KR100341797B1 - Isolation method of natural antioxidants(N-feruloylserotonin) from the seed of Carthamus tinctorius L - Google Patents

Abstract

The present invention relates to a method for separating and purifying N-feruloylserotonin, which is an antioxidant contained in safflower seeds, in order to provide a natural antioxidant that is harmless to the human body and has excellent antioxidant power. Extracting an antioxidant with methanol (MeOH) solution, and sequentially fractionating the extract with ethyl acetate, normal butanol and water, and dissolving the solvent in the ethyl acetate fraction organic solvent from which the solvent is removed. Passing through a predetermined amount, passing through the thin layer chromatography (TLC) and separating the fractions having the same height of the developed material, and separating the similar substances from each other; DPPH radical activity to measure and trace the antioxidant N-feruloylserotonin contained in Fahrenheit can be separated and extracted by a simple process. Purification of high purity is simple and quick because the active substance is traced and separated by measuring the DPPH radical scavenging activity of the fraction separated by chromatography. There is an advantage to obtain antioxidants.

Description

Isolation method of natural antioxidants (N-feruloylserotonin) from the seed of Carthamus tinctorius L}

The present invention relates to a method for separating and purifying N-feruloylserotonin, which is an antioxidant contained in safflower seed.

One of the chemical causes of poor quality during food processing or storage is the various oxides produced by the oxidation of lipids. These oxides inevitably occur in humans who need oxygen, and thus, ion transporters and proteins in cell membranes in the body. Attacking genes and the like causes various diseases such as aging and cancer.

Therefore, research on antioxidants that can prevent the degradation of food quality by preventing the oxidation of fats, prevent and treat human diseases and delay aging has been conducted for a long time, and many synthetic or natural antioxidants have been developed. It became.

However, many antioxidants are not actually used due to their effectiveness, economics and safety, and the synthetic antioxidants BHA (butylated hydroxyanisol), BHT (butylated hydroxytoluene) and natural antioxidants Jane tocopherol is generally used.

However, BHA and BHT, which are synthetic antioxidants, have excellent antioxidant power, but as their mutagenicity and toxicity are known recently, the development of safer and more potent natural antioxidants is required. Tocopherols, which are widely used as natural antioxidants, are vegetable For oil, there was a problem of low effect and high price.

The present invention has been made in order to solve the above problems of the prior art, and its object is to provide a natural antioxidant that is harmless to the human body and excellent in antioxidant power.

The above object of the present invention is achieved by providing a method for separating and purifying the antioxidant N-feruloylserotonin from safflower seed which has been used traditionally for a long time and confirmed its stability. The purification process is very simple and highly efficient by the follow-up chromatography and DPPH (1-diphenyl-2-picryl hydrazyl) method.

Safflower seed has long been known for its ability to quickly heal broken bones, prevent hypertension and atherosclerosis, and prevent aging.However, studies on safflower seed include analysis of general ingredients and mineral content, antioxidant effects of safflower flowers and Pigment separation was everything, and no studies on the purification of antioxidants from safflower seed have been attempted.

Therefore, the present inventors are the first to find that N-feruloylserotonin contained in safflower seed is an antioxidant during the follow-up study of safflower seed which has been widely used as a medicament for treating diseases and longevity food in folk medicine. Found and could be extracted and purified.

1 is a simplified process of separating active material from safflower seed.

The present invention comprises the steps of pulverizing safflower seed to extract the active substance with methanol (MeOH) solution and solvent fractionation, and separating and purifying only the active fraction from the fractionated solution using chromatography and DPPH method to obtain the active substance. do.

Hereinafter, the purification and structure confirmation of the antioxidant from safflower seed according to an embodiment of the present invention is as follows.

Example 1

<Extraction and Solvent Fraction of Active Substances>

1) The crushed safflower seed was put in 70-90% methanol solution and extracted and concentrated at room temperature for 1 day.

2) The extract was fractionated sequentially with ethyl acetate (EtOAc), normal butanol (n-BuOH) and water (H ₂ O) to fractionate the extract according to polarity.

3) The ethyl acetate fraction of the extract was put in a vacuum concentrator at a temperature of 40 to 50 ° C. to completely remove the solvent, and then an activity assay and an active substance separation fraction were prepared.

Example 2

<Preparation of Small Fractions and Material Separation Using Column Chromatography and Thin Layer Chromatography>

1) The ethyl acetate fraction obtained by completely removing the solvent obtained in Example 1 was dissolved in a 10: 1 solution of nucleic acid and ethyl acetate, passed through a silica gel column, and then sequentially prepared in a ratio of 5: 1, 3: 1, 2: 1. The ethyl acetate solution was passed through a silica gel column, and 70 ml aliquots were added to the test tube.

2) The fractions were concentrated by combining the same heights of the developed materials by thin layer chromatography (TLC) using a developing solvent having a ratio of chlorocuring and ethanol of 10: 1.

3) As a result of measuring DPPH radical activity for each of the fractions, the 8th fraction identified as active was repeated 1) and 2) to obtain 9 small fractions.

4) The four small fractions were obtained by repeating 1) and 2) for the sixth small fraction whose activity was confirmed by measuring DPPH radical activity among the nine small fractions.

5) Repeated steps 1) and 2) of the second small fraction whose activity was confirmed by measuring DPPH radical activity among the four small fractions yielded four small fractions.

6) NMR data of the third small fraction confirmed its activity by measuring DPPH radical activity among the four small fractions were structurally identified as N-feruloylserotonin.

Figure 1 shows the active material separation process from safflower seed according to Examples 1 and 2, the final fraction CTS-E-8-6-2-3 is N- feruloyl serotonin which is an antioxidant of safflower seed.

Example 3

<DPPH radical scavenging activity assay>

80 μl of the sample obtained in Example 2 and 5 ml of 1.5 × 10 ⁻⁴ M DPPH, each of which was dissolved in a 1 ml methanol solution, were added to a test tube, stirred, and reacted at room temperature for 30 minutes, followed by DPPH radical scavenging activity at 517 nm. The activity was assayed by searching.

Tables 1 to 4 show the results of DPPH radical scavenging activity of each fraction obtained from safflower seed shown in FIG. 1.

SiO ₂ column fraction 1 of the EtOAc layer Fraction number Absorbance at 517 nm CTS-E-1CTS-E-2CTS-E-3CTS-E-4CTS-E-5CTS-E-6CTS-E-7 CTS-E-8 CTS-E-9 1.6571.6381.6351.6211.6271.3571.009 0.432 0.816 control 1.687

SiO ₂ column fraction 2 of the EtOAc layer Fraction number Absorbance at 517 nm CTS-E-8-1CTS-E-8-2CTS-E-8-3CTS-E-8-4CTS-E-8-5 CTS-E-8-6 CTS-E-8-7CTS-E-8- 8CTS-E-8-9 1.3121.5121.4291.2631.008 0.468 0.8251.3091.569 control 1.644

SiO ₂ column fraction 3 of the EtOAc layer Fraction number Absorbance at 517 nm CTS-E-8-6-1 CTS-E-8-6-2 CTS-E-8-6-3CTS-E-8-6-4 1.180 0.139 1.5501.514 control 1.656

SiO ₂ column fraction 4 of EtOAc layer Fraction number Absorbance at 517 nm CTS-E-8-6-2-1CTS-E-8-6-2-2 CTS-E-8-6-2-3 CTS-E-8-6-2-4 1.2341.152 0.102 1.363 control 1.674

Example 4

<NMR Spectrum Analysis and Structure Identification>

Antioxidants finally separated in Example 2 were confirmed structural formula using ¹ H-NMR, ¹³ C-NMR.

The ¹ H magnetic resonance spectrum ( ¹ H-NMR) and the ¹³ C magnetic resonance spectrum ( ¹³ C-NMR) of the colorless crystals (MeOH-CHCl ₃ ), N-feruloylserotonin, having a melting point of 118 to 119 ° C. are as follows. same.

¹ H magnetic resonance spectrum (CD ₃ OD, 100MHz)

7.45 (1H, d, J = 15.9 kPa, H-7'0), 7.18 (1H, d, J = 8.8 kPa, H-3 '), 7.07 (1H, d, J = 2.4 kPa, H-4) , 7.03 (1H, s, H-2), 7.01 (1H, d, J = 2.2 Hz, H-6 '), 6.99 (1H, dd, J = 8.2, 2.4 Hz, H-6), 6.81 (1H , d, J = 8.2 ㎐, H-7), 6.70 (1H, dd, J = 2.2, 8.8 ㎐, H-2 '), 6.46 (1H, d, J = 15.9 ㎐, H-8), 3.83 ( 3H, s, -OCH ₃ ), 3.58 (2H, t, J = 7.3 ㎐, H-9), 2.93 (2H, t, J = 7.3 ㎐, H-8)

¹³ C magnetic resonance spectrum (CD ₃ OD, 100MHz)

169.16 (C-9 '), 151.05 (C-5), 149.63 (C-4'), 149.15 (C-5 '), 141.94 (C-7'), 133.05 (C-7a), 129.39 (C- 1 '), 128.27 (C-3a), 124.28 (C-2'), 123.14 (C-2), 118.89 (C-8 '), 116.41 (C-3'), 112.69 (C-6), 112.48 (C-7), 112.40 (C-3), 111.58 (C-6 '), 103.58 (C-4), 54.34 (-OCH ₃ ), 41.42 (C-9), 26.37 (C-8)

EI / MS (m / z)

352 (M +), 323, 177, 176, 159, 146

IR ν (KBr, max) cm ^-1

3396, 3185, 1662, 1605, 1590

Example 5

<Comparison of the Antioxidants (N-PeruloylSerotonin) Activity of Safflower Seed and Existing Antioxidants by DPPH Method>

Antioxidant activity of N-feruloylserotonin, an antioxidant active substance isolated from safflower seed, was measured by α-tocopherol, the most widely used natural antioxidant, and BHT, BHA, and DPPH, which are synthetic antioxidants, and the results are shown in Table 5 below. As can be seen from the N-feruloyl serotonin antioxidant activity of 6.6㎍ / ㎖ was the highest activity among BHT and BHA and the natural antioxidant α-tocopherol.

Comparison of N-feruloylserotonin Antioxidant Capacity with ECPH by DPPH Method (EC ₅₀ value) BHA BHT α-tocopherol N-feruloylserotonin 7.4 µg / ml 12.2 µg / ml 10.0 μg / ml 6.6 µg / ml

The present invention can separate and extract the N-feruloyl serotonin contained in safflower seed by a simple process, and by measuring the DPPH radical scavenging activity of the fractions separated by chromatography, the active substance is traced and separated, so that it is easy and quick. There is an advantage to obtain a high purity purified antioxidant.

In addition, N-feruloyl serotonin is a natural antioxidant that is superior to the conventional antioxidant antioxidants BHT and BHA, but harmless to the human body has a great effect on quality preservation during processing or storage of food.

Claims (4)

In the method of separating and extracting N-feruloylserotonin, a natural antioxidant contained in safflower seeds, crushing safflower seeds to extract antioxidants with methanol solution, and extracting the extracts sequentially with ethyl acetate, normal butanol and water. And dissolving the ethyl acetate fraction in which the solvent is removed from the fraction in an organic solvent, and then fractionating each fraction by passing through column chromatography, and passing each fraction through the thin layer chromatography (TLC). Natural antioxidants contained in safflower seed, comprising the steps of separating the similar substances by combining the fractions of the same height and measuring the DPPH radical activity of each of the analogous fractions. Isolation and Purification Method of Phosphorus N-feruloylserotonin. The concentration of the methanol solution is 70 to 90%, and the extraction of the antioxidant is carried out for 1 day at room temperature, the separation of N- feruloyl serotonin, which is a natural antioxidant contained in safflower seed Purification method. The method of claim 1, wherein the ethyl acetate fraction is placed in a reduced pressure concentrator at 40 to 50 ° C. to completely remove the solvent. The method for separating and purifying N-feruloyl serotonin which is a natural antioxidant contained in safflower seed. The method of claim 1, wherein the organic solvent is a 10: 1 solution of nucleic acid and ethyl acetate. The method for separating and purifying N-feruloyl serotonin, which is a natural antioxidant contained in safflower seed.