Constituent Analysis of Turmeric (Curcuma longa L) Extract andIts Reaction with m-Chloroperoxybenzoic Acid

울금 추출물의 구성 성분 분석과 m-Chloroperoxybenzoic Acid와의 반응

EXPERIMENTAL

Materials and measurements. Acetic acid (Showa Chemicals Inc., 99%) and ethanol (Hayman Limited, 99.9%) were used as the solvents in this experiment. m-Chloroperoxybenzoic acid (m-CPBA, Aldrich Chemical Company, Inc., 57-86%) was purified by literature methods18 and recrystallized from CH2Cl2. The turmeric was purchased from a herbal medicine store. The UV/VIS spectrum was measured on a Hewlett Packard HP 8452A diodearray UV/VIS spectrophotometer. The IR spectrum was measured by Bruker Vector-22 IR spectrophotometer with a KBr pellet. The GC/MS data was obtained by HP-6890 Plus gas chromatography and the mass spectrometer of the HP-5973 MSB in the Hazardous Substance Research Team of Korea Basic Science Institute Seoul Branch. The mass spectrum was analyzed using the mass spectral library search database Wiley 275000.

Preparation of turmeric extract. 1.06 g of turmeric and 130 mL of ethanol were placed in the 250 mL round bottomed flask with a reflux condenser and refluxing it for 33 hours. Ethanol extract was cooled at room temperature. The supernatant of ethanol extract was filtered and evaporated by a rotary vacuum evaporator to give a reddish orange colored turmeric extract. Turmeric extract was dried in a vacuum (below 10 mmHg) desiccator for 12 hours in room temperature, and it used as a reagent.

Preparation of stock solutions. The molecular weight of the turmeric extract was estimated to be about 210, which was calculated by considering the molecular weight and the relative abundance of the constituents from the GC/MS data (Table 1). The 1.0 × 10-2 M stock solution of turmeric extract was made by dissolving turmeric extract 0.0027 g (0.01 mmol) in acetic acid and making the amount of the solution 1 mL. The 1.0 × 10-1 M stock solution of m-CPBA was made by dissolving m-CPBA 0.0173 g (0.1 mmol) in acetic acid and making the amount of the solution 1 mL. Fresh stock solutions were always made before using.

Reaction of turmeric with m-CPBA. Reaction of turmeric with m-CPBA in acetic acid was monitored from measurements of the decrease in UV/VIS absorbance at 418 nm. UV/VIS absorbances were measured using a 1-cm quartz cell sealed by a rubber stopper. The temperature of the thermostated cell block in UV/VIS spectrophotometer was maintained at a constant temperature (± 0.01 ℃) using NESLAB model EX-210 constant temperature bath and circulator. After putting a small magnetic bar in 1-cm quartz cell and flushing it with dry nitrogen, 2.0 mL acetic acid was injected and the cell was sealed with a rubber stopper. Acetic acid in the cell was kept in the thermostated cell block for more than 20 minutes in the desired temperature of the reaction. For the reaction conditions, an appropriate amount of stock solutions was injected at the same time, and the decrease in UV/VIS absorbance was measured at 418 nm as a function of reaction time.

The reaction condition was a pseudo-first-order reaction, in which the concentration of m-CPBA was 20 times lager than the concentration of turmeric extract. The kinetic study of the reaction of turmeric extract and m-CPBA in the acetic acid was performed by varying the reaction temperature range of 30-50 ℃, when the mole ratio of turmeric extract and m-CPBA was varied to 1:20, 1:40, 1:80. The pseudo-first-order reaction rate constants were obtained from the slopes of plots of ln (A∞ - A0)/(A∞ - At) vs. time derived from k = 1/t ln (a/a - x). The values of the activation parameters were obtained by using the Arrhenius plot at three different temperatures and the Eyring equation.19

 

RESULTS AND DISCUSSION

The UV/VIS wavelength maximum of the turmeric extract appeared at 418 nm (εmax 370), because of a long chain conjugation or a polynuclear aromatic chromophore. The IR absorption peak of the turmeric extract was shown at 3378 cm-1 as a broad band, which corresponds to the hydrogen bonds of -OH bonds of the constituent and ethanol residue. The IR absorption peaks at the 3015-2925 cm-1 region correspond to C-H bonds. The IR absorption peaks shown at the 1600-1700 cm-1 region correspond to the C=O bonds and C=C bonds, and those at the 1600-1400 cm-1 region correspond to the aromatic rings of the aromatic compounds.

Table 1Gas chromatogram data and mass spectral data of turmeric extract

The GC/MS data of the turmeric extract is shown in Table 1. The gas chromatogram data revealed that there are at least 14 constituents in the turmeric extract. Only 7 compounds could be identified by MS database Wiley 275000, and it was difficult to identify for the other 7 constituents with the GC/MS data alone.20 Furthermore, the constituents C15H20O and C15H22O which had a retention time of 13.156 min and 13.672 min, respectively constituted 89.29% of the overall abundance (Table 1).

Fig. 1Mass spectrum and molecular structure of β-turmerone which gas chromatogram retention time was 13.156 minute.

Through the mass spectrum and the mass spectral library search database, the molecule structures of 7 constituents of turmeric extract could be identified. They were 2-methoxy-5-cinylphenol (C9H10O2), 1-(1,5-dimethyl-4-hexenyl)-4-methylbenzene (C15H22), 1-(2-methoxy-1-methylethyl)-2-methylbenzene (C11H16O), β-turmerone (C15H20O), a-turmerone (C15H22O), 2,3,5-trimethylfuran (C7H10O), and a-atlantone (C15H22O). Among these constituents, β-turmerone (Figure 1) constituted 80.67% abundance (Table 1).

In order to perform a kinetic study on the reaction between turmeric extract and m-CPBA, the decrease in UV/VIS absorbance was measured at 418 nm as a function of reaction time, The reaction temperature was 30 ℃, 40 ℃, and 50 ℃ when the mole ratio of turmeric extract and m-CPBA was 1:20, 1:40, and 1:80, respectively. As a function of reaction time, the absorbance decreased at the 418 nm and increased at the 340 nm, which showed the occurrence of oxidative cleavage reactions. The UV/VIS wavelength maximum of the reaction product shifted to the short wavelength region compared to that of the turmeric extract, which means that the molecular structures of the turmeric extract are cleaved by the reactions. In other words, the π electron conjugation system of the turmeric extract is cleaved by the oxidative reactions.

Table 2Pseudo first order reaction rate constants for the reaction of turmeric extracts with m-CPBA at mole ratio and temperature in acetic acid

The pseudo first order reaction rate constants were obtained from the first order reaction rate law by measuring the absorbance at the UV/VIS λmax 418 nm of the turmeric extract, varying the reaction temperature 30 ℃, 40 ℃, and 50 ℃ when the mole ratio of the turmeric extract and m-CPBA was 1:20, 1:40, and 1:80, respectively (Table 2).

The Arrhenius plot of the reaction of turmeric extract and m-CPBA in the acetic acid is shown on Figure 2. Using this plot the activation energy (Ea) was found to be 11.46 kcal/mol. 12.27 kcal/ mol, 11.83 kcal/mol, when the mole ratio was 1:20, 1:40, and 1:80, respectively. In addition, the thermodynamic activation parameters, which were the activation enthalpy △H‡, activation entropy △S‡, and the activation free energy △G‡, were determined according to the reaction conditions (Table 3).

Table 3Thermodynamic activation parameters for the reaction of turmeric extracts with m-CPBA at mole ratio and temperature in acetic acid

Fig. 2The Arrhenius plots for the reaction of turmeric extracts with m-CPBA at the mole ratio in acetic acid.

The activation energy was found to be 11.46-12.27 kcal/mol by varying the reaction temperature to 30 ℃, 40 ℃, and 50 ℃ when the mole ratios of the turmeric extract and m-CPBA in the acetic acid were 1:20, 1:40, and 1:80. The reaction rate constant increased 1.7-1.9 times when the reaction temperature increased 10 ℃. The activation entropy was a big negative, -35.89 ~ -40.70 e. u. to the reaction conditions (Table 3).

Reactions that have an activation energy of below 19 kcal/mol in organic chemical reactions occur spontaneously at room temperature or at below room temperature.21 Since the oxidation reaction of turmeric extract and m-CPBA can occur easily because of the low activation energy of 12.27 kcal/mol, turmeric extract can easily interact with oxidative reagent. This kinetically shows that turmeric extract has an antioxidative activity that can remove highly active oxygen. The activation entropy having a big negative of -35.89 ~ -40.70 e. u., shows that turmeric extract can form a stable transition state with active oxygen, which has a high degree of freedom.22 Furthermore, this shows that turmeric extract easily reacts with active oxygen and can act as an antioxidant. Whereas the detailed reaction mechanism of the antioxidative activity of the turmeric extract can be examined by a kinetic study of each constituent of the turmeric extract.