Plant kingdom harbors an inexhaustible source of active ingredients invaluable in the management of many diseases. These active components of herbal remedies have an advantage of being in combination with many other substances that appear to be inactive [1]. In recent years, multiple drug resiatance has developed due to indiscriminate use of existing antimicrobial drugs in the treatment of infectious diseases [2]. This has led to search for new antimicrobial substances from various sources like the Med plants. The screening of plant extracts and plant products for antimicrobial activity has shown that they represent a potential source of novel antibiotic prototypes [3]. A number of evidences have been accumulated to demonstrate the promising potentials of Med plants used in various traditional, complementary and alternative systems [4]. The need of the hour is to screen a number of Med plants for promising antimicrobial activity.

Cryptocoryne spiralis (Retz) Fischer ex Wydler var. spiralis and Cryptocoryne retrospiralis (Roxb) Kunth (Araceae) are commonly found at margins of ditches or sandy soil of the river, gravelly river beds also growing on open situations on plateaus of higher altitude. They are widely distributed in India from Maharashtra to North Karnataka, Kerala, Coromandel Coast, Pondicherry, Bengal and Assam.

The rhizome of these plants are used by the traditional healers for the treatment of diarrhoea, fever and jaundice, burns and boils [5,6]. Prasad et al. showed that, Cryptocoryne spiralis var. spiralis was widely used as a substitute of highly expensive Aconitum heterophyllum Wall. for the treatment of diarrhea as well as cough, abdominal complaints, fever and in case of remedy for infantile vomiting [7].

In the present investigation, two species of Cryptocoryne genus i.e., Cryptocoryne spiralis var. spiralis and Cryptocoryne retrospiralis were selected to screen bioactive phytochemical and their antibacterial property.

Extraction of plant material

The rhizome and leaves of C. spiralis var. spiralis (CSS) and C. retrospiralis (CR) were cleaned, chopped and dried and ground separately to fine powder. Solvent and aqueous extraction of these powders was done by using solvents like acetone, ethanol and methanol in 1:10 (w/v) ratio for 24 h with shaking on rotary shaker at 150 rpm speed. Extracted material was filtered through Whattman filter paper No. 1. After filtration, extracts were again dried and concentrated by evaporating the solvent completely in water bath at the range of boiling points of solvents. The dried extracts were resuspended in 10% DMSO and stored at 4˚C [8].

Test organisms

Total six bacterial strains were used for this study out of which two were Gram positive, Bacillus subtilis (NCIM 2045), Micrococcus aureus (NCIM 2802) while, four were Gram negative, Pseudomonas aeruginosa (NCIM 2036), Escherichia coli (NCIM 2832), Salmonella typhi (NCIM 2501) and Klebsiella pneumoniae (NCIM 2883). Bacterial strains were obtained from NCIM, Pune, Maharashtra (India). The test organisms were sub cultured on nutrient agar medium at 37˚C for 24 h and preserved at 4˚C as a stock culture

Screening of antibacterial activity

Sensitivity of different bacterial strains to extracts from rhizome and leaf of different Cryptocoryne species was measured in terms of zone of inhibition using agar diffusion assay [9]. The plates containing Mueller-Hinton agar media were spreaded with 0.2 ml of the respective inoculums equivalent to McFarland 0.5 (15 × 10⁷cfu/ml) turbidity standard. Wells were bored out from agar plates using a sterilized stainless steel borer and filled with 0.1 ml (500 μg) of the extract. The plates were incubated at 37˚C up to 48 h and diameter of resultant zone of inhibition was measured. The bacteria with a clear zone of inhibition were considered to be sensitive. The experiments were performed in triplicate.

Determination of minimum inhibitory concentration (MIC)

The MIC of Cryptocoryne rhizome and leaf extracts was determined by agar diffusion assay [9]. The plates containing Nutrient agar media were seeded with 0.2 ml of the respective inoculums (15 × 10⁷organisms/ml). Wells were prepared in agar plates using a sterilized stainless steel borer and extracts of different concentrations prepared by serial dilution technique in different solvents were loaded. Each plate contained three wells including control as 10% DMSO.

Growth pattern of test organisms

The effect of rhizome and leaf extracts of C. spiralis var. spiralis (CSS) and C. retrospiralis (CR) on growth pattern of test organisms was studied [10]. One set of experiment where nutrient broth contains the respective solvents added with test organism i.e., solvent extract, while in another set of experiment nutrient broth contains rhizome or leaf extract only. The flasks were incubated for 24 h at 37˚C in incubator shaker but growth was monitored by measuring absorbance at 540 nm at every 30 min interval.

Phytochemicals analysis

The qualitative tests for phytoconstituents tannins, alkaloids, coumarins, saponins, steroids, flavonoid and glycosides in the acetone, ethanol, methanol and aqueous extracts were performed according to Firdouse, Alam and De et al. with some modifications [11,12].

Gas Chromatography-Mass Spectroscopy (GC-MS) analysis

GC-MS analysis was carried out by Shimadzu 2010 MS Engine (Shimadzu Corporation, Japan). The temperature was enhanced to 200˚C with a rate 10˚C min^-1 and then linearly raised to 280˚C at a rate of 20˚C min^-1. Helium was used as sample carrier gas with flow rate of 1 ml min^-1. The initial temperature of column was stabilized at 80˚C for 2 min by oven. The spectrums of the components were compared with the database of spectrum of known components stored in the GC-MS library. Measurement of peak areas and data processing were carried out by Turbo-Mass.

Screening of antibacterial activity

It was found that both species have significant antibacterial activity with rhizome and leaf extracts, although the inhibitory activity was found to be strain specific (Table 1). Ethanol and methanol extracts showed relatively significant antibacterial activities against both Grampositive and Gram-negative bacterial strains tested. Ethanol extracts from rhizome of CSS (22 mm), ethanol and methanol extract from rhizome of CR (20 mm) showed highest activity against Micrococcus aureus among all the bacteria. Ethanolic extracts from leaf of CSS (20 mm) and both ethanolic and methanolic extract from leaf of CR (18 mm) showed highest activity against Micrococcus aureus among all the bacterial strains studied. Aqueous extracts from rhizome and leaf of both species showed very poor inhibitory activity against all bacterial cultures except M. aureus. Present study showed that ethanol was the better solvent for extraction as compared to methanol, acetone and water.

Table 1: Antibacterial activity of organic solvent extracts of CSS and CR rhizome and leaf. E: Ethanol, A: Acetone, M: Methanol.

Determination of minimum inhibitory concentration (MIC)

The lowest concentration of the extract which showed zone of inhibition against respective organisms was taken as MIC. Better efficacy of both ethanol and methanol extracts of CSS, and CR rhizome and leaf were further supported by MIC results. The MIC values of ethanol extracts of rhizome were ranging from 200-400 μg/ml for CSS and 250-450 μg/ml for CR. Methanol extracts showed MIC values ranging from 250-400 μg/ml for CSS while 250-450 μg/ml for CR (Table 2). The highest MIC values were observed for acetone extracts from rhizome (550 μg/ml) of CR against P. aeruginosa. The highest MIC value (450 μg/ml) was found with ehanol extracts from rhizome of CR against P. aeruginosa and acetone extract from rhizome of CR against B. subtilis . The lowest MIC value for ethanolic extract (200 μg/ml) was observed for CSS against M. aureus and B. subtilis while, (250 μg/ml) acetonic, methanolic extract of CSS and ethanolic, methanolic extract of CR against M. aureus.

Table 2: Minimum inhibitory concentration (MIC) (µg/ml) of organic solvent extracts of CSS and CR rhizome and leaf.

The MIC values of ethanol extracts of leaf were ranged from (250-400 μg/ml) for both CSS and CR. Methanol extracts showed MIC values ranged from (250-400 μg/ml) for CSS and (300-450 μg/ml) for CR. (Table 2). The highest MIC values were observed for acetone extracts of leaf (500 μg/ml) of both CSS and CR against P. aeruginosa . The lowest MIC value for ethanol extracts of leaf (250 μg/ml) for CSS and CR while, for methanolic extract of leaf of CSS (250 μg/ml) was observed against M. aureus . This highlights the equal effectiveness of ethanol and methanol extracts in comparison to agar diffusion assay.

Growth pattern of test organisms

The objective of this study was to find out exactly what was the response of the test organism test organisms growing in presence of acetone, ethanolic and methanolic extracts. The results are as shown in Figures 1 and 2.

Figure 1: Growth pattern of microorganisms A) B. subtilis B) M. aureus C) P. aeruginosa D) E. coli E) S. typhi F) K. pneumoniae in presence of acetonic, ethanolic and methanolic extracts from rhizome of C. spiralis var. spiralis (CSS) and C. retrospiralis , (CR).

Figure 2: Growth pattern of microorganisms A) B. subtilis B) M. aureus C) P. aeruginosa D) E. coli E) S. typhi F) K. pneumoniae in presence of acetonic, ethanolic and methanolic extracts from leaf of C. Spiralis var. spiralis (CSS) and C. retrospiralis , (CR).

It can be noted that all the organisms had retarded growth rate in presence of ethanol and methanol extracts from rhizome and leaf of C. spiralis var . spiralis and C. retrospiralis . However, the retardation of growth was higher in ethanolic extracts as compared to methanolic extracts. Further M. aureus is least inhibited by the acetone extracts from the rhizome of all species and in presence of the acetone extracts from the leaf of all species the growth of M. aureus , E. coli and S. typhi is not affected significantly except that it retards to some extent. Therefore, best antimicrobial activity is seen with the ethanolic extract, followed by methanolic extract and the least is by the acetone extracts from the respective rhizome and leaf of all species (Figures 1 and 2).

Phytochemicals analysis

The qualitative phytochemical analysis of acetone, ethanol, methanol and aqueous rhizome and leaf extracts of CSS and CR were presented in Table 3. All phytochemical compounds viz. glycosides, flavonoids, tannins, alkaloids, steroids and saponins were found present in all the solvent extracts of CSS and CR.

Table 3: Phytochemical analysis of aqueous organic solvent extracts of CSS and CR leaf and rhizome.

Gas Chromatography-Mass Spectroscopy (GC-MS) analysis

GC-MS analysis of ethanol extracts of Cryptocoryne species is represented in Table 4 [13-21]. The important bioactive compounds detected are cyclo hydrocarbon Bicyclo (2.2.1) heptanes, 2-methyl-3- methylene-2-(4-methyl-3-pentenyl)-(1S-exo), 3-Butoxy-1,1,1,7,7,7- hexamethyl-3,5,5-tris (trimethylsiloxy) tetrasiloxane, cyclohexasiloxane, dodecamethyl, 2-Ethoxy-3 chlorobutane, santalol, cis-alfa santalol, trans beta santalol, neomenthol, menthol. These phytocompounds were reported responsible for various pharmacological actions. The presence of various bio-active compounds detected in GC-MS analysis using the ethanolic extracts justifies the use of plant for pharmacological action. The antibacterial activity and phytochemical evaluation in alcoholic, aqueous and petroleum ether extract of Cryptocoryne spiralis reported by Das et al. [22]. Madhavan et al. and Prasad et al. also support the present investigation [7,23].

Table 4: Phytocomponents identified in ethanolic rhizome extract of Cryptocoryne species using GC-MS analysis.

The demonstrable antimicrobial activity of the rhizome extracts of the C. spiralis var. spiralis and C. retrospiralis implies that, these plants have a potential for preparation of alternative medicines for infections caused by microorganisms which have become resistant to the current therapeutic measures, which is an essential requirement of the present medical practice. This is more emphasized by its antibacterial properties against Micrococcus aureus which is a very common wound infecting organism and equally resistant to many of the common medicines used to treat its infections both in humans and in animals. Pseudomonas aeruginosa is the next wound infecting organism but notorious for its resistance to the medicines used, is also found susceptible to these extracts and this is very significant application. The results of the present study reveal the fact that the organic solvent extracts exhibited greater antimicrobial activity because the antimicrobial principles were extracted only through the organic solvent medium [24]. The present study supports the use of Cryptocoryne rhizome in the traditional system of medicine.

The authors are grateful to the Honorable Principal of Smt. K.W. College, Sangli, India for providing the necessary facilities for successful completion of this investigation.

No potential conflict of interest was reported by the authors.