Antimicrobial potential of the phytoextracts of some Nyctaginaceae members

The emergence of drug resistant pathogens is becoming a serious threat to humanity and has necessitated the search for new antimicrobial drugs. The present study was done to evaluate the potential antimicrobial efficacy of some common members of Nictagenaceae against four bacterial strains; Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Proteus vulgaris. Different concentrations of Soxhlet extracts (aqueous, petroleum ether and ethyl alcohol) of the plants in different solvents were tested by standard filter paper disk diffusion method. The efficacy of the extracts determined from the zone of inhibition was compared with that of two common antibiotics (amphicillin and erythromycin). The minimum inhibitory concentration (MIC) values of the extracts were also determined from most efficient extracts. The selected plant extracts exhibited good antibacterial activity. The crude phytoextracts showed high degree of antimicrobial activity and confirms the traditional therapeutic claims of these plants. The results confirm that the plant extracts have great potential as antimicrobial compounds against microorganisms. Thus, they can be used in the treatment of infectious diseases caused by resistant microbes.


INTRODUCTION
Plants not only provide food for man but also give a number of active compounds with potent and varied therapeutic value.The world health organization reported that 80% of world's populations rely chiefly on traditional medicine (WHO, 1993) and would be the best source to obtain a variety of drugs (Santos et al., 1995).Traditional therapy involves the use of plant extracts or their active constituents and is within the reach of common people.
Infections caused by multiple resistant strains are becoming common in the developing world.Bacteria are necessary for the existence of higher orders of life and majority of them are harmless.Relatively a small number of bacteria are pathogenic, causing infectious diseases to plants and animals.These are the second leading cause of worldwide mortality and the third leading cause in economically developed countries.The problem of microbial resistance is growing and the outlook for the use of

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author.E-mail: mukeshsharma_uniraj@yahoo.co.in antimicrobial drugs in the future is still uncertain.Surprisingly, despite increased bacterial resistance to existing drugs, antibiotic development in the pharmaceutical industry is steeply declining (Projan, 2003;Wenzel, 2004).The past few decades of modern medicines certainly helped man in controlling and even eliminating some of deadly diseases caused by microorganisms, but it paved the way for the formation of more deadly pathogens, which are antibiotic resistant.Therefore, actions must be taken to reduce this problem, for example, to control the use of antibiotic, understand the genetic mechanisms of resistance and to continue studies to develop new drugs, either synthetic or natural.The ultimate goal is to offer appropriate and efficient anti-microbial drugs to the patient.
In recent years, there is a great demand for plantbased products of broad biological activities, low impact on environment and safety to non-target organisms.The effects of plant extracts on bacteria have been studied by a number of researchers in different parts of the world.Plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines.
More than 80% of the population in developing countries depends on plants for their medical needs (Farnsworth, 1988;Balick et al., 1994).
The use of plant extracts and phytochemicals, both with known antimicrobial properties, can be of great significance in therapeutic treatments.In the last few years, a number of studies have been conducted in different countries to prove such efficiency (Almagboul et al., 1985;Artizzu et al., 1995;Ikram and Inamul, 1984;Izzo et al., 1995;Kubo et al., 1993;Shapoval et al., 1994).Many plants have been used because of their anti-microbial traits, which are due to compounds synthesized in the secondary metabolism of the plant.These products are known by their active substances, for example, the phenolic compounds which are part of the essential oils (Jansen et al., 1987) as well as in tannin (Saxena et al., 1994).
Boerhaavia diffusa L. (Common names, Hogweed, Pigweed, Punarnava, Thazhuthama) is also known as punarnava and it has a long history of use by indigenous and tribal people and in Ayurvedic herbal system of medicines.A perennial diffuse herb with stout root stock and many procumbent grows as common weed in wastelands and roadsides.The plant is bitter, astringent, cooling anthelmintic, diuretic, aphrodisiac, cardiac stimulant, diaphoretic, emetic, expectorant, anti-inflammatory, febrifuge, laxative and tonic.It is useful in all types of inflammations, strangery, leucorrhoea, opthalmia, lumbago, myalgia, scabies, cardiac disorders, jaundice, anaemia, dyspepsia, constipation, cough, bronchitis and general debility (Varier, 1997).Bougainvillaea spectabilis Willd. is a genus of flowering plants, native to the tropical and subtropical regions of South America (from Brazil west to Peru and south to southern Argentina Chubut province).It grows as a common garden plant throughout India, as indoor houseplants in temperate regions and they are kept small by bonsai techniques.They have anthelmintic, analgesic, anti-inflammatory, heaptoprotective, and anti-cancerous properties.Mirabilis jalapa Linn.(Common names, Marvel of Peru, Four O' clock, Anthimalari, Gulabbas, Beauty of the night, Clavillia) are large herbaceous plant grown in gardens throughout India.The plant is used as abortive, anti-bacterial, anticandidal, anti-fungal, anti-viral, anti-spasmodic, uterine stimulant, anti-dysenteric, anti-parasitic, carminative (expels gas), detoxifier, digestive stimulant, diuretic, purgative (strong laxative), tonic, vermifuge (expels worms), and wound healer.Hence, more studies pertaining to the use of plants as therapeutic agents should be emphasized, especially those related to the control of antibiotic resistant microbes.The objective of this research was to evaluate the potential of plant extracts on standard microorganism strains as well as multi-drug resistant bacteria, which were isolated from hospitals.Moreover, we investigated the synergistic effects of extracts with antimicrobial actactivity in association with antibiotics against drugs resistant bacteria.

Collection of plant materials
The fresh plant materials of B. diffusa, B. spectabilis and M. Jalapa were collected from Elackad, Kuravilangad, 23 km from Kottayam in October-December, 2006.The plants were collected when the species were in the fruiting stage.This was done because the fruits are important for species identification.The main author and coauthors collected the test plant species.The plants were identified using the herbarium collection at the Deva Matha College, Kuravilangad, Kerala, India and finally from the Herbarium, University of Rajasthan, Jaipur, India.All the voucher specimens are deposited in the herbarium, Deva Matha College, Kuravilangad, Kerala, India.

Extraction of plant material
Healthy plant parts were collected, dried in shade and powdered.About 30 g of powdered leaf, stem and root were extracted with 220 ml of hot water, ethanol and petroleum ether in a Soxhlet apparatus.The extracts were evaporated to dryness and solutions of 5, 10, 15, 20 and 100% were prepared in the solvent in which it was extracted.

Isolation and maintenance of test organisms
Four pathogenic bacteria species; Escherichia coli, Staphylococus aureus, Pseudomonas aeruginosa and Proteus vulgaris were used for the antimicrobial assays.Bacterial strains were procured from the Microbiology and Pathology Department, S.M.S. Medical College and S.M.S. Hospital, Jaipur for the present study.The test strains were raised in pure form on nutrient agar medium (Merck, g/l).

Antimicrobial assays
The filter paper disc diffusion method (Karaman et al., 2003) was used to study the effect of plant extracts on bacteria.Agar plates were prepared using nutrient agar (Hi-media, g/l).The plates, when half set were inoculated with bacteria using sterile cotton swabs, 3 sterile paper discs (1 cm diameter) of Whatman no. 1 filter paper, one dipped in pure solvent and 2 in the plant extracts were placed above the inoculated plates.The disc dipped in the solvent was taken as the control.The 3 other discs as the experiment, gave 3 readings for inhibition zone.The mean value was taken from 3 readings.All the above procedure was done in aseptic conditions provided by a laminar airflow chamber.The plates were then incubated in an inverted condition at 28°C for 24 h in a BOD incubator.The inhibition zone formed due to the allelopathic effect of the extracts was measured in millimeters.The mean of the 3 values of each plate was taken as the zone of inhibition.The above procedure was repeated for different concentrations of leaf, stem and root extracts of B. diffusa, B. spectabilis and M. jalapa.

Minimum inhibitory concentration (MIC)
The MIC was determined for the antimicrobially most efficient extracts using microdilution broth method (Bassolé et al., 2003).All the extracts were made in DMSO.The extracts were diluted to give one series with concentrations of 0.001 to 2.5 mg/ml.MIC was determined by incorporating different concentrations of extracts in test media.A control without extracts was also run along with the experiments.The lowest concentration of the extracts that did not The data given are mean (n=3) ± standard error.
show any viable growth after 48 h of incubation at 35°C (compared with control) was considered as MIC value.All the tests were made in triplicate.

Analysis of data
All values are expressed as means ± standard error.The data collected was analyzed using one-way analysis of variance (ANOVA) and the effect of the differences among group means were considered significant when P value was < 0.05.

RESULTS
The leaf, stem and root extracts in water, petroleum ether and ethyl alcohol of B. spectabilis, B. diffusa and M. jalapa were prepared and tested for their antibacterial effect against 4 strains of bacteria like S. aureus, E. coli, P. aeruginosa and P. vulgaris, after 24 h of incubation.The alcohol extracts of all plants showed inhibition towards all the 4 strain of bacteria, indicating clear inhibition zones around the paper discs.No inhibition was observed in the control.B. diffusa leaf extracts showed maximum inhibition against all the test bacteria and seems to contain more bactericidal compounds than stem and root extracts.The ethanol extracts of leaf showed maximum effect against S. aureus, E. coli and P. aeruginosa while the growth of P. vulgaris was inhibited maximally by the aqueous and petroleum ether extracts (Table 1).
Among the different extracts of B. spectabilis, leaf extracts in ethyl alcohol showed the maximum inhibition towards all the test bacteria.The aqueous and petroleum extracts of B. spectabilis leaves also showed inhibition towards all the bacteria studied.The stem and root extracts showed a lesser degree of inhibition towards the bacterial strains (Table 2).
M. jalapa leaf, stem and root extracts in water, petroleum ether and ethyl alcohol showed certain degree of inhibition towards the bacterial strains.Among the different extracts of M. jalapa the root extracts in ethyl alcohol showed maximum antibacterial potency against S. aureus, E. coli and P. vulgaris.The aqueous stem extract of M. jalapa showed maximum inhibition of P. aeruginosa The data given are mean (n=3) ± standard error.The data given are mean (n=3) ± standard error.The data given are mean (n=3) ± standard error.
Root extract of M. jalapa showed the lowest MIC values against all the pathogenic bacteria tested.Standard antibiotics also showed good MIC values against the pathogenic bacteria (Table 4).
The comparison of phytoextracts with antibiotics (Table 5) clearly indicates its efficacy in controlling the growth of pathogenic bacteria.Among all the phytoextracts used in the present study, 25% concentration of M. jalapa plant showed best antibacterial activity followed by B. spectabilis.The large zone of inhibition of M. jalapa was reported against P. aeruginosa (12.0 ± 0.12) and small was evaluated against E. coli (7.00 ± 0.12).Both standard antibiotics used for antibacterial activity repre-sented excellent antibacterial activity but large inhibition zone was presented by 25% concentration of erythromycin.E. coli was significantly affected (18.50 ± 0.16) and showed poor growth in test medium.The high anti-bacterial potency of the plants confirms their traditional therapeutic claims.

DISCUSSION
Plant extracts are able to restrict the growth of bacteria due to the presence of active principles in it.These active principles may inhibit protein synthesis of bacterial cell wall or alter the membrane function, inhibit protein synthesis or synthesis of purine and pyrimidines, hinder respiration or antagonize the metabolic pathways of microorganism leading to retardation of growth of bacteria.These active principles in these plants could be used as potent antibiotics.
The leaf, stem and root extracts in water, petroleum ether and ethyl alcohol of B. spectabilis, B. diffusa and M. jalapa showed antibacterial effect against 4 strains of bacteria; S. aureus, E. coli, P. aeruginosa and P. vulgaris.The result is in agreement with the earlier reports of Hiremath et al. (1996Hiremath et al. ( , 1997) ) indicating that dicot plants produce certain alkaloids which can control the growth of microbial pathogens.
During the present study, the extracts from the leaves, stems and roots of test plants exhibited strong antimicrobial activity greater than those from other parts that is bark, fruits, flowers ad seeds.The reason behind this point is that these plant organs, at the start, are more susceptible to microbial attack.And at a later stage, these parts naturally get enriched with bioactive compounds than some other parts to overcome this kind of problem.Because of their stronger innate immunity, these plant organs are protected against microbial attack.Some previous reports confirm this statement (Alwadi and Baka, 2001).
The phytoextracts showed a lesser degree of inhibition when compared to antibiotics.The antibiotics are in extra pure form when compared with the crude phytoextracts.The alkaloids responsible for the inhibitory effect of the phytoextracts have to be selected, purified and can be used as a broad-spectrum antimicrobial agent (Sakharkar and Pati, 1998;Nagendran and Thiruvalluvan, 1999).
The results suggest the presence of good antibacterial potency, and have a high potential as a source of antibacterial agent for therapeutic use.The use of antibiotics is not only expensive but also develops drug resistance in bacteria.The microbial inhibitory compounds that is alkaloids, peptides and glycosides present in the phytoextracts of B. spectabilis, B. diffusa and M. jalapa can be utilized for the effective control of pathogenic bacteria.The present study revealed that the root part of M. jalapa can be recommended for large scale production of active compounds due to its significant as well as excellent antibacterial efficiency.The high degree of antibacterial activity seems to confirm the folk therapy of infection and traditional therapeutic claims of these plants.
The antimicrobial results of this study also support several of the traditional medicinal uses of Nictagenaceae members in India and show that the ethnobotanical approach to screening plants as potential sources of bioactive substances is of great value.The potential for developing antimicrobial drugs from higher plants appears rewarding, as it will lead to the development of a phytomedicine to act against microbes.Therefore, such screening experiments form a primary platform for further phytochemical and pharmacological studies that may open the possibility of finding new clinically effective antibacterial compounds.The synergistic effect from the association of antibiotic with plant extracts against resistant bacteria leads to new choices for the treatment of infectious diseases.This effect enables the use of the respective antibiotic when it is no longer effective by itself during therapeutic treatment.
The present findings show that the plant extracts had a high antibacterial activity.Further research work is underway to purify and characterize the antibacterial proteins/ peptides from the plants.

Table 1 .
Antibacterial activity of phytoextracts of B. diffusa.

Table 2 .
Antibacterial activity of phytoextracts of B. spectabilis.

Table 3 .
Antibacterial activity of phytoextracts of M. jalapa.

Table 4 .
Minimal inhibitory concentration (MIC) of plant extracts against pathogenic bacteria.

Table 5 .
Comparison of phytoextracts with antibiotics.