. Shokeen et al. / International Journal of Antimicrobial Agents 33 (2009) 86–91 [2] Cohen MS. Sexually transmitted diseases enhance HIV transmission: no longer a hypothesis. The Lancet 1998;351(Suppl. 3):S5–7. [3] Bala M, Ray K, Kumari S. Alarming increase in ciprofloxacin and penicillinresistant Neisseria gonorrhoeae isolates in New Delhi, India. Sex Transm Dis 2003;30:523–5. [4] Tapsall JW. Antibiotic resistance in Neisseria gonorrhoeae. Clin Infect Dis 2005;41(Suppl. 4):S263–8. [5] Bala M, Ray K, Gupta SM, Muralidhar S, Jain RK. Changing trends of antimicrobial susceptibility patterns of Neisseria gonorrhoeae in India and the emergence of ceftriaxone less susceptible N. gonorrhoeae strains. J Antimicrob Chemother 2007;60:582–6. [6] Ahmad I, Beg AZ. Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens. J Ethnopharmacol 2001;74:113–23. [7] Shokeen P, Ray K, Bala M, Tandon V. Preliminary studies on activity of Ocimum sanctum, Drynaria quercifolia and Annona squamosa against Neisseria gonorrhoeae. Sex Transm Dis 2005;32:106–11. [8] Shokeen P, Bala M, Singh M, Tandon V. In vitro activity of eugenol, an active component from Ocimum sanctum, against multiresistant and susceptible strains of Neisseria gonorrhoeae. Int J Antimicrob Agents 2008;32:174–9.
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[9] World Health Organization. Laboratory diagnosis of gonorrhoea. WHO Regional Publications, South-East Asia Series, No. 33. Geneva, Switzerland: WHO; 1999. [10] Patwardhan B. Ethnopharmacology and drug discovery. J Ethnopharmacol 2005;100:50–2. [11] Subapriya R, Nagini S. Medicinal properties of neem leaves: a review. CurrMed Chem Anticancer Agents 2005;5:149–56. [12] Lanhers MC, Fleurentin J, Cabalion P, Rolland A, Dorfman P, Misslin R, et al. Behavioral effects of Euphorbia hirta L.: sedative and anxiolytic properties. J Ethnopharmacol 1990;29:189–98. [13] Singh N, Nath R, Gupta ML. A pharmacological evaluation of antistress activity of Diospyros peregrina Gurke. Indian J Pharmacol 1988;20: 102–8. [14] Caceres A, Mendez H, Mendez E, Cohobon E, Sanayoa BE, Jauregui E, et al. Antigonorrhoeal activity of plants used in Guatemala for the treatment of sexually transmitted diseases. J Ethnopharmacol 1995;48: 85–8. [15] Selvakumar N, Vanaja Kumar PV, Krishnamurthy Prabhakar R, Murthy PS. In vitro susceptibility of Mycobacterium tuberculosis to trifluoroperazine. Curr Sci 1997;73:10.
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
Evaluation of the activity of 16 medicinal plants against Neisseria gonorrhoeae P. Shokeen a , M. Bala b , V. Tandon a,∗ a b
Dr B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India Regional STD Teaching, Training and Research Centre, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India
a r t i c l e
i n f o
Article history: Received 17 May 2008 Accepted 17 July 2008 Keywords: Neisseria gonorrhoeae Antimicrobial susceptibility Medicinal plants
a b s t r a c t 50% Ethanolic extracts of various parts of 16 medicinal plants were evaluated for potential activity against clinical isolates and WHO strains of Neisseria gonorrhoeae, including multidrug-resistant (MDR) strains. Activity was calculated as percentage inhibition in comparison with penicillin and ciprofloxacin and strains were categorised as less sensitive, sensitive or highly sensitive to the extracts. The extracts caused differential inhibition of N. gonorrhoeae, with greater inhibition of the MDR strains. Among the extracts tested, 60% exhibited high activity whereas 20% showed moderate activity and 20% had little activity against N. gonorrhoeae. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Gonorrhoea affects more than 60 million people worldwide every year [1] and is a global health problem. Gonococci not only cause a high incidence of acute infections and complications but also play a major role in facilitating human immunodeficiency virus (HIV) acquisition and transmission [2]. Considering the expensive cost of treatment of gonorrhoea and the increase in international travel, gonococcal infections are a significant threat in developing countries. Although the frequency of asymptomatic infections, lack of innate or acquired immunity, and changes in human sexual behaviour have all contributed to the continued spread of gonococcal infections, the major contributing factor has been the development of antimicrobial resistance. Over the last several years, Neisseria gonorrhoeae strains have developed a high level of resistance to several antibiotics, including penicillin, tetracycline and quinolones [3,4]. Recently, isolates with reduced susceptibility to ceftriaxone as well as a substantial proportion (23.3%) of multiresistant strains have been reported from New Delhi, India [5]. Moreover, some of the newly investigated fluoroquinolones have reported problems of adverse drug reactions, some of which can be serious or fatal. Consequently, there is an urgent need for safe, alternative antigonococcal compounds that can be administered orally and have effective potency, allowing high therapeutic efficacy (>95.0% cure rate) preferably with a single-dose regimen. Plants produce a variety of secondary metabolites that have long been of interest to man. In recent years these are being used,
∗ Corresponding author. Tel.: +91 11 2766 6272/7151; fax: +91 11 2766 6248. E-mail addresses: [email protected], [email protected] (V. Tandon).
either directly as precursors or as lead compounds, in the pharmaceutical industry and it is expected that plant extracts showing target sites other than those used by antibiotics will be active against drug-resistant microbial pathogens. However, very little information is available on such activity of medicinal plants and, of the 400 000 plant species on Earth, only a small percentage has been systematically investigated for their antimicrobial activities. India is fortunate in possessing the world’s richest flora, with approximately 120 families of plants comprising 130 000 species. Additionally, there is a rich local ethnobotanical bibliography describing the species most frequently used by the population to cure various diseases. Although screening of Indian medicinal plants has revealed varying degrees of antimicrobial activity against pathogenic and opportunistic microorganisms [6], there is still a lack of experimental scientific studies confirming the possible antigonorrhoeal properties of a great number of these remedies. In a previous study, various extracts of Ocimum sanctum, Drynaria quercifolia and Annona squamosa showed considerable activity against N. gonorrhoeae [7], and bioassay-guided fractionation of the crude extract of O. sanctum led to the identification of eugenol as an active component [8]. Thus, it was considered worthwhile to screen a few of those medicinal plants that have been used in traditional medicine in one form or the other for activity against N. gonorrhoeae. 2. Materials and methods 2.1. Plant material The plant parts used were as follows: the roots of Adhatoda vasica; the stem bark of Albizzia lebbeck, Drynaria peregrina and Michelia champaca; the seeds of Alangium salviifolium; the fruits of
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P. Shokeen et al. / International Journal of Antimicrobial Agents 33 (2009) 86–91
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Table 1 Susceptibility of clinical isolates and WHO strains of Neisseria gonorrhoeae to various antibiotics WHO strain/clinical isolate
-Lactamase test
PEN
CFX
SPM
WHO A WHO B WHO C WHO G WHO K WHO L WHO O 1 2 3 4 5 6 7 8 9 10 11
−ve −ve −ve −ve −ve −ve +ve −ve +ve −ve −ve −ve −ve +ve −ve +ve −ve −ve
S LS LS LS R R R LS R LS LS LS LS R LS R S LS
S S S S LS LS S S S S S S S S S S S S
R S S S S S R S S S S S S S S S S S
TET CDS test
NCCLS method
Not TRNG Not TRNG Not TRNG TRNG Not TRNG Not TRNG Not TRNG Not TRNG TRNG Not TRNG Not TRNG TRNG Not TRNG Not TRNG Not TRNG Not TRNG TRNG Not TRNG
S S R TRNG R I R S TRNG S S TRNG S S LS S TRNG S
CIP
NAL
S S S LS R R S R R LS R R LS R R R R R
S S S R R R S R R R R R R R R R R R
PEN, penicillin; CFX, ceftriaxone; SPM, spectinomycin; TET, tetracycline; CIP, ciprofloxacin; NAL, nalidixic acid; CDS, calibrated dichotomous sensitivity; NCCLS, National Committee for Clinical Laboratory Standards; S, sensitive; R, resistant; LS, less sensitive; I, intermediate; TRNG, tetracycline-resistant N. gonorrhoeae.
Anethum sowa; the leaves of Azadirachta indica, Cedrela toona and Eugenia camaldulensis; the entire plant of Euphorbia hirta and Phyllanthus fraternus; the leaves, stem and stem bark of Plumeria rubra; the stem and roots of Ricinus communis; the leaves and stem bark of Salvadora persica; the tubers of Solanum tuberosum; and the bulbs of Urginea indica. Seeds of A. salviifolium and fruits of A. sowa were purchased from Abirami Botanical Corporation (Tuticorin, Tamil Nadu, India), whereas tubers of S. tuberosum were purchased from a local market. All other plant parts were collected from inside or places nearby the campus of the University of Delhi, Delhi, India, except bulbs of U. indica that were collected from Pushkar, Rajasthan, India. All plant materials were identified by taxonomist Prof. S.R. Yadav prior to their use.
1000 g of each extract were used instead of antibiotic disks. Autoclaved distilled water-impregnated disks were used as a control. To check the antimicrobial activity of plant extracts, samples were made in autoclaved distilled water and filtered through a 0.2-m filter. The activity of the plant extracts against N. gonorrhoeae was measured, indicated by clear zones of inhibition. Percentage inhibition of N. gonorrhoeae by the extracts was calculated by considering inhibition by each of penicillin (0.5 IU) and ciprofloxacin (1 g) to be 100% and then measuring the corresponding percentage inhibition by the extracts. Each experiment was performed in triplicate and the average value of inhibition and standard deviation were calculated.
2.2. Extraction of plant material
3.1. Antimicrobial susceptibility
As described previously [7], 100 g of each plant part was washed with distilled water, shade dried and extracted with 50% ethanol (E. Merck, Darmstadt, Germany).
WHO strains and clinical isolates showed varying susceptibilities to the different antibiotics. WHO strains K, L and O and Isolates 2, 5, 7, 9 and 10 were multidrug-resistant (MDR) as they were resistant to three or more antibiotics (Table 1).
2.3. Neisseria gonorrhoeae clinical isolates and World Health Organization (WHO) strains Seven WHO control strains (A, B, C, G, K, L and O) were used in the present study. In addition, 11 clinical isolates (Isolates 1–11) were used, which were isolated from patients with acute gonococcal urethritis and were identified using standard methods [7,9]. 2.4. Antimicrobial susceptibility testing Susceptibility testing of clinical isolates and WHO strains to penicillin (0.5 IU), ceftriaxone (0.5 g), spectinomycin (100 g), tetracycline (10 g), ciprofloxacin (1 g) and nalidixic acid (30 g) (Oxoid, Basingstoke, UK) was performed as described previously [7]. 2.5. Testing the activity of plant extracts Susceptibility of N. gonorrhoeae to the extracts was determined by the same method as used for testing antibiotics [7] except that Whatman filter paper disks (6 mm diameter) impregnated with
3. Results
3.2. Inhibition of N. gonorrhoeae by different extracts Autoclaved distilled water used as a control showed no inhibition of N. gonorrhoeae, whilst the 50% ethanolic extracts of different plant parts inhibited N. gonorrhoeae differentially. 3.2.1. Inhibition compared with penicillin All the WHO strains showed variable sensitivity to the extracts of different plant parts. WHO A was sensitive to penicillin but was less sensitive to the plant extracts. WHO strains B, C and G were less sensitive to penicillin. Of these three strains, WHO B was sensitive to extracts of A. salviifolium, A. indica and M. champaca and stem bark of S. persica, but was less sensitive to the remaining 16 extracts; WHO C was sensitive to 14 extracts and was less sensitive to A. vasica, A. lebbeck, D. peregrina, P. rubra (leaves and stem) and S. tuberosum; and WHO G was sensitive to 13 extracts whilst it was less sensitive to A. vasica, A. indica, C. toona, D. peregrina, S. tuberosum, U. indica and stem bark of P. rubra. The remaining three WHO stains (WHO K, L and O) were resistant to penicillin but were highly sensitive to all the plant extracts (Table 2).
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Table 2 Inhibition of various clinical isolates and WHO strains of Neisseria gonorrhoeae in comparison with penicillin Plant part
Resistanta
Less sensitiveb
Sensitivec
Highly sensitived
Adhatoda vasica roots Clinical isolates WHO strains
Nil Nil
1, 3, 4, 5, 6, 8, 10, 11 A, B, C, G
9 Nil
2, 7 K, L, O
Albizzia lebbeck stem bark Clinical isolates WHO strains
Nil Nil
1, 3, 5, 6, 8, 10, 11 A, B, C
4, 9 G
2, 7 K, L, O
Alangium salviifolium seeds Clinical isolates WHO strains
Nil Nil
4, 10, 11 A
1, 3, 5, 6, 8 B, C, G
2, 9, 7 K, L, O
Anethum sowa fruits Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B
1, 3, 4, 5, 6 C, G
2, 7, 9 K, L, O
Azadirachta indica leaves Clinical isolates WHO strains
Nil Nil
5, 6, 8, 10 A, G
1, 11 B, C
2, 3, 4, 7, 9 K, L, O
Cedrela toona leaves Clinical isolates WHO strains
Nil Nil
1, 5, 8, 10, 11 A, B, G
3, 4, 6 C
2, 7, 9 K, L, O
Drynaria peregrina stem bark Clinical isolates WHO strains
Nil Nil
1, 3, 4, 5, 6, 8, 10, 11 A, B, C, G
9 Nil
2, 7 K, L, O
Eugenia camaldulensis leaves Clinical isolates WHO strains
Nil Nil
8, 10 A, B
1, 3, 4, 5, 6, 11 C, G
2, 7, 9 K, L, O
Euphorbia hirta entire plant Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B
1, 3, 4, 5, 6 C, G
2, 7, 9 K, L, O
Michelia champaca stem bark Clinical isolates WHO strains
Nil Nil
8 A
1, 5, 6, 10, 11 B, C, G
2, 3, 4, 7, 9 K, L, O
Phyllanthus fraternus entire plant Clinical isolates WHO strains
Nil Nil
8, 10 A, B
1, 3, 5, 6, 11 C, G
2, 4, 7, 9 K, L, O
Plumeria rubra leaves Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B, C
1, 3, 5, 6 G
2, 4, 7, 9 K, L, O
P. rubra stem Clinical isolates WHO strains
Nil Nil
3, 6, 8, 10, 11 A, B, C
1, 4, 5, 9 G
2, 7 K, L, O
P. rubra stem bark Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B, G
1, 3, 4, 5, 6, 9 C
2, 7 K, L, O
Ricinus communis stem Clinical isolates WHO strains
Nil Nil
3, 6, 8, 10, 11 A, B
1, 4, 5 C, G
2, 4, 7, 9 K, L, O
R. communis roots Clinical isolates WHO strains
Nil Nil
1, 5, 8, 10 A, B
3, 4, 6, 9, 11 C, G
2, 7 K, L, O
Salvadora persica leaves Clinical isolates WHO strains
Nil Nil
8, 10 A, B
1, 3, 5, 6, 11 C, G
2, 4, 7, 9 K, L, O
S. persica stem bark Clinical isolates WHO strains
Nil Nil
8, 10 A
1, 3, 5, 6, 11 B, C, G
2, 4, 7, 9 K, L, O
Solanum tuberosum tubers Clinical isolates WHO strains
Nil Nil
1, 5, 6, 8, 10, 11 A, B, C, G
3, 4, 9 Nil
2, 7 K, L, O
Urginea indica bulbs Clinical isolates WHO strains
Nil Nil
8, 10, 11 A, B, G
1, 3, 5, 6 C
2, 4, 7, 9 K, L, O
a b c d
No inhibition. 1–50% inhibition. 51–99% inhibition. ≥100% inhibition.
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Table 3 Inhibition of various clinical isolates and WHO strains of Neisseria gonorrhoeae in comparison with ciprofloxacin Plant part
Resistanta
Less sensitiveb
Sensitivec
Highly sensitived
Adhatoda vasica roots Clinical isolates WHO strains
Nil Nil
1, 3, 4, 6, 7, 9, 11 A, B, C, G, O
5, 8 Nil
2, 10 K, L
Albizzia lebbeck stem bark Clinical isolates WHO strains
Nil Nil
1, 3, 6, 7, 11 A, B, C, G, O
4, 5, 8, 9 Nil
2, 10 K, L
Alangium salviifolium seeds Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 4, 6, 7, 8, 9, 11 Nil
2, 5, 10 K, L
Anethum sowa fruits Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 4, 6, 7, 8, 9, 11 Nil
2, 5, 10 K, L
Azadirachta indica leaves Clinical isolates WHO strains
Nil Nil
6 A, C, G, O
1, 5, 7, 8 B
2, 3, 4, 9, 10, 11 K, L
Cedrela toona leaves Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 4, 5, 6, 7, 8, 9, 11 Nil
2, 10 K, L
Drynaria peregrina stem bark Clinical isolates WHO strains
Nil Nil
1, 3, 6, 7, 9, 11 A, B, C, G, O
4, 5, 8 Nil
2, 10 K, L
Eugenia camaldulensis leaves Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8 Nil
2, 4, 5, 9, 10, 11 K, L
Euphorbia hirta entire plant Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 11 Nil
2, 4, 5, 9, 10 K, L
Michelia champaca stem bark Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 6, 7, 8, 9 Nil
2, 3, 4, 5, 10, 11 K, L
Phyllanthus fraternus entire plant Clinical isolates WHO strains
Nil Nil
7 A, B, C, G, O
1, 3, 6, 8 Nil
2, 4, 5, 9, 10, 11 K, L
Plumeria rubra leaves Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 9, 11 Nil
2, 4, 5, 10 K, L
P. rubra stem Clinical isolates WHO strains
Nil Nil
3, 6, 7 A, B, C, G, O
1, 4, 8, 9, 11 Nil
2, 5, 10 K, L
P. rubra stem bark Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 9, 11 Nil
2, 4, 5, 10 K, L
Ricinus communis stem Clinical isolates WHO strains
Nil Nil
3, 6, 7 A, B, C, G, O
1, 8, 9, 11 Nil
2, 4, 5, 10 K, L
R. communis roots Clinical isolates WHO strains
Nil Nil
1 A, B, C, G, O
3, 4, 5, 6, 7, 8, 9 Nil
2, 10, 11 K, L
Salvadora persica leaves Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 9 Nil
2, 4, 5, 10, 11 K, L
S. persica stem bark Clinical isolates WHO strains
Nil Nil
Nil A, C, G, O
1, 3, 6, 7, 8 B
2, 4, 5, 9, 10, 11 K, L
Solanum tuberosum tubers Clinical isolates WHO strains
Nil Nil
1, 6, 7, 9 A, B, C, G, O
3, 5, 8, 11
2, 4, 10 K, L
Urginea indica bulbs Clinical isolates WHO strains
Nil Nil
Nil A, B, C, G, O
1, 3, 6, 7, 8, 9, 11 Nil
2, 4, 5, 10 K, L
a b c d
No inhibition. 1–50% inhibition. 51–99% inhibition. ≥100% inhibition.
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Isolates 2, 7 and 9 were resistant to penicillin but were highly sensitive to all plant extracts, except Isolate 9, which was sensitive to A. vasica, A. lebbeck, D. peregrina, P. rubra stem and stem bark, R. communis roots and S. tuberosum. Isolate 10 was sensitive to penicillin as well as to stem bark of M. champaca, although it was less sensitive to all the other extracts. Isolates 1, 3–6, 8 and 11 were less sensitive to penicillin as well as to some extracts, whilst they were sensitive to the other extracts (Table 2). 3.2.2. Inhibition compared with ciprofloxacin WHO strains A, B, C and O were sensitive to ciprofloxacin and were less sensitive to all the plant extracts, except WHO B, which was sensitive to the leaves of A. indica and stem bark of S. persica. WHO G was less sensitive to ciprofloxacin as well as to all plant extracts. In contrast, WHO K and L were resistant to ciprofloxacin but were highly sensitive to all plant extracts (Table 3). Isolates 3 and 6 were less sensitive to ciprofloxacin, but Isolate 3 was highly sensitive to the extracts of leaves of A. indica and stem bark of M. champaca, sensitive to 13 extracts and less sensitive to the extracts of A. vasica, A. lebbeck, D. peregrina, P. rubra stem and R. communis stem. Similarly, Isolate 6 was sensitive to 13 extracts and less sensitive to the remaining 7. All of the other isolates were resistant to ciprofloxacin and showed different susceptibilities to different extracts. Isolates 2 and 10 were highly sensitive to all the extracts. Isolate 4 was highly sensitive to 12 extracts, sensitive to 7 extracts and less sensitive only to the roots of A. vasica. Isolate 1 was sensitive to 15 extracts, but was less sensitive to A. vasica, A. lebbeck, D. peregrina, R. communis roots and S. tuberosum. Isolate 5 was highly sensitive to 13 extracts and sensitive to A. vasica, A. lebbeck, A. indica, C. toona, D. peregrina, R. communis roots and S. tuberosum. Isolate 7 was sensitive to 13 extracts and less sensitive to A. vasica, A. lebbeck, D. peregrina, P. fraternus, stem of P. rubra and R. communis, and S. tuberosum. Isolate 8 was sensitive to all plant extracts. Isolate 9 was highly sensitive to A. indica, E. camaldulensis, E. hirta, P. fraternus and S. persica stem bark, sensitive to 12 extracts and less sensitive to A. vasica, D. peregrina and S. tuberosum. Isolate 11 was highly sensitive to 7 extracts and sensitive to 10 extracts, although it was less sensitive to A. vasica, A. lebbeck and D. peregrina (Table 3). Thus, the extracts of A. vasica, A. lebbeck, D. peregrina and S. tuberosum showed little activity, as most of the strains were less sensitive to these extracts; extracts of C. toona, stem of P. rubra and roots and stem of R. communis showed moderate activity as strains were less sensitive, sensitive or highly sensitive to these extracts. The remaining 12 extracts exhibited high activity against N. gonorrhoeae, as most of the strains were either highly sensitive or sensitive to these extracts (Tables 2 and 3). 4. Discussion The worldwide increase in resistance of N. gonorrhoeae to different antibiotics necessitates the search for alternative remedies for the treatment of gonorrhoea. Ethnopharmacology and natural product drug discovery remains a significant hope in the current target-rich, lead-poor scenario [10]. All the plant parts used in the present study have been used traditionally in various forms, especially for the treatment of various diseases including those caused by microorganisms, and have a very high margin of safety. For example, all parts of A. indica, especially the leaves, have been used traditionally for the treatment of inflammation, infections, fever, skin diseases and dental disorders. The aqueous extract of the leaf was not toxic to mice up to oral doses of 1000 mg/kg, and methanolic leaf extracts showed an
oral LD50 of about 13 g/kg in acute toxicity studies in mice [11]. In the present study, extract of A. indica leaves exhibited high activity against N. gonorrhoeae. The roots of R. communis had moderate activity in the present study and were found to have no toxicity even at a dose of 10 g/kg body weight when given via the oral route in rats (Shokeen et al., Manuscript Number- FCT-D-07-00743, accepted in Food and Chemical Toxicology). Euphorbia hirta is used in cough, asthma, colic, dysentery and genitourinary diseases and its aqueous extract did not induce any toxic effect when administered intraperitoneally and orally even with high doses (≥100 mg of dried plant/kg) in mice [12]. This plant showed good activity in the present study. The approximate LD50 of D. peregrina extract in mice was found to be 2570 ± 81 mg/kg by the oral route [13]. In a similar earlier study by Caceres et al. [14], tincture of plants used popularly in Guatemala for the treatment of gonorrhoea was tested for in vitro activity against five clinical strains of N. gonorrhoeae, of the 46 plants investigated, 13 (28.3%) showed evident inhibition zones, 7 (15.2%) showed little activity and 26 (56.5%) showed no activity. In the present study, of the 20 plant extracts tested, 4 extracts (20%) had comparatively less activity, 4 extracts (20%) exhibited intermediate activity, whereas 12 extracts (60%) were found to be highly active against N. gonorrhoeae. This is the first report of its kind on the screening of these plants for activity against N. gonorrhoeae. It is worthwhile noting that in the present study WHO strains K, L and O were MDR strains. Moreover, WHO K and L were highly sensitive to all plant extracts compared with penicillin and ciprofloxacin, whereas WHO O was highly sensitive to all the extracts only compared with penicillin. A similar pattern of greater inhibition of MDR clinical isolates was also observed. Such an observation has been made previously by us with different extracts of O. sanctum, D. quercifolia and A. squamosa [7] as well as purified fractions and the active component of O. sanctum [8]. A similar observation has been made by Selvakumar et al. [15] in studies with Mycobacterium tuberculosis, where trifluoroperazine was found to be more bactericidal to drug-resistant isolates than to sensitive isolates. The decreased growth rate of drug-resistant isolates compared with drug-sensitive isolates was found to be one of the reasons of the observed phenomenon. Also in the present study a change in the cell wall permeability of the organism may account for the observed interesting results. The sensitivity of N. gonorrhoeae (including MDR strains) to the plant extracts motivates us to isolate the active components from the highly active plants and to study their mechanism of action. Acknowledgments PS is indebted to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for a Senior Research Fellowship. The authors thank Dr J. Tapsall, Neisseria Reference Laboratory, Prince of Wales Hospital, Sydney, Australia, for supplying low-concentration antibiotic disks and World Health Organization strains, and to Prof. S.R. Yadav, Shivaji University, Kolhapur, Maharashtra, India, for providing bulbs of U. indica. They also acknowledge Mrs Leelamma Peter, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India, for excellent technical assistance. Funding: PS received a Senior Research Fellowship from the CSIR, New Delhi, India. Competing interests: None declared. Ethical approval: Not required. References [1] Ison CA, Dillon JAR, Tapsall JW. The epidemiology of global antibiotic resistance among Neisseria gonorrhoeae and Haemophilus ducreyi. Lancet 1998;351:8–11.
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