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Analisis Total Fenolat, Total Flavonoid dan Antioksidan pada Tiga Tanaman Menggunakan metode Spektrofotometri UV Vis dan TLC Densitometri
ABSTRACT
Secondary metabolite compounds classified in the phenolic group, including
flavonoids, are responsible for various pharmacological activities such as antibacterial,
antimocrobial, antidiabetic, antioxidant, etc. The compounds are able to absorb UV rays,
and some of them absorb in the visible area. It makes UV-Vis spectrophotometry a suitable
technique for determining the quantity of phenolic compounds and flavonoids. In vitro test
of plant extract antioxidants using the DPPH radical scavenging method (α, α-diphenyl-β-
picrylilhydrazyl) is also commonly carried out using this UV-Vis spectrophotometer. All
spectrophotometric analyzes for these three parameters were carried out in the visible area
between 350 - 800 nm. Plant extracts have various types of flavonoid compounds that have
absorption in the UV-Vis light area. The presence of non-phenolic compounds is also
thought to interfere with the analysis. Therefore a suitable and selective method for
quantitative analysis is needed. In this study, analysis of total phenolics, total flavonoids
and antioxidants was carried out using 2 methods simultaneously, namely the TLC
Densitometry (Thin Layer Chromatography) and UV-Vis spectrophotometry. As samples,
three plants were used, green meniran (Phyllanthus niruri), parsley (Petroselinum
crispum) and kersen (Muntingia calabura L) because they have the potential to be
developed as medicinal plants. Parsley plant samples were obtained from the Kopeng area,
Salatiga, while meniran and kersen were from Semarang, Central Java. The sample
extraction was carried out by maceration 3 x 24 hours using 96% ethanol.
Dechlorophylation extract samples were carried out by two-phase extraction using diethyl
ether. Evaporation was carried out on all extracts using a rotary evaporator, and followed
by freeze drying. The dechlorophylated dry extract was here referred to as sample A,
whereas the extract without dechlorophylation was called sample B. Spectroscopic
analysis of total phenolics, total flavonoids and antioxidants used commonly methods in
the literature. Total phenolics used Folin-Ciocalteu reagent with gallic acid standard, and
were measured at a wavelength of 765 nm. Total flavonoids used the complexing
compound AlCl3, and were measured at a wavelength of 510 nm. Antioxidation activity
using DPPH reagent at a wavelength of 516 nm. The analysis of the three parameters using
TLC Densitometry was carried out with silica gel plate G 60 F254, elution using chloroform
eluent for sugar and chloroform: methanol (4.5: 0.5) for meniran and parsley.
Quantification using the Camag TLC Scanner Densitometer. The total flavonoid analysis
of the elution results was carried out by spraying the TLC results with AlCl3, and stain
detection was carried out at a wavelength of 366 nm. The results of antioxidant analysis
using the TLC Densitometry method gave IC50 values which had the same tendency as the
UV-Vis Spectrophotometric method. The IC50 values obtained from the UV-Vis
Spectrophotometric method were 25.26 - 4913.74 ppm while the TLC Densitometry
method was 24.74 - 4674.61 ppm. In accordance with the theory, the peak area at
wavelength 516, which describes the DPPH radical structure, decreases along with the
increase in peak area at a wavelength of 366 nm. The increase in the maximum peak area
at 366 nm UV light was 56.08%. In the analysis using the TLC Densitometry method, the
TFC value is largely determined by the selection of the right eluent. Meanwhile, the use
of the TLC Densitometry method is not recommended for TPC analysis.
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