The aim of this study is to carry out preliminary investigation of bioactive compounds and bioautographic studies of whole plant extract of
The search for solutions to the global problems of antibiotic resistance in pathogenic microorganisms has often been driven on the isolation and characterization of new antimicrobial compounds from a variety of sources including medicinal plants [
The plant family Euphorbiaceae contains skin irritating and tumor promoting diterpenoids [
However, some species are used in folk medicine to treat skin diseases, gonorrhea, migraine, intestinal parasites, and warts [
The active principles of many drugs found in plants are secondary metabolites [
This present study focused on identification of bioactive compounds in whole plant extract of
The samples of
The
The crude methanol extract of whole plant of
The ethyl acetate extract used for the column chromatography was in crude form. The weight of the extract used was 4 g. This was eluted with 200 mL of 100% n-hexane first and then with varying ratios of hexane : ethyl acetate used. 20 mL of eluents was collected at intervals and similar compounds were pooled together after TLC.
A glass column of length 30 cm and width 3 cm parked with silica gel was used for the partitioning of the ethyl acetate fraction. The ethyl acetate fraction was first eluted with 200 mL of 100% n-hexane and two different sets of fractions were collected and labeled as A and B. The fraction obtained by eluting the column with 9 : 1 n-hexane : ethyl acetate was labeled as C. n-Hexane : ethyl acetate in the ratio 4 : 1 eluted fractions D, E, F, and G. 7 : 3 n-hexane : ethyl acetate mixture eluted the fraction labeled H. The fractions were further used to confirm the bioactivity of the ethyl acetate fraction.
The A, B, C, D, E, F, G, and H fractions obtained from the column chromatography were concentrated on rotary evaporator and analysed using GC-MS-QP2010 Plus Shimadzu and FT-IR model 8400S scanned in accordance with ATSM1252-98 to determine the probable compounds responsible for the bioactivity of the fractions.
The test organisms were typed cultures of
McFarland’s standard method was adopted from Cheesebrough [
The agar well diffusion method of the National Committee on Clinical Laboratory Standard [
The agar overlay method was adopted for the bioautographic studies. The agar media were applied directly onto the developed TLC plate of the whole plant ethyl acetate fraction, TLC chromatogram. 19 mL of molten nutrient agar was seeded with 1 mL of standardized overnight culture of the susceptible organism. This was poured over the developed chromatogram TLC plate kept in a Petri dish. This was allowed to solidify and prediffuse for 2 hrs before incubation at 37°C for 24 hrs for the bacterial isolates. The plates were sprayed with aqueous solution of methyl thiazolyl tetrazolium (MTT) chloride for the detection of dehydrogenase activity. Zones of inhibition were observed as clear spots against purple background.
The percentage yield for the
The thin layer chromatograms of the ethyl acetate fraction revealed the presence of eight compounds with different Rf values as shown in Table
Rf values for different compounds present in the ethyl acetate fraction.
Spots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
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Rf values | 0.16 | 0.42 | 0.54 | 0.65 | 0.71 | 0.78 | 0.83 | 0.89 |
The partitioning of the ethyl acetate fraction on silica gel column using 100% n-hexane, 9 : 1 hexane : ethyl acetate, 4 : 1 hexane : ethyl acetate, and 7 : 3 hexane : ethyl acetate also revealed sixty-eight fractions of different colours that were grouped into eight using the similarity in their Rf values (Table
Different fractions of the ethyl acetate fraction partitioned on silica gel column.
Serial number | Solvent system | Fractions eluted | Colour | Group |
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1 | n-Hexane | 1, 2, 3 | Black | A |
2 | n-Hexane | 4, 5, 6, 7, 8, 9, 10 | Light green | B |
3 | Hexane : ethyl acetate (9 : 1) | 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 | White | C |
4 | Hexane : ethyl acetate (4 : 1) | 21, 22, 23, 24 | Yellow | D |
5 | Hexane : ethyl acetate (4 : 1) | 25, 26, 27, 28, 29, 30, 31, 32, 33 | Dark brown | E |
6 | Hexane : ethyl acetate (4 : 1) | 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 | Dark green | F |
7 | Hexane : ethyl acetate (4 : 1) | 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 | Green | G |
8 | Hexane : ethyl acetate (7 : 3) | 63, 64, 65, 66, 67, 68 | Brown | H |
The results of the GC-MS analysis of fractions A, B, C, D, E, F, G, and H revealed different phytochemicals (terpenoids, flavonoids, alkaloids, saponin, and steroids) as shown in Tables
GC-MS chromatogram for
GC-MS chromatogram for
GC-MS chromatogram for
GC-MS chromatogram for
GC-MS chromatogram for
GC-MS chromatogram for
GC-MS chromatogram for
GC-MS chromatogram for
These functional groups were revealed by the FT-IR analysis of the ethyl acetate fraction (see Table
FT-IR spectra of the ethyl acetate extract.
Serial number | Peaks in ethyl acetate extract | Motion | Functional group |
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1 | 725.26 |
C–H bending out of plane | Aromatics |
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2 | 1041.6 |
C–N stretching |
Amines |
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3 | 1249.91 | C–C(O)–C | Esters |
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4 | 1373.36 | CH3 bend | Alkanes |
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5 | 1456 | CH3 bend | Alkanes |
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6 | 1612.54 |
N–H stretching | Nitrogenous compounds |
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7 | 3379.4 | O–H stretch | Alcohol/phenol |
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8 | 1041.6 | C–O stretch | Phenol |
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9 | 1724 | C=O stretching | Aliphatic aldehydes |
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10 | 2856.67 | Carboxylic acid O–H stretch | Acid |
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11 | 2924.18 | C–H stretching | Aliphatic hydrocarbons |
Phytochemicals in
Peak number | Name | Structure |
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4 | 3-Fluorophenyl 2-fluoro-6-(trifluoromethyl)benzoate |
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14 | 4-Bromophenyl heptyl phthalate |
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19 |
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34 | Bis(4-methylheptan-3-yl) phthalate |
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Phytochemicals in
Peak number | Name | Structure |
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8 | Menthyl acetate |
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10 | 2,4,4-Trimethylpentyl 2-ethylbutanoate |
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18 | 2,2,6-Trimethyl-7-(prop-1-en-2-yl)cyclooctane-1,5-dione |
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19 | Heptan-3-yl isobutyl phthalate |
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20 | 4-(((Tetrahydrofuran-2-yl)methyl)amino)-1-oxaspiro[4.5]dec-3-en-2-one |
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23 | 7,9-Di- |
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25 | Butyl heptan-3-yl phthalate |
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28 | 2-( |
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32 | 2-Methyl-2-(3-methyl-2-oxobutyl)cyclohexanone |
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34 | 1,2,3,5-Tetraisopropylcyclohexane |
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39 | Oleamide |
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44 | Bis(2-ethylhexyl) phthalate |
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Phytochemicals in
Peak | Name | Structure |
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1 | 2,6-Di- |
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3 | 2,4-Di- |
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12 | Diisobutyl phthalate |
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14 | 7,9-Di- |
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17 | Dibutyl phthalate |
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24 | 4,4,6a,6b,8a,11,11,14b-Octamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-ol |
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34 | Lup-20(29)-en-3-ol, acetate |
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35 | 4-(3-Hydroxy-3-methylpentyl)-3,4a,8,8-tetramethyldecahydronaphthalen-1-ol |
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Phytochemicals in
Peak | Name | Structure |
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5 | 1,2-Dimethyl-3-(prop-1-en-2-yl)cyclopentane |
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8 | Heptan-4-yl isobutyl phthalate |
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13 | Dibutyl phthalate |
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20 | 4,4,6a,6b,8a,11,11,14b-Octamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-ol |
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22 | 4,4,6a,6b,8a,11,11,14b-Octamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12b,13,14,14a,14b-icosahydropicen-3-ol |
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24 | (8 |
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25 | 4a,8,8a-Trimethyl-2-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,8a-octahydronaphthalene |
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30 | 3a-(Hydroxymethyl)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-1 |
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32 | 2a,5a,8,8-Tetramethyl-3-(6-methylheptan-2-yl)hexadecahydrocyclopenta[ |
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Phytochemicals in
Peak | Name | Structure |
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7 | Diisobutyl phthalate |
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26 | 5-Methyl-5-(4,8,12-trimethyltridecyl)dihydrofuran-2(3 |
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29 | Bis(2-ethylhexyl) phthalate |
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34 | ( |
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Phytochemicals in
Peak | Name | Structure |
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1 | 2-Phenylacetic acid |
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3 | 4-(2-Hydroxyethyl)-2-methoxyphenol |
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4 | 2,4-Di- |
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5 | 4,4,7a-Trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4 |
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9 | Triethyl 2-hydroxypropane-1,2,3-tricarboxylate |
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32 | ( |
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Phytochemicals in
Peak | Name | Structure |
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1 | 2,4-Di- |
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2 |
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6 | 7,9-Di- |
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19 | 4,4,6a,6b,8a,11,12,14b-Octamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-ol |
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Phytochemicals in
Peak | Name | Structure |
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4 |
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5 | 2,3,6-Trimethylnaphthalene |
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7 | 3-(2-Methylprop-1-en-1-yl)-1 |
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9 | 2,3,6-Trimethylnaphthalene |
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13 | 2-Phenylbicyclo[3.2.1]octa-2,6-diene |
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14 | Undecan-5-ylbenzene |
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18 | (4,5,5-Trimethylcyclopenta-1,3-dien-1-yl)benzene |
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21 | (2-Methyl-[1,1′-biphenyl]-3-yl)methanol |
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31 | Phenanthrene |
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39 | 4-Methylnaphtho[1,2- |
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43 | 7,9-Di- |
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49 | 5-Methyl-3,3-diphenyl-3 |
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The results of the bioautography tests of ethyl acetate extract against
Bioautography results of ethyl acetate extract against
The chromatogram of the
All the compounds A, B, C, D, E, F, G, and H showed good activity against the test organisms with zones of inhibitions ranging between 18 and 35 mm at the highest concentration level (100 mg/mL).
The bioautography results confirmed the presence of different antimicrobial compounds in the ethyl acetate fraction. All the compounds A, B, C, D, E, F, G, and H showed good activity against the test organisms with zones of inhibitions evident from the clearing of the spots from the TLC plates (Figure
The ethyl acetate fraction of the methanol extract of the whole plant of
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are grateful to Benedict O. Odjobo, Analytical Unit, Federal Institute of Industrial Research, Oshodi, for the GC-MS analysis and the National Research Institute for Chemical Technology, Zaria, for the FT-IR.