Sexually Transmitted Diseases:
Preliminary Studies on Activity of Ocimum sanctum, Drynaria quercifolia, and Annona squamosa Against Neisseria gonorrhoeae
Shokeen, Poonam MSc*; Ray, Krishna MD†; Bala, Manju MD†; Tandon, Vibha PhD*
From the * Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India; and the † Regional STD Teaching, Training, and Research Centre, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India.
One of the authors, Poonam Shokeen, is indebted to CSIR, New Delhi, for a Junior Research Fellowship. The authors thank Dr. J. Tapsall, Neisseria Reference Laboratory, Prince of Wales Hospital, Sydney, Australia, for supplying low-potency antibiotic discs and World Health Organization strains; Dr. S. Hegde, St. Aloysius College, Mangalore, India, for providing the leaves and rhizomes of Drynaria quercifolia; Dr. S. Chatterjee, Indian Herbs Research and Supply Company Limited, Saharanpur, India, for providing the leaves of Ocimum sanctum; and acknowledge Mrs. Leelamma Peter, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India, for excellent technical assistance. The authors also acknowledge Ajeet Narayan, Institute of Genomics and Integrative Biology, Delhi, India, for preparing the extracts of Drynaria quercifolia, Rajesh Kumar Gupta for preparing the extracts of Annona squamosa, and Dr. Uma Choudhary for invaluable suggestions and kind support.
Correspondence: Vibha Tandon, PhD, Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India. E-mail: firstname.lastname@example.org.
Received for publication May 26, 2004, and accepted November 22, 2004.
Background: Despite the progressive increase of antimicrobial resistance of Neisseria gonorrhoeae worldwide, there are limited reports of alternative remedies from plants.
Goal: The aim of the current study was to screen 3 plants, Ocimum sanctum, Drynaria quercifolia, and Annona squamosa, for activity against Neisseria gonorrhoeae.
Study: By disc diffusion method, extracts of these 3 plants were screened for activity against Neisseria gonorrhoeae; their antimicrobial activity was calculated as percentage inhibition and compared with penicillin and ciprofloxacin.
Results: The extracts of all 3 plants caused inhibition of Neisseria gonorrhoeae clinical isolates and World Health Organization (WHO) strains, more so than the multidrug resistant Neisseria gonorrhoeae.
Conclusion: Neisseria gonorrhoeae clinical isolates and WHO strains were sensitive to extracts of Ocimum sanctum, Drynaria quercifolia, and Annona squamosa. This motivates us to isolate the active component/second from the 3 plants.
Gonorrhea is one of the common sexually transmitted diseases in the developing countries and a global health problem. It has far-reaching social and economic consequences. Control of gonococcal infection is a difficult and complex issue. Several authors have reported the emergence of strains resistant to different antibiotics.1–5 The development of antibiotic resistance by Neisseria gonorrhoeae may involve both chromosomal- and plasmid-mediated mechanisms, and for some antibiotics, both may be implicated.6 The problem of increasing resistance is further compounded both by wide dissemination of resistant clones and emergence of strains with novel resistance mechanisms. This increased resistance has resulted in reduced effectiveness of penicillin and tetracycline and increased costs of treatment when quinolones and third-generation cephalosporins are used. In view of all these facts, there is an urgent need for alternate antimicrobial substances, which are cheap, readily available for the population, and have minimum side effects.
Herbal remedies obtained from plants have been used from time immemorial for treatment of many diseases, including bacterial infections. Ocimum sanctum (O. sanctum) belongs to the family Labiatae and is locally known as “Tulsi.” It is worshipped widely and considered sacred by the Hindus. There are numerous reports about the antimicrobial activity of essential oil and leaf extract of this plant.7–16 The inhibitory activity of essential oil of O. sanctum towards different microbes such as Staphylococcus aureus, Shigella boydii, Salmonella typhi, Bacillus subtilis, and Epidermophyton cresens has been reported.7,10,15 The aqueous leafy extract of O. sanctum was found to completely inhibit the growth of all 3 H37Rv (sensitive), SmR10, and INHRRv6 (resistant) strains of Mycobacterium tuberculosis.11 Ethanolic extract (80%) of the leaves of O. sanctum inhibited the growth of Alternaria alternata.12 The antibacterial, antifungal, and insecticidal activity and inhibition of germination by the volatile fraction and residue of extract of O. sanctum has been investigated.13 Essential oil of O. sanctum was more effective than clove oil against Gram-positive bacteria than Gram-negative organisms and was also found to be effective against pathogenic, as well as nonpathogenic, fungi.14 The antibacterial activity of extracts of O. sanctum against pathogenic Gram-positive and Gram-negative bacteria has also been studied.16
Drynaria quercifolia (D. quercifolia) is locally known as “Attukalkizhangu.” Traditionally, it is used in the treatment of diarrhea, typhoid, cholera, chronic jaundice, fever, headache, skin diseases, and syphilis. The methanol extract of rhizomes of D. quercifolia has shown concentration-dependent inhibitory activity against many microorganisms.17
Annona squamosa, commonly known as “custard apple”, belongs to the family Annonaceae. It is a native of the West Indies and is cultivated throughout India, mainly for its edible fruit. The ethanolic extract of seeds of A. squamosa showed no appreciable antibacterial effect in vitro against 5 Gram-positive and 5 Gram-negative bacteria in one study.18 But significant inhibition of S. aureus, Streptococcus pyogenes, corynebacterium species, Escherichia coli, and Pseudomonas aeruginosa by different extracts of A. squamosa and O. sanctum was observed in another study.8 n-Alkanols, 16 hentriacontanone, and sterols obtained from the petroleum ether extracts of leaves of A. squamosa showed inhibitory activity against Staphylococcus albus, S. aureus, Streptococcus viridans, E. coli, Pseudomonas pyocyanea, and Klebsiella species.19
Although a number of attempts have been made to investigate the activity of plants against N. gonorrhoeae, there is no report on inhibition of N. gonorrhoeae by these 3 plants.20–25 In the present manuscript, we have presented an organized study of the inhibition of N. gonorrhoeae by O. sanctum, D. quercifolia and A. squamosa. This is the first report of its kind on the antigonorrhoeal activity of these 3 Indian plants.
Leaves of O. sanctum were kindly provided by Dr. S. Chatterjee (Indian Herbs Resesarch and Supply Company Limited, Saharanpur, India). Leaves and rhizomes of D. quercifolia were kindly provided by Dr. S. Hegde, St. Aloysius College, Mangalore, India, whereas leaves of A. squamosa were collected from the gardens of Indian Agricultural Research Institute, Delhi, India. Parts of all these 3 plants were identified by taxonomist Prof. C. R. Babu of the Botany Department, University of Delhi, Delhi. Seeds of A. squamosa were obtained from fruits purchased from the local market.
Preparation of Plant Extracts
Extraction of each plant was done thrice, and thus 3 batches of extracts were prepared. Each time, 1 kg of the required plant part of each of O. sanctum and D. quercifolia was cut into small pieces, put in a round-bottom flask, and was subjected to exhaustive serial extraction using a mechanical stirrer by using the solvents of increasing polarity index starting from nonpolar to polar. Leaves of O. sanctum were extracted with hexane, benzene, chloroform, ethyl acetate, acetone, 70% ethanol (all solvents from Spectrochem Private Limited, Mumbai, India), distilled water, and distilled hot water. Fertile leaves of D. quercifolia were extracted with hexane, methanol, and distilled water and infertile leaves with hexane, ethyl acetate, methanol, and distilled water, whereas rhizomes of D. quercifolia were extracted with hexane, benzene, methanol, and distilled water. Similarly, the whole extracts of leaves of A. squamosa were prepared using ethanol and methanol. Whole extract of intact seeds of A. squamosa were prepared using hexane and chloroform.
The extracts were filtered with Whatman filter paper (type 4), and the filtrate was concentrated under reduced pressure on a rotavapor (BÜCHI, R-3000, Switzerland) at 40°C temperature. The extracts were further dried under reduced pressure. Water and hot-water extracts were lyophilized after concentration on a rotavapor. All the extracts were obtained in powder form, except both extracts of seeds of A. squamosa, which were obtained in oil form. The extracts were stored at 4°C for future use.
To check the antimicrobial activity, required amount of water and hot-water extracts of O. sanctum, water extracts of all parts of D. quercifolia, and methanol and ethanol extracts of leaves of A. squamosa were dissolved in autoclaved distilled water. The rest of the extracts were dissolved in dimethyl formamide (DMF) (Spectrochem Private Limited, Mumbai, India) as these extracts were insoluble in water and the best solubility was found in DMF. The extracts were then filtered through a 0.2-μm filter and were used at a concentration of 1000 μg/disc, except the seed extracts of A. squamosa, which were in oil form, of which 10 μL was used as such per disc.
N. gonorrhoeae Clinical Isolates
N. gonorrhoeae was isolated from 24 male patients with acute gonococcal urethritis attending the Regional STD Teaching, Training and Research Centre, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, which were direct smear-positive cases. For isolation of N. gonorrhoeae, urethral swabs were inoculated onto chocolate agar (Columbia agar base [Hi Media, India] and sheep blood) and saponin-lysed blood agar with vancomycin, colistin, nystatin, and trimethoprim (VCNT) supplement (Hi Media, India). The inoculated culture plates were incubated at 36°C in a moist atmosphere containing 5% CO2 (candle extinction jar) for 24 to 48 hours. These consecutive clinical isolates were identified on the basis of colony morphology, gram staining, oxidase, superoxol, and rapid carbohydrate utilization tests.26
N. gonorrhoeae World Health Organization (WHO) Strains
Six WHO strains (WHO B, C, F, G, L, and O), kindly provided by Dr. J Tapsall, Neisseria Reference Laboratory, Prince of Wales Hospital, Sydney, Australia, were used.
Antimicrobial Susceptibility Testing
The susceptibility testing of all the clinical isolates and WHO strains to penicillin (0.5 IU), ceftriaxone (0.5 μg), spectinomycin (100 μg), tetracycline (10 μg), ciprofloxacin (1 μg), and nalidixic acid (30 μg) (Oxoid) was done by the Australian Gonococcal Surveillance Programme (AGSP) method based on the Calibrated Dichotomous Sensitivity (CDS) test on chocolate agar plates.26 Since the CDS method recognizes only plasmid-mediated resistance and does not recognize chromosomally mediated resistance to tetracycline, susceptibility testing of all the clinical isolates and WHO strains of N. gonorrhoeae to tetracycline was also done by the National Committee for Clinical and Laboratory Standards (NCCLS) method using GC agar base with vitamino growth supplement (HI Media, India) instead of isovitalex.27Quality control of this medium was assured by using WHO reference strains A to E. β-Lactamase production was detected by the chromogenic cephalosporin method with use of nitrocefin freeze-dried powder (Oxoid).26
Testing the Activity of Plant Extracts
Susceptibility testing of N. gonorrhoeae to the extracts was done by the same method as used for testing antibiotics,26 except that Whatman filter paper discs of 6-mm diameter, impregnated with the required concentration of each extract, were used instead of antibiotic discs. DMF or autoclaved, distilled-water–impregnated discs were used as control for extracts dissolved in DMF or water, respectively. The activity of the plant extracts against N. gonorrhoeae was measured, indicated by clear zones of inhibition. The zone of the control if present was subtracted from the zone of the respective extracts. Percentage inhibition of N. gonorrhoeae by the extracts was calculated by considering inhibition by each of penicillin (0.5 IU) and ciprofloxacin (1 μg) 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. Student 2-tailed test was used for calculation of statistically significant difference between the extracts.
The results of susceptibility of clinical isolates and WHO strains to different antibiotics are shown in Table 1. Among the 24 clinical isolates, 14 were resistant, 8 were less sensitive, and 2 were sensitive to penicillin. Three isolates were penicillinase-producing N. gonorrhoeae (PPNG). Four and 8 isolates exhibited plasmid and chromosomally-mediated resistance to tetracycline, respectively. Three isolates were found to exhibit chromosomally mediated resistance to both penicillin and tetracycline (CMRNG). All the clinical isolates were sensitive to ceftriaxone and spectinomycin and were resistant to ciprofloxacin and nalidixic acid.
Among the 6 WHO strains, 2 were resistant, 2 were less sensitive, and 1 was sensitive to penicillin. Only 1 strain was PPNG. Two strains exhibited chromosomally mediated resistance to tetracycline, and 1 was tetracycline-resistant N. gonorrhoeae (TRNG). One strain was less sensitive to ceftriaxone and 1 was less sensitive to ciprofloxacin. Resistance to spectinomycin was observed in 1 strain. Similarly, resistance to ciprofloxacin and nalidixic acid was observed in 1 and 2 strains, respectively.
Inhibition of N. gonorrhoeae by Extracts of the 3 Plants
Autoclaved distilled water used as control showed no inhibition of N. gonorrhoeae. Water and hot-water extracts of O. sanctum and water extracts of all 3 parts of D. quercifolia had no inhibitory activity against N. gonorrhoeae. No statistically significant difference was found between the extracts of the 3 plants.
Inhibition as Compared to Penicillin
The inhibition by the plant extracts as compared to penicillin is given in Table 2.
Penicillin-resistant isolates (1–11, 15, 20, and 23) were either sensitive or highly sensitive to all the extracts of the 3 plants, except isolates 7 and 9, which were surprisingly resistant to the extracts of A. squamosa. Isolates less sensitive to penicillin (12–14, 17–19, 22, and 24) were either less sensitive or sensitive to the plant extracts. Isolates sensitive to penicillin (16 and 21) were less sensitive to all the extracts of the 3 plants.
Strains WHO L and WHO O (resistant to penicillin) showed very high sensitivity to all the extracts, sensitivity of WHO L being more than that of WHO O for most of the extracts. Among the strains less sensitive to penicillin (WHO B, C, and G), WHO B was less sensitive to a few extracts and sensitive to the other extracts, while the other 2 were sensitive to all the extracts. WHO F, which was sensitive to penicillin, showed less sensitivity to extracts of all the 3 plants.
Inhibition as Compared to Ciprofloxacin
The inhibition by plant extracts as compared to ciprofloxacin is shown in Table 3.
All the 24 clinical isolates were resistant to ciprofloxacin but exhibited different sensitivities to the plant extracts. Two isolates (7 and 9) were resistant to A. squamosa, a few were less sensitive, some sensitive, while the others showed very high sensitivity to the plant extracts. This might be due to difference in the mechanism of inhibition of ciprofloxacin and the plant extracts.
WHO L was the only strain resistant to ciprofloxacin, but it was highly inhibited by all the extracts. WHO G was less sensitive to ciprofloxacin. The rest of the WHO strains were sensitive to ciprofloxacin, and all of these strains showed sensitivity to the plant extracts but to a less extent as compared to WHO L.
There has been a progressive increase in resistance of N. gonorrhoeae to antimicrobials used for the treatment of gonorrhea in many parts of the world, including India, and the number of such reports is increasing day by day.1–5,28–34 Resistance of 1.15% of the total isolates tested to ceftriaxone was reported in one study.35 Recently, resistance of 0.57% and of 0.46% of the total isolates tested to ceftriaxone has been reported in 2 different studies.36,37 This resistance cannot be ignored and is of grave concern.
In the current study, all 6 WHO strains tested were sensitive to extracts of all 3 plants. The most encouraging part of this study was that 1 of the WHO strains, WHO L, was highly sensitive to extracts of all 3 plants in spite of being resistant to 3 antibiotics– penicillin, ciprofloxacin, and nalidixic acid, and having intermediate sensitivity to tetracycline (Table 1). The most important point is that this was the only strain among the WHO strains being less sensitive to ceftriaxone and still exhibited the highest inhibition by most of the extracts of O. sanctum. Chloroform extract of the leaves of O. sanctum showed a very high percentage of inhibition of this strain (267%) at 1000-μg concentration that was 167% more than caused by both ciprofloxacin (1 μg) and penicillin (0.5 IU) (Fig. 1). Similarly, strain WHO O was found to be highly inhibited by most of the extracts when the results were compared with penicillin, even though it was resistant to penicillin, spectinomycin, and exhibited chromosomally mediated resistance to tetracycline. Another interesting observation is that strain WHO C, which was less sensitive to penicillin and sensitive to ceftriaxone, spectinomycin, ciprofloxacin, and nalidixic acid, was not highly inhibited by extracts of the 3 plants. Thus, the WHO strains that were showing multiresistance to the antibiotics tested were more sensitive to the extracts of the 3 plants, whereas those that were sensitive to the antibiotics were not highly sensitive to the extracts of the 3 plants.
Similarly, all the 24 clinical isolates were sensitive to the extracts of O. sanctum and D. quercifolia, and 22 clinical isolates were sensitive to the extracts of A. squamosa, though all these 24 isolates were resistant to 2 or more antibiotics (Table 1).
Clinical isolate 7 was inhibited the maximum by 2 extracts of D. quercifolia. Isolate 8 was inhibited the maximum by 7 extracts as compared to penicillin and by 2 extracts as compared to ciprofloxacin. Similarly, isolate 11 was inhibited the maximum by 9 extracts as compared with penicillin and by 7 extracts as compared with ciprofloxacin, and isolate 23 was inhibited the maximum by 4 extracts as compared with penicillin and by 3 extracts as compared to ciprofloxacin. Interestingly, all these 4 isolates (isolates 7, 8, 11, and 23) were resistant to penicillin, ciprofloxacin, and nalidixic acid. Isolate 23 exhibited intermediate sensitivity to tetracycline, isolates 7 and 11 exhibited chromosomally mediated resistance to tetracycline, and isolate 8 was a TRNG.
Even the 3 CMRNG isolates, isolate 1, 5, and 6, were inhibited by the extracts of the 3 plants, all 3 isolates exhibiting high inhibition as compared to penicillin. It can be observed from the above results that the multiresistant isolates are highly sensitive to the plant extracts, and therefore, probably the mechanism of inhibition of N. gonorrhoeae by the plant extracts is different from antibiotics like penicillin, ciprofloxacin, tetracycline, and nalidixic acid
The plant products have been found less toxic and a safer way for treatment of various diseases. LD50 of aqueous extract of leaves of A. squamosa was found to be more than 50 times the ED50 (dose = 350 mg/kg body weight; unpublished data). In acute toxicity study of 70% ethanol extract of O. sanctum, no gross behavioral, neurologic, and autonomic effects were observed in mice, and the acute LD50 was found to be 4950 mg/kg by the oral route. From the mean LD50 and ED50 values, the safety ratio of O. sanctum was found to be 12.93 in normal mice.38
Since all the clinical isolates and WHO strains are sensitive to the plant extracts, this study presents the utility of medicinal plants for the development of new chemical entities against N. gonorrhoeae. As the extracts of these 3 plants are showing high inhibition of N. gonorrhoeae WHO strains and clinical isolates, more so the multidrug resistant N. gonorrhoeae, we are purifying these plant extracts to get a single active component/second.
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