Antimicrobial activity of ethanolic extracts of Justicia neesii
Abstract
Agar well diffusion method was used to evaluate the antimicrobial potential of Justicia neesii extract. The maximum activity index (AI) values are observed against Klebsiella pneumonia (1.208) and low AI value for Streptococcus faecalis (0.963) compared to other bacterial species. The maximum AI values are observed against Saccharomyces cerevisiae (1.147) and low AI value for Fusarium axisporum (0.986) compared to other fungal species. The MIC and MBC/MFC values indicated the bacteriostatic/ fungistatic nature of the extract and also having good correlation with the zone of inhibition values. The total activity values indicated that J. neesii extract can show antimicrobial activity even at higher dilutions, except for Gram negative bacteria.
Introduction
Wide range of plants from tropical and sub-tropical regions of India were reported to posses antimicrobial properties (Prasannabalaji et al., 2012). J. neesii is one of such plant belongs to the family Acanthaceae grows in tropical regions of India as a small tropical herb. In the previous studies on this plant reported the presence of various types of lignans. Three β-apolignans including 1,4-dihydotaiwanin C, jusneesiin, jusneesiinol (Rajasekhar, 1998) and two arylnaphthalide lignans including jusmicranthin and justirumalin are found to be present (Rajasekhar, 1999; Gopalaiah, 2001). The plant was also found to contain diphyllin glycosides like neesiinoside A and neesiinoside B (Subbaraju, 2001). But, the review of scientific literature reviled that there is no significant pharmacological work done on J. neesii. So it was consider worthwhile to elucidate the antimicrobial properties of J. neesii plant extracts.
Materials and Methods
Collection and identification of plant: Plant material free from infection was collected from different areas of East Godavari district, Andhra Pradesh during the month of February 2014 on day time. The plant was taxonomically identified by the experts of Botanical Survey of India, Hyderabad (BSI/DRC/2013-14/Tech./915-A).
Extraction of plant material: Whole plant parts including leaves, stem, twigs, flowers, seeds, roots were separated and made free from soil matter. They were dried and powdered by using hand pulveriser to a course powder. Then the powder was extracted with ethanol by using sohxlet apparatus at a temperature of 50-55ºC for 8 hours. The extracts were concentrated using vacuum evaporator and the semisolid mass was dried in vacuum desiccators. The yield of plant extract was found to be 10.58 % (w/w).
Preparation of nutrient agar: The weighed amount of NaCl (5 g), peptone (10 g), beef extract (10 g) are dissolved in 1,000 mL of the water, then agar (20 g) is added slowly on heating with continues stirring until agar is completely dissolved and pH is adjusted to 7.2 to 7.4. This nutrient agar medium is then sterilized by moist heat sterilization method using autoclave at temperature of 120°C at 15 lb pressure maintained for 15 min.
Preparation of potato dextrose agar (PDA): A potato was peeled and 100 g was measured, finely chopped and boiled to a mash in distilled water. The dextrose was measured (12.5 g) and placed in a 1L measuring cylinder. Agar was measured (12.5 g) and added to the measuring cylinder (with the dextrose). The potato mash was stirred and strained into the cylinder. Hot distilled water was added to make up 500 mL. The contents was continuously poured and stirred until consistency was achieved. The content was then poured into a conical flask, plugged with cotton wool, over which aluminum foil was tightly wrapped. The flask was then autoclaved at 121°C for 24 hours (Murray, 1995).
Microbial cultures: Bacterial cultures of Gram positive bacteria- Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 3160), S. faecalis (MTCC 459), Gram negative bacteria- Escherichia coli (MTCC 46), Pseudomonas aeruginosa (MTCC 1688), Klebsiella pneumonia (MTCC 4032) and fungal cultures of Aspergillus flavus (MTCC 277), Aspergillus niger (MTCC 2723), Candida albicans (MTCC 183), Fusarium axisporum (MTCC 1755), Saccharomyces cerevisiae (MTCC 4742) were procured from Institute of Microbial Technology, Chandigarh, Punjab, India. All the test organisms were maintained on nutrient/potato dextrose agar slopes and sub-cultured once in every two weeks.
Preparation of inoculums: The test inoculums are prepared as per the McFarland method. McFarland standard was prepared by adding 0.5 mL of 1.175%w/v barium chloride to 85 mL of 1%v/v sulfuric acid and mixed. Then the volume was made up to 100 mL with 1% sulfuric acid. The optical density of the prepared solution was checked at 625 nm range gives an absorbance ranging from 0.08 to 0.10. The bacterial inoculums are prepared from 24 hour old cultures by taking 3-5 morphologically similar colonies of respective micro organisms and transferred into 5 mL sterile saline solution and adjusted to 0.5 McFarland turbidity standards equivalent to the cell density of 1-5 x 108 CFU/mL. The fungal inoculums are prepared from seven days old culture plates by taking 3-5 morphologically similar colonies of respective micro organisms and transferred into 5 mL sterile saline solution and adjusted to 0.5 McFarland turbidity standards. From this solution 1:10 dilution are preformed three times with growth medium to get inoculums density of 1-5 x 103 CFU/mL (Mcfarland, 1907).
Antimicrobial assay: The antimicrobial assay was carried out by agar well diffusion assay (Perez, 1990). The sterilized microbial medium was cooled to 50°C. 20 mL of the microbial media was taken into sterile universal bottles and seeded with 0.2 mL of respective cultures of standard inoculums size aseptically. Then the seeded media was transferred to the sterile petri dishes under aseptic conditions. The wells are created by using sterile cork borer of 6 mm diameter at equidistance points. The respective wells are supplied with 200, 400 and 800 µg of test drug in 25 µL volume. The petri plates were then kept in refrigerator for diffusion and then transferred to biological incubator and bacterial culture plates are incubated at 37°C for 24 hours and fungal culture plates at 27°C for 48 hours. Ciprofloxacin and nystatin are used as positive controls at 10 µg/well concentration to compare the antibacterial and antifungal affects respectively. Each experiment was performed in triplicate and the zone of inhibition values are noted after incubation period by using Antibiotic zone reader. Activity index for each extract was calculated by using following formula.
Activity index (AI) = Inhibition Zone of the sample/ Inhibition Zone of the standard.
Minimal inhibitory concentration (MIC): The minimum inhibitory concentration (MIC) is the concentration required to inhibit growth of a specific isolate in vitro under standardized conditions. The MIC of test extract on each organism was determined by using serial dilution method. For this purpose 10 mg/mL concentration of test solution was prepared and two fold serial dilutions are made to get 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, 0.019 mg/mL concentrations of solutions. 1 mL of test solution is then taken into each test tube and diluted with 1 mL of sterile nutrient agar media (for bacteria) or potato dextrose agar media (for fungi). The test tubes were then inoculated with 0.1 mL of microbial suspension of standard size. The tubes were incubated at 37°C for 24 hours for bacteria and 28°C for 48 hours for fungi in a biological incubator and observed for change in turbidity and compared with the growth in control tubes which contains 75% ethanol in water (Pelczer et al., 2004).
Minimum bactericidal/ fungicidal concentration (MBC/MFC): The MBC/MFC is the lowest concentration at which the culture has been completely sterilized. 50 µL of the media from each test tube showing no visible growth was taken and inoculated into 2 mL of sterile broth media and incubated as done in MIC determination.
Total activity (TA) determination: Total activity is the volume at which the test extract can be diluted with the ability to kill microorganisms. It is calculated by dividing the amount of extract from 1 g plant material by the MIC of the same extract or compound isolated and is expressed in mL/g (Eloff, 2004).
Total activity = Extract per gram dried plant part/MIC of extract
Results
The antimicrobial nature of ethanolic extract of J. neesii was estimated by the zone of inhibition and activity index values. The plant extracts at all the concentrations showed significant antibacterial activity compared standard drug ciprofloxacin in dose-dependent manner (Table I, II).
Table I: Antibacterial activity of J. neesii against Gram positive bacteria
| Treatment | Test organism | |||||
|---|---|---|---|---|---|---|
| Bacillus subtilis | Staphylococcus aureus | Streptococcus faecalis | ||||
| IZ (mm) | AI | IZ (mm) | AI | IZ (mm) | AI | |
| Ciprofloxacin (10 µg/well) | 23.3 ± 0.7 | - | 27.0 ± 0.6 | - | 27.3 ± 0.3 | - |
| J. neesii (200 µg/well) | 12.7 ± 0.7 | 0.543 | 13.7 ± 0.3 | 0.506 | 12.7 ± 0.9 | 0.464 |
| J. neesii (400 µg/well) | 18.0 ± 0.6 | 0.771 | 19.3 ± 0.7 | 0.716 | 17.3 ± 0.3 | 0.634 |
| J. neesii (800 µg/well) | 26.0 ± 0.6 | 1.114 | 28.0 ± 0.6 | 1.037 | 26.3 ± 0.9 | 0.963 |
| Values are expressed in mean ± SEM; n = 3 | ||||||
Table II: Antibacterial activity of J. neesii against Gram negative bacteria
| Treatment | Test organism | |||||
|---|---|---|---|---|---|---|
| Escherichia coli | Pseudomonas aeruginosa | Klebsiella pneumonia | ||||
| IZ (mm) | AI | IZ (mm) | AI | IZ (mm) | AI | |
| Ciprofloxacin (10 µg/well) | 17.7 ± 0.3 | - | 18.0 ± 0.6 | - | 16.0 ± 0.6 | - |
| J. neesii (200 µg/well) | 09.0 ± 0.6 | 0.509 | 10.7 ± 0.3 | 0.593 | 10.0 ± 0.6 | 0.625 |
| J. neesii (400 µg/well) | 14.7 ± 0.3 | 0.830 | 16.0 ± 0.6 | 0.889 | 15.7 ± 0.9 | 0.979 |
| J. neesii (800 µg/well) | 20.3 ± 0.3 | 1.150 | 20.7 ± 0.3 | 1.148 | 19.3 ± 0.9 | 1.208 |
| Values are expressed in mean ± SEM; n = 3 | ||||||
The maximum AI values are observed against K. pneumonia (1.208) and low AI value for S. faecalis (0.963) compared to other bacterial species. However, the maximum zone of inhibition values are observed for Gram positive bacteria compared to Gram negative bacteria. The minimal inhibitory concentration values have shown good correlation with IZ values. The Gram positive bacteria showed lowest MIC of 0.039 mg/mL compared to the other species. Higher MBC values are observed for most of the bacteria compared to their MIC. The plant having higher antibacterial potential against Gram positive bacteria (TA: 2712.82 mL/g).
The plant extracts at all the concentrations showed significant antifungal activity compared standard drug nystatin in dose-dependent manor (Table III).
Table III: Antifungal activity of J. neesii against pathogenic fungi
| Treatment | Test organism | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Aspergillus flavus | Aspergillus niger | Candida albicans | Fusarium axisporum | Saccharomyces cerevisiae | ||||||
| IZ (mm) | AI | IZ (mm) | AI | IZ (mm) | AI | IZ (mm) | AI | IZ (mm) | AI | |
| Nystatin (10 µg/well) | 23.7 ± 0.3 | - | 24.3 ± 0.3 | - | 27.0 ± 0.6 | - | 24.3 ± 0.9 | - | 25.0 ± 0.6 | - |
| J. neesii (200 µg/well) | 13.0 ± 0.6 | 0.549 | 14.7 ± 0.9 | 0.603 | 13.0 ± 0.6 | 0.481 | 11.7 ± 0.3 | 0.480 | 13.3 ± 0.7 | 0.533 |
| J. neesii (400 µg/well) | 18.7 ± 0.3 | 0.789 | 18.0 ± 0.6 | 0.740 | 18.7 ± 0.7 | 0.691 | 15.7 ± 0.9 | 0.644 | 19.3 ± 0.9 | 0.773 |
| J. neesii (800 µg/well) | 25.0 ± 0.6 | 1.056 | 25.0 ± 0.6 | 1.027 | 28.7 ± 0.3 | 1.062 | 24.0 ± 0.6 | 0.986 | 28.7 ± 0.9 | 1.147 |
| Values are expressed in mean ± SEM; n = 3 | ||||||||||
The maximum AI values are observed against S. cerevisiae (1.147) and low AI value for F. axisporum (0.986) compared to other fungal species. Low MIC values are observed for Candida albicans and S. cerevisiae (0.039 mg/mL) compared to other fungi. The plant having higher antifungal potential against Gram positive bacteria Candida albicans and S. cerevisiae (TA: 2712.82 mL/g). The plant has shown good MIC and MBC/MFC values against all microorganisms (Table IV).
Table IV: MIC, MBC/MFC and TA values of J. neesii
| Microorganism | MIC | MBC/MFC | Total activity (TA) |
|---|---|---|---|
| Bacillus subtilis | 0.039 | 0.156 | 2712.82 |
| Staphylococcus aureus | 0.039 | 0.078 | 2712.82 |
| Streptococcus faecalis | 0.039 | 0.156 | 2712.82 |
| Escherichia coli | 0.312 | 1.250 | 339.10 |
| Pseudomonas aeruginosa | 0.156 | 0.650 | 678.20 |
| Klebsiella pneumonia | 0.156 | 0.312 | 678.20 |
| Aspergillus flavus | 0.078 | 0.312 | 1356.47 |
| Aspergillus niger | 0.078 | 0.312 | 1356.47 |
| Candida albicans | 0.039 | 0.156 | 2712.82 |
| Fusarium axisporum | 0.156 | 0.625 | 678.20 |
| Saccharomyces cerevisiae | 0.039 | 0.312 | 2712.82 |
| TA was calculated by taking the yield: 105.8 mg/g dried plant and values are expressed in mL/g | |||
Discussion
The activity index values are helpful in estimating the potential of antimicrobial activity quantitatively compared to the respective standards. The plant extract has shown higher AI values against Gram negative bacteria which means that the extracts are having good activity against the Gram negative bacteria compared to the standard. However, when we observe the IZ values the highest inhibition was observed against Gram positive bacteria and fungi. So we can say that the plant extract is having good antibacterial activity against both the Gram strains and showing higher activity against Gram negative bacteria compared to the standard ciprofloxacin. The low IZ values of Gram negative bacteria may be due to its resistant cell wall composition. The higher values of MBC/MFC than that of MIC indicated that bacteriostatic/fungistatic nature of the extracts, which were observed for the active extracts. Total activity indicates the volume at which extract can be diluted which still having ability to kill microorganism. From the results we can say that the plant extract can show antimicrobial activity even at higher dilutions, except for Gram negative bacteria like E. coli.
Our preliminary phytochemical investigation shows the presence of flavonoids, glycosides, lactones, lignins, phenols, phytosterols, quinins, reducing sugars, saponins and terpinoids. The ability of flavonoids in forming complexes with cell walls of micro organisms and changing the structural integrity, positioned them in class of antibiotics. The antibiotic nature of flavonoids also increases with their lipophilicity (Jeyaseelan, 2012). The terpenes and phenols also exhibit antimicrobial properties (Shabir, 2011). The antimicrobial potential of triterpinoid saponins and lignans are also found in recent studies (Khan, 2011; Vasilev, 2005). The significant antimicrobial properties observed in this screening may be due to the presence of one of these phytochemicals in the ethanolic extract of the J. neesii.
Conclusion
J. neesii is having potential antimicrobial activity.
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