THE ANTIMICROBIAL ACTIVITY OF n-C6H14 AND CH3CH2OH EXTRACT OF SOLANUM MELONGENA FRUIT AND LEAVES OF MORINGA OLEIFERAHTML Full Text
THE ANTIMICROBIAL ACTIVITY OF n-C6H14 AND CH3CH2OH EXTRACT OF SOLANUM MELONGENA FRUIT AND LEAVES OF MORINGA OLEIFERA
R.C. Jagessar* and N. Ramchartar
Department of Chemistry, University of Guyana, Georgetown, Guyana
ABSTRACT: As part of a research initiative to evaluate plants used for their nutritional and herbal values, the antimicrobial activity of Solanum Melongena’s fruit and Moringa oleifera’s leaveswere investigated. Each plant part was subjected to selective extraction using solvents of varying polarity: n-C6H14, CH2Cl2, EtOAc and CH3CH2OH. The n-C6H14 and CH3CH2OH extract of these two plants were tested for their antimicrobial activity at three different concentrations of 5%, 10% and 20% of crude extracts against three pathogenic bacterial strains: Eschericia coli, Staphyloccocus aureus and Klebsiella pneumoniae using the Disc diffusion assay. Both n-C6H14 and CH3CH2OH extracts showed antibacterial activity at a higher concentration of 20% of crude extract. The order of bacteria susceptibility to Moringa oleifera extract been S. aureus > K. pneumoniae > E. coli whereas that for Solanum Melongena extract been S. aureus > E. coli > K. pneumonia. The area of zone of inhibition ranges from 44.15 mm2 to 53.55 mm2. These investigations suggest that the extracts of Moringa oleifera and Solanum Melongena can be used as antibacterial agents in addition to their nutritional value.
Antimicrobial, Solanum Melongena, Moringa oleifera, E. coli, S. aureus and K. pneumonia, Bacteria susceptibility
INTRODUCTION: Guyana has a rich bio diversified flora whose organic and aqueous extract have been shown to possess potent and selective antimicrobial activity compared with standard antibiotics such as penicillin, nystatin and ampicillin 1-7 etc. Research in the design and syntheses of antimicrobials will be everlasting endeavours on our planet considering the fact that bacteria and fungus developed resistance to antimicrobials over a period of time 8-13. Antibiotic resistance has become a global concern 13. This is primarily due to indiscriminate use of commercial antimicrobial drugs used for the treatment of infectious diseases.
This has led to the search for new antimicrobials, both herbal and synthetic.
However, synthetic drugs/medicine has several adverse side effects which are usually irreversible when administered and the cost of synthesizing drugs in most cases is an expensive endeavour 10-12. In addition, phytochemical screening and natural products isolation can lead to novel and know natural products whose in vitro antimicrobial activity can be correlated with that of the crude plant extract 14-15.
There is also a need to assess the medicinal values of plant used as food source. Thus, efforts should be made to intensify the production of food crops in the agro-industry that have antimicrobial properties in addition to their nutritional properties. As such, the antimicrobial activity of the n-C6H14 and CH3CH2OH extract of Solanum melongena (Solanaceae) and Moringa oleifera (Moringaceae)were investigated in vitro against pathogenic Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. Such an endeavour will be a boost to the agro industry and Health Sector.
Moringa oleifera is the most widely cultivated species of the genus Moringa, the only genus in the Moringaceae. This plant is rich in unique compounds such as glucosinolates and isothiocyanates. Natural products such as 4-(4'-O-acetyl-a-L-rhamno pyranosyloxy)benzyl isothiocyanate, 4-(a-L-rhamno pyranosyloxy)benzyl isothiocyanate, niazimicin, pterygospermin (2), benzyl isothio cyanate (1) and 4-(α-L-rhamnopyranosyloxy)benzyl glucosinolate isolated from Moringa species have been reported to have hypotensive and anticancer activity. Phytochemicals such as the carotenoids (β-carotene or pro-vitamin A have also been isolated 16, 17. The structures of two of these compounds are shown in Fig. 1.
FIG. 1: BENZYL ISOTHIOCYANATE (1) AND PTERYGOSPERMIN (2) FROM MORINGA OLEIFERA
The leaves are the most nutritious and contain significant amount of vitamin B6, vitamin C, provitamin A, β-carotene, magnesium and protein. Calcium in Moringa oleifera leaves are usually complexed as crystals of calcium oxalate.
Moringa oleifera provides a rich and rare combination of zeatin, quercetin, kaempferom and many other phytochemicals such as hexadecanoic acid, ethyl palmitate, palmitic acid, ethyl ester, 2,6-Dimethyl-1, 7-octadiene-3-ol, 4-Hexadecen-6-yne, 2-hexanone, and 3-cyclohexyliden-4-ethyl - E2- Dodecenylacetate 17.
It is very important for its medicinal value. Various parts of the plant such as the leaves, roots, seed, bark, fruit, flowers and immature pods act as cardiac and circulatory stimulants, possess anti-tumour,anti-pyretic, anti-epileptic, anti-inflammatory and anti-ulcer activity 17.
Moringa oleifera preparations have been used for its anti-trypanosomal, hypotensive, antispasmodic, antiulcer, anti-inflammatory, hypocholesterolemic, and hypoglycemic activities, as well as having considerable efficacy in water purification by flocculation, sedimentation, antibiosis and even reduction of Schistosome cercariae titer 18.
A new biflavonol glycoside, Solanoflavone was isolated from aerial part of Solanum melongena. The chemical structure was elucidated as isorhamnetin-3-O-beta-D-glucopyranoside-(4'->O->4''')-galangin-3''-O-beta-D-glucopyranoside on the basis of physico-chemical and spectroscopic methods, including 2D NMR spectral techniques 19.
Flavonoids were isolated from Solanum melongena and shown to have potent antioxidant activity. Concentrations of malondialdehyde, hydroperoxides and conjugated dienes were lowered significantly 20.
Phenylethyl cinnamides as potential alpha-glucosidase inhibitors were isolated from the roots of Solanum melongena (Solanaceae). Bioassay-guided fractionation against alpha-glucosidase resulted in isolation and identification of six phenolic compounds from the 70% EtOH extract of the roots. Three of the phenylethyl cinnamides, N-trans-feruloyl tyramine, N-trans-p-coumaroyl tyramine and N-cis-p-coumaroyl tyramine possessed inhibitory activity against alpha-glucosidase with IC50 values of 500.6, 5.3 and 46.3 microM, respectively. Mechanisistic studies revealed these phenylethyl cinnamides as non-competitive inhibitors. The above is the first study of the alpha-glucosidase inhibitory activities of the roots of Solanum melongena, suggesting potential medicinal use of this herb 22.
Phytochemical examination of the methanolic and aqueous extracts of the fruit and crown of Solanum Melongena showed the presence of alkaloids, saponins, steroids, tannins/ phenolics, flavonoids, proteins and carbohydrates. Ascorbic acid and phenolics both which are powerful antioxidants were present in fruit, the presence of saponins and glycoalkaloids which were also found in the fruit protects plant from microbial pathogens.
Various parts of Solanum melongena (Solanaceae) are useful in the treatment of inflammatory conditions, cardiac debility, neuralgia, ulcers of nose, cholera, bronchitis and asthma. Roots are used as anti-asthmatic and general stimulant, juice is employed for otitis, applied to ulcers of the nose. Leaves are used in the treatment of bronchitis, asthma and dysuria, also given in liver complaints and they stimulate inter hepatic metabolism of cholesterol. The fruit of Solanum melongena is a high valued vegetable all over the world because of its taste and higher percentage of Vitamin B2. The fruit is also used in the treatment of diabetes 22.
MATERIALS AND METHODS:
Reagents and materials: Antibiotics, Ampicillin, Mueller Hinton Agar, agar plates were purchased from the International Pharmacy Association (IPA) in Guyana. Bacterial culture was obtained from the Georgetown Public hospital.
Collection of Plant material: Fresh leaves of Moringa oleifera and fruits of Solanum meologena were handpicked at Friendship village on the Corentyne, Berbice, and placed in bags. These were washed with tap, distilled water and were dried for four (4) hours. They were further air dried for one week and sent for authentication at the Centre for the Study of Biological Diversity, University of Guyana.
Grinding and Extraction: Approximately six hundred grams (600 g) of the fruit of the Solanum melongena were cut into small pieces and blended thrice in six hundred milliliters (600 ml) of n-C6H14. The contents were then filtered into air tight glass containers. The procedure was repeated using the more polar CH3CH2OH solvent. Leaves of Moringa oleifera, 600 grams in weight was also blended thrice in 600 ml of n-C6H14. The contents were filtered into air tight glass containers. The procedure was repeated using freshly distilled CH3CH2OH. The contents for each extraction was filtered, solvents dried over anhydrous Na2SO4 and removed in vacuo using a rota vapor, resulting in viscous oils.
Reference and Control: Ampicillin was chosen as the reference for all bacteria species used: E. coli, S. aureus and Klebsiella pneumoniae. TheControl experiment consists of a plate of solidifying agar onto which was inoculated pure solvent with microorganism mixed in a 1:1 portion 23-24.
Antimicrobial tests: Plant extracts were investigated for their antimicrobial activity using the Disc Diffusion assay 23-24.
Source of microorganisms: Gram negative (-) E. coli, Klepsiella pneumoniae and Gram positive (+) strains Staphylococcus aureus (ATCC 25923) were obtained from the Georgetown Public Hospital, GPH and stored in a refrigerator until required.
Positive control: In this study, tetracycline was used as a positive control to screen and analyze the antimicrobial properties of the different medicinal plants. This antimicrobial drug is clinically effective against both gram- negative as well as gram positive microbes. Side-effects from tetracycline are not common. Use of the antibiotic have known to cause stomach or bowel upsets and on rare occasions, allergic reactions.
In vitro Antimicrobial Susceptibility Tests: The Disc diffusion method was used to screen plant extracts for its in vitro antimicrobial activity. Plates were labeled according to extract, concentration and bacteria. Using the Disc diffusion assay 24,an inoculum containing bacteria cells were applied onto Mueller Hinton agar plates. A sterile swabbed was dipped into the bacteria culture and was uniformly spread on the surface of the Mueller Hinton agar. This was allowed to dry for 10 minutes. On each plate, four discs were placed equidistant using a sterilized tweesor. One of these is the reference disc onto which antibiotic was also applied and was used as the positive control: ampicillin for the bacteria. The reference antibiotic disc contained 200mg antibiotic/ml.
The discs were made by cutting discs (5-6mm) from a filter paper with a sterilized perforator. Each disc was impregnated with the anticipated antimicrobial plant extract of Solanum Melongena and Moringa oleifera at appropriate concentrations of 5%, 10% and 20 % of n-C6H14 or CH3CH2OH extract using a microlitre syringe. The plates were then incubated with the test organism: Bacteria at 37ºC for 24 hours. The antimicrobial compound diffuses from the disc into the medium. Following overnight incubation, the culture was examined for areas of no growth around the disc (zone of inhibition, ZOI). The diameter of the zone of inhibition was measured using a transparent plastic ruler. Each experiment was done in triplicates (table 1).
TABLE 1: MEAN, STANDARD DEVIATION AND AREA OF ZONE OF INHIBITION FOR THE n-C6H14 AND CH3CH2OH EXTRACT OF SOLANUM MELONGENA AND MORINGA OLEIFERA
|Sample||Pathogenic Microorganism||Concentration(%)||Mean Diameter||Mean Diameter with Standard deviation||Area of Zone of Inhibition(mm2)|
|Solanum melogena Hexane||E. coli||5||4.43||4.43 ±3.85||15.04|
|10||4.46||4.46 ± 2.97||15.65|
|20||7.03||7.03 ± 0.25||38.79|
|S. aureus||5||6.77||6.77 ± 1.04||35.87|
|10||7.1||7.1 ± 0.22||39.57|
|20||5.03||5.03 ± 2.53||19.86|
|Klebsiella spp||5||2.33||2.33 ± 1.04||4.26|
|10||7.97||7.97 ± 3.87||48.99|
|20||7.17||7.17 ± 0.25||40.24|
|Solanum melongena Ethanol||E. coli||5||7.2||7.2 ± 0.71||40.69|
|10||7.43||7.43 ± 0.30||43.33|
|S. aureus||5||7.87||7.87 ± 0.32||48.49|
|10||7.73||7.73 ± 0.64||46.9|
|20||8.27||8.27 ± 0.21||53.55|
|Klebsiella spp||5||7.03||7.03 ± 0.11||38.79|
|10||7.53||7.53 ± 0.32||44.51|
|Moringa oleifera Hexane||E. coli||5||4.4||4.4 ±3.81||15.19|
|S. aureus||5||4.66||4.66 ±4.07||17.04|
|klebsiella spp||5||7.33||7.33 ± 0.28||42.17|
|10||7.26||7.26 ± 0.20||41.37|
|20||4.86||4.86 ± 4.23||18.54|
|Moringa oleifera Ethanol||E. coli||5||6.73||6.73 ±0.25||33.55|
|10||4.76||4.76 ± 4.12||17.78|
|20||7.73||7.73 ± 0.11||46.9|
|S. aureus||5||5||5 ± 4.35||38.46|
|Klebsiella spp||5||6.93||6.93 ±0.05||37.69|
Positive control: (Table 2)
TABLE 2: AREA OF ZONE OF INHIBITION, ZOI FOR THE POSITIVE CONTROL, TETRACYCLINE AGAINST PATHOGENS
|Microorganism||Area of zone of inhibition (mm2)|
DISCUSSION: Antimicrobial properties of Solanum melogena and Moringa oleifa C2H5OH and n-C6H14 extracts were investigated in vitro at concentrations of 5%, 10% and 20% using the Disc diffusion assay. Investigations were done against three pathogenic microorganisms: E. coli, S. aureus and Klebsiella pneumonia using the Disc diffusion assay. The area of zone of inhibition was used as the guage of the plant’s antimicrobial properties. Larger the diameter of zone of inhibition, greater is the plant’s antimicrobial activities. It is anticipated through the antimicrobial activity of plant extract, no area of growth will be induced around the disc. Bacteria colonies sensitive to the antimicrobial are inhibited at a distance from the disc whereas resistant strains grow up to the edge of the disc. Discs applied to the plates already streaked with bacteria and the fungus.
A comparison of the effect of the various solvent extracts against the three pathogenic microorganisms at three different concentrations can be discussed. In general, there seem to be an increase in the plant’s extract antimicrobial activity as the concentration of the extract is increased. For example, Solanum melongena C2H5OH extract induces area of zone of inhibition (ZOI) of 40.69, 43.33 and 45.7 mm2 against E.coli as the concentration of the plant extract increased from 5% to 20%. Likewise Moringa oleifera CH3CH2OH extract induces area of zone of inhibition of 37.69, 42.17 and 49.36 mm2 against Klebsiella pneumoniae at concentration of 5, 10 and 20% of extract respectively.
However, there were exceptions to the above general increase in bacterial activity. For example, Solanum melogena n-C6H14 extract showed an increase in antimicrobial activity against S.aureus followed by a decrease at the 20% concentration. For example, area of zone of inhibition of 35.87 mm2, 39.57 mm2 and 19.86 mm2 was observed at concentration of 5, 10 and 20% of extract. Moringa oleifera C2H5OH extract also showed a decreased in antimicrobial activity followed by an increase.
For example, against Klebsiella species value of 33.35 mm2, 17.78 mm2 and 46.0 mm2 were obtained at the respective concentrations of 5, 10 and 20 % of extract. Of significance, there was a decrease in the area of zone of inhibition for Moringa oleifera hexane extract against Klebsiella species at all three concentrations. Area of zone of inhibition of 42.17 mm2, 41.37 mm2 and 18.54 mm2 were obtained against Klebsiella species at concentrations of 5, 10 and 20% of extract. The highest area of zone of inhibition of 53.55 mm2 induced by Solanum melogena C2H5OH extract against S. aureus at 20% concentration of extract.
The smallest area of zone of inhibition of 15.04 mm2 was induced by Solanum melogena n-C6H14 extract against E. coli, where values of 15.04 mm2, 15.65 mm2 and 38.79 mm2 were registered at the respective concentration. The C2H5OH extract of either plant seems to be more antimicrobial than the n-C6H14 extract, suggesting greater localisation of plant natural products antimicrobial agents or the interactions of natural products via non covalent interactions to produce novel antimicrobial systems or assemblies. For example, Solanum melogena n-C6H14 extract induces area of zone of inhibition of 35.37 mm2, 39.57 mm2 and 19.86 mm2 against S. aureus. However, Solanum melogena CH3CH2OH extract induced area of zone of inhibition of 48.49 mm2, 46.9 mm2 and 53.53 mm2 against S. aureus at concentration of 5%, 10% and 20% concentration respectively.
FIG. 2: AREA OF ZONE OF INHIBITION (mm2) OF PLANT EXTRACTS AGAINST E. COLI AT CONCENTRATION of 5, 10 and 20%
Graph 1 shows the area of ZOI (mm2) at 5%, 10%, & 20% concentrations of both plant extracts against colonies of E. coli.
From the graph it can be observed that the n-C6H14 extract of Moringa oleifera was more antibacterial. Values of 38.79 mm2 and 39.12 mm2 were recorded against E. coli. Also, at the 20% concentration, Moringa oleifera C2H5OH extract was more antimicrobial than Solanum melogena. Values of 46.9 mm2 and 45.7 mm2 were registered respectively.
FIG. 3: AREA OF ZONE OF INHIBITION (mm2) OF PLANT EXTRACTS AGAINST S. AUREUS AT CONCENTRATION OF 5, 10 AND 20%.
Graph 2 shows the area of ZOI (mm2) at 5%, 10%, & 20% concentrations of both plant extracts against colonies of S. aureus. From the graph, the n-C6H14 extract of Moringa oleifera is more antimicrobial than that of Solanum melogena. Values of 44.51 mm2 and 19.86 mm2 were observed respectively. However, Solanum Melongena C2H5OHextract is more antimicrobial against S. aureus than Moringa’s C2H5OH extract at the 20% concentration. Values of 53.55 mm2 and 51.5 mm2 were observed respectively.
FIG. 4: AREA OF ZONE OF INHIBITION (mm2) OF PLANT EXTRACTS AGAINST KLEPSIELLA SPECIES AT CONCENTRATION OF 5, 10 AND 20%.
Graph 3 shows the area of ZOI (mm2) at 5%, 10% and 20% concentrations of plant extract against colonies of Klebsiella pneumoniae. From the graph it can be observed that the n-C6H14 extract of Solanum melogena induces a higher area of zone of inhibition against Klebsiella pneumoniae compared with Moringa oleifera at the 20% concentration. Values of 40.24 cm2 and 18.54 cm2 were registered respectively. Likewise, C2H5OH extract of Solanum Melongena were more antimicrobial than Moringa oleifera at 20% concentration of plant extract. Areas of ZOI registered were 44.15 mm2 and 49.36 mm2 respectively.
Antimicrobial activity was also investigated for the positive control, tetracycline against the pathogens. It is found that the diameter of the zone of inhibition, ZOI is less than that induced by the n-C6H14 and CH3CH2OH extract of both plants. This suggests and justifies the use of these fruits as potent antimicrobial agent in addition to their nutritional status.
CONCLUSION: From this study, it can be concluded that n-C6H14 and CH3CH2OH extract of Solanum melogena and Moringa oleifera possess antibacterial activity as significant zone of inhibition were observed. The area of ZOI ranges from 19.86 mm2 to 53.55 mm2. The CH3CH2OH extracts showed more potential antimicrobial properties than the n-C6H14 extract. The n-C6H14 and CH3CH2OH extract of both plants showed selective antimicrobial activity against the three pathogens: E. coli, S. aureus and Klebsiella pneumoniae. Against, E. coli and S. aureus, Moringa oleifera n-C6H14 is more resistant than S. melogena. Solanum melogena extract is more resistant against Klebsiella pneumonia compared to Moringa oleifera extract. For CH3CH2OH extract, against E. coli and Klebsiella pneumoniae, Moringa oleifera extract is more resistant. However, Solanum melogena extract is more resistant against S. aureus.
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How to cite this article:
Jagessar RC and Ramchartar N: The antimicrobial activity of n-C6H14 and CH3CH2OH extract of Solanum melongena fruit and leaves of Moringa oleifera. Int J Pharm Sci Res 2013; 4(6); 2214-2220. DOI: 1
R.C. Jagessar* and N. Ramchartar
Department of Chemistry, University of Guyana, Georgetown, Guyana
14 January, 2013
23 April, 2013
29 May, 2013
01 June, 2013