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Keywords:

  • antibacterial activity;
  • bactericidal;
  • Curcuma amada;
  • difurocumenonol;
  • mango ginger;
  • MIC;
  • rhizome

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Aim:  The aim of the present work was to purify and characterize potential natural antibacterial compound from mango ginger (Curcuma amada Roxb.) rhizome.

Methods and Results:  The mango ginger rhizome powder was sequentially extracted and screened for antibacterial activity by agar well diffusion method and broth dilution method. Nonpolar extracts of mango ginger showed high antibacterial activity against gram-positive bacteria with low minimum inhibitory concentration (60–180 ppm). Among five extracts of mango ginger, the chloroform extract demonstrated highest antibacterial activity. Antibacterial activity-guided fractionation of the chloroform extract by repeated silica gel column chromatography yielded pure compound. The purified antibacterial compound was analysed by UV, IR, LC-MS and 2D-HMQCT NMR spectra and was identified as a difurocumenonol, a novel compound not reported previously.

Conclusions:  Mango ginger extracts and isolated difurocumenonol demonstrated high antibacterial activity against gram-negative and gram-positive bacteria.

Significance and Impact of the Study:  A novel and natural antibacterial compound as well as mango ginger extracts can be used as food preservative to control the growth of food-borne pathogens and as a source of mango flavour.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Food-borne illness remains a major concern even in industrialized countries (Gould et al. 1995). Therefore, there has been a growing interest in new and effective techniques to reduce the cases of food-borne illness (Otshudi et al. 1999). Antimicrobials from natural sources like plants have been used for food safety since antiquity (Alzoreky and Nakahara 2003). There is an increasing interest in the use of plant-derived antimicrobial compounds as natural food preservative. In addition, plant derivatives have unique structural diversity. This has led to a renewed interest in bioactive compounds from fruits, vegetables and spices.

Spices are used in foods primarily because they impart desirable flavours but they may fulfil more than one function to the food when they are added (Nasar-Abbas and Kadir Halkman 2004). Mango ginger (Curcuma amada Roxb.) belongs to the family Zingiberaceae. It is a perennial herb with modified fleshy stem called rhizome below the ground. The main use of mango ginger rhizome is in the manufacture of pickles. It has a morphological and phylogenic resemblance with ginger (Zingiber officinale) but imparts mango (Mangifera indica) flavour. Mango flavor is mainly attributed to car-3-ene and cis-ocimene among the 68 volatile aroma components present in the essential oil of mango ginger rhizome (Achut and Bandyopadhyaya 1984; Srinivas Rao et al. 1989; Singh et al. 2002, 2003). The mango ginger rhizome has been extensively used as appetizer, alexteric, antipyretic, aphrodisiac, laxative, and also, in the ancient Indian system of medicine known as Ayurveda, to cure biliousness, itching, skin diseases, bronchitis, asthma, hiccough and inflammation as a result of injuries (CSIR 1950; Kirtikar and Basu 1984; Warrier et al. 1994). In spite of various medicinal properties, high food value for its exotic flavour, there are no reports on purification and characterization of bioactive molecules from mango ginger in the literature.

An attempt has been made in the present investigation to isolate and characterize an antibacterial compound from mango ginger rhizome and to test its activity on a wide range of bacteria.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Plant material

Fresh and healthy mango ginger (C. amada Roxb.) rhizomes were procured from the local market, Mysore, India during December 2003. The plant was identified by Prof. Shivamurthy, head, Department of Botany, University of Mysore, Mysore, India. Rhizomes were washed, sliced and dried in a hot air oven at 50°C for 72 h and powdered to 60 meshes in an apex grinder (Apex Constructions, London).

Chemicals

All the chemicals used for extraction and column chromatography were of AR grade from Merck Limited, Mumbai, India; high-performance liquid chromatography (HPLC)-grade methanol from Ranbaxy fine chemicals limited, Mumbai, India. Silica gel (60–120 mesh) used for column chromatography was from Qualigens fine chemicals, Mumbai, India; Silica gel (100–200) used for column chromatography was from Loba Chemie Pvt. Ltd., Mumbai, India. Silica gel used for thin-layer chromatography (TLC) was from Glaxo Laboratories, Mumbai, India. Nutrient agar and nutrient broth were from HiMedia Laboratories Limited, Mumbai, India.

Bacterial strains and inoculum preparation

The antibacterial activity was tested against Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus mirabilis, Yersinia enterocolitica, Micrococcus luteus, Staphylococcus aureus, Enterococcus fecalis, Bacillus subtilis, Bacillus cereus and Listeria monocytogenes. These bacterial strains isolated from clinical samples were obtained from the Department of Microbiology, Mysore Medical College, Mysore, India. Their cultural characteristics and morphological features were reconfirmed and also subjected to standard biochemical tests (Krieg and Holt 1984; Sneath et al. 1986) before experimentation. The test organisms were maintained on nutrient agar slants.

Isolation of bioactive compound from chloroform extract

Preparation of extracts

The sequential extraction was carried out with the same powder using solvents of increasing polarity (Fig. 1). About 100 g of dry mango ginger powder was sequentially extracted using n-hexane, chloroform, ethyl acetate, acetone and methanol at room temperature (27°C) and at atmospheric pressure by shaking at 100 rev min−1. Solvent extraction was carried out for 48 h in total. After each solvent extraction step, the extracts were filtered and concentrated by using rotary evaporator (Buchi Rotavapor R-124, Switzerland). The concentrated extracts were freeze-dried to remove the solvent and stored in refrigerator. The yield of each extract was noted. All the extracts were screened for antibacterial activity by agar well-diffusion method. As the chloroform extract showed high antibacterial activity, it was selected for the isolation and purification of the bioactive compound. The entire procedure for extraction and purification is shown in Fig. 1.

image

Figure 1.  Flow chart for the isolation of bioactive compound from chloroform extract of mango ginger rhizome.

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Fractionation of the chloroform extract

Activated silica gel (60–120 mesh) was packed onto a glass column (450 × 40 mm) using n-hexane solvent. For large-scale isolation of compound, 15 g of crude chloroform extract was loaded on the top of silica gel. The column was eluted stepwise at a flow rate of 1 ml min−1 with 500 ml of hexane, 2000 ml of hexane : chloroform (75 : 25 to 0 : 100 v/v), 2000 ml of chloroform : ethyl acetate (75 : 25 to 0 : 100 v/v), 2000 ml of ethyl acetate : acetone (75 : 25 to 0 : 100 v/v) and 1500 ml of acetone : methanol (75 : 25 to 0 : 100 v/v). About 82 fractions measuring 100 ml each were collected and concentrated by using the rotary evaporator.

Thin-layer chromatography

An aliquot of all the concentrated fractions were loaded on the activated silica gel TLC plates (20 × 20 cm). The plates were developed using hexane : chloroform (80 : 20), chloroform : ethyl acetate (90 : 10) and ethyl acetate : methanol (90 : 10) solvents. The spots were located by exposing the plate to iodine fumes. Fractions having the same number of spots with similar Rf values on the TLC plate were pooled. The pooled fractions were numbered (Fr.1–Fr.5). All the five pooled fractions were tested for antibacterial activity as described in the following.

Further purification of bioactive fraction

As fraction three (Fr.3) obtained from the first step of column chromatography (Fig. 1) showed high antibacterial activity, it was selected for further purification. About 3·5 g of bioactive Fr.3 was further purified using silica gel (60–120 mesh) column (450 × 20 mm). The column was eluted stepwise at a flow rate of 1 ml min−1 with 100 ml of hexane, 200 ml of hexane : chloroform (90 : 10 to 0 : 100 v/v), 800 ml of chloroform : ethyl acetate (90 : 10 to 0 : 100 v/v), 600 ml of ethyl acetate : acetone (90 : 10 to 0 : 100 v/v) and 400 ml of acetone : methanol (90 : 10 to 0 : 100 v/v). About 21 fractions measuring 100 ml each were collected and concentrated in a rotary evaporator. An aliquot of all the fractions were loaded on the TLC plate, fractions having similar Rf values were pooled and numbered (Fr.1′–Fr.4′). These four fractions were tested for antibacterial activity.

Fraction two (Fr.2′) obtained from the second step (Fig. 1) showed high antibacterial activity, hence selected for further purification. About 800 mg of bioactive Fr.2′ was further purified on a silica gel (100–200 mesh) column (600 × 15 mm). The column was eluted stepwise at a flow rate of 1·5 ml min−1 with 100 ml of hexane : chloroform (90 : 10 to 0 : 100 v/v), 400 ml of chloroform : ethyl acetate (95 : 05 to 0 : 100 v/v) and 200 ml of ethyl acetate : acetone (95 : 05 to 0 : 100 v/v). About 28 fractions measuring 25 ml each were collected and concentrated. Fractions having similar Rf values on the TLC plate were pooled and numbered (Fr.1′′–Fr.3′′). Among these, fraction number two (Fr.2′′) obtained from the third step (Fig. 1) showed a single spot in the TLC profile. This pure compound was subjected to various spectroscopic techniques for elucidation of the structure.

High-performance liquid chromatography

The purified compound was tested for its purity using HPLC, using LC-10AT liquid chromatograph (LC; Shimadzu, Singapore) equipped with C-18 column (300 × 4·6 mm 5 μ Thermo Hypersil) and methanol : water (60 : 40) as a mobile phase with a flow rate of 1 ml min−1. Ultraviolet (UV) detection was carried out with a diode array detector (Shimadzu).

Characterization of bioactive compound

UV spectrophotometry

UV-visible spectrum of the isolated compound was recorded on a Shimadzu UV-160A instrument (Shimadzu) at room temperature. About 1 mg of the isolated compound dissolved in 20 ml of chloroform was used to record the spectrum (200–800 nm).

Infrared spectrometry

Infrared (IR) spectrum of the isolated compound was recorded on a Perkin-Elmer FT-IR Spectrometer (Spectrum 2000) at room temperature. About 1 mg of the isolated compound dissolved in 10 ml of dimethylsulfoxide (DMSO) was used to record the spectrum (frequencies between 4000 and 400 cm−1).

LC-mass spectrometry

Mass spectrum of the isolated compound was recorded on instrument HP 1100 MSD series (Palo Alto, CA, USA) by electrospray ionization (ESI) technique with a flow rate of 0·2 ml min−1 on the C-18 column and at a total run time of 40 min. Diode array was used as a detector. About 1 mg of the isolated compound dissolved in 5 ml of methanol was used for recording the spectrum.

Two-dimensional heteronuclear multiple quantum coherence transfer spectroscopy

Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX 500 NMR instrument (Rheinstetten, Germany) operating at 500 MHz for 1H and 125 MHz for 13C at room temperature. A region from 0 to 12 ppm for 1H and 0 to 200 ppm for 13C was employed. Signals were referred to internal standard tetramethylsilane. About 45 mg of the isolated compound dissolved in 0·75 ml of CDCl3 was used for recording the spectra.

Determination of antibacterial activity

Agar well-diffusion method

In vitro antibacterial activity was determined by agar well-diffusion method (Mukherjee et al. 1995). The overnight bacterial culture was centrifuged at 8000 rev min−1 for 10 min at 40C. The supernatant was discarded and the bacterial cells were resuspended in the saline to make the suspension 105 CFU ml−1 and used for the assay. The plating was carried out by transferring bacterial suspension (105 CFU ml−1) to sterile petri plate and mixed with molten nutrient agar medium (HiMedia Laboratories Limited, Mumbai, India) and allowed to solidify. About 75 μl of the sample (2 mg ml−1) was placed in the wells and allowed to diffuse for 2 h. Plates were incubated at 37°C for 48 h and the activity was determined by measuring the diameter of the inhibition zones. Solvent control and amoxicillin (Galpha Lab., Mumbai, India) were also maintained. The assay was carried out in triplicate.

Minimum inhibitory concentration

The minimum inhibitory concentration (MIC) was determined according to the method described by Jones et al. (1985). Different concentrations (20–300 ppm) of hexane, chloroform, ethyl acetate, acetone, methanol extracts, isolated compound (difurocumenonol) and 100 μl of the bacterial suspension (105 CFU ml−1) was placed aseptically in 10 ml of nutrient broth separately and incubated for 24 h at 37°C. The growth was observed both visually and by measuring OD at 600 nm at regular intervals followed by pour plating as described earlier. The lowest concentration of the test sample showing no visible growth was recorded as the MIC. Triplicate sets of tubes were maintained for each concentration of the test sample.

Determination of minimum bactericidal concentration

Minimum bactericidal concentration (MBC) was determined according to the method of Smith-Palmer et al. (1998). Test tubes containing nutrient broth with different concentrations of hexane, chloroform, ethyl acetate, acetone, methanol extracts, isolated compound (difurocumenonol) were inoculated with 100 μl of the bacterial suspension (105 CFU ml−1). Inoculated tubes were incubated for 24 h at 37°C and growth was observed both visually and by measuring OD at 600 nm. About 100 μl from the tubes not showing growth were plated on nutrient agar as described earlier. MBC is the concentration at which bacteria failed to grow in nutrient broth and nutrient agar inoculated with 100 μl of suspension. Triplicate sets of tubes were maintained for each concentration of the test sample.

Statistical analysis

The experiments were carried out in triplicates. Significant differences (P < 0·05) were determined by Duncan's Multiple Range Test (DMRT).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Antibacterial activity of mango ginger extracts

Sequential extraction of 100 g of mango ginger powder using hexane, chloroform, ethyl acetate, acetone and methanol yielded 11, 8, 0·9, 0·8 and 10 g of extract, respectively (Fig. 1). Among the five extracts of mango ginger, chloroform extract showed highest antibacterial activity against maximum number of bacteria, i.e. B. cereus, B. subtilis, M. luteus, Staph. aureus, L. monocytogenes, Enterococus fecalis and Salm. typhi. Hexane extract showed inhibition to all the bacteria as in chloroform extract except Salm. typhi. Ethyl acetate extract showed inhibitory effect as in chloroform extract except Salm. typhi and L. monocytogenes. Acetone extract inhibited M. luteus, L. monocytogenes while methanol extract showed inhibition only against Enterococcus faecalis. The chloroform extract of mango ginger exhibited a greater range of antibacterial activity compared with other solvent extracts (Table 1). However, E. coli, Enterobacter aerogenes, K. pneumoniae, Ps. aeruginosa, Pr. mirabilis and Y. enterocolitica were not inhibited by any of the solvent extracts of mango ginger. The high antibacterial activity with low MIC for different species of gram-positive bacteria exhibited by chloroform extract prompted to select for further purification and characterization of the antibacterial compound. The antibacterial activity of all the fractions (Fr. 1–Fr. 5 and Fr. 1′–Fr. 3′) obtained by repeated silica gel column chromatographic technique is shown in Tables 2 and 3.

Table 1.   Minimum inhibitory concentrations (MIC) for different extracts of mango ginger
BacteriaMIC (ppm)*
Hexane extractChloroform extractEthyl acetate extractAcetone extractMethanol Extract
  1. *Each value represents mean of three different observations. Mean values with different superscript letters differ significantly at P < 0.05.

  2. –, No zone of inhibition.

Pseudomonas aeruginosa
Escherichia coli
Salmonella typhi180a
Klebsiella pneumoniae
Enterobacter aerogenes
Proteus mirabilis
Yersinia enterocolitica
Micrococcus luteus80a80a100b200c
Staphylococcus aureus120c80a100b
Enterococcus fecalis180b140a140a220c
Bacillus cereus160c60a80b
Bacillus subtilis120b60a60a
Listeria monocytogenes80a100b140c
Table 2.   Antibacterial activity of different fractions of chloroform extract
BacteriaInhibition zone (mm)* exhibited by five fractions obtained from chloroform extract
Fr.1†Fr.2Fr.3Fr.4Fr.5
  1. *Each value represents mean of three different observations. Mean values with different superscripts differ significantly at P < 0·05.

  2. †Five fractions (Fr.1–Fr.5) are obtained from the chloroform extract by silica gel column chromatography. Each fraction was having unique spots on thin-layer chromatography plate.

  3. –, No zone of inhibition.

Pseudomonas aeruginosa
Escherichia coli
Salmonella typhi
Klebsiella pneumoniae
Enterobacter aerogenes
Proteus mirabilis
Yersinia enterocolitica
Micrococcus luteus15 ± 0·8b13 ± 0·5a17 ± 1·0c12 ± 0·3a
Staphylococcus aureus15 ± 0·5a
Enterococcus fecalis12 ± 0·2a12 ± 0·6a14 ± 0·5b
Bacillus cereus12 ± 0·5a13 ± 0·4a16 ± 0·6b
Bacillus subtilis13 ± 0·3a14 ± 0·8a16 ± 0·7b
Listeria monocytogenes14 ± 0·7a15 ± 0·9a13 ± 0·8a
Table 3.   Antibacterial activity of four fractions collected by column chromatography
BacteriaInhibition zone (mm)* exhibited by four fractions (Fr.1′–Fr.4′) obtained by second step of chromatography of fraction three (Fr.3)
Fr.1′†Fr.2′Fr.3′Fr.4′
  1. *Each value represents mean of three different observations. Mean values with different superscripts differ significantly at P < 0·05.

  2. †Four fractions (Fr.1′–Fr.4′) were obtained by second step of chromatography of ‘active fraction’ (Fr.3).

  3. –, No zone of inhibition.

Pseudomonas aeruginosa
Escherichia coli
Salmonella typhi
Klebsiella pneumoniae
Enterobacter aerogenes
Proteus mirabilis
Yersinia enterocolitica
Micrococcus luteus14 ± 0·6a18 ± 1·015 ± 0·8a14 ± 0·5a
Staphylococcus aureus14 ± 0·8a16 ± 0·313 ± 0·4a
Enterococcus fecalis13 ± 0·5a13 ± 0·3a12 ± 0·7a
Bacillus cereus12 ± 0·4a16 ± 0·513 ± 0·5a
Bacillus subtilis12 ± 0·9a16 ± 0·614 ± 0·7a
Listeria monocytogenes15 ± 0·8a

Purification and identification of bioactive compound

The pure compound was subjected to various spectroscopic analysis, i.e. UV, IR, LC-mass spectroscopy (MS) and two-dimensional heteronuclear multiple quantum coherence transfer spectroscopy (2D-HMQCT NMR) to deduce the structure. The compound exhibited UV maxima at 242 nm corresponding to ππ* transition of C = C double bonds. IR spectral data showed O–H stretching at 3442 cm−1, alkyl stretching at 2995 cm−1 and carbonyl stretching at 1670 cm−1 indicating the presence of OH and olefinic carbonyl groups. LC-MS data showed parent molecular ion peak at 498 and other major M/e fragments at 219 and 279 (Table 5). 2D-HMQCT spectrum showed as many as three CH3 groups as singlets (Table 6). Other three CH3 signals were observed as doublets and indicated that they were attached to CH carbons. The corresponding 13C signals for the remaining six CH3 groups were also observed. The region between 1·1 and 2·4 ppm indicated quite a lot of CH and CH2 signals with complex multiple splittings. The region between 3·5 and 4·5 ppm in 1H spectrum showed CH signals attached to OH groups along with the corresponding 13C signal. A carbonyl signal at 197·8 ppm was also observed. Some quaternary carbons and aromatic carbons in the region 97·2–116·2 ppm were observed. Olefinic carbon signals were observed at 136·1 ppm. A furan signal at 144·1 ppm was also observed. Based on all these spectral data, the structure was deduced to be a probable precursor of difurocumenone and was designated as a ‘difurocumenonol’ [13,15,23,25-tetrahydroxy-1,5,10,14,17,21-hexamethyl-7,19-dioxahexacyclo (13.9.1.0 2,14.0 4,8.0 16,24.0 18,22) pentacosa-4 (8), 5, 11, 16 (24), 18 (22), 20-hexaen-3-one] (Fig. 2).

Table 5.   Spectral data for the isolated compound
UV λmax (Chloroform)242 nm
  1. UV, ultraviolet; IR, infrared; LC-MS, liquid chromatography-mass spectroscopy.

IR data3442 cm−1 (–OH stretching), 2995 cm−1 (Alkyl –CH stretching), 1670 cm−1 (C = O stretching), 1437 cm−1 and 1055 cm−1
LC-MS498 M+4, 279 M/e and 219
Molecular formulaC29H34O7
Table 6. 1H and 13C data* (in ppm) of the isolated difurocumenonol
Signal13C (ppm)1H (ppm)
  1. *Some of the assignments are interchangeable; b, could not be detected

1-CH319·01·48
5-CH314·50·65
10-CH321·80·83
14- CH314·40·75
17 -CH320·22·10
21- CH314·40·72
9-CH238·32·40
2-CH55·51·15
6-CH144·07·05
10-CH29·51·25
11-CH136·14·45
12-CH115·56·75
13-CH56·53·95
17-CH38·02·40
20-CH144·07·10
23-CH50·83·50
25-CH65·54·55
3-CO197·8
C-143·1
C-4b
C-5b
C-8b
C-1442·0
C-1597·2
C-16b
C-18142·1
C-21b
C-22106·9
C-24116·2
image

Figure 2.  Structure of difurocumenonol.

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Minimum inhibitory concentration

Different solvent extracts of mango ginger showed MIC values ranging from 60 to 220 ppm (Table 1). The high antibacterial activity with low MIC for wide range of bacteria was exhibited by chloroform extract. Chloroform extract was very effective against B. subtilis, B. cereus with MIC of 60 ppm and also inhibited the growth of M. luteus and Staph. aureus at 80 ppm. While Salm. typhi, Enterococcus fecalis and L. monocytogenes were completely inhibited at 180, 140 and 100 ppm, respectively. However, all the mango ginger extracts and isolated difurocumenonol were not effective against E. coli and P. mirabilis (Tables 1 and 4). Antibacterial activity against gram-negative bacteria (Ps. aeruginosa, E. coli, Salm. typhi, K. pneumoniae, Enterobacter aerogenes, Pr. mirabilis and Y. enterocolitica) was observed in ‘active fraction-2′′’ (Fr.2′′) obtained from repeated column chromatographic technique (Fig. 1).

Table 4.   Antibacterial activity of difurocumenonol
BacteriaDifurocumenonol
MIC (ppm)*Bactericidal activity
  1. *Each value represents mean of three different observations. The minimum inhibitory concentration (MIC) values also represent the bactericidal concentrations for all the bacteria.

  2. –, No inhibition.

Pseudomonas aeruginosa100No
Escherichia coliNo
Salmonella typhi80No
Klebsiella pneumoniae160No
Enterobacter aerogenes120No
Proteus mirabilisNo
Yersinia enterocolitica60Yes
Micrococcus luteus60Yes
Staphylococcus aureus60Yes
Enterococcus fecalis120No
Bacillus cereus40Yes
Bacillus subtilis40Yes
Listeria monocytogenes40Yes

The most striking increase in antibacterial activity of difurocumenonol was observed against B. cereus, B. subtilis, and M. luteus with MIC 40, 40 and 60 ppm, respectively. In addition, difurocumenonol also inhibited the growth of five gram-negative bacteria, i.e. Ps. aeruginosa, Salm. typhi, K. pneumoniae, Enterobacter aerogenes and Y. enterocolitica with MIC of 100, 80, 160, 120 and 60 ppm, respectively (Table 4). However, these were not inhibited by the chloroform extract.

Determination of bactericidal effect

Difurocumenonol was found to be bactericidal against a wide range of bacteria tested (Table 4). MBC ranged from 40 to 60 ppm for gram-positive bacteria (M. luteus, Staph. aureus, B. cereus, B. subtilis and L. monocytogenes). Interestingly, difurocumenonol also showed bactericidal activity against only one of the six gram-negative bacteria (Y. enterocolitica) at 60 ppm. It appeared that effective MIC also represents the effective bactericidal concentration for the bacteria tested.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The results indicated differential activity between polar and nonpolar solvent extracts of mango ginger. It appeared that, with the increase in polarity of the solvent there was a decrease in antibacterial activity and range of bacterial strains. High resistance of gram-negative bacteria to mango ginger extracts may be because of the presence of their outer layer composed of lipopolysaccharides, which acts as an effective barrier for many hydrophobic molecules. In contrast, lack of outer polysaccharides layer in gram-positive bacteria may be responsible for more permeability to amphipathic compounds (Cowan 1999).

The repeated antibacterial activity-guided fractionation of chloroform extract by silica gel column chromatography yielded pure compound. The structure of the isolated compound was deduced as ‘difurocumenonol’ after extensive analysis of spectroscopic data. This is the first antibacterial compound isolated and characterized from mango ginger rhizome.

Difurocumenonol is a novel compound exhibiting an interesting antibacterial activity. Testing of active chloroform extract fractions and difurocumenonol with a representative panel of bacteria showed that difurocumenonol was more effective against wide spectrum of bacteria, i.e. Ps. aeruginosa, Salm. typhi, K. pneumoniae, Enterobacter aerogenes, Y. enterocolitica, M. luteus, Staph. aureus, Enterococcus fecalis, B. cereus, B. subtilis and L. monocytogenes.

High antibacterial activity of difurocumenonol against a wide range of bacteria may be attributed to its structural components. Difurocumenonol possesses four-hydroxyl, six-methyl and one-carbonyl groups along with two furan rings. Difurocumenonol by virtue of possessing two furan rings, which are aromatic in nature, thus possesses units, which are capable of exhibiting delocalization of electrons, a feature that has been proposed to be responsible for increased antibacterial activity (Ultee et al. 2002). These may account for the enhanced activity of difurocumenonol compared with its source extract. The bioactivity of difurocumenonol may be similar to several other compounds like curcumin, capsaicin, caffeic acid, carvacrol, eugenol and menthol (Apisariyakul et al. 1995; Cichewicz and Thorpe 1996; Ali-shtayeh et al. 1997; Cowan 1999). In addition, presence of hydroxyl groups in plant derivatives has been associated with many biological activities (David 1995; Haliwell et al. 1995; Tess et al. 1999; Laurence et al. 2001; George et al. 2002; Adewole et al. 2004; Sara 2004). Hydroxyl group may be actively responsible for depletion of ATP-dependent metabolic functions, ultimately leading to cell death (Ultee et al. 2002). Further, presence of oxygen function in the framework of the compound increases the antibacterial properties (Naigre et al. 1996). Further investigations are in progress to test the mode and site of action of the difurocumenonol.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Difurocumenonol is a novel antibacterial compound that was successfully isolated and characterized from mango ginger rhizome. This is the first report on antibacterial activity of mango ginger extracts and difurocumenonol. Difurocumenonol in contrast to its source extract demonstrated a pronounced increase in its antibacterial activity to a wide spectrum of bacteria including gram-negative bacteria. Screening of difurocumenonol for other functional properties like antiviral, anticancer and antioxidant activities are to be explored for its efficacy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

We thank Dr V. Prakash, Director, Central Food Technological Research Institute, Mysore for his keen interest in the work and encouragement. Mr Policegoudra R.S. thanks the Council of Scientific and Industrial Research, New Delhi, India, for awarding the Senior Research Fellowship. We also thank the Sophisticated Instruments Facility, IISc, Bangalore, for the NMR analysis and Molecular Biophysics Unit, IISc, Bangalore for the LC-MS analysis and Mr Ravi R., Scientist, Department of Sensory Science, CFTRI, Mysore for his help in statistical analysis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
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