Sri Astuti, et al.
Fingerprint Study of Foenicullum Vulgare Mill...
METHODOLOGY Material
Foeniculum vulgare Mill. fruit were collected from Indonesian Medicinal and Aromatic Corps Research Institute and was determined in Herbarium Bogoriense, Research Centre for Biology, Indonesian Institute of Science, Bogor, Indonesia with determination letter number 439/IPH.1.02/If.8/VI/2011. Solvent used in this research were n-hexane, chloroform, ethyl acetate, ethanol and methanol (Merck Co.)
Equipment
Evaporator Buchi R-200 (Buchi Co.), 60 GF254 silica plates (Merck Co.) , Linomat 5 TLC Spotter (CAMAG), TLC Scanner 3 (CAMAG), TLC Documentation System Reprostar3 (CAMAG) and UFLC LC-20 series (Shimadzu, Kyoto, Japan)
Methods Extraction
Dried powder material (30 g) of Foeniculum vulgare Mill. leaves were macerated in 4 kinds of solvents (n-hexane, chloroform, ethyl acetate, and ethanol) each was 300 mL for 24 hours (three times repeated). Each macerate was evaporated (by rotary evaporator), then weight the extracts and determined the yields.
Analysis
TLC analysis of each extract was carried out on silica gel GF 254 as stationary phase, and there were 3 mobile phases which were applicable for Foenicullum vulgare Mill extract : a) n-hexane : ethyl acetate (7 : 3) v/v; b) chloroform : ethyl acetate (9 : 1 v/v); c) toluene : chloroform : ethyl acetate (3 : 6 : 1 v/v/v). The TLC analysis results were detected by TLC Scanner and recorded TLC Documentation System.
RESULTS AND DISCUSSIONS
The yields of n-hexane, chloroform, ethyl acetate, and ethanol extracts were showed on Table 1.
The chloroform extract gave the highest yield. It was caused by the polarity of chloroform. Chloroform has a low polarity index, and the main compound of Foenicullum vulgare Mill. (anethol) also has a low polarity index. Therefore, chloroform could bind the main compound of F. vulgare Mill.
The TLC analysis profiles of each extract on silica gel GF 254 using mobile phase: a) n-hexane : ethyl acetate (7 : 3) v/v; b) chloroform : ethyl acetate (9 : 1 v/v); c) toluene : chloroform : ethyl acetate (3 : 6 : 1 v/v/v) which were recorded by photo documentary system can be seen on Figure 1 and 2. All eluents above had a low polarity index or classified as a non polar – semi polar eluents. Those eluents could separate the compounds contained in the extract clearly therefore it can be concluded that F.
vulgare Mill extracts had the best separation result in a non polar to semi polar eluent/mobile phase.
The chromatograms profile (254 nm and 366 nm) of TLC scanner can be seen at Figure 3 and 4.
The chromatograms profile showed that n-hexane: ethyl acetate (7: 3 v/v) had the best separation which had the specific retention factor (1) λ = 254 nm : at 0.21, 0.32, 0.37, and 0,90 for n-hexane extract; at 0.20, 0.31, 0.37, and 0.91 for chloroform extract; at 0.22, 0.33, 0.39, and 0.87 for ethyl acetate extract; and 0.23; 0.34; and 0.41 for ethanol extract; (2) λ = 366 nm at 0.32, 0.68, 0.75, and 0,90 for n- hexane extract; at 0.31, 0.64, 0.72, and 0.90 for chloroform extract; at 0.33, 0.67, 0.72, and 0.91 for ethyl acetate extract; and 0.35, 0.70, and 0.48 for ethanol extract.
Elution with chloroform : ethyl acetate (9 : 1 v/v) showed the specific retention factor (1) λ = 254 nm : at 0.12, 0.43, and 0,48 for n-hexane extract; at 0.04, 0.12, 0.44, and 0.75 for chloroform extract; at 0.13, 0.44, 0.50, 0.56, and 0.87 for ethyl acetate extract; and 0.04; 0.12, 0.15, 0.46, and 0.79 for ethanol extract; (2) λ = 366 nm at 0.16, 0.50, 0.54, 0.76, 0.84, and 0,93 for n-hexane extract; at 0.05, 0.16, 0.35, 0.53, 0.84, and 0.97 for chloroform extract; at 0.05, 0.35, 0.58, 0.80, 0.85, and 0.94 for ethyl acetate extract; and 0.05, 0.37, 0.49, 0.56, 0.80, 0.88, and 0.97 for ethanol extract.
Sri Astuti
Figure 1. Chromatogram profile (λ = 254 nm) on TLC silica plates of n-hexane, chloroform, ethyl acetate, and ethanol extracts after elution with A) n-hexane : ethyl acetate (7 : 3 v/v); B) chloroform : ethyl acetate (9 : 1 v/v); C) toluene : chloroform : ethyl acetate (3 : 6 : 1 v/v/v).
Figure 2. Chromatogram profile (λ = 366 nm) on TLC silica plates of n-hexane, chloroform, ethyl acetate, and ethanol extracts after elution with A) n-hexane : ethyl acetate (7 : 3 v/v); B) chloroform : ethyl acetate (9 : 1 v/v); C) toluene : chloroform : ethyl acetate (3 : 6 : 1 v/v/v).
Fingerprint Study of Foenicullum Vulgare Mill...
Figure 3. The chromatograms profile (254 nm) of TLC scanner n-hexane, chloroform, ethyl acetate, and ethanol extracts eluted with A) n-hexane : ethyl acetate (7 : 3) v/v; B) chloroform : ethyl acetate (9 : 1 v/v); C) toluene : chloroform : ethyl acetate (3 : 6 : 1 v/v/v).
Figure 4. The chromatograms profile (366 nm) of TLC scanner n-hexane, chloroform, ethyl acetate, and ethanol extracts eluted with A) n-hexane : ethyl acetate (7 : 3) v/v; B) chloroform : ethyl acetate (9 : 1 v/v); C) toluene : chloroform : ethyl acetate (3 : 6 : 1 v/v/v).
Sri Astuti
And elution with toluene : chloroform : ethyl acetate (3 : 6 : 1 v/v/v) showed the specific retention factor (1) λ = 254 nm : at 0.10, 0.12, 0.38, 0.42, 0.60, 0.74 and 0,95 for n-hexane extract; at 0.10, 0.12, 0.38, 0.60, 0.75, and 0.94 for chloroform extract; at 0.08, 0.13, 0.39, 0.45, 0.61, 0.65, 0.90, and 0.96 for ethyl acetate extract; and 0.11, 0.13, 0.40, 0.62, 0.77, and 0.92 for ethanol extract; (2) λ = 366 nm at 0.42, 0.61, 0.65, 0.71, and 0,80 for n-hexane extract; at 0.24, 0.40, 0.61, 0.72, 0.81, and 0.94 for chloroform extract; at 0.24, 0.45, 0.61, 0.72, 0.80, and 0.96 for ethyl acetate extract; and 0.27, 0.40, 0.63, 0.73, and 0.97 for ethanol extract.
Every chromatogram of all extracts in the same eluent has the similar pattern. It means that they have some similar retention factors value. ompare to others, ethyl acetate extract had the best TLC profile; therefore it can be used for standardization of F. vulgare Mill. extract.
CONCLUSION
According to the results above, it can be concluded that the fingerprint/ chromatogram profile of ethyl acetate extract of F.vulgare Mill. can be used for standardization of its traditional medicine extract.
ACKNOWLEDGEMENT
This paper is ackowledgement to Recearch Centre for Drug and Food - The National Agency of Drug and Food Control for funding this research.
REFERENCES
Chun, X. J., S. B. Guo, Z. F. Ping, Li. L, 2005, Analysis of the Volatile Constituents from Foeniculum Vulgare Mill by Gas Chromatography—Mass Spectrometry Coupled with Solid Phase Microextraction, Journal of Fine Chemical, 7, 9 – 15.
Gulfraz, M., et al, 2008, Composition and antimicrobial properties of essential oil of Foeniculum vulgare, African Journal of Biotechnology, 7(24), 4364–4368.
Hua-ze, D., et al, 2009, Effect of Foeniculum vulgare Mill on the Immunological Functions in Mice, Journal of Anhui Agricultural Science, 27, 201 -210.
Nassar, M. I., E. A. Aboutabl, Y.A. Makled, E.D.A. El-Khrysi, and A.F Osman, 2010, Secondary Metabolites and Pharmacology of Foenicullum vulgare Mill Subs. Piperitum, Rev. Latinoamer Quim, 38/2, 103-112.