KR100757086B1 - Antioxidative composition containing extract of Carthamus tinctorius sprout - Google Patents

Abstract

The present invention relates to an antioxidant composition comprising methanol extract of safflower as an active ingredient. The present invention safflower methanol extract prepared by adding methanol as an extractant to safflower ( Carthamus tinctorius L.) has an excellent hydrogen donating and antioxidant activity. There is.

Safflower seed, antioxidant activity, safflower petal extract, lipid peroxidation inhibitory effect

Description

Antioxidant composition containing extract of Carthamus tinctorius sprout with methanol extract of safflower sprout as an active ingredient

1 is a graph showing the results of measuring the hydrogen donating ability by DDPH for the antioxidant activity in water-soluble and methanol extract extracted from safflower seed, sprout and petal (A: safflower petals, B: safflower seed, C: safflower seed). BHT: butylhydroxy toluene, DW: distilled water, MeOH: methanol.

Figure 2 is a graph showing the results of investigating the effects of safflower seed, sprout and petal water solubility and methanol extract by adding Fe ⁺⁺ / ascobate to rat brain microsomes to induce peroxidation non-enzymatically.

Figure 3 is a graph showing the results of measuring the antioxidant activity of the water-soluble and methanol extract of safflower seed, sprouts and petals by the method of sulfuric acid (thiocyanate) using linolenic acid, an unsaturated fatty acid. BHT: butylhydroxy toluene, DW: distilled water, MeOH: methanol, flower DW: safflower flower extract, flower MeOH: safflower flower methanol extract, sprout DW: safflower sprout water extract, sprout MeOH: safflower methanol extract, seed DW: safflower seed water Extract, seed MeOH: safflower seed methanol extract.

The present invention relates to a composition for antioxidants containing safflower methanol extract as an active ingredient. More specifically, the present invention relates to a methanol extract of safflower ( Carthamus tinctorius L.), which has excellent antioxidant activity.

Antioxidants are widely distributed in animals and plants. Antioxidants such as phenolic compounds, flavone derivatives, tocopherols, ascorbic acid and selenium in fruit and vegetables delay or prevent the oxidation of fats. It also plays an important role in preventing aging by preventing and delaying cardiovascular disease. Natural antioxidants present in the plant family are widely used in foods, medicines, and cosmetics. The rancidity of fats and oils or foods is mainly caused by the combination with oxygen in the air. To prevent this, many synthetic or natural antioxidants have been developed. However, due to its effectiveness and economic efficiency, BHT ( Butylated hydroxytoluene (BH), butylated hydroxyanisole (BHA), propyl gallate (PG), and the like. Synthetic antioxidants, however, are most commonly used in commercial foods because of their high antioxidant power, and their use is legally regulated due to safety concerns when used in foods. Natural products such as tocopherol, although safe, alone have low oxidative chain inhibition ability and are expensive. Therefore, in recent years, many studies have been actively conducted to develop natural antioxidants which are safer and have excellent antioxidant effects in various herbal medicines and edible plant extracts.

Safflower ( Carthamus tinctorius L.), also called safflower, is a perennial herb belonging to the Asteraceae family, with reddish yellow flowers in July-August and fruiting in September-October. The flowers and seeds of safflower are used for medicinal purposes. Flowers are natural pigments, which have been used for a long time, and pigments such as safflower yellow and carthamin (C ₂₁ H ₂₂ O ₁₁ ) and glycerides of various fatty acids. It is contained as a main component. In particular, cartamine has a warm weakness and reigns over blood, has been widely used in oriental medicine as a blood and pain medicine. Safflower seed contains 75% of polyunsaturated fatty acid linolenic acid, which lowers blood cholesterol levels and shows excellent effects in treating circulatory diseases such as arteriosclerosis, hyperlipidemia and hypertension, and has been used as a raw material for osteoporosis and arthritis. So far, research on this plant has only reported effective isolation and chemical structure analysis of safflower red pigment carthamin, physicochemical properties of safflower seed, isolation of N-feruloylserotonin from safflower seeds and measurement of antioxidant activity. Systematic research on physiological activity is insufficient.

In view of the above, the present inventors have separated water-soluble and methanol extracts from safflower seeds, sprouts, and flowers as a means for effectively utilizing the bioactive substances of safflower, and compared their total polyphenol compound contents and antioxidant effects. The water extract and the methanol extract of safflower petals confirmed that the lipid peroxidation inhibitory effect and the antioxidant effect was excellent and completed the present invention.

Accordingly, it is an object of the present invention to provide an antioxidant composition comprising methanol extract of safflower ( Carthamus tinctorius L.) order having excellent antioxidant activity as an active ingredient. Another object of the present invention to provide a method of producing the methanol extract of safflower sprouts.

The object of the present invention is to obtain an extract by adding water or methanol in an amount of 10 times the total weight ratio of safflower seed and sprouts and petals as an extraction solvent, the polyphenol compound content, hydrogen donating ability, lipid peroxidation inhibition rate and By investigating and comparing the antioxidant activity, the methanol extract of safflower sprout of the present invention was achieved by confirming that the antioxidant activity was excellent.

Hereinafter, the configuration and operation of the present invention.

The present invention comprises the steps of repeatedly extracting the dried safflower petals and safflower seeds and net weight ratio 10 times distilled water twice in a water bath in a water bath for 3 hours and then vacuum concentrated and lyophilized to prepare a water extract; Preparing a methanol extract by adding 10 times the total weight ratio of methanol to safflower petals and dried safflower seeds and petals, extracting at 60 ° C., and concentrating under reduced pressure; The polyphenol compound content of the safflower petal extract and methanol extract of the present invention was measured by the modified Folin-Denis method, and the content of the polyphenol compound of safflower seed and sprout extract and the methanol extract were compared. ; Measuring hydrogen donating ability of the safflower petal water extract and methanol extract of the present invention by DDPH (α, α'-diphenyl-β-picrylhydrazyl.) And comparing the safflower seed and sprout water extract with methanol extract; Preparing a microsomal fraction from male rats and measuring the lipid peroxide content induced by Fe ⁺⁺ / ascorbate; The water-soluble and methanolic extracts of safflower seed, sprouts and petals consisted of investigating the antioxidant activity of thiocyanate using linolenic acid, an unsaturated fatty acid, and comparing the values.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1: Preparation of water extract and methanol extract of safflower petals

The dried safflower petals were purchased and used in June 2000 at Honghwa Garden, Sancheong-gun, Gyeongnam.

Using safflower petals as it is, the solution which was extracted twice in a water bath with 10-fold amount of distilled water by weight ratio was concentrated in vacuo. The concentrate was lyophilized at -80 ° C to be a water-soluble extract, and the methanol extract was extracted at 60 ° C and concentrated under reduced pressure to be used in all experiments.

Comparative Example 1 Preparation of Seed and Sprout Water Extracts and Methanol Extracts of Safflower

Safflower seed was purchased and used in Honghwawon, Sancheong-gun, Gyeongnam in June 2000. After soaking pure silver safflower seed in water for 3-4 hours, it is sown evenly in the cultivator (Korean bio sprout cultivator) to block the light and through the vent of the lid. Cultivation was done with moderate air conditioning. Two weeks later, safflower sprouts were grown to about 10 cm and lyophilized and used for the experiment.

Seed and pure silver of safflower were dried finely pulverized, and the solution was repeatedly concentrated in vacuo for 3 hours in a water bath with 10-fold amount of distilled water in a weight ratio. The concentrate was lyophilized at -80 ° C to be a water-soluble extract, and the methanol extract was extracted at 60 ° C and concentrated under reduced pressure to be used in all experiments.

Experimental Example 1: Analysis of polyphenolic compound content in water extracts of safflower petals, sprouts and seeds and methanol extract

The polyphenol compound contents of the safflower extracts prepared in Examples and Comparative Examples were measured.

The content of the polyphenol compound was measured by slightly modifying the Folin-Denis method (Gutfinger, T. J. Am. Oil Chem. Soc ., 58 , 966-968, 1981). That is, water-soluble extracts of safflower seeds, sprouts and flowers and methanol extracts are dissolved in 1 mg / ml, 0.2 ml of the solution is added to a test tube, distilled water is added to make 2 ml, and 0.2 ml Folin-ciocalteu ^and s phenol reagent are added thereto. After mixing well, it was allowed to stand at room temperature for 3 minutes. After exactly 3 minutes, 0.4 ml of saturated Na ₂ CO ₃ solution was added, mixed, distilled water was added to make 4 ml, and the supernatant was measured at 725 nm for 1 hour at room temperature. At this time, the total polyphenol compound was determined from the standard curve prepared using tannic acid. The standard curve using tannic acid is dissolved in 1 mg of tannic acid in 1 ml of 50% methanol solution and the final concentration is taken to be 0, 50, 100, 150, 200 and 300 ㎍ / ml solution. Was prepared by measuring the absorbance at 725 nm.

 Phenolic material is a secondary metabolite widely distributed in the plant system and has various structures and molecular weights. Since they have phenolic hydroxyl groups, they have the property of binding to proteins and other macromolecules, as well as bioactive functions such as antioxidant effects. The tannic acid standard curve was used here to measure the water solubility of safflower seeds, sprouts and flowers, and the content of polyphenols in methanol extracts.

As shown in Table 1 below, the polyphenol content was 12.70% in the safflower petal water-soluble extract and 12.34% in the methanol extract of safflower seed, followed by 8.75% in the safflower extract water extract and 8.05% in the safflower petal methanol extract. , Safflower seed water-soluble extract was 6.96%, safflower sprout methanol extract was 5.10%. The results of analysis of total polyphenolic compound content in domestic edible foods are 0.27%, 0.83%, 0.23%, 0.12%, 0.12 for sesame, perilla, acorn, apricot seed, almond, sunflower seed, walnut and pumpkin seed, respectively. %, 0.14%, 2.02%, 2.06%, 0.13%, and reported by Lee and Lee (Maxson, ED et al. Cereal Chem ., 49 , 719-729, 1972., Lee, JH et al. , Korean J. Food Sci. Technol ., 26 , 310-316, 1994) 1.47% for cactus seeds, polyphenol compounds according to the heat of leek seeds reported by Cha et al. At 20 ℃, 40 ℃ and 60 ℃, respectively Compared with these results, 3.64%, 4.62%, and 5.51%, water soluble in safflower seeds, sprouts and flowers, and methanol extracts contained a considerable amount of polyphenols. Polyphenol content was higher than that.

 Determination of the Water Solubility of Seeds, Sprouts and Flowers of Safflower and the Content of Polyphenols in Methanol Extracts  sample Extraction solvent Total Polyphenols (%)  Safflower Seed Extract  6.96             Methanol extract 12.34  Safflower Extract  8.75             Methanol extract  5.10  Safflower petal extract 12.70             Methanol extract  8.05

Experimental Example 2: Measurement of hydrogen donating ability by DPPH

In this experimental example, the hydrogen donating ability by DDPH of safflower extracts obtained in Examples and Comparative Examples was measured.

DPPH (α, α'-diphenyl-β-picrylhydrazyl) solution was dissolved DPPH 16 mg in 100 ml ethanol, and then mixed with 100 ml of distilled water. It was made by filtration to 2. 5 ml of this solution was mixed with 1 ml of a sample solution at a constant concentration (0.05, 0.1%), and then the absorbance was measured at 528 nm at 5 minute intervals.

The DPPH method is known as the hydrogen donating ability of antioxidants to reduce the dark purple color by reduction by tocopherol, ascorroic acid, polyhydroxy aromatic compounds and aromatic amines. The antioxidant activity of the water-soluble and methanol extracts from safflower seed, shoots and petals was measured as shown in FIG. By area, the effect increased as the concentration increased.In seeds, 0.1% methanol extract> 0.1% water soluble extract> 0.05% water soluble extract> 0.05% methanol extract, and in flowers, 0.1% water soluble extract> 0.05% water soluble extract> In order of 0.1% methanol extract> 0.05% methanol extract, in order, 0.1% methanol extract> 0.05% methanol extract> 0.1% water soluble extract> 0.05% water soluble extract. In particular, 0.1% methanol extract of safflower sprouts showed very high hydrogen donating activity, almost similar to the control 0.05% BHT (butylhydroxytoluene). In the case of safflower petals, the antioxidant activity was higher in the water-soluble extract than in the methanol extract, which is related to the hydrogen donating ability as well as the high content of polyphenol compounds due to the red pigment of safflower flower dissolving well in water. I think there is. N-feruloylserotonin, an antioxidant active substance extracted from safflower seed in safflower seed (Baeg, NI, et al. J. Korean Soc. Agric. Chem. Biotechnol ., 42, 366-368, 1999), was extracted from BHT, BHA. And superior antioxidant activity than commercial antioxidants such as tocopherol. In this experiment, however, crude extract from safflower seed was considered to have lower antioxidant activity than the control BHT.

Experimental Example 3: Preparation of Brain Microsome Fraction and Fe ⁺⁺ Investigation of Lipid Peroxide Content Induced by / ascorbate

In this experimental example, the lipid peroxide content induced by Fe ⁺⁺ / ascorbate of safflower extracts prepared in Examples and Comparative Examples was investigated.

Six-week-old Sprague-Dawley male rats (Kyudo Experimental Animal Co. Tosu, Japan) bred on a commercial diet were lightly anesthetized with diethyl ether, and then opened and washed immediately with cold saline and drained with filter paper. After absorption, 1.15% KCl-10 mM phosphate buffer (pH 7.4) was added and homogenized with a homogenizer. The solution was centrifuged at 12,000 rpm for 20 minutes with a cooling centrifuge (Kubota, KR-20000 T, Tokyo, Japan) set at 4 ° C, and the supernatant was filtered with 4 layers of gauze, and the filtrate was ultracentrifuged at 4 ° C. The fraction precipitated by centrifugation at 45,000 rpm for 45 minutes in a separator (Hitachi 55p-72, Tokyo, Japan) was added to the precipitated fraction to a microsomal fraction by adding a certain amount of 1.15% KCl-10 mM phosphate buffer (pH 7.4).

Lipid peroxide content was measured in 1.5 ml of 50 mM Tris-HCl buffer (pH 7.5) according to Wong et al. (Wong, SF, et al., J. Inorganic. Biochem., 14, 127-134, 1981). The reaction solution was added by adding 0.2 ml (6.0 mg / ml), 0.1 ml of brain microsomal fraction (containing 1 mg of protein in 1 ml), 0.1 ml of 0.1 mM ascorbate, and 0.1 ml of 5 mM FeSO ₄ . After mixing well, incubation was performed in a shaking water bath at 37 ° C. for 1 hour to induce peroxidation. The control was carried out in the same manner as above without adding a sample. After incubation, 0.5 ml of a mixture of 3 M trichloroacetic acid and 2.5 N HCl was added according to Esterbauer et al. (Esterbauer, H., et al., In ` Methods in Enzymology ', Academic Press, New York, USA, 1981). After centrifugation at 3,000 rpm for 10 minutes, 1 ml of supernatant was added, 1 ml of 0.67% TBA was added, mixed and heated for 30 minutes in boiling water for color development. After cooling, the absorbance was measured at 535 nm, and the inhibition of lipid peroxidation was compared with the inhibition rate (%) for the absorbance of the control.

Lipid peroxidation reaction is recognized as the most important mechanism that indicates the pathological physiological phenomena and tissue damage caused by various toxic compounds, drugs or diabetes. In general, cell membrane damage of biological tissues is pointed to as one factor due to the peroxidation of polyunsaturated fatty acid, a component of cell membrane, which is caused by oxygen free radicals produced by internal factors as well as in vitro factors of lipid peroxidation. This is caused by cascading peroxidative damage to biological membranes in which large amounts of unsaturated fatty acids are present. In addition, when a living body causes an imbalance between free radical scavenging systems that prevents the action of free radicals, various harmful effects such as tissue damage, carcinogenesis, inflammation, adult disease, and aging are caused. In vivo, especially brain tissues appear to have a higher content of lipid peroxide than other tissues, and it is known that the incidence of diseases related to such lipid peroxides is high. Therefore, it is assumed that brain tissue is more sensitive to oxidative stress than other tissues in vivo. In this experiment, Fe ⁺⁺ / ascorbate was added to brain microsomes to induce peroxidation non-enzymatically, so that safflower seed, sprout and petals were soluble. And the effect of methanol extract was investigated.

As shown in FIG. 2, lipid peroxidation inhibition was determined by the petal water soluble extract of safflower (97.19%)> pure methanol extract (81.38%)> petal methanol extract (80.97%)> seed water extract (68.86%)> pure water soluble. Extract (64.99%)> seed methanol extract (64.95%) showed strong antioxidant activity in the water-soluble extract of the petals.

Experimental Example 4 Investigation of Antioxidant Activity by Thiocyanate

Antioxidant activity by thiocyanate was performed by modifying Nakatani's method (Nakatkani, N: Recent advance in the study on nature antioxidants. Nippon Shokuhin Kogyo Gakkaishi, 37 , 569-576, 1990). In other words, 9 ml of 40 mM phosphate buffer (pH 7.0) was added to 2.88 ml of linolenic acid (25 mg / ml in EtOH), 0.12 ml of each sample solution (2 mg / ml) was mixed, and then incubated at 40 ° C. at regular intervals. Measured by. Take 0.1 ml of this reaction solution into a test tube, mix 9.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate solution, and 0.1 ml of 20 mM ferrous chloride / 3.5% HCl solution. After exactly 3 minutes absorbance at 500 nm was measured. At this time, BHT, a synthetic antioxidant, was used as a BHT addition group for the comparison of activity using 1/10 of the sample addition amount.

Aqueous and methanolic extracts of safflower seed, shoots and petals were measured for antioxidant activity by the method of thiocyanate using linolenic acid, an unsaturated fatty acid.

Experimental results As shown in FIG. 3, as in the experiments using DPPH, saponified methanol and water-soluble extracts showed relatively high antioxidant activity compared to other extracts. In addition, like the safflower seed extract, safflower sprout methanol extract showed a strong antioxidant activity compared to the control until day 8 reaction. The high antioxidant activity of safflower seed in linolenic acid oxidation test system is not related to the presence of N-feruloylserotonin. However, BHT, which is more commonly used than these extracts, showed strong antioxidant activity. These results showed that the water-soluble extracts of safflower petals had higher contents of polyphenol compounds and inhibition of brain membrane peroxidation, but showed higher antioxidant activity in safflower seed extracts in linolenic acid oxidation test system.

From the above results, the DPPH method, which shows the degree of antioxidant activity, showed high antioxidant effect in each methanol extract and water soluble extract in safflower petals, sprouts and seeds. Inhibition of biomembrane microsomal lipid peroxidation effect was higher in safflower petal extract and linolenic acid oxidation test than safflower seed extract.

As described above, the safflower sprout methanol extract of the present invention prepared by using safflower ( Carthamus tinctorius L.) sprout as an extract solvent has excellent effects of antioxidant activity and hydrogen donating action, and thus, food and pharmaceutical products. It is a very useful invention in the industrial and cosmetic industry.

Claims (4)

Safflower seed soaked in water is sown in a sprout planter equipped with aeration to block light and added 10 times the amount of methanol to safflower sprouts obtained by cultivation for two weeks, and contains 0.1% safflower methanol extract obtained by extracting at 60 ° C. An antioxidant composition. delete delete delete

Images