No. |
Chemical Compound |
Reagent or Treatment |
Results (color / precipitate) |
Conclusion |
1. |
Alkaloids |
Dragendorff Bouchard at Mayer |
(+) orange-brown (+) yellow-brown (+) turbidity and a white precipitate |
(+) |
2. |
Anthraquinone glycosides |
NaOH |
not occurred intensively redness color on NaOH layer |
(-) |
3. |
Flavonoids |
Zn add concentrated hydrochloric acid Mg add concentrated hydrochloric acid |
(+) red |
(+) |
4. |
Glycosides |
Molisch Fehling Liebermann-Burchard |
(+) purple rings (+) orange-red precipitate (+) red |
(+) |
5. |
Saponins |
Hot water and shaken |
(+) stability foam |
(+) |
6. |
Cyanogenic glycosides |
Pikrat sodium |
not occurred orange color in Whatman paper |
(-) |
7. |
Tanins |
FeCl3 1% |
(+) green-black |
(+) |
8. |
Triterpenoids / Steroid |
Liebermann-Burchard |
(+) old purple |
(+) triterpenoids |
Note: (+) = positive compound, (-) = negative compound
Results of simplicia phytochemistry screening on outer layers of onion (Allium cepa L.) bulbs indicated the existence of chemical compounds in methanol extract from directly maceration using methanol, this showed methanol as a solvent that can dissolve various compounds.
Result of Chemical Compound Analysis by Thin Layer Chromatography Results of chemical compounds analysis of hexane extract, ethyl acetate extract and methanol extract from stratified maceration method yield carried out thin layer chromatography (TLC). Stationary phase used aluminum plate of TLC silica gel 60 F254 (Merck®). Mobile phase used hexane:ethyl acetate (7:3 for hexane extract and 3:7 for ethyl acetate and also methanol extract) (pro analysis, Merck®) with visualization agent was 50% sulfuric acid in methanol to display the entire class of chemical compounds contained in each extract, Liebermann-Bourchard to detect the presence of free compounds of triterpenes and steroids, Dragendorff to detect alkaloid class of compounds and 5% iron (III) chloride to detect a class of polyphenolic compounds. Results variety chromatograms and Rf value for each extract can be seen in Table-2, 3 and 4 below.
Chromatograms result of hexane extract with using hexane:ethyl acetate (7:3) as mobile phase and 50% sulfuric acid in methanol as visualization was provided 13 spots that showed the presence of at least 13 compounds. Chromatogram results using Liebermann-Bourchard as visualization was obtained 7 spots (6 of them was purple and 1 green) which mean there was triterpenoids compound (purple) and steroids compound (green) whereas visually, FeCl3 5% and Dragendorff was not visible spots, it means that there were not alkaloids compounds detected by Dragendorff and no polyphenol compounds detected by FeCl3 5%.
Table-2: Results of Chromatogram and Rf Value of Hexane Extract of Outer Layers of Onion (Allium cepa L.) Bulbs
Chromatograms result with using mobile phase hexane:ethyl acetate (7:3) |
||||
visually |
H2SO4 50% reagent |
Liebermann-Burchard reagent |
FeCl3 5% reagent |
Dragendorff reagent |
not visible |
0,90(p) 0,80(p) 0,76(p) 0,70(g) 0,67(v) 0,55(y) 0,51(p) 0,48(y) 0,38(v) 0,36(g) 0,31(y) 0,20(b) 0,15(v) |
0,76(p) 0,67(p) 0,55(g) 0,48(p) 0,38(p) 0,31(p) 0,20(p) |
not visible |
not visible |
Note: b = brown, g = green, p = purple, v = violet, y = yellow
Table-3: Results of Chromatogram and Rf Value of Ethyl Acetate Extract of Outer Layers of Onion (Allium cepa L.) Bulbs
Chromatograms result with using mobile phase hexane:ethyl acetate (3:7) |
||||
visually |
H2SO4 50% reagent |
Liebermann-Burchard reagent |
FeCl3 5% reagent |
Dragendorff reagent |
not visible |
0,90(br) 0,88(p) 0,82(p) 0,80(br) 0,64(br) 0,61(p) 0,59(b) 0,46(br) 0,44(b) 0,38(br) 0,35(g) 0,29(y) |
not visible |
0,90(g) 0,80(g) 0,64(yg) 0,46(yg) 0,38(yg) 0,35(bl) |
0,59(y) 0,44(y) 0,29(b) |
Note: b = brown, bl = blue, br = brownish red, g = green, p = purple, y = yellow, yg = yellowish green
Chromatograms result of ethyl acetate extract (in table 3) with using hexane:ethyl acetate (3:7) as mobile phase and 50% sulfuric acid in methanol as visualization was provided 12 spots that showed the presence of at least 12 compounds. Chromatogram results using FeCl3 5% as visualization was obtained 6 spots (5 of them was green and yellowish green and 1 blue) which means there was polyphenols compound. Chromatogram results using Dragendorff has obtained 1 spot (brown) that indicated alkaloids compounds, whereas visually and Liebermann-Bourchard was not visible spots, it means that there were not triterpenes and steroids compounds detected.
Chromatograms result of methanol extract (in table 4) with using hexane:ethyl acetate (3:7) as mobile phase and 50% sulfuric acid in methanol as visualization was provided 16 spots that showed the presence of at least 16 compounds. Chromatogram results using Liebermann-Bourchard as visualization was obtained 3 spots which means there was triterpenoids compound. Chromatogram results using FeCl3 5% as visualization was obtained 8 spots (7 of them was variation green and 1 blue) which mean there was polyphenols compound. Chromatogram results using Dragendorff has obtained 1 spot (brownish yellow) that indicated alkaloids compounds.
Table-4: Results of Chromatogram and Rf Value of Methanol Extract of Outer Layers of Onion (Allium cepa L.) Bulbs
Chromatograms result with using mobile phase hexane:ethyl acetate (3:7) |
||||
visually |
H2SO4 50% reagent |
Liebermann-Burchard reagent |
FeCl3 5% reagent |
Dragendorff reagent |
not visible |
0,92(b) 0,87(b) 0,81(b) 0,76(o) 0,72(b) 0,67(b) 0,60(o) 0,56(b) 0,52(o) 0,50(b) 0,46(o) 0,37(b) 0,30(b) 0,22(y) 0,17(y) 0,11(b) |
0,61(p) 0,31(p) 0,17(p) |
0,92(yg) 0,81(yg) 0,77(g) 0,71(g) 0,68(bl) 0,56(g) 0,52(g) 0,37(g) |
0,46(o) 0,22(o) 0,11(by) |
Note: b = brown, bl = blue, by = brownish yellow, g = green, o = orange, p = purple, y = yellow, yg = yellowish green
Results of Antimicrobial Activity
Antibacterial Activity of Extract of Outer Layers of Onion Bulbs (Allium cepa L.) for Staphylococcus aureus (SA) ATCC 29737 can be seen in Fig.-1. Antibacterial activity against SA showed increasing inhibitory diameter when the extract concentration was increased. However, for extracts, SMH was not showed antibacterial activity against SA in various extract concentrations. Based on the data presented of the identification of the component chemical compounds on the hexane extract containing dominant of triterpenoid (Table-2). This data showed that hexane extract from outer layers of onion bulbs (Allium cepa L.) produced from maceration extraction using hexane solvents contains triterpenoid compounds that were not had antibacterial activity against SA. Antibacterial activity against SA showed that extraction by stratified maceration method using methanol solvent (SMM) at a concentration of 150 mg / ml provided the greatest antibacterial activity with the highest inhibitory diameter values ??(d = 12.00 mm). The antibacterial activity SMM also has a wider inhibitory diameter compared to the methanol extract produced from directly maceration method (DMM). The results of the analysis of chemical compounds for DMM extracts (Table-1) and SMM (Table-4) also showed the components of each chemical compound. DMM extract has a variety of plant chemical compounds (secondary metabolites), such as the class of chemical compounds alkaloids, anthraquinone glycosides, flavonoids, glycosides, saponins, tanins and triterpenoids caused by using of methanol solvents by directly maceration method. The SMM extract used methanol solvents but extraction had been done before using hexane and ethyl acetate solvents in a row, this causes the number of chemical compound components in the SMM extract to decrease because previously the other chemical compounds had been drawn by solvents hexane and ethyl acetate. The SMM extract had triterpenoid and phenol chemical compounds (Table-4). These results provided the possibility that extracts had fewer chemical compounds so gave greater antibacterial activity. The results obtained also caused to the presence of triterpenoid and phenol compounds from the SMM extract having greater antibacterial activity against SA than these compounds in the DMM extract which had many other chemical compounds.
Antibacterial Activity of Extract of Outer Layers of Onion Bulbs (Allium cepa L.) for Escherichia coli (EC) ATCC 10536 can be seen in Fig.-2. Antibacterial activity against EC showed increasing inhibitory diameter when the extract concentration was increased. However, for extracts SMH was not showed antibacterial activity against EC in various extract concentrations. This result was the same as the antibacterial activity against SA. Antibacterial activity against EC showed that extraction by stratified maceration method using methanol as solvent (SMM) at a concentration of 150 mg / ml provided the greatest antibacterial activity (d = 11.16 mm) then followed by SME (d = 10.50 mm) and DMM (d = 9.60 mm). SME extract provided stronger antibacterial activity against EC than DMM extract. The analysis of the components of the chemical compounds of the SME extract showed that the SME extract containing phenol compounds (Table 3) which were indicated stronger in inhibiting EC growth compared with DMM extracts. DMM extract has a variety of plant chemical compounds (secondary metabolites), such as the class of chemical compounds alkaloids, anthraquinone glycosides, flavonoids, glycosides, saponins, tanins and triterpenoids caused by the use of methanol solvents by directly maceration method. The SME extract used ethyl acetate solvents, but previously extracted using hexane solvents, this resulted indicated was not detection of triterpenoid and steroid compounds because the compounds such as free triterpenoids and free steroids were withdrawn by hexane solvents. These results provided the possibility that extracts had a component of chemical compounds in the phenol group provided stronger antibacterial activity against EC than these phenol compounds combined with other chemical compounds such as those found in DMM extracts.