Abstract
The bactericidal activities of the aqueous and methanol extracts of the leaves of three plants used as popular medicine in India were studied. The dried leaf extracts of Vangueria spinosa. B. (Rubiaceae), Cestrum nocturnum. L. (Solanaceae), and Nyctanthes arbortristis. L. (Oleaceae) were tested in vitro. by the disk diffusion method against four bacterial strains, namely, Staphylococcus aureus. (MTCC 2940), Bacillus subtilis. (MTCC 441), Escherichia coli. (MTCC 739), and Pseudomonas aeruginosa. (MTCC 2453). Both the aqueous and the methanol extracts of V. spinosa. showed the strongest activity, followed by C. nocturnum. and N. arbortristis.. The only exception was P. aeruginosa., which, in turn, was found to be resistant against the aqueous extract of N. arbortristis.. Aqueous extracts of the leaves of all the plants appeared to have less antibacterial activity than the methanol extracts.
Introduction
In the field of medicine, antibiotics are important, providing treatment of sepsis and infections. With some exceptions, most of the antibiotic drugs are chemically produced and have some adverse effects on the human body and non-target organisms. Moreover, pathogenic strains of bacteria, over time, develop resistance to certain antibiotics.
Plants and plant products have been used extensively throughout history to treat medical problems. Numerous studies have been carried out on various natural products screening antimicrobial activity (Rai, Citation1989; Negi et al., Citation1993; Reddy et al., Citation2001; Sakagami et al., Citation2001; ErdoÛrul, Citation2002; Nita et al., Citation2002; Ates & ErdoÛrul, 2003; Velickovic et al., Citation2003; Bhattacharjee et al., Citation2005Citation2006). It has been suggested that the aqueous and alcohol extracts of plants used in allopathic medicines are potential sources of antiviral, antitumor, and antimicrobial agents (Chung et al., Citation1995; Vlietinck et al., Citation1995). The selection of crude plant extracts for screening programs has the potential of being more successful in its initial steps than the screening of pure compounds that are isolated from natural products (Kusumoto et al., Citation1995).
Vangueria spinosa. B. (Rubiaceae) is a deciduous shrub or small tree that varies in height from 3–7 m, depending on the habitat. It can be single or multistemmed. The bark is grayish to yellowish brown in color, smooth and peeling in irregular small strips. The leaves are single, oppositely arranged, as is typical of this family. The leaves are light green in color, covered with soft, velvety short hairs, particularly when the plant is young. Antelopes graze upon the leaves of this tree. This plant has some medicinal value as well. An infusion of the roots and leaves has been used to treat malaria, chest ailments such as pneumonia, as a purgative, and to treat ringworms. An infusion of the leaves is used to relieve a toothache. A decoction of the pounded leaves and small twigs is remarkably effective to treat swollen limbs, especially in children (Vlietinck et al., Citation1995; Thakre, Citation2004).
Cestrum nocturnum. L. (Solanaceae), commonly known as night-blooming Jessamine, is a sprawling shrub with glossy, smooth, simple leaves; vine-like stems; and greenish, creamy white tubular flowers. C. nocturnum. is an integral part of the butterfly gardens as it provides food for the larvae of some caterpillars. Mature leaf contains a calcinogenic glycoside that leads to vitamin D toxicity and is responsible for elevated serum calcium level (Mello, Citation2003).
Nyctanthes arbortristis. L. (Oleaceae), commonly known as bruschia, is a large deciduous shrub or small tree. Its branches are quadrangular, and it is rough all over with an uneven epidermis and stiff white hairs. Leaf juice is used to treat loss of appetite, piles, liver disorders, biliary disorders, intestinal worms, chronic fever, obstinate sciatica, rheumatism, and fever. The seeds are used as anthelminthics. The bark is used for the treatment of bronchitis and snakebite (Drury, Citation1873; Kirtikar & Basu, Citation1993).
The present study was designed to determine the role of leaf extracts (aqueous and methanol extracts) of V. spinosa, C. nocturnum., and N. arbortristis. for potential antibacterial activity, if any, against two Gram-positive bacteria (Staphylococcus aureus. MTCC 2940, and Bacillus subtilis. MTCC 441) and two Gram-negative bacteria (Escherichia coli. MTCC 739 and Pseudomonas. aeruginosa. MTCC 2453). The observed inhibition zones were measured (in mm) and compared against standard antibiotic nystatin.
Materials and Methods
The plant material used in this study consisted of mature leaves of V. spinosa., C. nocturnum., and N. arbortristis., collected from the outskirts of Burdwan (23°16′N, 87°54′E), West Bengal, India, during the spring season (mid-March to mid-April 2005). The leaves were initially rinsed with distilled water and dried on paper towels in the laboratory at 37±1°C for 24 h.
Preparation and preservation of plant extract
Aqueous extract
Each of the three samples, which consisted of mature leaves of V. spinosa., C. nocturnum., and N. arbortristis., were weighed out (50 g) and soaked separately in 200 ml of cold water contained in conical flasks stoppered with rubber corks and left undisturbed for 24 h. They were then filtered off using sterile filter papers (Whattman No.1) into clean conical flasks and subjected to water bath evaporation, where the aqueous solvents were evaporated at boiling temperature of 100°C. The standard extracts thus obtained were then stored at 4°C in a refrigerator until further use (Akueshi et al., Citation2002).
Methanol extract
After drying, the plant materials were ground separately in a grinding machine (MX-110 PN, Japan) in the laboratory. Exposure to sunlight was avoided to prevent the loss of active components. Methanol extraction fluid (200 ml) was mixed with 50 g each of powdered plant materials. The mixtures were then kept for 24 h in tightly sealed vessels at room temperature, protected from sunlight, and stirred thoroughly several times a day with sterile glass rods. The mixtures thus obtained were filtered through Whatman No. 1 filter papers and the residues adjusted to the required concentration (50 ml of methanol for the residue of 50 g of powdered plant material) with the extraction fluid for further extraction. This was repeated three-times, and a clear colorless supernatant extraction liquid was finally obtained. The extracted liquids were subjected to rotary evaporation in order to remove the methanol. The semisolid extracts produced were kept at − 80°C (REVCO model No. ULT 790-3-V 32) in a freezer overnight and then subjected to freeze-drying for 24 h at − 60°C at 200 ml vacuum. Then the extracts were stored in an airtight container at 4°C in the refrigerator until further use. All the dried extracts were exposed to UV rays (200–400 nm) for 24 h and checked frequently for sterility by streaking on nutrient agar plates (Chessbrough, Citation2000).
Antibacterial assay
Disc diffusion method
Antibiogram was done by disc diffusion method (NCCLS M2-A5, 1993; Bauer et al., Citation1966) using plant extracts and commonly used antibiotics. The test quantity of specific extracts, as shown in Tables and Citation2, were dissolved in either distilled water or dimethylsulphoxide (DMSO), depending upon the solubility of the extracts. The dissolution of the organic extracts (methanol) was aided by 1% (v/v) DMSO and that of the aqueous extracts with water, which did not effect the growth of microorganisms, in accordance with our control experiments. The surfaces of media were inoculated with bacteria from a broth culture. High-potency bio-discs (Himedia) were placed on the agar. After 18 h of incubation at a specific temperature [(30±1)°C for B. subtilis. and 37°C for S. aureus, E. coli., and P. aeruginosa.], the plates were examined and the diameters of the inhibition zones were measured to the nearest millimeter.
Table 1.. Antibacterial activity of specific concentration of aqueous extract of medicinal plant compared to control by disc diffusion method.
Table 2.. Antibacterial activity of specific concentration of methanol extract of medicinal plant compared to control by disc diffusion method.
Dilution method for minimum inhibitory concentration
Of the three plants tested, the ones that showed antibacterial activity against some of the selected pathogens were selected for further tests to calculate their minimum inhibitory concentration (MIC) by dilution method. These tests were performed in sterile 96-well microplates and macroplates. The microdilution was performed in 96-well microtiter plates with U-shaped wells, while the macrodilution technique was as described by the National Committee for Clinical Laboratory Standards was followed (NCCLS, Citation1993; Paiva et al., Citation2003). In brief, the cultures were diluted in Müeller-Hinton broth at a density adjusted to 0.5 McFarland turbidity. The final inoculum was 5 × 105 CFU/ml of bacterial colony. Controls with 0.5 ml of culture medium only or others with plant extracts were used in the tests. The wells were filled with 100 µl of sterile H2O, and 100 µl of the plant extracts were added to the wells by serial two-fold dilution from the suspension of plant extract stock solution. Each well was inoculated with 100 µl of 0.5 McFarland standard bacterial suspensions so that each well received 5 × 105 CFU/ml. The plates were covered, placed in plastic bags, and incubated at 37°C for 24 h. In this study, the MIC was the lowest concentration of plant extracts that exhibited no growth of the organisms in the wells by visual reading.
Test microorganisms
Four bacterial strains were used for the study. Gram-positive bacteria include S. aureus. MTCC 2940 and B. subtilis. MTCC 441, and Gram-negative bacteria include E. coli. MTCC 739 and P. aeruginosa. MTCC 2453. All the tested strains are reference strains and were collected from the Microbiology Laboratory of Burdwan Medical College. The bacterial cultures were maintained in nutrient broth (Himedia, M002) at 37°C and maintained on nutrient agar (Himedia, MM012) slants at 4°C.
Statistical analysis
Since the readings of control (distilled water) experiments in the in vitro. antibacterial studies of those plants were zero, the data were analyzed by simple arithmetic means of the different extracts, and the standard errors were compared with the control.
Results
The antibacterial activities of the leaf extracts of V. spinosa., C. nocturnum., and N. arbortristis. in different solvents (aqueous and methanol) against E. coli, P. aeruginosa, B. subtilis., and S. aureus. are shown in Tables and , respectively.
In , all the bacterial strains were found to be effective against aqueous extract except P. aeruginosa., which was not effective against aqueous extract of N. arbortristis.. reveals that the methanol extracts of the plants were comparatively more effective than their aqueous counterparts against all the bacterial strains. Thus, from Tables and , it is evident that the organic extracts are comparatively more effective than the aqueous extracts. The organic and aqueous extracts of V. spinosa. are comparatively more effective than those of C. nocturnum., followed by N. arbortristis.. Antibiogram of the commonly used antibiotics is shown in , and the MIC value of the tested plant extracts against the tested microorganisms is shown in .
Table 3.. Susceptibility of four reference bacterial strains to some antibiotics in nutrient agar.
Table 4.. Minimum inhibitory concentration (MIC) of different extracts by dilution method.
Discussion
The results indicate that the crude extracts of all plants studied showed antibacterial activities toward the Gram-positive bacteria (S. aureus. and B. subtilis.). These results are consistent with previous reports on related plants with respect to Gram-positive bacteria (Cowan, Citation1999). The resistance of Gram-negative bacteria (E. coli. and P. aeruginosa.) to plant extracts was expected as, in general, this class of bacteria is comparatively more resistant than the Gram-positive bacteria. Such resistance could be due to the permeability barrier provided by the cell wall or to the membrane accumulation mechanism (Adwan & AbuHasan, Citation1998).
Infections caused by these bacteria, especially those with multidrug resistances, are among the most difficult ones to treat with conventional antibiotics (CDC NNIS System, Citation1999). Therefore, it seems likely that the antibacterial compounds extracted from these plants might inhibit bacteria by a different mechanism than that of currently used antibiotics and have therapeutic values as antibacterial agents.
It appears that the microorganisms were not as sensitive to the aqueous extracts compared to the methanol extracts as determined by diffusion. The reasons for this could be that all of the identified components from plants active against microorganisms, aromatic, or saturated organic compounds are most often obtained through initial methanol extraction (Cowan, Citation1999).
In conclusion, it is suggested that these plants may be used to discover natural bioactive products that might lead to the development of new drugs. Such screening of various natural organic compounds and identification of active agents must be considered as a fruitful approach in the search for new pharmaceuticals.
Acknowledgment
The authors acknowledge the help of Department of Science and Technology, New Delhi, for providing infrastructure through FIST program.
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