KR101449377B1 - Extraction method of green tea acetone extracts - Google Patents

Abstract

The present invention relates to a method of extracting green tea acetone extracts, and more particularly, to an invention of confirming antioxidant activity and anticancer activity of green tea acetone extracts extracted with acetone as a solvent and having antioxidant and anticancer activities.
According to the present invention, besides existing catechins, it is possible to present a new source of the anticancer composition while revealing the new physiological activity function of the green tea, and the effect of presenting a new function as the anticancer composition of green tea.

Description

{Extraction method of green tea acetone extracts}

The present invention relates to a method for confirming that an acetone extract of green tea extracted with acetone exhibits antioxidative and antitumor activities.

Recently, it has been proven that the intake of phytochemicals contained in crops has a strong antioxidant activity and plays an important role in the prevention and treatment of various diseases. I various anti cancer agent and wherein the mutant has been to make to reduce the proliferation and the frequency of the intermediate parameters biomarker (biomarker) of the tumor (tumor), cancer (cancer) on the in vivo, antitumor through genetic toxicity test in a short period of time on in vitro It has been confirmed that it exhibits efficacy.

In various animal experiments, the anticancer efficacy has been verified at the level or above that the phytochemical is ingested into the human body. However, the investigation on chlorophyll found in the nutrients such as the leaves and fruits of the crops is very limited to be. In addition, the contribution of the antioxidant activity of the active ingredients of the crops to the treatment of diseases and health promotion contributes to the development of phytochemicals such as vitamin C, E, and polyphenol, or by pigment compounds such as chlorophyll and carotenoid , And most of the studies are limited to other phytochemicals except chlorophyll.

Chlorophyll is an intrinsic pigment that represents the green color of plants, and it plays an important role in absorbing the red and blue parts in the electromagnetic spectrum and converting solar energy into chemical energy through photosynthesis. During photosynthesis, electrons are directly delivered to the oxygen or energized to generate reactive oxygen species (ROS, reactive oxygen species) such as superoxide radicals (O ₂ ^- ) or singlet oxygen ( ¹ O ₂ ) oxygen species, which can act as an important signaling pathway for plant defense mechanisms under stress (Van Breusegem et al., 2001). Excess levels of reactive oxygen species are harmful to photosynthesis mechanisms It can provide a bad environment (Alscher et al. 1997). In this respect, chloroplasts need to develop effective defense mechanisms against photo-oxidative damage and photo-inhibition (Foyer et al., 1994; Asada, 2000). For this reason, plants contain various antioxidants and chlorophyll derivatives may be a source of potent oxidizing agents or antioxidants.

However, the physiological activity of such chlorophyll derivatives has not been studied at present and there is almost no use for them. The reason for this is considered to be due to the difficulty in obtaining chlorophylls in purified form and the decomposition pattern due to structural instability. For this reason, there is almost no report on antioxidant activity and anticarcinogenicity of chlorophyll.

As a pigment component present in plants, lutein, one of the carotenoids present in plants, is a hydroxyl carotenoid present in green vegetables.

Zeaxanthin, a compound associated with lutein, has two hydroxy groups as dihydroxy xanthophyll family carotenoids, while hydrocarbons such as β-carotene and lycopene (hydrocarbon carotenoids) do not have oxygen atoms. The hydroxy groups of lutein and giacastine show greater polarity than the hydrocarbyl carotenoids, which are carotenoids that do not contain it, and contribute to the beneficial effects of the human visual system.

Lutein is known to function only as a filter for high energy blue light, or to remove photocatalytic radicals and reactive oxygen species (Krinsky, 1989). It has been known that the intake of lutein in relation to disease is effective for eye diseases such as age-related macular degeneration and cataract.

Green tea was produced by deactivating oxidizing enzyme present in tea leaves with steam or heat by using shoots or leaves of tea plant ( Camellia Sinensis ), and it has been used as a symbol tea since BC. The difference in the search in green tea varies depending on the content of the chlorophyll compound including chlorophyll and its derivatives contained in the leaf. The higher the content of chlorophyll and derivatives thereof, the greener the search, the better the quality.

Recently, as the pharmacological mechanism of various components contained in green tea has been gradually found, methods for using green tea extract for various purposes as well as for edible use have been developed.

However, most of the researches related to green tea use the characteristics of catechin, and studies on components other than catechin have not been made sufficiently.

The inventors of the present invention have studied the physiological activity of chlorophyll derivatives and luteins for human health, and found that the use of chlorophyll a, chlorophyll b, pheophytin a, pheophytin b The present inventors completed the present invention by confirming that the acetone extract of green tea has an antioxidant and anticancer activity as a result of obtaining antioxidative and antitumor activities of green tea acetone extracts containing chlorophyll derivatives and chitosan derivatives.

Accordingly, it is an object of the present invention to provide an anticancer composition and a health supplement containing the green tea acetone extract.

As an example for achieving the above object, the method of extracting green tea acetone extract of the present invention is characterized by containing chlorophyll of chlorophyll a, chlorophyll b, pheophytin a and pheophytin b having anticancer activity.

The green tea acetone extract preferably has a chlorophyll content of 0.1 to 20 mg / g.

In another aspect of the present invention, the present invention provides a health supplement for cancer prevention and prevention, which comprises the anti-cancer composition.

Hereinafter, the anticancer composition of the present invention will be specifically described focusing on an example of obtaining green tea acetone extract. Also, in the following specification, chlorophyll means chlorophyll a, b and pheophytin a, b.

First, in order to prepare the green tea powder, the leaves of the green tea are dried and pulverized to make dry powder. The thus-dried green tea leaves are pulverized by various methods and powdered.

The green tea powder is extracted with chlorophyll and derivatives thereof using acetone as a solvent. The acetone may be used at a weight of 5 to 20 times the weight of the green tea powder. The extraction may be carried out by various methods such as low temperature dark condition extraction, room temperature extraction, reflux extraction, and ultrasonic extraction.

In the case of low temperature dark condition extraction, the extraction was carried out under refrigeration conditions, specifically at 3 to 5 DEG C, and when extracted for 10 to 24 hours, the extraction efficiency of chlorophyll a, b and pheophytin a and b was high. The extraction efficiency of chlorophyll a, b and pheophytin a and b was high when the extraction was carried out at room temperature for 20 to 25 ° C for 22 to 24 hours. The extraction efficiency of chlorophyll a, b and pheophytin a and b was high when the extraction time was 160 to 180 minutes when the extraction was performed at 37 to 40 ° C in the case of ultrasonic extraction. In the case of reflux extraction, the extraction efficiency of chlorophyll a, b and pheophytin a and b was high when the extraction time was 120 to 180 minutes at 75 to 80 ° C.

When the low-temperature dark condition extraction, the room temperature extraction, the ultrasonic extraction and the reflux extraction are compared, reflux extraction is more preferable in order to obtain a large amount of chlorophyll, and ultrasonic extraction is also possible for convenience of work.

As described above, in order to obtain the extract by immersing the dry powder of green tea directly in acetone, the green tea is extracted several times with hot water to remove the phenolic and polar substances contained in the green tea, and the filtered residue is dried and dried Compared with the conventional method of pulverizing and immersing in acetone to obtain an extract, it is possible to suppress the structural destruction due to pyrolysis and oxidation of chlorophyll caused by high temperature and water when extracting polar substances using hot water, There is an advantage that the operation other than the stepwise extraction, such as drying and re-pulverization after hot water extraction, can be simplified.

In addition, in the conventional method, several times of hot water extraction performed for elution and removal of the polar material involves the structural destruction of chlorophyll by pyrolysis and oxidation due to use of high temperature and water.

On the other hand, in the present invention, by using the low-temperature immersion and hexane distribution described below, the polar substances are completely removed from the acetone extract and the decomposition of chlorophyll, which may be generated by high temperature and water, by heat and oxidation is prevented, So that a large amount of chlorophyll can be extracted with high purity.

As described above, the filtrate extracted with acetone is allowed to stand at low temperature (4 to -20 DEG C) for 12 to 24 hours to filter insoluble precipitate. The insoluble precipitate contains various polar substances such as sugar, glycoside, phenolic substance, polar protein, and vitamin binding substance which are recrystallized due to extremely low solubility in a low-temperature acetone state. The precipitate is left at low temperature to form a precipitate, There is an advantage in that a part of the polar substance whose solubility is lowered in acetone can be removed through specific heat treatment.

Since the filtrate in which the insoluble precipitate is removed by leaving at a low temperature as described above coexists with some polar and intermediate polar substances, it is possible to use the solvent distribution, which is a specific heat treatment method that minimizes the structural destruction, Is completely removed.

That is, saturated saline and distilled water were added to the acetone filtrate from which the insoluble precipitate was removed, hexane with a low specific gravity was added thereto with a nonpolar solvent, and the mixture was vigorously shaken to induce layer separation. The upper layer, The chlorophylls can be separated and recovered without any inclusion of the polar material.

Acetone is an organic solvent and is well mixed with other organic solvents or distilled water. When distilled water or a polar solvent is simply added to remove polar substances from the acetone extract, acetone and other organic solvents and distilled water Are mixed with each other without inducing the layer separation. Therefore, it is impossible to remove the intermediate polar and polar materials from the acetone extract through layer separation using the polarity of the solvent.

Thus, in the present invention, saturated saline is used to remove some intermediate polarity and polar substances mixed in the acetone extract. Since the method using saturated saline solution is a method by non-heat treatment, the problems caused by the heat treatment can be solved. In addition, when saturated saline solution and distilled water are simultaneously added to the acetone extract, the saturated saline used together with the distilled water acts as a polar solvent. However, since the distilled water is almost saturated with the solute by the saturated salt added, The polar material can dissolve, but the solubility at such a level as to dissolve the non-polar material is completely lost. When nonpolar solvent hexane is added, chlorophyll and its derivatives, which are nonpolar materials contained in the acetone extract, naturally shift to a hexane layer having a relatively higher polarity affinity than a saturated saline solution, and a polar material Is transferred to the saline solution layer, it is possible to exclude the polar material from being mixed into the hexane layer.

The intermediate polar and polar substances are removed using the above hexane distribution, and hexane as a solvent is removed in a hexane solution containing high purity chlorophyll to obtain a green tea acetone extract.

On the other hand, the green tea acetone extract was concentrated and then subjected to column chromatography to obtain individual fractions of chlorophyll. The fractions were concentrated to dryness by re-dissolution and purified by preparative HPLC.

The individual structures of purely separated chlorophylls were identified by various spectroscopic methods such as NMR and MS. As a result, chlorophyll a, b and pheophytin a and b were confirmed.

Chlorophyll is the most widely found pigment in nature, and many researchers have been interested in the health effects of chlorophyll and metabolites, such as high content in plants, human digestion processes, and degradation during food processing.

The anticancer composition comprising the green tea acetone extract as an active ingredient can be administered orally or parenterally at the time of clinical administration, for example, intravenously and intraarterially, intramuscularly, subcutaneously, intraperitoneally, mucosally or topically, transdermally.

The compositions may be formulated for oral administration, for example as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs, And the like. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation into formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrating agents such as corn starch or sweet potato starch; Lubricating oil such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the composition of the present invention can be administered parenterally, and parenteral administration is applied to subcutaneous injection, intravenous injection, intramuscular injection, intra-thoracic injection injection method and mucosa or local application. Nasal spray or inhalant), gel, suspension (aqueous or non-aqueous liquid suspension, oil-in-water emulsion or water-in-oil emulsion), solution, and the like. For formulation into parenteral administration formulations, the composition may be formulated as a solution in the form of a unit dosage form of an ampoule or vial by mixing it with water in combination with a stabilizer or buffer.

The effective dose of the green tea acetone extract of the present invention may vary depending on the patient's age, physical condition, body weight, etc. Generally, 1 to 20 mg / day, preferably 5 to 10 mg / Day, and may be administered several times a day, preferably two to three times a day, at regular intervals according to the judgment of a doctor or pharmacist.

In addition, since the green tea acetone extract of the present invention is extracted from edible green tea, it is safe for human body and suitable for use in food, so it can be developed as a health supplement food having cancer prevention and prevention effect.

When the green tea acetone extract of the present invention is contained in the health supplement, it may be used in the range of 0.01 to 50% (w / w), preferably 1 to 30% (w / w) of the total weight.

That is, various foods can be prepared by a method commonly known to be added to ordinary palatable foods such as noodles such as ramen noodles, noodles, tofu, cereals, breads, chewing gum, candies, confectionery, etc., And can also be formulated into a common formulation such as tablets, granules, pills, hard capsules, soft capsules or liquid formulations, and can be made into juice, pouches, beverages, or dairy products, May be appropriately selected and blended by those skilled in the art according to the formulation.

Carotene bleaching analysis was performed on chlorophyll-a, chlorophyll-b, pheophytin-a, and pheophytin-b isolated from green tea to inhibit lipid peroxidation and water / linoleic acid emulsion (β-carotene bleaching (DPPH radical scavenging activity, hydroxyl radical scavenging activity, and superoxide anion scavenging activity) by applying the electron microscope (SEM) and the electron spin resonance (ESR) The antioxidant activities of the compounds were assayed by the activity and reducing power assays.

According to the present invention, it can be expected that an acetone extract of green tea can be used as an anticancer composition in addition to the well-known green tea catechins.

According to the present invention, an effect of presenting a new source of anticancer composition can be expected.

1 shows the antioxidant activity (A) and the inhibition rate (B) of the acetone extract of green tea ( Camellia sinensis , GT) against the production of peroxidase by the oxidation of linoleic acid.
Figure 2 shows the antioxidant activity of green tea acetone extracts measured by the? -Carotene bleaching assay method in a water / linoleic acid emulsion.
Fig. 3 shows DPPH group-clearing activity of green tea acetone extract and? -Tocopherol. α-tocopherol was used as a positive control and no antioxidant system was used in the control. Absorbance was measured at 517 nm and the results were expressed as mean ± SD [n = 3].
FIG. 4 shows the reducing power of acetone extract of BHA and Camellia sinensis (GT). BHA was used as a positive control and the results were expressed as mean ± SD [n = 3].
FIG. 5 shows the inhibition rate of superoxide formation on NBT reduction of green tea acetone extracts, and α-tocopherol was used as a positive control, and the results were expressed as mean ± SD [n = 3].
6 shows the hydroxyl-scavenging activity of green tea acetone extract (GT, 100 占 퐂 / ml). BHA (butylated hydroxyl anisole) and α-tocopherol were used as positive control and acetone without antioxidant was used as negative control. Results were expressed as mean ± SD [n = 3].

Below. The present invention will be described in detail based on examples and the like, but the present invention is not limited to the following examples and the like.

Example 1. Preparation of green tea acetone extract

The green tea leaves were dried and pulverized and passed through a 60 mesh sieve. 2 L of acetone was added to 20 g of the pulverized sample, and extracted with a sonicator at 40 ° C for 3 hours. The same procedure was repeated three times. The extracted solutions were all combined. 2 filter paper, and insoluble precipitate was re-filtered while standing at -20 캜 low temperature for 24 hours.

600 mL of the filtered acetone extract was transferred to a separatory funnel, and 200 mL of saturated saline and 1,000 mL of distilled water were added thereto. 300 mL of hexane was further added thereto and vigorously shaken to induce layer separation. After the layer was separated, The saline layer was discarded and the hexane layer in the upper layer was completely recovered.

The filtered hexane layer was concentrated in a 40 [deg.] C vacuum condenser to prepare a black tea gelatin acetone extract in which the hexane solvent was removed.

Experimental Example 1. Lipid peroxidation inhibitory activity of green tea acetone extract

Antioxidant activity of green tea acetone extract was tested by Nagai et al. (2005). 0.208 ml of sodium phosphate buffer (pH 7.0) was mixed with 100 μg / ml of green tea acetone extract and 0.208 ml of 2.5% (w / v) linoleic acid was added. The start of the oxidation reaction was carried out by adding 0.021 ml of 0.1 M 2,2'-azobis (2-amidinopropane) dihydrochloride [30] And the degree of oxidation of linoleic acid was checked every 12 hours from 72 hours to 72 hours. To 0.1 ml of the above reaction solution every 12 hours after the start of the oxidation reaction induction, 0.07 M ferrous chloride solution containing 4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, 3.5% 0.1 ml was added and the mixture was allowed to stand for 3 minutes. The degree of peroxide formation over time was measured by measuring the absorbance at 500 nm using a UV-1200 UV / VIS spectrometer (Shimadzu, Kyoto, Japan). Control group was prepared by adding linoleic acid alone without adding green tea acetone extract and using α-tocopherol (VE) at the same concentration level. After 72 hours, the final inhibition rate (%) was calculated as the ratio of the final absorbance to the treatment absorbance to the control absorbance. The results are shown in Fig.

Lipid peroxidation is initiated as hydrogen atoms (H ·) are taken away from methylene (--CH ₂ -) groups of unsaturated fatty acids by free radicals. The common radicals are OH, RO, ROO, HO ₂ , etc. O ₂ ^- and H ₂ O ₂ do not have this capability in their own right. The alkyl group (R ·) formed by the deodorization of hydrogen is converted into a diene form through molecular reconfiguration, which again binds to oxygen to form peroxy radical. As described above, the peroxidation of the peroxidic group leads to a rapid increase of the peroxide because a kind of chain reaction proceeds by deodorizing hydrogen from the other unsaturated fatty acid.

In case of green tea acetone extract, the absorbance at 100 ㎍ / ㎖ showed a low absorbance at 12 hours, and the increase rate of absorbance increased from 24 hours to 31.7% after 72 hours And exhibited lipid peroxidation inhibitory activity lower than that of vitamin E. In addition, the antioxidative activity of linoleic acid at the early stage of peroxidation was examined. As a result, the absorbance of the acetone extract of green tea increased rapidly from 12 hours in the control group, but the rate of increase in the rate of increase was significantly decreased in the acetone extract of green tea.

Experimental Example 2. Analysis of β- carotene bleaching assay of green tea acetone extract

The antioxidative activity of green tea acetone extracts was examined by modifying the delayed ability of β-carotene bleaching in water / linoleic acid emulsion by Miller (1971). β-carotene exhibits rapid fade in the absence of other antioxidants, and the free linoleic acid attacks β-carotene to dissociate the double bond and lose its specific color characteristics. Green tea acetone extract was treated at a concentration of 100 ㎍ / ㎖, and the same concentration of BHT was used as a control. To prepare the emulsion, 1 ml of β-carotene solution (1 mg / ml in chloroform), 40 μl of linoleic acid (20 mg) and 400 μl of Triton X-100 (100 mg) were placed in a flask and chloroform was removed in the presence of nitrogen , 100 ml of ionized water oxidized with oxygen for 30 minutes in advance was slowly added thereto while stirring, and a stable emulsion was formed. 3 ml of the emulsion solution was placed in a spectrophotometric cuvette (light path 10 mm), and 0.2 ml of the prepared green tea acetone extract was added and the absorbance was measured at 470 nm. After the initial absorbance was measured, the reaction solution was stored at 50 ℃ for 15 min in a dark place on a water bath for up to 120 min. The degree of antioxidant activity was decreased in the optical density (DOINIT - DO final) The amount of oxidation was calculated as 100% oxidation and expressed as the inhibition rate of oxidation for the control without pigments.

As shown in FIG. 2, the acetone extract of green tea showed an oxidation inhibition rate of 48.3% when treated at a concentration of 100 μg / ml, but no significant difference was observed at the concentration above 100 μg / ml.

Experimental Example 3: DPPH radical scavenging activity of green tea acetone extract

DPPH radical scavenging activity of green tea acetone extract was assayed by Schimada et al. (1992). 1 ml of a 0.5 mM DPPH ethanol solution and 2 ml of 0.1 M acetate buffer were mixed and treated with green tea acetone extract (concentration: 10 ~ 50 ㎍ / ml), and allowed to stand for 30 minutes in a dark state at room temperature And the absorbance was measured at 517 nm using a UV-1200 UV / VIS spectrometer (Shimadzu, Kyoto, Japan). The positive control group was compared with 10 to 50 μg / ml of α-tocopherol (VE).

As shown in Fig. As a result of comparing DPPH radical scavenging activity of green tea acetone extract with α-tocopherol, green tea acetone extract showed 26% inhibition rate when treated with 10 μg / ㎖, similar to positive control α-tocopherol showing 28% inhibition rate The inhibition rate was 41% at 20 ㎍ / ㎖ treatment and 46% at 30 ㎍ / ㎖ treatment. However, the inhibition rate was only maintained at a constant level at 20 ㎍ / ㎖ treatment.

Experimental Example 4. Comparison of Reducing Ability of Green Tea Acetone Extract

The reducing power of the green tea acetone extract was measured by Oyaizu (1986) method and the reducing power of iron ion to antioxidant was measured. 1 ml of 0.2 M sodium phosphate buffer (pH 6.6), 1 ml of a concentration-dependent sample (10-100 μg / ml) and 1 ml of 1% potassium ferricyanide were mixed and the mixture was mixed with 50 After incubation for 20 min., 1 ml of 10% TCA (triobarbituric acid) was added. After the reaction mixture was centrifuged at 13,000 × g for 5 minutes, 2 ml of methanol was added to 2 ml of the supernatant, and 0.1 ml of 0.1% iron chloride was added thereto. The absorbance was measured using a UV / VIS spectrophotometer Lt; / RTI > BHA was used as a control, and the erasure rate was calculated by calculating (%) = [1- (A0 / A1)] × 100 [A ₀ = blank, A ₁ = extract].

As shown in FIG. 4, when the reducing power of the green tea acetone extract was compared with that of BHA, the reduction power of the green tea acetone extract was increased proportionally with increasing concentration, and when 20 to 80 ㎍ / BHA, but it showed lower activity than BHA at over 100 ㎍ / ㎖.

Experimental Example 5. Superoxide anion scavenging activity of acetone extract of green tea

The superoxide anion scavenging activity of green tea acetone extract was investigated by the superoxide anion generated by the reaction of xanthine and xanthine oxidase and nitrone spin trap reacting rapidly with these superoxide anion. DMPO (5,5-dimethyl-1-pyrroline-N-oxide). 20 μl each of green tea acetone extract and ascorbic acid concentration samples (5, 10, 25, and 50 μg / ml), 20 μl of 3 M DMPO and 10 μl of 10 mM 20 μl of xanthine and 20 μl of 0.25 U xanthine oxidase were added and mixed to make the total volume 200 μl. The mixture was left at room temperature for 2.5 minutes, transferred to a quartz capillary tube and analyzed by an ESR spectrometer (JES-FA ESR spectrometer, JEOL, Tokyo, Japan). The control blank was prepared by adding 20 μl of acetone instead of the sample. The magnetic field of the ESR was 336.5 mT, the power was 20 mW, the frequency was 9.8 GHz, and the modulation amplitude was 1.0 was adjusted to gauss, gain was 200, scan time was 0.5 minute, scan width was 10 mT, time constant was 0.03 sec, temperature was 25 Lt; 0 > C. Calculation of the scavenging activity of the superoxide anions on the samples was carried out by the following formula using the difference in mean height to the signal intensities of the treatment and control.

Activity (%) = [1- (ESR signal intensity for medium containing the additives / ESR signal intensity for the superoxide anion)] × 100

The results of the superoxide anion scavenging activity (%) of the acetone extract of green tea and ascorbic acid by concentration using ESR were shown in comparison with the following Table 1.

Extract ([mu] g / ml) Camelliasinensis (GT) Ascorbic acid 5 15.16 ± 1.33 5.14 ± 0.22 10 35.11 ± 2.15 17.26 + - 0.58 25 48.52 + 1.64 25.17 ± 1.08 50 63.28 + - 2.53 36.15 ± 1.86

As shown in Table 1 above, the superoxide scavenging activity of the acetone extract of green tea was superior to that of ascorbic acid, and the scavenging activity was constantly increased as the concentration increased.

The superoxide radical scavenging activity of the acetone extract of green tea using ESR was investigated using nitro-blue tetrazolium (NBT) reduction (Nagai et al., 2005) . In other words, 0.02 ml of 3 mM xanthine, 0.48 ml of 0.05 mM sodium carbonate buffer, pH 10.5, 0.02 ml of 3 mM EDTA, 0.15% bovine serum albumin, 20, 40 and 60 ㎍ / ㎖ of green tea acetone extracts were added to the mixture consisting of 0.02 ml of water and 0.02 ml of 0.75 mM NBT. The mixture was incubated at 25 ° C for 10 minutes, Of XOD (6 mU / mL) was added to initiate the reaction, followed by reaction at 25 DEG C for 20 minutes. Then, 0.02 ml of 6 mM CuCl was added to terminate the reaction, and the absorbance was measured at 560 nm. The inhibition rate against NBT reduction according to the amount of sample added by concentration was shown, and α-tocopherol was used as a positive control. The results are shown in FIG.

As shown in FIG. 5, when the superoxide-scavenging activity of the green tea acetone extract was examined using NBT (nitro-blue tetrazolium) reduction method using ESR, green tea extract showed about 26% super And showed a tendency to increase continuously up to 60 ㎍ / ㎖.

Experimental Example 6. Hydroxy radical scavenging activity test of green tea acetone extract

The inhibitory activity of hydroxyl group formation by ESR was tested for the hydroxy group scavenging activity of green tea acetone extract. Hydroxy group formation was carried out using Fenton reaction (H ₂ O ₂ + FeSO ₄ ) and DMPO, which can react rapidly with hydroxyl group, was used. 20 μl each of the green tea acetone extract and ascorbic acid concentration samples (5, 10, 25 and 50 μg / ml) in 0.1 M phosphate buffer (pH 7.4), 0.3 M DMPO (5,5- 1-pyrroline-N-oxide (0.2 mL), 10 mM FeSO _{4 (} 0.2 mL) and 10 mM H ₂ O _{2 (} 0.2 mL) were added to the mixture, and the mixture was allowed to stand at room temperature for 2.5 minutes, transferred to a quartz capillary tube, (JES-FA ESR spectrometer, JEOL, Tokyo, Japan). The control was prepared by adding 20 μl of acetone instead of the sample. The magnetic field of ESR was 336.5 mT, the power was 20 mW, the frequency was 9.8 GHz, the amplitude modulation was 1.0 Gaussian, the gain was 200, and the scan time was 0.5 minute The scan width was 10 mT, the time constant was 0.03 sec, and the temperature was fixed at 25 ° C. Calculation of scavenging activity of hydroxy groups on the samples was carried out by using the difference in average height of the signal intensities between the treatment and the control.

Activity (%) = [1- (ESR signal intensity for medium containing the additives / ESR signal intensity for the superoxide anion)] × 100

The hydroxy-scavenging activity (%) of green tea acetone extract and ascorbic acid using ESR was shown in Table 2 below.

Concentration (占 퐂 / ml) Camellia sinensis (GT) Ascorbic acid 5 7.68 ± 2.14 3.56 + - 0.54 10 29.14 + 1.15 8.65 0.75 25 34.64 + 1.31 9.25 + 1.56 50 39.22 + - 2.25 9.85 ± 1.44 Activity (%) = [1- (ESR signal intensity for medium containing the additives / ESR signal intensity for the superoxide anion)] × 100

As shown in Table 2, it was confirmed that the acetone extract of green tea had an excellent hydroxyl-scavenging activity as compared with ascorbic acid, and the scavenging activity was constantly increased as the concentration increased.

The hydroxy-scavenging activity of green tea acetone extracts using ESR was evaluated by converting the 2-deoxyribose by Fenton reaction into malonaldehyde by oxidation with hydroxyl group to obtain chromagen ) Was formed by the method of measuring the degree of formation. To the solution containing 2.8 mM 2-deoxyribose, 100 μM FeCl ₃ , 104 μM EDTA and 1 mM H ₂ O ₂ , 20 mM sodium phosphate buffer (pH 7.4) was added to prepare 1 ml of the mixture Respectively. The reaction was terminated by adding 0.75 ml of 1.0% (w / v) TBA to the reaction mixture for 1 hour at 37 ℃ in an Eppendorf tube. The reaction solution was treated with water at 98 ° C for 20 minutes and then cooled at room temperature and 1 ml of acetone was added thereto to stabilize the color. The color was stabilized by using a UV-1200 UV / VIS spectrometer (Shimadzu, Kyoto, Japan) And the absorbance was measured. The hydroxy-scavenging activity test was calculated as a ratio that inhibits the oxidation of 2-deoxyribose by the hydroxyl group upon addition of the green tea acetone extract and was compared with that of the existing antioxidants α-tocopherol (VE) and BHA . The results are shown in FIG. 6 below.

As shown in FIG. 6, in order to confirm the hydroxyl-scavenging activity of the green tea acetone extract by ESR, the hydroxy-scavenging activity of the green tea acetone extract was confirmed by the Fenton reaction. As a result, the green tea acetone extract showed about 150 μg / Ml, whereas a similar level of hydroxy group scavenging activity was observed in the positive control BHA (90 μg / ml) (concentration 400 μM) and in the case of α-tocopherol 180 μg / Respectively.

Generally, hydroxy groups are derived from H ₂ O ₂ and are the radicals that plants often produce under severe stress, which is the most reactive mechanism for the formation of reactive oxygen species and the reaction of these reactive oxygen species with other reduced forms of molecules It is known to be highly toxic (Foyer et al., 1994; Asada, 2000). In the elimination and elimination of reactive oxygen species, the elimination activity of the acetone extract of green tea against high hydroxy groups indicates that the green tea acetone extract as an antioxidant has a high therapeutic and prophylactic effect on various diseases caused by oxidative damage .

Experimental Example 7. Inhibition of cancer cell proliferation of green tea acetone extract

The acetone extracts of green tea were tested for inhibition of cell proliferation of cancer cell lines of green tea acetone extracts according to the concentrations (A549, AH49, LNCaP, HCT-15 and MCF-7) 200, 100, 50 / / ml), and cell viability was assayed by MTT assay. Each cancer cell line was cultured in a 96-well plate (1 × 10 ⁴ / well), and then the sample was treated for each concentration and further cultured for 36 hours. Cell viability was assayed using a commercially available kit (Cell Titer 96 non-radioactive cell proliferation assay kit, Promega, Madison, Wis.). The tetrazolium compound MTS [3- (4,5- 20 μl of a mixture containing an electron donating reagent phenazine methosulfate (PMS) was added to each well, and the mixture was again allowed to react for 1 hour . After incubation at 37 ° C in 5% CO ₂ , the absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay plate reader. The results are shown in Table 3 below [n = 3].

Extract Concentration (占 퐂 / ml) Viable Cell (% of Control) / Human Cancer Cell line ^* A549 ACHN LNCaP HCT15 MCF-7 GT 200 18.0 8.0 32.0 ± 4.0 24.0 ± 5.0 25.0 + - 4.0 16.0 ± 2.0 100 21.0 ± 6.0 46.0 ± 6.0 35.0 + - 4.0 36.0 ± 2.0 29.0 ± 4.0 50 54.0 + - 7.0 61.0 + - 6.0 41.0 ± 6.0 51.0 ± 4.0 58.0 + - 5.0

(A549), kidney cancer cell line (ACHN), colon cancer cell line (HCT15), and carcinoma cell line (HCT15) to test the growth and proliferation inhibitory effect of the green tea acetone extract on the human cancer cell line, (LNCaP) and breast cancer cell line (MCF-7). As a result, green tea acetone extract showed 50% cancer cell survival rate at 100 ㎍ / ㎖ for all cell lines, The cancer cell survival rate of A549 lung cancer cell line, LNCaP prostate cancer cell line and MCF-7 breast cancer cell line was 18%, 24% and 16% at 200 ㎍ / ㎖ treatment.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventions. It will be apparent to those of ordinary skill in the art.

In the figure, GT means green tea extract, VE means vitamin E (α-tocopherol), α-toco means α-tocopherol (vitamin E), BHA means butylated hydroxyl anisole).

Claims (3)

Drying and grinding the green tea leaves to prepare green tea powder; Immersing the green tea powder in acetone and filtering the filtrate at a low temperature of 4 to -20 占 폚 for 12 to 24 hours to remove insoluble precipitates; Adding a saturated saline solution, distilled water, and hexane to the acetone extract from which the insoluble precipitate has been removed, shaking it, and then taking a hexane fraction; And extracting the green tea acetone extract containing chlorophyll a, chlorophyll b, pheophytin a and pheophytin b chlorophyll having anticancer activity, which comprises concentrating the hexane fraction under reduced pressure to remove hexane. Way.
The method according to claim 1,
Wherein the green tea acetone extract comprises chlorophyll in a range of 0.1 to 20 mg / g.
The method according to claim 1,
Wherein the green tea acetone extract has activity against one or more cancer selected from lung cancer, prostate cancer, and breast cancer.

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