Research Article - (2020) Volume 11, Issue 1
Civet is an important commodity of commerce because of its use in perfume industry. It is also used to some extent in traditional medicine. Civetone is the main component of civet that is responsible for its characteristic odor. The amount of civetone in the musk of civet cat is not determined yet. In this project, rapid, simple and sensitive quantitative thin-layer chromatography method for the quantification of civetone in civet was developed and validated. The method involved extracting samples with dichloromethane by sonication, and analysis by CAMAG TLC scanner 3, after using a civetone dervatizing reagent. The densitometric scanning and analysis was done in absorbance mode at 371 nm. A new method for quantifying civetone in civet was developed in the course of this study. This involved taking a known amount of civet adding to it, a dervatizing agent 2,4-dintrophenylhydrazine, which now raises the absorption maximum to the visible range, thus making quantification work possible. The linear regression analysis data for calibration plots showed good linear relationship with r2=0.999 in the concentration range of 1-5 μg spot-1 with respect to peak area. The method was validated for accuracy, precision and recovery. The civetone value in crude civet samples, quantified by TLC was 0.8-1.2%.
Civet; Civet musk; Civetone; Limu genet; Zibad
Civet samples, standards and chemicals
Analytical grade chloroform (99.98%), acetone (99.99%), petroleum ether, CH2Cl2, H2SO4, EtOH, and 2,4-dinitrophenylhydrazine (DNPH) , caffeine , CDCl3, CCl4, DMSO anisaldehyde reagent (anisaldehyde, H2SO4, EtOH and AcOH [0.5:0.5:9:0.1]) were used. Seven different civet samples were used in this study. One civet sample obtained from Ato Hamza Siraj, Jimma and the other six samples were obtained from Quality and Standard Authority of Ethiopia (QSAE) through W/r Genet.
Isolation of civetone
Three different methods were used in this work to isolate civetone from civet. First, 8 g of civet was taken and socked with CHCl3 (80 ml) and sonicated for 20 min filtered and concentrated to give 6 g (75%) of CHCl3 extract. All the CHCl3 extract was packed on silica gel (70-230 mesh) and eluted using petroleum ether: CHCl3. By increasing polarity, nine fractions were collected. Of these nine fractions, Fr-7 showed one yellow spot on TLC after spraying by anisaldehyde reagent and concentrated to give 120 mg mainly civetone (78% pure). Second, 1.3 g civet was taken and socked with CHCl3 and sonicated for 20 min, filtered and concentrated to give 1.1 g (84%) of CHCl3 extract. Acetone (10 ml) was added to CHCl3 extract then sonicated for 4 min, filtered and washed to give 430 mg. This was applied on silica gel column (70-230 mesh) using petrol: CHCl3 by increasing polarity, two fractions were collected. Fr-2 gave one yellow spot on TLC after spraying by anisaldehyde reagent and concentrated to give 13 mg (80% pure). Finally, 20 g of civet musk from one of the samples was taken, socked with CH2Cl2, then sonicated for 20 min, filtered and concentrated to give 17 g (85%). The CH2Cl2 extract was dissolved by CHCl3 (5 ml) and applied on VLC by using VLC grade silica gel (100 g) and then eluted using petrol/CHCl3 by increasing polarity, three fractions were collected. TLC of these three fractions was shown that the presence of civetone in Fr-2 and Fr-3. These two fractions were separately packed on column using the same solvent system as mentioned above and those fractions containing civetone were collected and weighed to be 146 mg and 280 mg respectively. The two were combined and washed by acetone; the acetone soluble and insoluble portions were separated and concentrated to get 200 mg (1% of civet sample) and 103 mg respectively. NMR shows that the acetone soluble portion was mainly civetone (86% pure). This was taken as a reference for analysis in this work.
Preparation of standards
For TLC analysis, two standards were prepared:
Standard solution I: To 75 mg of reference civetone in 25 ml volumetric flask CHCl3 was added up to the mark to get 3 mg/ml or 3 μg/ μl Stock Solution.
Standard solution II: 1 ml of Solution I was diluted to 10 ml in a volumetric flask to give 0.3 mg/ml or 0.3 μg/ μl by CHCl3. Both solutions were stored at 4c.
Preparation of Brady’s reagent and civetonide
DNPH was dissolved in EtOH at elevated temperature, and filtered while it is still hot. The solution was allowed to cool; this cause crystallizes out. The crystal was separated from the solution and the melting point was measured (197-198°C) using Thiel tube. 30 mg of the recrystallized DNPH, 3-drops of H2SO4 and 2.5 ml of EtOH were mixed in 10 ml volumetric flask and CHCl3 was added to get 3 mg/ml solution of DNPH. 1 ml of this solution was taken using 2 ml measuring pipette and then transferred into 10 ml volumetric flask and CHCl3 was added up to the mark in order to get 0.3 mg/ml solution of a reagent known as Brady’s reagent [11-14]. 20 g of civet sample was taken and socked with CH2Cl2, and sonicated for 20 min, filtered and concentrated. Brady’s reagent was added to fraction one (Fr-1) allowed to stand for 10 min to react, then applied on column, and eluted by the same solvent system as mentioned above and five fractions were collected. Fr-3 and Fr-4 showed one yellow spot on TLC and the two were recombined and concentrated to give 300 mg civetonide.
Preparation of Civet samples for quantification analysis
1.5 g of each sample was taken and dissolved by CH2Cl2 (30 ml) and sonicated for 1 h, and then concentrated to give CH2Cl2 extract. To the CH2Cl2 extract, acetone (10 ml) was added, then filtered and concentrated. Two solutions were prepared for analysis:
Stock solution I: All acetone soluble portions transferred to 25 ml volumetric flak and filled by CHCl3 up to the mark.
Stock solution II: 5 mg/ml solution of each sample was prepared by taking small amount and diluted farther.
The developed quantitative thin-layer chromatography techniques are simple, rapid, accurate and precise methods for the determination of civetone in civet samples. The method showed appropriate recoveries and repeatability ’ s and the proposed mobile phase effectively resolves civetone and thus, the method can be used for quantitative as well as qualitative analysis of civetone in different samples. The analysis results demonstrate that civetone can be effectively resolved and quantified in civet samples. For the revitalization of civet trade stakeholders of civet should share the problem of civet farmers by controlling the quality of civet, improve the handling of civet cat.
Ethiopian ministry of education should be acknowledged for funding this work.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Ethics approval was not required for this research.
The authors hereby declare there is no conflict of interests.
Chemical Sciences Journal received 912 citations as per Google Scholar report
