INTRODUCTION:

The use of medicinal herbs in the treatment of infection is an age-old practice and several natural products are used as phytotherapic for treatment of many diseases. Human infections constitute a serious problem and most frequent pathogens are microorganisms such as bacteria and fungi. On the other hand, the development of resistant strains of pathogenic bacteria to antibiotics currently in use is a problem of continuing concern to public health. Increase prevalence of multidrug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial infections and adds urgency to the search for new infection-fighting agents.

Recent years have witnessed a renewed interest in exploring natural resources for developing such compounds. Medicinal plants are the natural and safer source of phytochemicals to fight against new strains of microorganism[1, 2].

Ipomea trupethum is a common medicinal herb used by ethno medical practitioners, belonging to the family Convolvulaceae [3] .This plant has several medicinal properties and has been extensively used in Indian traditional medicine for hemorrhoids, chancres and ulcerations ophthalmia. Unfortunately, this important medicinal plant has been enlisted as endangered species. But due to rapid destruction of habitat they become extinct day by day [4]. Among the different approaches, in vitro culture method provides new means of conserving and rapidly propagating valuable, rare and endangered medicinal plants [5].

Evidently, there are no sufficient scientific studies that confirm the antimicrobial properties of these plants. So, this study also looks into the in vitro antimicrobial activity of Operculina turpethum leaves and callus crude extract against five pathogenic microorganisms. The present study reports a reliable protocol for the in vitro initiation of callus from leaves of Operculina turpethum.

MATERIALS AND METHODS:

Callus induction and maintenance:

Ipomea turpethum plants were collected from the campus of Sahrdaya College of Engineering and Technology, Kerala. Callus initiation from leaves of Ipomoea turpethum has been carried out [6-8]. Initially, the collected leaves were washed under running tap water for 30 min and treated with Tween-20 for 6 min followed by treatment with 70% v/v in ethanol for 5 min in shaking. Finally the explants were sterilized 0.1% w/v mercuric chloride solution with 3-4 drops of Tween-20 for10 min with gentle shaking. Sterilized explants were washed 5-7 times with sterile distilled water to remove the effect of mercuric chloride. Then the explants were cultured in MS medium [9] containing different concentrations and combinations of BAP, Kin, for in vitro callus induction. The cultures were incubated in a growth chamber at28±2°C under 16 h photoperiod regimes and provided by white fluorescent lights with 40-45 μmolm^-2s^-1 illumination. Callus cultures were maintained on solid MS medium, subcultured every 30 days at 28±2ºC with a daily photoperiod of 16 hours. Callus cultures were harvested at day 30 of cultivation and dried at 60ºC.

Extraction of antimicrobial components from leaf and callus culture:

Dried and powdered leaves and callus were soaked separately in ethanol (1:10 w/v) for 4-5 days at room temperature with occasional shaking. After filtration of the extracted solutions they were concentrated in a rotary evaporator at 40^oC to dryness. For aqueous extraction, leaves and callus were pasted using a mortar and pestle and soaked into sterile distilled water (1:10 w/v) in a conical flask and kept for 24 h with gentle agitation and then filtered off using sterile filter paper. This extract was subjected to water bath for evaporation and resulted crude extract. Collected solid residues (crude extract) were stored at 4 °C and were dissolved freshly in respective solvents prior to screening for antimicrobial activity [8, 10-12].

Determination of antibiotic resistance:

The pathogenic bacteria were used in order to evaluate the antibiotic susceptibility. The clinical isolates were obtained from the department of microbiology of Jubilee Mission Hospital. The bacteria were cultured on nutrient agar medium at 37°C and maintained by sub culturing periodically on agar slant at 4°C[13].

Comparative study of antimicrobial activity of plant extract with callus extract:

Antibacterial activity was tested by well-diffusion method [8, 10-12]. Extracts were prepared by reconstituting in respective solvents. A 100 μl of 24 h old broth culture of each test bacteria was spread on the nutrient agar plate. The standard antibiotic discs (Hi-Media, Mumbai, India) of ampicillin and neomycin (10 μg) was served as positive control and ethanol, water used as negative control. The assay plates were incubated at 37 °C for 24 h. The diameter of the zones of inhibition around each of the well was considered as measure of the antimicrobial activity. Each experiment was carried out in triplicate and the mean diameter of the inhibition zone was recorded. The extracts, which showed antimicrobial activity, were subjected to the MIC assay.

Minimum inhibitory concentration:

A series of culture tubes (micro dilution assays) (25) were prepared all containing the same volume of medium inoculated with test microorganisms. The lowest concentration of sample at which the subculture from test dilution yielded no viable organisms was recorded as minimum bactericidal concentration. Serial two-fold dilutions of the extracts ranging from 20 to 2000µg/ml were prepared in 2 ml of nutrient broth. Then 40 μl of the test organism was added and incubated for 24 hours. The MIC values were interpreted as the lowest concentration of the extracts, which showed clear fluid with no development of turbidity. Blanks and positive controls were also included.

RESULT:

Callus induction and maintenance:

Explants inoculated in BAP containing medium showed better response than kinetin hormones or combinations of hormones used, and gave highest frequency of callus (Figure 1). Among the various concentrations of BAP, 2.0 mg/l showed the best results (Table1) producing highest callus per explants within 4 weeks of culture. Explants inoculated in Kin containing medium doesn’t give callus[14, 15].

Figure. 1. Callus produced from leaves of medicinal plant Ipomea turpethum in MS +2BAP medium

Table 1. Effects of plant growth hormone on initiation of callus from leaves of I turpethum.

Plant hormones (mg/ml)	Callus formation
BAP
1.00	+
1.50	++
2.00	+++
Kin
1.00	-
1.50	-
2.00	-
BAP+ Kin
1.00+0.5	-
1.00+1.0	-
2.00+0.5	+

- = no callus, + = little callus, ++ =moderate callus, +++ = huge callus

Determination of antibiotic resistance

The antibiotic resistance offered by the microorganisms obtained from the hospital is shown in Table 2. The readings were taken to the nearest millimeter [16-18]. It was found that all the microorganisms were resistant for the given antibiotics.

Table 2. Antibiotic susceptibility test for various Microorganisms.

Microorganisms	A	P	C	T	S
Escherichia coli	-	-	-	-	-
Proteus sp	-	6	-	-	-
Staphylococcus sp	-	-	10	12	-
Klebsiella sp	-	7	11	8	6
Pseudomonas sp	-	-	-	-	9

A-ampicillin, P-penicillin, C-chloramphenicol, T-tetracyclin, S-streptomycin

Comparative study of antimicrobial activity of plant extract with callus extract:

Plant substances continue to serve as exclusive source of drugs for the majority of the world population and several plant based drugs are in extensive clinical use .The antimicrobial activities of two solvent extracts compared with those of ampicillin, neomycin as positive control and ethanol and water used as negative control are summarized in Table 3. Assay of the extracts revealed that ethanol extracts showed a varied degree of inhibition against pathogenic bacteria while aqueous extract did not show significant zone of inhibition. In general, ethanol extracts showed higher activity than aqueous extract and produced inhibition zones ranging from 10 to 16 mm in diameter [19][20].

The comparative study of antibacterial activity of leaf extracts with callus extract against pathogenic bacteria revealed an increase in inhibitory activity of leaf derived callus compared to the whole leaf extracts. Table 4.

Table 3. Antibacterial activity of various leaf extract of I.turpethum against pathogenic microorganisms

Microorganisms	Ethanol extract	Water extract	Ampicillin
Escherichia coli	10	-	-
Proteus Sp	13	-	-
Staphylococcus sp	12	-	-
Klebsiella sp	14	-	-
Pseudomonas sp	16	-	-

No zone of inhibition was observed upon treatment with ethanol or water (negative control)

Table 4. Antimicrobial activity of ethanol extracts of Ipomea turpethum from leaves and callus against pathogenic microorganisms

Microorganism	Zone of inhibition (mm)	Zone of inhibition (mm)
Microorganism	Leaf	Leaf callus
Escherichia coli	10	12
Proteus sp	13	15
Staphylococcus sp	12	13
Klebsiella sp	14	17
Pseudomonas sp	16	18

No zone of inhibition was observed upon treatment with ethanol or water (negative control)

Minimum inhibitory concentration:

The Minimum inhibitory concentration (MIC) values of active extracts were determined by the broth dilution method and presented in Table 5. The MIC value of extracts ranged from 0.10 to 0.72 mg/ml. Ethanol extracts from callus had lower MIC values comparable with ethanol extract from leaves against the tested pathogenic strains[21]. Moreover, the therapeutic efficacy was found to be higher even in low concentration. This clearly exhibits the advantage of administering the ethanolic extract of I.turpethum .

Table 5. MIC of ethanolic extract from leaves and callus of Ipomea turpethum against pathogenic microorganisms

Organisms	MIC (µg/ml)	MIC (µg/ml)
Organisms	Leaf Extract	Callus Extract
Escherichia coli	720	690
Proteus sp	150	100
Staphylococcus sp	520	460
Pseudomonas sp	250	210
Klebsiella sp	220	190

DISCUSSION:

Ipomea turpethum is large evergreen perennial climbing plant with fragrant flowers conspicuous flowers. The active principle of the leaves is a substance called cardiotonic Oleandrin. It has anti-inflammatory and stimulant properties. The roots, bark and seeds contain cardio-active glycosides, which have been designated earlier as neriodorein carbine, and the anti-inflammatory and stimulant, are good painkillers. A paste of the root is used as an external application for piles, chancres and ulcers. Oil from the root bark is used for skin diseases a scaly nature. The fresh juice of the leaves is in the eye to induce tearing down in ophthalmia. Unfortunately, this important medicinal plant is now has appeared as endangered species. There is a growing demand for these plants, which received either in vivo or in vitro, because of the uniqueness of this species

In vitro culture method offers new ways to conserve and multiply rapidly valuable, rare and endangered medicinal plants. An established protocol including plant regeneration would be helpful for preventing extinction. A review of available literature revealed no previous report on callus induction from plant leaf of this important species. In this study we successfully induced callus from leaf of I. turpethumim MS +BAP media. These callus cells were used testing antimicrobial activity.

Antimicrobial activities of ethanolic and aqueous extract of I. turpethum on various antibiotic resistant pathogenic strains were carried out. Ethanol extracts exhibited a higher degree of antimicrobial activity as compared with aqueous extracts. It was found that the leaf extracts were bioactive against Gram-positive and Gram negative bacteria. The plants extracted with ethanol were shown to be active against five pathogenic strains of microorganisms with predominance for Pseudomonas sp. The inhibition zone produced for ethanolic extracts of leaves were in the range of 10 to 16 mm. The plant cell callus culture extracts were also bioactive and with bioactivity, in general, against the same strains of adult plants extracts, in the range of12 to 18 mm. The antimicrobial agents in the extracts are not known yet. However, previous chemical analysis of the plants extracts, indicated the presence of flavonoids, shows antimicrobial activity as reported by Chattopadhyay et al.(26)

The objective of antimicrobial activity was to analyze past, present and future of medicinal plants to suggest as fundamental the research on plant extract mechanism of action, interactions with antibiotics or with other medicinal plants.The screening of plant extracts and plant products for antimicrobial activity has shown that higher plants represent a potential source of novel antibiotic prototypes. The appeal of using natural products for medicinal purposes is increasing, and at present, researchers aim to produce substances with anti-tumor, anti-viral, hypoglycemic, anti-inflammatory, anti-parasite, antimicrobial tranquilizer and immunomodulating activities through tissue culture technology[22]. Advances in the area of cell cultures for the production of medicinal compounds has made possible the production of a wide variety of pharmaceuticals like alkaloids, terpenoids, steroids, saponins, phenolics, flavonoids [23,24].

In conclusion, we described here a simple and reproducible callus induction method for I. turpethum that would be helpful for large scale isolation of components from this endangered species. Evidence of strong antimicrobial components in the leaves and callus extracts would be useful in developing antimicrobial substances in medicinal industries. .

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Received on 04.05.2012 Modified on 31.05.2012

Research J. Pharm. and Tech. 5(6): June 2012; Page 805-808