KR20030021640A - Extracts of Phyllostachys edulis leaf having antioxidant activity and the process for preparation thereof - Google Patents

Abstract

PURPOSE: A method of preparing a leaf extract of Phyllostachys pubescence having excellent antioxidant activity, in particular, an active oxygen scavenging function is provided. Thus obtained extract can be applied to a pharmaceutical composition for prevention and treatment of aging and adult disease and a functional food and cosmetic for prevention of aging. CONSTITUTION: Phyllostachys pubescence is washed, freeze-dried and crushed to produce powder. Next, the dried powder is extracted under reflux, in an organic solvent selected from hexane, chloroform, ethylacetate and butanol and then solvent fractionated. Additionally, the solvent fraction is purified using column chromatography and thin layer chromatography. The antioxidant composition contains the Phyllostachys pubescence extract as an effective component.

Description

Extract of Bacterium japonica with antioxidant activity and its preparation method {Extracts of Phyllostachys edulis leaf having antioxidant activity and the process for preparation

The present invention relates to an extract of the dead bamboo leaf with antioxidant activity and a method for producing the same. More particularly, the present invention relates to an extract of an antioxidant activity with an antioxidant activity and a method for producing the same, which are extracted using an organic solvent and purified using column chromatography and thin layer chromatography.

All aerobic organisms, including humans, survive through energy metabolism using oxygen (O ₂ ). However, when oxygen in vivo undergoes various physical, chemical and biological stresses, superoxide anion radical (O ₂ ), hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (OH), etc. It turns into harmful active oxygen species, causing fatal physiological disorders in the human body and, in severe cases, causing disease and losing life. In particular, if the harmful effects of free radicals and free radicals continue to accumulate as the cells age, diseases related to various adult diseases such as carcinogenesis, arteriosclerosis, heart disease, and skin aging, as well as the overall aging of cells It has been suggested as a cause of human disease occurrence and aging (Harman, D., Free Radicals in Biology, Academic Press, New York, 255-275 (1982)).

As a result, research into the development of antioxidants known to control free radicals and reactive oxygen species, which have been identified as the cause of aging and geriatric diseases, has been actively conducted, and superoxide dismutase, peroxidase, and catalase have been developed. ), Many studies have been conducted on antioxidants such as glutathione peroxidase and low molecular weight antioxidants derived from natural products such as tocopherol, ascorbate, carotenoid, and glutathione. (Chang, SS, et al., J. Food Sci. 42: 1102, 1977; Hammerschmidt, PA and Pratt, DE, J. Food Sci. 43: 556, 1977; Pratt, DE and Watts, BW, J. Food Sci. 29: 17, 1964), 2,6'-di-tert-butyl-4-hydroxy toluene (2,6'-di-tert-butyl-4-hydroxytoluene, BHT), 2,6'- Di-tert-butyl-4-hydroxyanisole (2,6-di-tert-butyl-4-hydroxyanisole, BHA), etc. Many synthetic antioxidants have been developed and used in medicine and food (Kitahara, K., et al., Chem. Pharm. Bull. 40: 2208, 1992; Hatano, T., Natural Medicines 49: 357,1995; Masaki , H., et al., Biol. Pharm. Bull. 18: 162, 1995).

Butyl hydroxy anisole (BHA) and butyl hydroxytoluene (BHT), the synthetic antioxidants are relatively excellent in antioxidant capacity, but their usage and use are strictly limited. Therefore, there is an active attempt to find an antioxidant that is high in natural products and does not harm the human body. For example, catechin family compounds of green tea leaves (Nanjo, F .; Goto, K .; Seto, R .; Suzuki, M .; Hara, Y. Free Radic. Biol. Med. 21: 895-902, 1996), carrots Beca carotene-based compounds of the well-known, in the case of domestic flavone-based antioxidants isolated from the was studied by the present inventors (Kweon, MH; Kwak, JH; Sung, HC; Yang, HCJ Biochem. Mol. Chem. 29: 423-428, 1996).

Therefore, the present inventors, while researching to develop a new natural antioxidant, a large amount of antioxidants excellent in free radicals and free radical scavenging activity and lipid peroxidation inhibitory activity in the extract of the leaves of the bamboo shoots, one of the bamboos inhabiting in Korea It was found to contain.

Bamboo is widely distributed in subtropical and temperate regions. Especially, bamboo is an evergreen tree belonging to the genus Japonica, which is common in Asian monsoon zones. Bamboo is mainly cultivated in the southern part of Korea. . Kings and cotton pads are used for furniture, rather than food. Mengjongjuk (孟宗竹) is the thickest among bamboos, with a diameter of about 20cm in Japan. It is also used for making furniture, but it is commonly called edible bamboo because it is thick and can be eaten. In addition to the parts used for wood, all the dead leaves are discarded. In addition, the dead leaves have been used as a treatment for vomiting, anti-inflammatory, miscarriage, sweating, and intermediates (consent of consent, Chinese herbal medicine dictionary) and other substances that exhibit antimicrobial properties. It has been used as a bamboo container in Japan since ancient times, and has been used in food storage for meat and dumplings in Korea.

Most studies on bioactive substances of bamboo including bamboo shoots have been reported in Korea and Japan mainly on antibacterial activity. Already, 2,6-dimethylbenzoquinone and benzoic acid, which are antimicrobial substances in bamboo leaves, have been identified by Japanese researchers. In Korea, the antimicrobial activity of bamboo leaf extracts (Kim Mi-Jung et al., Korean Journal of Nutrition and Nutrition, Vol. 25 No. 1, p.135-142, 1996) and the general characteristics of bamboo shoots Very limited studies have been reported, including reports on component analysis (Jung Jong Sung et al., Korean Journal of Forestry, vol. 78, No. 1, p. 55-60, 1989). In addition, using a bamboo extract extracted from bamboo bamboo bamboo, bamboo leaves or a mixture thereof, a meat product processing method that can remove the off-flavor of meat material and enhance the taste of meat (Korean Publication No. 98-082956), there is a domestic patent (Korean Publication No. 2000-074538) that improves the quality of food by simply adding bamboo leaf powder or bamboo leaf extract (paper of bamboo or habitat unknown), but it is mainly discarded. It was first discovered by the present inventors that the leaf extracts of the bamboo shoots have antioxidant activity.

Therefore, it is an object of the present invention to provide an extract of the dead leaves of antioxidant. It is another object of the present invention to provide a method for preparing the extracts of the Blind bamboo leaf having the antioxidant activity.

The present invention is lyophilized by lyophilization and then crushed and powdered, extracted with ethanol, and then hexane, chloroform, ethyl acetate, butanol using an organic solvent partitioned fractions sequentially, using column chromatography and thin layer chromatography The free radical scavenging activity, free radical scavenging activity, and lipid peroxidation inhibitory activity of the purified bamboo blind leaf extract were measured.

Hereinafter, the configuration and operation of the present invention.

1 is a graph showing the antioxidant activity of the ethanol extract and organic solvent fraction of the present invention.

The manufacturing method according to the present invention comprises the steps of reflux extraction at 80 ± 2 ° C. using 70% ethanol after lyophilizing or drying and crushing the dead bamboo leaves after crushing; Ethanol precipitation of the extract to remove the polymeric compound, and then sequentially partitioning fractions using hexane, chloroform, ethyl acetate, butanol with different polarities; Dissolving the extract in distilled water to purify the column with four steps of adsorptive HP-20 column, polar silica gel column, LH-20 gel filtration column, ODS reversed phase chromatography; The column purification active fractions are dissolved in methanol and finally purified by thin layer chromatography followed by normal and reverse phases.

Scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, xanthine and xanthine oxidase, for fractions obtained in each step of the process As a result of measuring the scavenging activity of the active oxygen generated by the superoxide and the lipid peroxidation inhibitory activity in the microsomes isolated from the rat liver, it was confirmed that each fraction has antioxidant activity. A comparative study with antioxidants showed that the activity was better.

Therefore, the refractory extract, concentrate and dry the dried powder of the bamboo shoots with ethanol to obtain a water-soluble dark brown extract, which has DPPH free radicals and active oxygen activity and lipid peroxidation inhibitory composition as an antioxidant composition Can be used.

In addition, the ethanol extract is subjected to ethanol precipitation to remove the polymeric compound, and then sequentially partitioned and fractionated using organic solvents having different polarities, and solvent-soluble and lyophilized water-soluble brown powder is obtained for the highest active butanol fraction. It can also be used as an antioxidant composition.

In addition, the water-soluble pale yellow powder obtained by dissolving the brown powder, which is the butanol fraction in distilled water, through four-stage purification of an adsorptive HP-20 column, a polar silica gel column, an LH-20 gel filtration column, and an ODS reversed phase column chromatography was also used as an antioxidant composition. Can be used.

After dissolving the column-purified slightly yellow powder in methanol, finally performing thin layer chromatography of the normal phase and reverse phase, taking a separation band showing strong activity, eluting the active substance in methanol, and centrifuging the silica gel in the remaining eluate. And after removal through filtration concentrated and lyophilized water-soluble milky white powder can be used as an antioxidant composition.

In addition, the antioxidant composition derived from the stem of the present invention includes all the extracts of the previous step.

The blind bamboo leaf extract according to the present invention was shown to have an excellent antioxidant effect as compared to the conventional natural antioxidants as well as synthetic antioxidants. Therefore, the composition for antioxidants containing the extracts of the stem of the present invention can be used as a pharmaceutical composition or functional food and cosmetics for preventing and treating aging and geriatric diseases. In addition, since the dead bamboo leaf has been used for a long time as a herbal medicine and edible extract of the present invention can be expected that there will be no problems such as toxicity and side effects.

Hereinafter, the specific configuration and operation of the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these embodiments.

Example 1 Preparation of Bacterium Leaf Antioxidant Extract

Bamjongjuk leaf used in the present invention is a leaf of Bamjongjuk inhabiting Hacheongmyeon, Geoje-do, Korea, collected and washed with water, lyophilized or crushed after drying, and used as an extract.

Experimental Example 1 Ethanol Extraction of the

500 g of the dried bamboo leaf powder was added to a 5 L volume round bottom flask, and 3 L of 70% ethanol was added to the mixture at 80 ± 2 ° C. for 6 hours at reflux for 3 to 4 times to extract about 10 L of extract. The obtained residue was removed by centrifugation (4 ° C, 5000 rpm, 25 minutes) and reduced pressure filter. The filtrate was removed using a rotary evaporation pressure reducer and lyophilized to obtain about 90 g of antioxidant extract.

Experimental Example 2: Organic Solvent Distribution of

After dissolving the extract in Experimental Example 1 in 1 L of distilled water, 3 L of cold 95% ethanol was added thereto, stirred, and left to stand at 4 ° C for 12 hours. The precipitate produced in this process is removed by centrifugation (4 ° C., 6000 rpm, 30 minutes) and reduced pressure filter filtration, and the filtrate is dried under reduced pressure and redissolved in 2 L of distilled water. Then, 2 L of n-hexane was mixed in a 5 L separatory funnel, and the resultant was separated twice into a hexane layer and an aqueous layer. 2 L of chloroform was added to the aqueous layer, and the mixture was partitioned twice into a chloroform layer and an aqueous layer, and ethyl acetate was further added to the aqueous layer, twice separated into an ethyl acetate layer and an aqueous layer, and 2 L of n-butanol was finally added to the aqueous layer. Fraction was performed twice with butanol layer and aqueous layer.

Each fraction obtained above was dried under reduced pressure to remove each organic solvent, followed by lyophilization. As a result, 15.2 g, 6.9 g, 2.1 g, 9.3 g, and 16.1 g of hexane fraction, ethyl acetate fraction, butanol fraction, and final aqueous layer fraction were obtained. Got it.

Experimental Example 3: Column Chromatography Purification

The n-butanol fraction (9.3 g) of Experimental Example 2 was dissolved in 50 mL of alkaline water (pH 10) and adsorbed onto the upper part of the first column (4 x 28 cm) filled with adsorptive diion HP-20 resin. 2.2 g of the active fraction eluted with% methanol is eluted in a second column (5 x 12 cm) filled with polar silica gel using a chloroform solvent with increasing methanol concentration. The active fraction was dried under reduced pressure and dissolved in 1 mL of methanol, and introduced into a third column (2 x 95 cm) filled with low molecular gel filtration Sephadex LH-20. Fractions were made in 4 mL portions per minute. The active fractions (tubes 48-60) were mixed and concentrated in a fourth column (1.5 x 8.5 cm) filled with hydrophobic octadecyl silane (ODS) and flown at a concentration gradient of 30 to 70% methanol. Fractionation of 2 mL under mL / min yielded 357 mg of strong antioxidant activity fraction (tubes 10-18).

Experimental Example 4 Thin Layer Chromatography Purification

After dissolving the active fraction of Experimental Example 3 in a small amount of methanol and dropping onto a thin layer chromatography plate (20 × 20 cm) coated with polar silica gel, n-butanol: methanol: water = 4: 1: 2 Develop with solvent and scrape off the active band at the Rf value of 0.7. This component was eluted with methanol and redeposited on a reversed phase plate (20 x 10 cm) coated with hydrophobic octadecsilane (ODS) silica to develop 30% methanol as a mobile phase solvent, and the final purified dead leaf leaf antioxidant at the Rf 0.3 position. 108 mg of the composition was obtained.

Example 2 Investigation of Antioxidant Activity of Extracts from

In order to investigate the antioxidant activity prepared in Example 1, scavenging activity, free radical scavenging activity, and lipid peroxidation inhibitory activity on free radicals were measured, respectively.

The free radical scavenging activity was measured using 1,1-diphenyl-2-picryl hydrazyl (1,1-diphenyl-2-picryl hydrazyl, DPPH) according to the method of Bullos. That is, 20 mg of DPPH was dissolved in 150 mL of ethanol to make a DPPH solution, and then a sample dissolved in 0.5 ml of methanol was added at a concentration of 0.1-1 mg / mL, immediately shaken for 5 seconds, and then 10 to 30 minutes later. , The absorbance decrease for the control group without the sample at 517 nm is shown as free radical scavenging activity.

Superoxide anion scavenging activity was measured by photooxidation of nitroblue tetrazolim (NBT) by active oxygen using an active oxygen generator using xanthin / xanthin oxidase enzyme reaction. It was measured using a change in absorbance (530 nm).

Lipid peroxidation inhibitory activity was determined by inducing lipid peroxidation by Fe ²⁺ / ascorbate system using microsomes obtained by ultracentrifugation (77000g, 60 minutes, Hitachi RP 30) of isolated rat hepatocytes. Malondialdehyde (malonaldehyde, MDA) was reacted with thiobarbituric acid (TBA) to quantify the amount of MDA reduced by the sample. Add 200 μL of the liver microsome solution containing the sample and 225 μL of sodium dodesyl sulfate (SDS) solution to the test tube, shake-mix for 5 seconds, and then add 20% acetic acid, 75 μL distilled water, and 1.2% TBA solution. 1 mL each was added and warmed in a water bath for 30 minutes. After cooling at room temperature for 30 minutes, the supernatant was measured for absorbance at 532 nm by centrifugation at 3000 rpm for 20 minutes to determine the lipid peroxidation inhibitory activity (%) of the control without the sample.

Experimental Example 5: Determination of antioxidant activity of each extract of organic solvent

Ethanol extract ( 1 ), n-hexane fraction ( 2 ), chloroform fraction ( 3 ), ethyl acetate fraction ( 4 ), n-butanol fraction ( 5 ) and final aqueous layer fraction ( 6 ) obtained in Experimental Examples 1 and 2 DPPH free radical scavenging activity, active oxygen scavenging activity and lipid peroxidation inhibitory activity at the concentration of 1 mg / mL were measured by the method of Example 2, and the results are shown in FIG.

As shown in Fig. 1, ethanol extract, which is a crude extract of the bamboo shoots, also has strong antioxidant activity due to its high DPPH free radical scavenging activity, active oxygen scavenging activity and lipid peroxidation inhibitory activity, and particularly, n-butanol fraction has excellent antioxidant effect. Appeared to be.

Experimental Example 6 Comparison of Purification Efficiency of the Final Purified Fraction of Ethanol Extract and Ethanol Extract According to the Present Invention

In order to confirm the purification efficiency of the thin layer chromatography final purified extract of Experimental Example 4, the DPPH free radical scavenging activity, active oxygen scavenging activity and lipid peroxidation inhibitory activity of Experimental Example 1 and Experimental Example 4 extract 2 It was measured by the method of and the results are shown in Table 1.

Comparison of Antioxidant Activity of Final Tablets from Ethanol Extracts and Ethanol Extracts Antioxidant activity (IC ₅₀ Value, μg) 70% Ethanol Reflux Extract Fraction Thin layer chromatography purification fraction DPPH radical scavenging activity 613.7 9.9 Free radical scavenging activity 579.3 2.9 Lipid peroxidation inhibitory activity 645.6 19.5

In Table 1, the IC ₅₀ value means a concentration of a substance that causes 50% of activity, and thus a lower value means a stronger antioxidant activity. As shown in Table 1, the final tablet extract of the dead bamboo leaf leaves showed about 62 times more DPPH free radical scavenging activity, 200 times more active oxygen scavenging activity, and 33 times higher lipid peroxidation activity than ethanol extract.

Experimental Example 7 Comparison of Antioxidant Activity of Purified Fractions of Blind Bamboo Leaves according to the Present Invention and Conventional Antioxidants

DPPH free radical scavenging activity, active oxygen scavenging activity, and lipid peroxidation inhibitory activity were investigated using the method of Example 2 under the same conditions as the final purified fraction of the bamboo shoot obtained in Experimental Example 4 and the conventional antioxidant. Conventional antioxidants include alpha-tocopherol, vitamin C, epigallocatechin, a green tea antioxidant, and other natural antioxidants, caffeic acid, chlorogenic acid and ferulic acid, and butylhydroxytoluene (BHT), a synthetic antioxidant. . The comparison results are shown in Table 2 below.

Comparison of Antioxidant Activity between the Antioxidant Compositions of Tablets Antioxidant activity (IC ₅₀ Value, μg) DPPH radical scavenging activity Free radical scavenging activity Lipid peroxidation inhibitory activity Blind Bamboo Antioxidant Composition 9.9 2.9 19.5 Alpha-tocopherol > 50 45.0 > 50 Vitamin c 25.3 > 50 - Epigallocatechin 8.6 > 50 26.3 Caffeic Acid 13.7 6.8 38.3 Chlorogenic acid 12.0 13.8 35.5 Ferulic acid 36.5 28.3 > 50 BHT > 50 > 50 19.2

As shown in Table 2, the final bamboo leaf extract showed a superior antioxidant activity compared to the conventional natural and synthetic antioxidants, especially active oxygen scavenging ability was very strong.

In addition, the antioxidant activity of the present invention is because the activity is not changed even at high temperature and strong light during the extraction process, so the resistance to heat and light is strong, it can be seen that it is a water-soluble stable compound because it is well soluble in water.

As described above through the above examples and experimental examples, the present invention extracts natural antioxidants having excellent antioxidant activity, in particular, active oxygen scavenging function, from the dead bamboo leaves, which have been disposed of in the past. Bacterium japonica extract of the present invention is a natural antioxidant, water soluble and resistant to heat and light, so it can be used as a pharmaceutical composition for preventing and treating aging and adult diseases, functional food for anti-aging, functional cosmetics, etc. It is a very useful invention in the field industry.

Claims (6)

Bleached bamboo leaf extract with antioxidant activity obtained by refluxing the bamboo leaf leaves with ethanol. [Claim 2] According to claim 1, In addition to the ethanol reflux extraction process is extracted with an organic solvent selected from hexane, chloroform, ethyl acetate, butanol and extracted from each of the solvent fractions. The antioxidant activity according to claim 2, wherein the butanol fraction is obtained by adsorptive HP-20 column, polar silica gel column, LH-20 gel filtration column, and ODS reversed phase chromatography in addition to the organic solvent partitioning fraction. With locust leaf extract. [4] The method of claim 3, wherein the extract of the columnar leaves having antioxidant activity, wherein the column fraction is obtained by thin layer chromatography in addition to the column chromatography process. Antioxidant composition comprising as an active ingredient the extract of any one of claim 1 selected from claim 1. Washing the seedling leaves with lyophilization and then crushing the dried powder to reflux with ethanol; Ethanol precipitation of the extract to remove the polymeric compound, and then sequentially partitioning fractions using hexane, chloroform, ethyl acetate, butanol with different polarities; And Method for producing a seed dead bamboo leaf extract comprising the step of purifying the organic solvent fractions by column chromatography and thin layer chromatography