AJ – 2013 - 667 Article-25 Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 16, No. (1) : 2014 : 145-154 © Global Science Publications ISSN-0972-3005
COMPARATIVE EVALUATION OF ANTIMICROBIAL ACTIVITIES OF ROOT, STEM AND LEAVES OF HOLOPTELEA INTEGRIFOLIA AGAINST PATHOGENIC BACTERIA KAVITHA ALLI AND LAKSHMI NARASU MANGAMOORI* Centre for Biotechnology, IST, Jawaharlal Nehru Technological University, Hyderabad 500 072, India. (Received : 27 July, 2013; accepted : 9 September, 2013) Key words: Holoptelea integrifolia, Zone of inhibition, Agar well diffusion method, Minimum inhibitory concentration, Phytochemical components, GC-MS analysis.
Abstract–Holoptelea integrifolia, a large spreading glabrous tree belonging to the family of ulmaceae is widely used in traditional medicine for treatment of cancer, gastritis, diabetes, wound healing and is known to possess many phytochemicals. The nature of phytochemicals present in the stem, leaf and roots were analyzed with different solvents using GC-MS analysis and evaluated for antimicrobial activity. The antimicrobial activity of these extracts were checked against a range of gram positive and gram negative pathogens using agar well diffusion method. The minimum inhibitory concentration and minimum bactericidal concentration was determined by two-fold broth dilution method and compared against the standard antibiotic chloramphenicol. The antimicrobial activities were comparable to those obtained with standard antibiotics. Root extracts had exhibited highest antimicrobial activity followed by stem and leaves. Acetone extracts of root and ethyl acetate extract of stem had exhibited good activity against the pathogens tested. The GC-MS analysis also revealed phenols, carboxylic acids, esters and other phytochemical components for Holoptelea integrifolia, which assured that it has high antimicrobial activity against pathogens. This study clearly demonstrates that Holoptelea integrifolia has good antimicrobial properties and the root has better antimicrobial properties than leaves and stem. All extracts except the aqueous extract had significant broad-spectrum inhibitory activity. Our study is the first to present the evidence of better antimicrobial properties of root over stem and leaves.
INTRODUCTION The quest for plants with antimicrobial properties continues to thrive as scholars survey plants for various phytochemical compounds. Nature has been a source of medicinal plants for thousands of years and the use of medicinal plants for relief from illness can be traced back to five thousand years of written documents of early civilization in India, China and the near east. WHO advocated traditional medicines as remedies for ailments of both microbial and non-microbial origins (WHO Technical Report, 1978).The development and spread of multidrug resistance among pathogens (Cohen, 1992; Neu, 1992; Yurdakok et al. 1997) has led to the search for novel antimicrobials of plant origin (Olukoya et al. 1993). The use of plant extracts and phytochemicals for treatment of microbial infections is a major step towards handling of *Corresponding author’s email:
[email protected]
Abbreviation GC-MS : Gas Chromatography- Mass Spectrometry, WHO: World Health Organization HPLC : High Performance Liquid Chromatography, NIST: National Institute of Standards and Technology MHA : Muller-Hinton Agar, MHB: Muller-Hinton Broth, MIC : Minimum Inhibitory Concentration, MBC: Minimum Bactericidal Concentration
multidrug resistant pathogens. Earlier reports indicate that Caryophyllus aromaticus (clove), Syzygyum joabolanum (jambolan), Punica granatum (pomegranate) and Thymus vulgaris (thyme) extracts inhibit the growth of multidrug resistant forms of pseudomonas aeruginosa (Nascimento et al. 2000) . Plants synthesize a number of phytochemicals that possess medicinal properties. A number of
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phytochemicals have been identified as active principles for treatment of various ailments (Taylor et al. 2001; Ncube et al. 2008). The study of pharmacological activity of compounds paves the way for the synthesis of more potent drug with reduced toxicity (Pamplona et al. 1999; Manna et al. 2000). As per WHO estimates nearly80 percent of the population of developed countries use traditional medicines derived from medicinal plants for primary healthcare. There is an urgent need for a thorough investigation of the phytochemicals and their properties (Ellof, 1998). In view of this fact, current study has been carried out on Holoptelea integrifolia. Its vernacular name is Nemali chettu (Telugu) and commonly known as Indian Elm tree. Holoptelea integrifolia belongs to the family of Ulmaceae and it is widely distributed all over tropical and temperate regions of northern hemisphere including Indian peninsula, China, Burma and Srilanka. The leaves and stem of this plant are used by local people for gastritis, skin diseases, obesity (Bambhole et al. 1985), cancer (Graham et al. 2000) and for wound healing in the form of paste. The fresh material, stem or leaves of plant is applied as paste externally twice or thrice a day for wound healing purpose (Sidhu et al. 1999). The bark and leaves of the plant are reported to contain anti-inflammatory, carminative, anthelmintic, depurative, astringent, dyspepsic and antidiabetic properties (Warrier et al. 1995). Wound healing activities of leaves and stem of Holoptelea integrifolia was studied extensively (Reddy et al 2008) and the antibacterial activity of stem bark using methanol, petroleum ether, benzene, chloroform was also reported (Nadella et al 2011). Chloroform extract has shown better antimicrobial properties against the tested microorganisms. A comprehensive Study of all the phytochemical extracts has not been done till date with reference to their antimicrobial properties. The present communication reports the antimicrobial activities of leaves stem and roots extracts obtained with different solvents and also reports various phytochemicals responsible for this antimicrobial activity. MATERIALS AND METHODS Materials Plant Material Holoptelea integrifolia was collected from Narsapur
forest area, Medak district, of Andhra Pradesh, India. Plant is authenticated by Dr.Rasingham, Botanical Survey of India, Hyderabad,India. Chemicals and Drugs Methanol, Acetone, Ethyl acetate, Muller-Hinton Agar and Muller-Hinton broth were bought from Himedia Mumbai, India. Microorganisms All the microorganisms used for antimicrobial screening were procured from Global Hospitals, Hyderabad, India. A total of ten microorganisms were selected for screening, of which four were gram positive - Staphylococcus aureus,Bacillus subtilis,Enterococcus faecalis and Micrococcus luteus and six were gram negative - Pseudomonas aeruginosa,Escherichia coli,Salmonella typhimurium, Klebsilla pneumoniae,Enterobacter cloacae and Proteus vulgaris. Bacterial stock cultures were maintained on Muller Hinton agar slants and were stored at 4oC. Preparation of extracts Fresh leaves, stem and root were taken and washed with free flowing clean water and later washed with distilled water. Washed leaves, stem and root were shade dried, to retain the active components of plant material. After drying, the plant material was chopped into small pieces and then powdered using motor and pestle. 30gms of powdered material was dissolved in 300ml of solvent in a glass Stoppered round bottom flask. The mixture was shaken well and kept at room temperature in shaking incubator for 72 hrs. The extracts were filtered by using Whatman no.1 filter paper. Then the extracts were concentrated in a rotavapor at reduced pressure below 40 oC and evaporated to dryness in vacuum oven at 40 oC. Water extract of Holoptelea intigrifolia was prepared as above by soaking dried powder in distilled water and stirred with a magnetic stirrer at a low speed for 24hr. The extract was filtered using Whatman no.1 paper and was subsequently lyophilized in a lyophilizer at 5µm Hg pressure at -50oC. The extracts obtained were stored at 4oC until further use. Antimicrobial activity Sensitivity test by agar well diffusion method: Testing for antimicrobial activity was done on different microorganisms (four gram positive, six gram negative) according to the method described by National committee for clinical laboratory
Comparative Evaluation of Antimicrobial Activities of Root, Stem and Leaves of Holoptelea standards M7-A4 1997 ( Perez et al. 1990) . Culture media was prepared using 38g/L Muller Hinton agar (MHA) and autoclaved at 121oC; 15psi for 20 min. 20mL of MHA was poured on petri dish and allowed to solidify. 0.1ml of an actively growing culture (approximately 1x108 CFU/ml) was spread with sterile swab evenly on the surface of a MullerHinton Agar plate. 10mg of dried extract of leaves stem and root of Holoptelea integrifolia was dissolved in 1mL of HPLC grade solvents ethyl acetate, acetone, methanol and distilled water separately. A well of 8mm diameter was cut in the medium and 0.1ml of Methanol, Acetone, Ethyl acetate and aqueous extracts were added to the wells. The plates were incubated at 37oC for 24h. The results were recorded by measuring the zones of inhibition surrounding the well. Similar method was followed to measure the zone of inhibition for the antibiotic chloramphenicol at a concentration of 1mg/mL. Minimum Inhibitory Concentration (MIC) The tube broth dilution assay described by (Muroi and Kubo, 1996) was followed for determining MIC. The two-fold serial dilution assay was performed in Muller-Hinton broth (MHB). The stock solution of the extracts and antibiotic chloramphenicol were prepared to a final concentration of 5 mg/mL. The stock solution was serially diluted by two fold to obtain concentrations ranging from 5 mg/mL to 0.005mg/mL. Each tube was filled with 2.5mL MHB containing 50µL inoculum. The turbidity of the suspension was adjusted to match 0.5McFarland standard using a spectrophotometer at 600nm, which corresponds to 1.6x108cfu/mL. 500µL of each dilution was added to the tube containing suspension and was incubated at 37oC for 24h.The lowest concentration showing inhibition of growth of bacteria was determined as MIC. This assay was repeated three times and final MIC was determined based on mean of the three values. Minimum Bactericidal Concentration (MBC) For determination of MBC, a portion of 5µL from each tube that showing no turbidity was diluted with drug-free MHB and incubated at 37oC for 48h. The lowest concentration of the tube with no visible growth was regarded as MBC. MBC assay was also performed in triplicate. GC-MS analysis The bioactive compounds in all extracts of Holoptelea integrifolia were analyzed by GC-MS using an
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Agilent 6890 N gas chromatograph coupled to an Agilent 5975 N mass spectrometer. An HP-5MS capillary column (30m X 0.25mm ID, 0.25µm film thickness) was used for gas chromatographic separation. The instrument was set to an initial temperature of 60 oC and maintained at this temperature (isothermal) for 2 minutes. The GC oven temperature was programmed from 60oC to 280oC at a rate of 10oC/min and ending with a 9min isothermal at 280oC. The injection volume was 2µL with a split ratio of 1:10, the injection temperature was held at 220oC. Helium (99.999%) was used as carrier gas at 1mL/min. The mass spectrometer was operated in electron impact (EI) mode with ionization energy of 70 eV and source temperature was held at 280oC.MS spectra were obtained in the mass range of m/z 43-450. Interpretation of mass spectrum of GC-MS was done using database of NIST (National Institute Standards and Technology). Compound identification was done by comparing the retention times with those of authentic compounds and the spectral data obtained from NIST Ver2.0 MS library data of the corresponding compounds. RESULTS Antimicrobial sensitivity test The methanol, acetone and ethyl acetate extracts of leaves, stem and root has shown inhibition effects on all the ten microorganisms. Aqueous extracts have shown no inhibition or less than 9mm inhibition on all ten microorganisms. Acetone extract of root showed the maximum zone of inhibition of 22.6mm. Methanol extracts have shown zone of inhibition ranging from 11.8mm to 19.9mm, acetone extracts have shown zone of inhibition from 12.4mm to 22.6mm and ethyl acetate has zone of inhibition from 12.2mm to 21.9mm. Chloramphenicol has shown Zone from 21mm to 24mm. All the values of zone of inhibition are shown in Table 1. Ethyl acetate extract of stem was effective against Pseudomonas aeruginosa and Salmonella typhimurium. Ethyl acetate extracts of leaf and root were found to be sensitive to Klebsiella pneumonia. Acetone extracts of root was effective against salmonella typhimurium and klebsiella pneumonia. Acetone extract of stem is effective against Pseudomonas aeruginosa and leaf against Bacillus subtilis. Methanol extracts of stem and roots are effective against Klebsiella pneumonia and Bacillus subtilis. Staphylococcus aureus and E.coli
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are less sensitive to leaf extracts of all solvents and Proteus vulgaris is least sensitive to root extracts of all solvents. MIC and MBC Comparison of MIC, MBC values shows the extent of inhibitory activities of different extracts of leaves, stem. The inhibitory activities of ethyl acetate extract of stem, acetone extract of root on Proteus vulgaris, Salmonella typhimurium were close to that of standard drug Chloramphenicol than other extracts. The MIC’s of leaves ranged from 0.156mg/mL to 1.250mg/mL. stem ranged from 0.03906mg/mL to 1.250mg/mL and root ranged from 0.039mg/mL to 1.250mg/mL. The MIC and MBC values varied from organism to organism and depending on the type of extracts used. The MIC and MBC’s of different microorganisms for all solvents were given in Table 2. The tables also include the MIC, MBC’s for
standard drug Chloramphenicol. GC-MS analysis This report is the first of its kind to analyze the chemical constituents responsible for the antimicrobial activity of Holoptelea Integrifolia. GCMS analysis of each sample contained 55-75 compounds that matched NIST library data. GC-MS phytochemical screening results of all extracts showed presence of 110 compounds whose % peak area were greater than 3%. Table 3 gives the retention times and the relative percentages of the compounds present in respective extracts. Some of the main compounds like n-Hexadecanoic acid, phytol and some more fatty acids, carboxylic acids were seen to be present in all the extracts, but differed in their relative amounts, indicating their role in antimicrobial activity. Some of the GC-MS peaks remained unidentified because lack of authentic samples and library data of corresponding
Table 1. Antimicrobial sensitivity of all extracts and chloramphenicol – Zone is in mm and extracts is 10mg/mL Pathogenic organism
Part tested
Aqueous
S.aureus
Leaf Stem Root Leaf Stem Root Leaf Stem Root Leaf Stem Root Leaf Stem Root Leaf Stem Root Leaf Stem Root Leaf Stem Root Leaf Stem Root Leaf Stem Root
-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-NIL-
B.subtilis
E.faecalis
M.luteus
P.aeuruginosa
E.coli
S.typhimurium
K.pneumonia
E.cloacae
P.vulgaris
Methanol
Acetone
Ethyl acetate
Chloromophenicol (1 mg/mL)
14.8 16.6 15.1 18.3 15.4 17.2 15.4 14.6 12.6 16.6 15.4 11.8 15.2 16.6 17.2 13.6 14.1 14.8 17.4 17.3 18.2 16.5 18.6 19.9 17.2 15.8 15.4 14.7 14.3 13.6
13.4 15.6 21.2 18.7 17.9 19.6 14.5 14.8 16.4 14.9 13.2 18.2 16.4 19.4 20.8 12.4 16.5 19.4 15.6 17.1 22.6 15.2 17.9 22.2 17.0 16.9 17.8 15.2 14.4 16.8
13.6 17.8 17.2 16.4 17.0 16.1 13.1 15.3 12.8 15.1 13.6 12.2 16.8 19.7 18.4 14.8 19.1 16.9 16.2 21.9 17.6 20.1 15.3 18.4 16.2 17.4 16.2 16.6 15.6 16.6
21.4
22.4
21.1
23.2
22.8
21.6
22.5
22.0
22.6
21.6
Comparative Evaluation of Antimicrobial Activities of Root, Stem and Leaves of Holoptelea compounds. The best results in terms of peak quality and clarity of the extracts were obtained with acetone extract of root and ethyl acetate extract of stem aligning with the antimicrobial results obtained. Fig 1, 2, 3 shows typical total ion chromatograms of good quality chromatograms among all results. The main compounds in methanol extracts of leaves were n-Hexadecanoic acid , 9 12 15Octadecatrienoic acid methyl ester (Z,Z,Z)- and Octadecanoic acid, stem were Tridecanoic acid, Stigmasterol 22 23-dihydro- and 18-Nonadecenoic acid and that of roots were 1 3 4Trimethyladamantane, allyl ethyl ester, 2-Propenal 3-(2,6,6-trimethyl-1-cyclohexen-1-yl)- and 1 2Benzenedicarboxylic acid dipropyl ester. Some
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compounds present in acetone were also found in methanol extracts like n-Hexadecanoic acid, 9 12 15Octadecatrienoic acid methyl ester (Z, Z, Z) - and tetradecanoic acid. New compounds in acetone extracts of leaves were Isophytol, 7,10,13Hexadecatrienoic acid, methyl ester, in stem were 6Tetradecyne, Z-5-Nonadecene and Cyclotetracosane, in root were Octadecane, 1 2Benzenedicarboxylic acid butyl cyclohexyl ester and Cyclodecasiloxane eicosamethyl-. Compounds which are common to ethyl acetate extracts and other extracts were n-Hexadecanoic acid, 9 12 15Octadecatrienoic acid methyl ester (Z, Z, Z)-, Tetradecanoic acid and Isophytol. New compounds in ethyl acetate extracts of leaves were 9 12 15Octadecatrienal, in stem were 9 17-Octadecadienal
Table 2. MIC, MBC (values in brackets) of extracts and chloramphenicol against test microorganisms. All values are measured in mg/mL Pathogenic organism
Part tested
Methanol
Acetone
Ethyl acetate
Chloromophenicol
Staphylococcus aureus
Leaf Stem Root
0.625(1.250) 0.625(1.250) 1.250(1.250)
1.250(1.250) 0.625(1.250) 0.078(0.312)
1.250(1.250) 0.156(0.312) 0.312(0.625)
0.0125(1.250)
Bacillus subtilis
Leaf Stem Root
0.156(0.625) 0.625(1.250) 0.312(0.625)
0.156(0.312) 0.312(0.625) 0.156(0.312)
0.312(0.625) 0.156(0.312) 0.312(0.625)
0.0200(0.020)
Enterococcus faecalis
Leaf Stem Root
0.312(0.625) 0.625(1.250) 0.625(1.250)
0.156(0.312) 0.312(0.625) 0.312(0.625)
0.625(0.625) 0.156(0.312) 0.625(0.625)
0.0062(0.078)
Micrococcus luteus
Leaf Stem Root
0.156(0.312) 0.156(0.312) 0.312(0.625)
0.625(1.250) 0.312(0.625) 0.156(0.312)
0.312(0.625) 0.312(0.625) 0.312(0.625)
0.0310(0.312)
Pseudomonas aeuruginosa
Leaf Stem Root
0.625(1.250) 0.625(1.250) 0.312(0.625)
0.312(0.625) 0.156(0.312) 0.078(0.312)
0.312(0.625) 0.078(0.156) 0.156(0.625)
0.0780(0.625)
Escherichia coli
Leaf Stem Root
0.625(1.250) 0.312(0.625) 0.156(0.312)
0.625(0.625) 0.312(0.625) 0.156(0.312)
0.312(0.625) 0.156(0.312) 0.312(0.625)
0.0062(0.312)
Salmonella typhimurium
Leaf Stem Root
0.312(0.625) 0.312(0.625) 0.156(0.312)
0.625(1.250) 0.312(0.312) 0.039(0.078)
0.625(0.625) 0.039(0.156) 0.312(0.625)
0.0025(0.312)
Klebsiella pneumonia
Leaf Stem Root
0.312(0.625) 0.312(0.625) 0.156(0.312)
0.312(0.625) 0.156(0.312) 0.039(0.156)
0.078(0.156) 0.078(0.312) 0.156(0.312)
0.0125(0.156)
Enterobacter cloacae
Leaf Stem Root
0.312(0.625) 0.625(1.250) 0.312(0.625)
0.312(0.312) 0.625(0.625) 0.312(0.625)
0.312(0.625) 0.312(0.625) 0.625(1.250)
0.0125(0.312)
Proteus vulgaris
Leaf Stem Root
0.625(0.625) 0.625(1.250) 0.625(0.625)
0.625(1.250) 0.625(0.625) 0.312(0.625)
0.312(0.625) 0.312(0.625) 0.625(1.250)
0.0390(0.078)
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Fig. 1. Total Ion Chromatograph of Acetone extract of root from GC-MS Analysis
Fig. 2. Total Ion Chromatograph of ethyl acetate extract of stem from GC-MS Analysis
(Z)-, benzyl alcohol and in root were 2-Allylphenol, Benzoic acid 2-(hydroxymethyl)-. Analysis results also showed lot of compounds with less than 10% area and presence in multiple extracts. DISCUSSION AND OBSERVATIONS The results in Tables 1, 2 show that Holoptelea
integrifolia root, stem and leaf extract had an excellent anti-bactericidal activity against both Gram-positive and Gram-negative bacteria. Results also showed that the root had shown higher zone of inhibition compared to stem, leaves against most of the microorganisms, but leaf extract was found to be more effective against Bacillus subtilis and stem extract is found to be more effective against
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 31 32 33 34 35 36
2,3,5,6-Tetrafluoroanisole 2,5-Dimethoxy-4-ethylamphetamine 1(3H)-Isobenzofuranone 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol Pyrazine, 2-methoxy-3-(1-methylethyl) 3,5-Dihydroxytoluene 4-Methylimidazole-5-[1,1-dimethylb utyric acid amide] Bicyclo[3.1.1]heptane, 2,6,6-trimethyln-Hexadecanoic acid Tridecanoic acid Phytol 9,12,15-Octadecatrien-1-ol, (Z,Z,Z )9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)Cyclooctene, 3-ethenylOctadecanoic acid Hexadecane Benzoic acid, 2-(hydroxymethyl)Tetradecanoic acid Octadecane 3-Butylindolizidine 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester 1-Ethyl-3-propyladamantane 1,3,4-Trimethyladamantane 2-Propenal, 3-(2,6,6-trimethyl-1-c yclohexen-1-yl)6-Octadecenoic acid, (Z)9-Octadecenoic acid, (E)9,12-Octadecadienoic acid (Z,Z)Stigmasterol, 22,23-dihydro.beta.-Sitosterol 1,2-Benzenedicarboxylic acid, dipropyl ester Azulene, 1,4-dimethyl-7-(1-methylethyl)3,5-di-tert-Butyl-4-hydroxybenzaldehyde Nonadecane 4,8,12-Tetradecatrienal, 5,9,13-trimethylHexadeca-2,6,10,14-tetraen-1-ol, ,7,11,16-tetramethyl-, (E,E,E)-
Compound name 11.0 11.0 11.09 13.01 13.01 13.84 13.84 13.97 15.28 15.28 16.62 16.95 16.95 16.95 17.11 19.73 12.05 16.20 14.31 14.78 15.10 15.42 15.42 15.42 17.85 17.85 18.24 20.06 20.06 10.16 11.18 11.82 12.41 12.85 12.85
RT 4.22 4.22 2.71 3.26 3.26 4.76 4.76 1.37 9.70 9.70 9.82 19.09 19.09 19.09 8.06 1.86 -
1.11 3.81 19.5 19.5 3.70 2.79 19.5 1.09 3.81 2.63 3.06 3.06 3.06 12.6 12.6 2.32 10.9 10.9 -
10.8 1.18 0.90 8.58 12.2 12.2 12.2 15.5 3.24 1.51 0.90 5.94 5.94
Methanol (Area %) Leaves Stem Root
Table 3. Chemical compounds found in Hopotelea integrifolia extracts using GC-MS analysis
0.63 0.85 4.38 12.86 18.93 21.63 21.63 7.41 12.86 2.03 2.03 2.03 0.31 -
2.62 6.12 13.1 0.19 3.07 24.6 24.6 10.0 13.14 0.50 -
2.80 4.73 2.80 20.1 14.3 5.14 3.87 -
Acetone (Area %) Leaves Stem Root 0.39 0.35 10.1 18.2 18.2 14.7 26.9 28.5 8.41 -
4.07 0.71 17.6 0.54 11.9 6.87 1.31 4.07 26.3 17.8 -
8.16 1.09 4.23 4.23 8.02 8.16 2.78 4.46 8.2 -
Ethyl acetate (Area %) Leaves Stem Root
Comparative Evaluation of Antimicrobial Activities of Root, Stem and Leaves of Holoptelea 151
7.42 14.7 8.41 0.15 7.40 2.90 9.23 9.23 1.70 5.07 24.6 9.23 6.48 6.48 6.48 1.30 18.93 21.63 7.41 5.34 5.34 0.22 0.22 0.09 0.51 14.40 16.62 16.90 17.09 19.34 19.34 16.53 16.53 17.86 19.54 19.67 19.67 19.67
0.54 15.5 6.10 6.12 4.38 4.38 9.33 7.04 13.17 14.39 13.97 13.97
41 42 43 44 45 46 47 48 49 50 51 52 53
1,2-Benzenedicarboxylic acid, butyl decyl ester 1,2-Benzenedicarboxylic acid, bis(2-ethylbutyl) ester 1,19-Eicosadiene Bicyclo [3.1.1] heptane, 2, 6, 6-trimethyl-, [1R-(1.alpha. 2. beta., 5.alpha.)]3,7,11,15-Tetramethyl-2-hexadecen-1-ol Isophytol 7,10,13-Hexadecatrienoic acid, methyl ester Pentadecanoic acid Cyclotetracosane 9-Tricosene, (Z)Heptadecane Eicosane 6-Tetradecyne Z-5-Nonadecene Disulfide, di-tert-dodecyl Tetrapentacontane, 1,54-dibromoTetradecane, 1-bromo37 38 39 40
Ethyl acetate (Area %) Leaves Stem Root Acetone (Area %) Leaves Stem Root Methanol (Area %) Leaves Stem Root RT Compound name
Table 3. Continued..
1.23 3.59 -
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Pseudomonas aeuruginosa and Salmonella typhimurium. In this study it was observed that extraction of the plant with the solvents ethyl acetate, and acetone resulted in much greater antibacterial activity against all the pathogenic bacteria than extraction with methanol. In particular, the ethyl acetate extract of stem and acetone extract of root was found to be more effective. Micrococcus luteus and Proteus vulgaris are less susceptible to methanolic, ethyl acetate extracts. Acetone extracts were more effective against Salmonella typhimurium, Klebsiella pneumonia and Pseudomonas aeruginosa. This study recorded a notable susceptibility of these resistance strains, especially to root extract, suggesting that the components contained in that particular extract may provide an alternate strategy for combating these organisms and thus could improve the treatment of infections caused by these organisms. The above results indicate that root has very good antimicrobial properties, which was ignored till now by research community. Ethyl acetate extracts have higher zone of inhibition and better antimicrobial properties than methanol. The MIC, MBC values varied from one microorganism to another, but showed the same trend as that of zone of inhibition in most of the cases. Good results with acetone extract of roots and ethyl acetate extract of stem indicate a difference in chemical nature of the constituents due to their solubility in different solvents. GC-MS analysis results showed the same trend observed in zone of Inhibition and MIC results. All the samples contained fatty acids and carboxylic acids which were known to possess antimicrobial properties. Samples also showed presence of terpenes, phenols, sterols, Quinone’s, siloxanes, alkanes, alkaloids and small quantities of aromatic alcohols, proteins. The acetone extract of root, ethyl acetate, methanol extracts of stem possessed vide varieties of compounds and quantitatively fatty acids, carboxylic acids were the major category of compounds. Isolated 1,4-Naphthalenedione whose antimicrobial properties were validated in (Vinod et al. 2010) was found in acetone extracts of root. Fatty acids like 6-
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Fig. 3. Total Ion Chromatograph of methanol extract of stem from GC-MS Analysis
Octadecenoic acid, (Z)-, Tridecanoic acid, nHexadecanoic acid, Tetradecanoic acid, 9,12,15Octadecatrienoic acid, methyl ester, (Z,Z,Z)- were present in good quantities in most of the extracts. These components have reported anti-inflammatory, antimicrobial activities. Analysis also revealed presence of terpenes like Bicyclo[3.1.1] heptane, 2,6,6-trimethyl-, phytol, Hexadeca-2,6,10,14-tetraen1-ol, ,7,11,16-tetramethyl-, (E,E,E), Hexadeca2,6,10,14-tetraen-1-ol, ,7,11,16-tetramethyl-, (E,E,E), Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-, [1R(1α,2β,5α)]-, α- Amyrin and β- Amyrin. Samples contained small quantities of phenols. All the root extracts had significant quantities of alkanes like eicosane, heptadecane, tetracosane, 9-Tricosene, (Z)and Octadecane. Siloxanes present in methanol extract of leaves are also established as antimicrobial phytochemical components. Hexadecane which is present in most of the extracts was known to be effective against Pseudomonas aeruginosa. Better antimicrobial properties of acetone extract of root, ethyl acetate extract of stem can be attributed to fatty acids, terpenes and other phytochemicals with known antimicrobial activities. These extracts also had phytochemicals with known medicinal properties like anti-inflammatory, anti-oxidant and anti-cancer activities. However phytochemical composition of each extract is different from the other extracts. Studies on Holoptelea integrifolia done
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