EFFECT OF COLEUS AMBOINICUS LEAF EXTRACT AND OIL ON CLINICAL ISOLATES OF PSEUDOMONAS AND PROTEUS
Vasavi Dathar1* and Afrojahan2=
1. Department of Microbiology, Palamuru University, Mahabubnagar
2. Department of Zoology, Adarsh Junior College, Mahabubnagar
Herbal plant extracts are potential sources of antibiotic prototypes. Numerous studies have demonstrated the antimicrobial activity of active compounds in herbal plant extracts against commonly found bacteria. Hence, the search for novel plants with medicinal value that are potential alternatives to currently used antibiotics (tetracycline, chloramphenicol etc.) is an important area of research. In this paper, we investigate the antimicrobial activity of Coleus amboinicus or Plectranthus amboinicus by preparing an aqueous and hexane extract and leaf oil and testing against clinical isolates of Pseudomonas aeruginosa and Proteus mirabilis. Pseudomonas aeruginosa is a medically important, multidrug resistant pathogen causing nosocomial and opportunistic infections whereas Proteus mirabilis causes 90% of Proteus infections. Both the extracts and components of leaf oil of Coleus amboinicus leaves showed antibacterial activity against these two isolates at all the concentrations tested by agar well diffusion method, using Streptomycin as standard. The inhibition zone diameters were found to be in the range of 4.7 – 15.3 and 4.7 – 12.7 mm, 11.00 – 21.00 and 10.00 – 19.00 mm , with a standard error on the order of 0.0 to 1.73 as a function of increasing concentration of aqueous extract and hexane extract for Pseudomonas aeruginosa and Proteus mirabilis respectively. On the other hand, the components of leaf oil of Coleus amboinicus showed a maximum zone of inhibition of 17.00 ± 1.73 and 20.33 ± 0.58 mm for Pseudomonas aeruginosa and 20.67 ± 0.58 and 18.33 ± 1.53 mm for Proteus mirabilis with Eluent 1 and Eluent 2 respectively. The minimum inhibitory concentration (MIC) of the extract was found to be 60 mg/ml for Pseudomonas aeruginosa and 120 mg/ml for Proteus mirabilis. These findings indicate that the plant has a potential for the treatment of infections caused by these two bacteria.
Key words- Coleus amboinicus, clinical isolates, Pseudomonas aeruginosa, Proteus mirabilis, leaf oil, antibacterial effect
Screening of plant extracts and plant products for antimicrobial activity has shown that herbal plants represent a potential source of antibiotic prototypes . The increasing failure of chemotherapy and antibiotic resistance exhibited by bacterial pathogens has prompted researchers for screening of plants for their antimicrobial activity . Numerous studies have identified compounds within herbal plants that are effective antibiotics . In India, about 2000 drugs used are of plant origin and the search for phytomedicines that are safe and affordable is still on-going .
Coleus amboinicus or Plectranthus amboinicus is a member of the family Lamiaceae, which is an ornamental plant and is also known for its medicinal value. The leaves are traditionally used for the treatment of coughs, sore throats and nasal congestion and other problems such as infections, rheumatism and flatulence, malarial fever, hepatopathy, renal calculi, asthma, hiccoughs, bronchitis, helminthiasis, colic, convulsions and epilepsy [5,6]. The leaf oil also exhibits antibacterial and antifungal activity .
Pseudomonas aeruginosa is a common Gram-negative bacterium that can cause disease in animals and humans. It is motile, oxidase, citrate and catalase positive. It is ubiquitous and found in soil, water, skin flora, and most man-made environments throughout the world. The bacterium infects damaged tissues as it utilizes a wide range of organic materials. It may be fatal if the organism colonizes in critical body parts like lungs, urinary tract and kidneys . The bacterium was found in bacteremia  associated with cystic fibrosis and acute appendicitis in adults . A multi drug resistant strain has been isolated from a patient admitted in intensive care unit with highly invasive procedures and being treated with broad spectrum cephalosporins and aminoglycosides . Pseudomonas aeruginosa is increasingly recognized as one of the important nosocomial pathogens leading to severe infections especially in hospitalized patients in burn wards .
Proteus mirabilis is a Gram-negative, facultatively anaerobic rod-shaped bacterium. The bacterium is a common cause of urinary tract infections in individuals with functional or structural abnormalities or with long-term catheterization and forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis . Most cases of P. mirabilis bacteremia originate from a UTI . It also causes nosocomial infections, being the second most frequently isolated Enterobacteriaceae species after Escherichia coli in hospitals . P. mirabilis producing extended-spectrum β-lactamases has been found to cause blood stream infections in hospitalized patients using bladder catheters . P. mirabilis is generally susceptible to most apart from and , but 10–20% of P. mirabilis strains are also resistant to first-generationand .Our previous study on the methanolic extract of the leaves of Coleus amboinicus has shown antibacterial activity against eleven clinical isolates viz. methicillin resistant Staphylococcus aureus, Staphylococcus aureus, Enterococcus, E.coli, Klebsiella pneumoniae, Citrobacter divergens, Shigella flexneri, Salmonella paratyphi A, Salmonella paratyphi B, Proteus mirabilis and Pseudomonas aeruginosa . Among these, Pseudomonas aeruginosa was resistant to the control antibiotics tetracycline and chloramphenicol and Proteus mirabilis was resistant to tetracycline whereas the methanolic extract of Coleus amboinicus has shown antibacterial effect against these two bacteria. Interested by these facts, an attempt was made to study the antibacterial activity of aqueous extract of the plant against these two clinical isolates and the results are presented in this paper.
MATERIALS AND METHODS
Preparation of an aqueous extract of the leaves
Healthy leaves of Coleus amboinicus were collected and washed with distilled water. The leaves were shade dried at room temperature and ground uniformly into powder using a mechanical grinder. 10 grams of the leaf powder was soaked in 100 ml of distilled water in a conical flask and loaded on an orbital shaker at a speed of 120 rpm for 24 hours. The mixture was filtered using Whatman filter paper number 1. The filtrate was concentrated using rotary evaporator and dried using lyophilizer. The dried extract was collected in an air tight container and stored at 4°C . Various concentrations of the extract were prepared and used for antibacterial activity.
Preparation of hexane extract of the leaves
Fresh leaves of Coleus amboinicus were taken and grinded. The mixture was soaked with pure hexane in a conical flask and loaded on an orbital shaker for 24 hrs. The solution was filtered using Whatman filter paper number 1 and the filtrate was concentrated to obtain a crude extract. The extract was collected and stored at 4°C for further use. Various concentrations of the crude extract were prepared in ethanol and used for antimicrobial activity.
Separation of leaf oil by column chromatography
Fresh leaves of Coleus amboinicus were soaked in pure hexane for 24 hrs and the filtrate was collected. The process was repeated for 3 days. The filtrate was concentrated and the extract was run through a column of silica gel. Fraction 1 (eluent 1) was obtained with pure hexane solvent and a solvent of 5% hexane and ethyl acetate were used to get Fraction 2 (eluent 2). The eluents were concentrated and dissolved in ethanol to prepare different concentrations and antimicrobial assay was performed.
Preparation of the bacterial culture
Pseudomonas aeruginosa and Proteus mirabilis isolated from clinical samples were obtained from SVS Medical College, Mahabubnagar. Conventional bacteriological methods such as colony morphology, gram staining and biochemical tests were used for identification of isolates  (Table 1). The test organisms were inoculated in Mueller Hinton broth (pH 7.4.) for 8 hours. The concentration of the suspensions was adjusted to 0.5 Mc Farland standard  to reach an optical density of 0.08 – 0.10 at 625 nm by adding sterile distilled water. This gives a bacterial suspension containing 1.5 x 108 CFU/ml . Isolates were seeded on Mueller Hinton agar plates by using sterilized cotton swabs.
Antimicrobial sensitivity testing
Antimicrobial sensitivity testing was done by Agar well diffusion method. Mueller Hinton agar plates were prepared and wells of diameter 5mm were cut. The bacterial culture was spread with a sterilized cotton swab and 100 μl of various concentrations of the leaf extract viz. 3 mg, 6 mg, 12 mg and 24 mg per well were added to the wells. Streptomycin was used as a control antibiotic. Effect of the leaf extract on growth of the organisms was studied by inoculating the bacteria in the nutrient broth containing various concentrations of the extract and measuring the optical density at 625 nm. The minimum inhibitory concentration of the extract was tested by using the concentrations of 7.5 to 240 mg per ml.
Determination of Activity Index (AI)
Activity index of all the extracts was calculated using following formula 
Determination of relative percentage inhibition (RPI)
The relative percentage inhibition of all the test extracts with respect to the positive control was calculated by using the following formula .
Where X= Total area of inhibition of the test extract; Y= Total area of inhibition of the solvent and Z= Total area of inhibition of the standard drug. The total area of the inhibition was calculated by using the area = πr2; where r = radius of the zone of inhibition.
RESULTS AND DISCUSSION
The leaf extract of Coleus amboinicus showed antibacterial activity against clinical isolates of Pseudomonas aeruginosa and Proteus mirabilis at all the concentrations tested. The Inhibition Zone Diameters were found to be in the range of 4.67 – 15.33 and 4.67 – 12.67 with a standard error on the order of 0.6 to 1.2 as a function of increasing concentration of aqueous extract for Pseudomonas aeruginosa and Proteus mirabilis respectively. (Fig.1a and 1b). The values of antimicrobial activity of the leaf extract of Coleus amboinicus were expressed as mean ± standard deviation (n=3) for each sample. The Inhibition Zone Diameters showed a quadratic dependence (R2 > 0.98) on the concentration of the aqueous extract with a significant increase after 120 mg/ml for both the organisms.
Similar inhibitory effect of leaf extracts of Ocimum sanctum and Eucalyptus globulus against Pseudomonas was reported earlier . Leaf extracts of Artemisia nilagirica were also reported to have inhibitory effect on a clinical isolate of Pseudomonas aeruginosa . On the other hand, methanolic extract of Aloe vera gel did not show any inhibitory effect on Pseudomonas aeruginosa , whereas, acetone and ethanolic extracts have inhibited the growth of Pseudomonas aeruginosa .
Table 1: Basic characteristics of the two bacteria
|Bacterium||Pseudomonas aeruginosa||Proteus mirabilis|
|Gram Morphology||Gram negative rod||Gram negative,
|Methyl Red test||Negative||Positive|
|Voges Proskauer test||Negative||Negative|
Fig.1a. Effect of Coleus amboinicus leaf extract on Pseudomonas aeruginosa
Similarly, the extracts of mangrove leaves Sonneratia alba and Exoecaria agallocha showed inhibitory effect on Proteus mirabilis . On the other hand, the aqueous extract of Acalypha wilkesiana did not show inhibitory effect on Proteus mirabilis, but its ethanolic extract was active . The bacterium was also found to be resistant to leaf extract of Cajanus cajan .
Fig.1b. Effect of Coleus amboinicus leaf extract on Proteus mirabilis
The hexane extract of Coleus amboinicus is more effective compared to the aqueous extract on the clinical isolates of Proteus mirabilis and Pseudomonas aeruginosa as evidenced by the zone of inhibition against them (Table 2). The zone of inhibition increased with increase in concentration against the two bacteria. Pseudomonas aeruginosa is more sensitive to the hexane extract than Proteus mirabilis as with aqueous extract. On the other hand, hexane extracts of Trigonella foenum-graecum could not show any inhibitory activity on Pseudomonas aeruginosa whereas the aqueous extract could inhibit the bacterium .
Table 2: Effect of Hexane extract of Coleus amboinicus on the two bacteria
|Concentration of hexane
|Zone of inhibition in mm|
|Proteus mirabilis||Pseudomonas aeruginosa|
|30||10.00 ± 1.73||11.00 ± 1.00|
|60||13.67 ± 1.52||12.67 ± 0.58|
|120||16.00 ± 1.00||17.33 ± 0.58|
|240||19.00 ± 0.00||21.00 ± 1.00|
Increase in concentration of the leaf extract has shown a negative effect on the growth of organisms. This is indicated by the decrease in the turbidity of the broth with increase in the concentration of the leaf extract. The inhibition was more on Pseudomonas aeruginosa (Fig. 2a) when compared to Proteus mirabilis (Fig. 2b).
Fig. 2a. Effect of Coleus amboinicus leaf extract on the growth of Pseudomonas aeruginosa
Fig. 2b. Effect of Coleus amboinicus leaf extract on the growth of Proteus mirabilis
The same trend was observed in the inhibition zones produced against the two organisms, viz. the zone diameters were more for Pseudomonas aeruginosa when compared to those of Proteus mirabilis. The minimum inhibitory concentration of the aqueous extract of Coleus amboinicus was found to be 60 mg/ml and 120 mg/ml for Pseudomonas aeruginosa and Proteus mirabilis respectively.
The activity indices and relative percentage inhibition of the aqueous and hexane extracts with Streptomycin as control antibiotic are depicted in Table 3. The solvent used was ethanol for hexane extract with activity of 7.0 mm towards Proteus mirabilis and 8.0 mm towards Pseudomonas aeruginosa and distilled water for aqueous extract. Both the extracts have shown increased activity with increase in concentration against Pseudomonas aeruginosa and Proteus mirabilis. But hexane extract exhibited 2 to 4 times more activity compared to aqueous extract against both the bacteria.
Table 3: Activity of leaf extracts of Coleus amboinicus on the two bacteria
|Concentration of the Leaf extract (mg/ml)||Pseudomonas aeruginosa||Proteus mirabilis|
|Aqueous extract||Hexane extract||Aqueous extract||Hexane
AI: Activity Index; RPI: Relative Percentage Inhibition
Chromatographic separation of the components of leaf oil using silica gel with hexane as a solvent and hexane plus ethyl acetate yielded two components, Eluent 1 and Eluent 2 respectively. Different concentrations of the oil components were prepared in ethanol and their antimicrobial activity was tested against the two bacteria. The activity index of Eluent 1 ranged from 0.22 to 0.60 and Eluent 2 from 0.32 to 0.72 as the concentrations increased from 30 to 240 mg/ml for Pseudomonas aeruginosa (Table 4). The relative percentage inhibition was negative with lower concentrations of Eluent 1 but gradually increased with increase in concentrations. The zone of inhibition, activity index and relative percentage inhibition with Eluent 2 increased with increasing concentrations and Eluent 2 exhibited more activity against the bacterium compared to Eluent 1.
Table 4: Effect of Leaf oil of Coleus amboinicus on Pseudomonas aeruginosa
|Concentration of the
|Eluent I||Eluent II|
|ZOI (mm)||AI||RPI||ZOI (mm)||AI||RPI|
|30||6.33 ± 1.15||0.22||-3.22||9.33 ± 0.58||0.32||3.14|
|60||11.00 ± 1.00||0.39||7.71||14.33 ± 0.58||0.51||19.16|
|120||13.67 ± 1.53||0.48||16.16||16.67 ± 1.15||0.59||28.98|
|240||17.00 ± 1.73||0.60||30.44||20.33 ± 0.58||0.72||47.31|
ZOI: Zone of Inhibition; AI: Activity Index; RPI: Relative Percentage Inhibition
Table 5: Effect of Leaf oil of Coleus amboinicus on Proteus mirabilis
|Concentration of the
|Eluent I||Eluent II|
|30||5.0 ± 0.0||0.21||-4.84||7.33 ± 0.58||0.31||0.98|
|60||8.0 ± 0.0||0.34||3.02||12.33 ± 0.58||0.52||20.83|
|120||11.0 ± 1.0||0.47||14.52||14.00 ± 1.00||0.60||29.65|
|240||20.67 ± 0.58||0.89||75.70||18.33 ± 1.53||0.79||57.96|
ZOI: Zone of Inhibition; AI: Activity Index; RPI: Relative Percentage Inhibition
Similar increase in the activity with increase in concentration was found with the two eluents on Proteus mirabilis (Table 5). The zone of inhibition, activity index and relative percentage inhibition were higher with Eluent 2 upto a concentration of 120 mg/ml but there was a steep rise in the activity of Eluent 1 at a concentration of 240 mg/ml against the bacterium compared to Eluent 2. Similar results were observed with methanol and aqueous stem extracts of Withania somnifera against E.coli, Serratia marcescens and Bacillus cereus whereas hexane extract was inactive against Pseudomonas aeruginosa . Thus, biologically active constituents from Plectranthus amboinicus or Coleus amboinicus can be extracted with different solvents like hexane, ethyl acetate and methanol, where the ethyl acetate extract showed higher phenolics and higher antioxidant activity . The antibacterial activity of the plant may be due to the phenolic compounds which also possess other functional attributes like antimicrobial, anti-inflammatory, antimutagenic, hypocholestemic and antiplatelet aggregation properties.
The aqueous and hexane extracts of Coleus amboinicus leaves showed inhibitory effect on clinical isolates of Pseudomonas aeruginosa and Proteus mirabilis at concentrations of 30 mg to 240 mg/ml with a minimum inhibitory concentration of 60 mg and 120 mg/ml for Pseudomonas aeruginosa and Proteus mirabilis respectively. Among the two organisms, Proteus mirabilis required a higher concentration of the leaf extract for inhibition. This bacterium is resistant to many leaf extracts as previously reported [31, 32] and to the antibiotic tetracycline [17, 19]. Pseudomonas aeruginosa is also resistant to tetracycline and chloramphenicol . But both the organisms are sensitive to the aqueous extract of Coleus amboinicus. The leaf oil and its components are also active against the two bacteria at different concentrations. Pseudomonas aeruginosa is sensitive to the hexane extract of the plant whereas the bacterium is resistant to hexane extracts of other plants . This is an advantage of this plant over other plants and antibiotics, and can be exploited for the treatment of nosocomial infections caused by these bacteria.
Authors are thankful to The Principal, University P.G. College, Palamuru University; Head, Department of Microbiology, Palamuru University for their encouragement; and Mr. P. Srinivas Rao, Correspondent, Adarsh Degree & PG College, Mahabubnagar, for providing the facilities to carry out this work.
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20) University of Manchester, Oxford Road, Manchester, M13 9PL, UK
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