WO2005067404A2

WO2005067404A2 - Health food containing distilled pine-needle extract

Info

Publication number: WO2005067404A2
Application number: PCT/KR2005/000173
Authority: WO
Inventors: Gwang Seok Bae; Seung Si Ham
Original assignee: Gwang Seok Bae; Seung Si Ham
Priority date: 2004-01-20
Filing date: 2005-01-19
Publication date: 2005-07-28
Also published as: KR20050076325A; WO2005067404A3

Abstract

The present invention relates to health food containing clear extract of pine needles as an effective ingredient, more precisely, health food containing clear extract of pine needles as an effective ingredient which is prepared by the primary heating stage; the secondary heating stage; the third heating stage; the forth heating stage; draining stage; cooling stage; filtering stage. The clear extract of pine needles of the present invention has excellent anti-oxidation and anticancer activity, so that it can be effectively used for the preparation of health food having anticancer and anti-aging effect.

Description

[DESCRIPTION] [invention Title] HEALTH FOOD CONTAINING DISTILLED PINE-NEEDLE EXTRACT

[Technical Field] The present invention relates to health food containing clear extract of pine needles as an effective ingredient, more precisely, health food having anticancer and/or anti-aging effect containing clear extract of pine needles showing excellent anti-oxidation and anticancer activity as an effective ingredient.

[Background Art] Pine tree ( Pinus densiflora Sieb. et Zuccarini ) is an evergreen coniferous tall tree belonging to pinaceae, and is a wild plant found in every mountain ii. Korea

(KIM IL-HYUK, medicines botany particulars, Jinmyung publisher, 71, 1981) . Pine needles have been known to have stomach-strengthening effect and heamatic activity, and to prevent cerebral apoplexy, atherosclerosis, hypertension, diabetes mellitus, etc. Thus, they have been processed as Sunsik and/or tea for being taken (KIM WAN-HEE, the newest Dongeubogam, Pacific public corporation, 960, 1983) . Terepene, a volatile antibiotics, is one of active ingredients of pine needles and is included therein 7 - 12%. It is also called as monoterpene, seguiterpene or diterpene because it contains huge amount of isoprene. In the meantime, terepene is used for aromatherapy owing to its characteristic pharmaceutical effects such as anti-microbial, anticancer and anti-oxidative effects and lowering blood pressure, promoting hormone secretion, etc. Pine needles contain 16 kinds of amino acids, composing a protein, including 8 essential amino acids, so they can be an aid for the growth of a body, the increase of vitality, smooth blood flow, and improvement of hormone secretion, that is pine needles can be effectively used for anti-aging and the treatment of hyperlipidemia and high cholesterol. Among effective ingredients of pine needles, vitamin C shows activities of blood purification and anti-scurvy and has resistance against stress. Vitamin A has the function of strengthening mucous membrane so as to be used for the treatment of night blindness. Resin and tannin help the functions of digestive system. Alcohol and ester discharge waste products in body to hasten metabolism and have factors that can dissolve calcium compounds, indicating that they are effective ingredients to treat atherosclerosis. Iron is effective for the treatment of anemia. Chlorophyll reduces the accumulated cholesterol, and glycoginin has blood glucose lowering effect, making it a good candidate for the treatment of diabetes mellitus. Apiethinic acid helps detoxication of opium and nicotine and the treatment of pulmonary tuberculosis, pleuritis and other gastrointestinal diseases. In particular, abundant vitamin C and iron in pine needles are effective for the treatment of anemia, terpene promotes blood circulation by dissolving cholesterol causing atherosclerosis, and vitamin P (rutin) makes capillary vessels strong to prevent aging. In variety of health food, prepared by the processes of extracting effective ingredients which are physiologically active and show health improving effect and formulating those ingredients in the form for easy taking, are on market. In order to maintain the effect of such active ingredients included in food, the extracting process is the most important.

[Disclosure] [Technical Problem] A juice extractor has been generally used for extraction. The conventional juice extractor is designed for simultaneous pulverization and compression, and consumes electricity a lot. In addition, the extractor itself is made of metal, which might affect or induce reaction with extracted ingredients. It is also very noisy because it is an oil pressure system. Nutrients might be lost during the complicated extracting processes. It costs a lot to repair since the components are not itemized. Yield is unsatisfactorily low. Therefore, a novel extraction process has been required to overcome such disadvantages. In order to overcome the above problems of the conventional extraction of pine needles, the present inventors have developed a novel extracting device for pine needle extract which is composed of a steamer comprising an inner bucket and an outer bucket, a water tube boiler for heating the steamer, a distilled water concentration line for condensation of the steamed air, a water supply tube for the supply of water to the steamer, a cooler, in which the outside of cooling tube is surrounded by a water tank, that is attached to the end of the distilled water concentration line, and a filter for the filtration of the cooled solution. And the present inventors have applied for a patent with the device (Korean Patent Publication No: 2002-0029461, see Fig. 1) and also confirmed that clear extract of pine needles can be prepared by the device without change or loss of active ingredients of pine needles. The present inventors confirmed that clear extract of pine needles prepared by the above extracting device had excellent anticancer and anti-oxidative activities, and so the present inventors completed this invention by verifying that the extract can be effectively used as an active ingredient of health food for anticancer and anti-aging. It is an object of the present invention to provide health food containing clear extract of pine needles showing excellent anticancer and anti-oxidative activities as an effective ingredient.

[Technical Solution] In order to achieve the above object, the present invention provides anti-oxidative health food containing clear extract of pine needles as an effective ingredient. The present invention also provides anticancer health food containing clear extract of pine needles as an effective ingredient.

[Advantageous Effects] As explained hereinbefore, clear extract of pine needles of the present invention shows excellent anti- mutagenicity effect in addition to the genotoxicity inhibiting and anticancer effects, so that it can be successfully added to health food for anticancer and anti-aging.

[Description of Drawings] Fig. 1 is a schematic diagram showing the extracting device for the preparation of clear extract of pine needles of the present invention. <Explanation of marks of the diagram> 1: Washer, 2: Dehydrator, 3: Cutter, 5: Steamer, 6: Inner bucket, 7: Outer bucket, 8: Water tube boiler, 9: Distilled water concentration line, 10: Water supply tube, 11, 12: Valves, 13: Cooling tube, 14: Water tank, 15: Cooler, 16: Filter Fig. 2 is a graph showing the result of GC-MS total ion chromatography of volatile materials included in clear extract of pine needles of the present invention. Fig. 3 is a graph showing the anti-mutagenicity effect of clear extract of pine needles of the present invention on each mutagenic substance in a TA100 strain. Each bar represents the concentration of 50 /λP./plate, 100 μβ/plate, 150 £/plate, and 200 / /plate, from the left.

Fig. 4 is a graph showing the anti-mutagenicity effect of clear extract of pine needles of the present invention on each mutagenic substance in a TA98 strain. Each bar represents the concentration of 50 //-C/plate, 100 μJlJplate , 150 μJt/ plate , and 200 /plate, from the left. Fig. 5 is a graph showing the human tumor cell growth inhibiting effect of clear extract of pine needles of the present invention. Each bar represents the concentration of 12.5 /^6/well, 25 μJIJviell , 37.5 //f./plate, and 50 μJlJplate , from the left.

Fig. 6 is a graph showing the 293 cell growth inhibiting effect of clear extract of pine needles of the present invention. Fig. 7 is a graph showing the effect of clear extract of pine needles of the present invention on weight changes in rats. o: Clear extract of pine needles, •: Control

Fig. 8 is a graph showing the inducement of micronucleus formation after the administration of MNNG.

Fig. 9 is a graph showing that micronucleus formation induced by the administration of MNNG is inhibited by clear extract of pine needles of the present invention. •: Number of micronucleus per 1000 cells, A: Inhibition rate of micronucleus formation

[Best Mode] Hereinafter, the present invention is described in detail . Clear extract of pine needles of the present invention included as an effective ingredient in health food for anti-oxidation and anticancer is prepared by using the extraction device for clear extract of pine needles of the invention declared in Korean Patent Publication No. 2002-29461, and is prepared more preferably by the following steps: 1) Water and pine needles cut by a cutter (3) are put together in a steamer (5) at the ratio of 1:1 and the temperature of the steamer (5) is raised to 90^°C by a boiler (8) . Then, the boiler is turned off and the mixture is cooled down for 10 hours, which is the primary heating stage; 2) The steamer (5) is heated for 1 hour and 20 minutes at 100 ^°C under the pressure of 2 kg/cii, which is the secondary heating stage; 3) The steamer is heated again at 110^°C under the pressure of 2.7 kg/cilf for one hour, which is the third heating stage; 4) The steamer is heated for 40 minutes at 120^°C under the pressure of 3.4 kg/cilf, which is the forth heating stage; 5) The first heated vapor is passed through distilled water concentration line for 30 minutes and then drained out; 6) The vapor passed through the distilled water concentration line is cooled down in a cooler; and 7) The extract cooled down above is filtered by a filter.

Clear extract of pine needles of the present invention was tested to investigate its suitability for drinking water, aromatic constituents, anti-oxidative activity, UV-protecting effect, and whitening effect. As a part of in vi tro anticancer activity test, anti- mutagenicity test and cytotoxicity test to human cancer cell lines were performed. In addition, in vivo genotoxicity test and solid tumor cell growth inhibiting test using cancer cell lines were also performed. As a result, neither microorganisms including E. coli nor harmful heavy metals such as arsenic (As), lead(Pb), mercury(Hg), and cadmium(Cd) were found in clear extract of pine needles of the present invention. The contents of other ingredients in the extract were also under the permitted limit of drinking water. Thus, clear extract of pine needles of the present invention is safe for drinking. Anti-oxidative activity of clear extract of pine needles of the present invention was also investigated. While RC₅₀ of ethanol extract of pine needles, a control, was 69.91 β$, , RC₅₀ of clear extract of pine needles was 18.4 t, indicating that it has very strong anti- oxidative activity (see Table 4). UV-protecting effect of clear extract of pine needles was also investigated, resulting in 69.91 n and 18.4 nm at medium wavelength (308 nm) and long wavelength (350 nm) , respectively. Anti-mutagenicity activity of clear extract of pine needles was also investigated, resulting in the confirmation of no mutagenicity (see Fig. 3 and Fig. 4) . From the investigation of tumor cell growth inhibiting effect of clear extract of pine needles of the present invention was confirmed that the extract had relatively high inhibiting effect on various cancer cell lines, and in particular, 90.8%, 93.7% and 90.0% inhibiting effect on AGS, HeLa, and HepG2 cells were confirmed (see Fig. 5 and Fig. 6) . In order to investigate the effect of clear extract of pine needles on the growth of a small animal, weight changes of SD (Sprague-Dawley) rats were measured. As a result, significant weight gaining was observed after the administration of clear extract of pine needles (see Fig. 7 and Table 6) . In order to investigate the effect of clear extract of pine needles on organs, clear extract of pine needles was administered to SD rats for one month, then total weights of liver, kidney, spleen and heart were measured. The weight of each organ per 100 g of body weight was calculated and compared with that of a control to which distilled water was administered. As a result, significant weight changes of organs were not observed according to the administration of clear extract of pine needles of the present invention (see Table 7). Genotoxicity inhibiting effect of clear extract of pine needles of the present invention was also investigated in vivo. As a result, dose-dependent genotoxicity inhibiting effect was confirmed, in particular, 200 nig/kg of administration resulted in 72.4% inhibiting effect (see Fig. 9 and Table 8) . In addition, in order to investigate anticancer effect of clear extract of pine needles of the present invention, sarcoma-180 cell line was inserted in Balb/c mice to induce cancer and then solid tumor growth inhibiting effect was examined. As a result, the administration of clear extract of pine needles at the concentration of 200 μJlJday resulted in 26.9% solid tumor growth inhibiting effect, indicating that clear extract of pine needles could inhibit the growth of a tumor dose- dependently (see Table 9) .

The above results indicate that clear extract of pine needles of the present invention has excellent anti-mutagenicity and anticancer effects as well as in vivo genotoxicity inhibiting effect. As explained hereinbefore, clear extract of pine needles of the present invention has excellent anti- oxidative and anticancer effects, so that it can be effectively used for the production of health food promoting health condition. The applicable health food is exemplified by tea, Saeng-sik, meat, sausages, bread, chocolate, candies, snacks, cookies, pizza, ramyun, noodles, gums, dairy products, soups, beverages, or drinks, but not always limited thereto. In the preferred embodiments of the present invention, tea, beverages, Saeng-sik, candies and chewing gums were prepared as health food containing clear extract of pine needles of the present invention.

[Mode for Invention] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

<Manufacturing Example 1> Preparation of clear extract of pine needles The present inventors prepared clear extract of pine needles by using the extracting device that has been declared in Korea Patent Publication No. 2002-29461. Pine needles were taken from pine trees { Pinus densiflora Sieb. et Zuccarini ) in Hengseong and Inje provinces, Korea. The samples were washed with water thoroughly, and then completely dried. Pine needles were cut by a cutter (3) , and mixed with spring water in a steamer (5) at the ratio of 1:1. The temperature of the steamer (5) was raised to 90^°C by a boiler (8) (the primary heating) . The boiler was turned off to cool down the steamer for 10 hours. The boiler (8) was turned on again. The steamer (5) was heated at 100^°C for 1 hour and 20 minutes with keeping the pressure as 2 kg/cuf (the secondary heating) . The temperature was raised to 110^°C, followed by heating for one hour under the pressure of 2.7 kg/cuf (the third heating). The temperature was raised again to 120 ^°C, followed by heating for 40 minutes under the pressure of 3.4 kg/oil' (the forth heating) . Heating with different temperatures made terpene oil and other ingredients of pine needles dissolved thoroughly in water. The heated vapor was passed through distilled water concentration line by the automatically set valve (11) and then drained out for 30 minutes (draining stage) . By the above processes, terpene oil and pharmaceutical components of pine needles were concentrated and attached to distilled water concentration line. From then on, distilled water would be passed through the distilled water concentration line in the form of gas.

The gas hit the wall of the concentration line while being passed through. At this time, the gas loses some of terpene oil and pharmaceutical components but at the same time it gets some from the wall-attached terpene oil and the components, so in results, the gas keeps the original contents of terpene oil and pharmaceutical components of pine needles. Next, the gas was cooled down in a cooler (15) (cooling stage), resulting in concentrate. The concentrate was filtered by a filter

(16) to eliminate lumps (filtering stage) , resulting in the preparation of transparent clear extract of pine needles. Yield of the extract was 40%.

<Example 1> Analysis of ingredients of clear extract of pine needles <!-!> Analysis of suitability for food Ingredients included in distilled water were examined to investigate suitability for food of clear extract of pine needles of the present invention. Inductively coupled plasma spectrometer (ICP, Perkin Elmer DV4300) and atomic absorption spectrophotometer (AAS, Verian SpwctraAA 300) were used to analyze cation like heavy metal, and ion chromatography (IC, Dionex Model 120) was used to analyze anion. Gas chromatography (GC, Hewlett Packard HP5890Series II) was used to analyze volatile substances including residual pesticides, and BRAN LUEBBE autoanalyzer was utilized to detect cyanides, phenols and detergents. All the ingredients were investigated in the aspects of bacteriology and chemistry according to food evaluation clauses. As a result, no harmful components were detected. In particular, heavy metals that do harm human, for example arsenic, lead, mercury and cadmium, were not detected at all. The contents of other ingredients were all under the permitted limit. Thus, clear extract of pine needles of the present invention was confirmed to be safe as food.

<l-2> Analysis of aromatic constituents Aromatic constituents of clear extract of pine needles of the present invention were investigated by dynamic headspace method. Ingredients of the sample were investigated by GC and GC-Mass according to the conditions presented in Table 2 and Table 3. The contents of ingredients were calculated by integrator with external standard method, for which α-pinene (Sigma Co. U.S.A.) was used as a standard material. Aromatic constituents were confirmed by comparing them with mass spectrum of standard material and the spectrum of GC- Mass Wiley/NBS database.

[Table l]

GC conditions for aromatic constituents

[Table 2]

GC-Mass conditions for aromatic constituents

As a result, 34 kinds of aromatic constituents were determined. Among those ingredients, the contents of fenchol, bomeol, β-fenchyl alcohol and bomyl acetate, which were confirmed to be relatively excellent aromatic constituents, were 6.73, 13.05, 26.63 and 16.16%, i respectively. Alcohols and aldehydes were the two major ingredients (Table 3 and Fig. 2) . According to Kang and his colleague' s report on aromatic constituents of pine needles obtained by steam distillation, 44 kinds of ingredients were separated and identified. The major aromatic constituents were hydrocarbons, and β-cubebene, trans-caryophillene, 2-hexenal, T-murolol, δ-cadinene were next, in that order (KANG SEONG-KU etc, the fragrance ingredients from pine needles by solvent fraction, Korean J. Dietary Cul . , 11(3), 1996). More ingredients were detected by the steam distillation of the present invention, comparing when solvent extraction was used, and further, the contents and the compositions of those ingredients were also different from those extracted by the conventional method. Extraction method also affected the composition of aromatic constituents. According to Choi and her colleges, major components of pine tree, separated by steam distillation, were α- pinene, bomyl acetate, β-pinene and β-phellandrene, which are different from the result of the present invention, though (CHOI KYUNG-SUK etc, the fragrance ingredients of the refined oil from Ligida pine and pine needles KOREA FOOD SCIENCE CONFERENCE REPORT, 20(6): 773, 1998) . It is judged that the difference is resulted from that even same kind of trees raised under the same climate have different contents and compositions of ingredients according to season, age, a part of needles, climate, soil, the picking season and the picking method (Zavarian E., Monoterpenoid differentiation in realtion to the morphology of Pinus discolor and Pinus j ohannis . , Phytochem . , 10(12) :3107, 1986).

[Table 3]

Aromatic constituents of clear extract of pine needles

14 26.38 Catene 229 89 0.64 15 27.23 Fenchol 2414 96 6.73

Tβ^' 27.51 Nerol 381 45 1.06 IT 28.66 Camphor 629 98 1. 74 18 28.94 (Unknown) 676 27 1. 19 29.49 Isobomeol 841 53 2. 35 20 30.21 Bo eol 4681 95 ^■13 .05 21 30.61 Terpineol 1965 95 5. 48 22 31.66 β-fenchyl alcohol 9549 91 26 .63 23 31.78 Myrtenol 357 53 1. 00 2^_4^" 32.72 Isopropylbezaldehyde 99 0. 28 25 33.27 Thymyl methyl ether 433 95 1. 21 26 33.63 Cuminal 98 95 0. 27 27 35.40 Phellandral 455 98 1. 27 28 36.05 Bomyl acetate 5796 99 16 .16 2.4-bι-tert- 29 46.03 132 95 0. 37 butylphenol α-caryophyllene 30 48.92 52 64 0.14 alcohol 31 49.48 Aromadendrene 83 92 0.23 32 51.20 Cardina-1, 4-diene 105 97 0.29 33 51.77 γ-muurolen 179 90 0.48 34 52.28 -amorphene 66 98 0.18 100

<Example 2> Anti-oxidative activity of clear extract of pine needles

<2-l> DPPH radical scavenging activity 80 Dig of DPPH solution (0.4 mM : α, α-diphenyl-β- picrylhydrazyl, Cι₈H₂N₅0₆, FW 394.3, stored under 0 ^°C ) was dissolved in 500 ni-E of methanol, and the mixture was filtered by a filtering paper (Toyo No. 5A) . The solution is not stable, so it has to be prepared right before use and kept in a dark cold room until it is used. The zero point of spectrophotometer was reset by using methanol as a blank. Based on the method of Choi and his colleges, samples were dissolved in 4 niP, of methanol at different concentrations, then 1 ml of 1.5><10^"4 M DPPH solution (in methanol) was added thereto. The solution was left for 30 minutes, followed by measurement of 0D₅ι₇ (Choi J.S. et al . , Screening for antioxidant activity of plants and marine algae and its active principles from

Prunus daviana . , Kor. J. Pharmacology, 24, 299-303,

1993) . Each experiment was repeated three times to produce an average OD and then reducement of OD comparing to that of a control was calculated by the below mathematical formula 1 to determine hydrogen donating ability. RC₅o means the amount of sample reducing OD to 1/2 of a control.

[Mathematical Formula l]

A ; OD of a sample, B ; OD of a control As a result, while RC₅₀ of ethanol extract of pine needles was 69.91 μl, RC₅₀ of clear extract of pine needles was 18.4 μl , indicating that clear extract of pine needles of the present invention has very strong anti-oxidative activity, which is similar to the radical scavenging activity of BHA or α-tocopherol (Table 4).

[Table 4] Radical scavenging activity of clear extract of pine needles

<2-2> UV-protecting effect In order to investigate UV-protecting effect of clear extract of pine needles, absorption coefficient (E%cm) was measured at each medium wavelength region (UVB area, 280 ~ 320 nm) and long wavelength region (UVA area, 320 ~ 400 nm) of UV. OD₃₀8 and OD₃₅₀ of the sample were measured by using UV/VIS spectrophotometer (8452A, HP Co.), and also OD of distilled water in UV cell (1 cm in diameter) was measured for the comparison of UV- protecting effect. As a result, absorption coefficients (E%cm) at medium wavelength region (UVB area, 280-320 nm) and at long wavelength region (UVAarea, 320-400 nm) were 69.91 E%cm and 18.4 E%cm, respectively (Table 5). The results indicate that UV-protecting effect of clear extract of pine needles of the present invention is higher than those of the ethanol extract of pine needles (33.1, 15.2 E%cm) and the ethanol extract of pine tree stem (22.3, 6.3% E%cm) , although it is lower than those of dioxybenzone (synthetic filter) and oxybenzone {synthetic filter), standard UV-blocking agents.

[Table 5]

UVA/UVB absorbance of clear extract of pine needles Sample E%cm 308 nm E%cm 350 nm Clear extract of 69.91 18.4 pine needles Ethanol extract of 33.1 15.2 pine needles Ethanol extract of 22.3 6.3 pine tree stem Dioxybenzone 412.6 208.5 Oxybenzone 423.8 216.1

<2-3> Whitening effect 0.5 ml of undiluted solution of the sample was mixed with tyrosinase (200 unit), 0.5 mft of L-tyrosine

(0.1 g/mC) and 0.5 ml of 50 mM phosphate buffer (pH 6.8), followed by reaction at 37 ^°C for 10 minutes. OD₄₇₅ was measured and tyrosinase activity inhibition rate was calculated by the below mathematical formula 2.

[Mathematical Formula 2] % Inhibition Rate = A - B / A X 100 A ; Sample not treated, B ; Sample treated

As a result, the tyrosinase activity inhibition rate of clear extract of pine needles of the present invention was 8.61%.

<Example 3> In vi tro anticancer activity <3-l> Anti-mutagenicity effect <3-l-l> Frozen permanent As histidine auxotrophs of Salmonella typhimurium

LT-2, TA98 and TA100 were used, which were provided from B.N. Ames, University of California, USA. The experimental strains were prepared as a frozen permanent according to the method of Ames, and then stored at - 80^°C in a deep freezer. The mentioned frozen permanent means frozen stock of a strain. Precisely, Salmonella typhimurium TA98 and TA100 strains, provided from Ames, were cultured in nutrient medium (Difco) for 10 hours

(37^°C, shaking). The sterilized DMSO (dimethyl sulfoxide) was added into the medium at the concentration of 0.09 μ jl μl.. Upon complete mixing, the medium was distributed in sterilized 1.7 /? microtube (AxyGen, Inc.), followed by rapid freezing in dry ice.

<3-l-2> Preincubation of test strain 0.8 g of nutrient medium (Difco) and 0.5 g of NaCl were dissolved in 100 in?, of distilled water, which was distributed in L-test tubes by 10 ιn per each test tube, and autoclaved (121^°C, 20 minutes) . The test tubes were inoculated respectively with TA98 and TA100 strains cultured in master plate by using a platinum loop, followed by shaking culture at the speed of 60 times/minute in a 37 ^°C water bath. Time-dependent OD₆₆o and the number of total cell number were measured. When ODs of TA98 and TA100 reached 0.24 and 0.27, respectively, the strains were used for the experiments. The culture time was not longer than 12 hours.

<3-l-3> Preparation of master plate Each of frozen TA98 and TA100 strains was thawed at room temperature, followed by streaking with sterilized platinum loop on the plate supplemented with ampicillm to separate single colony, then the plate was cultured at 37 ^°C for 48 hours. The colony on the plate containing ampicillin was picked up with platinum loop and dissolved in a tube supplemented with 0.3 ill?, of PBS

(phosphate buffered saline) . The solution was absorbed into a cotton ball, which was then squeezed by pressing down on the inside wall of the test tube. 4-5 parallel lines were made on the ampicillin plate with the cotton ball, followed by further culture at 37 ^°C for 12 hours.

And the plate was stored at a 4 ^°C refrigerator.

<3-l-4> preparation of S-9 mixture In order to activate indirect mutagens, S-9 mixture, rat liver microsomal enzyme mixture, was prepared by the method of Maron and Ames (Ames B.N., and Maron D.M., Revised methods for the S . typhimuri un mutagenicity test., Muta tion Res . , 113, 173-215, 1983). Particularly, PB (phenobabital) and BF (5,6-benzo- flavuone) were used to induce liver enzyme of male SD (Sprague Dawley) rats having 200 g of weight. PB was dissolved in sterilized saline at the concentration of 20 liig/niC, and BF was dissolved in corn oil at the concentration of 10 nig/ill?, resulting in the preparation of inducers. At day 1, 30 mg/kg of PB was injected to the abdominal cavity of a rat. Likewise, 60 mg/kg of PB was injected at day 2, day 3 and day 4, respectively, and then the animal was fasted. At day 5, the animal was sacrificed by decapitation and the liver was taken at 4 ^°C germ-free condition. The extracted liver was washed with 0.15 M KCl several times and weighed in a container. 0.15 M KCl solution was added at the volume of triple the liver weight, followed by mashing it with a homogenizer. Centrifugation was performed with the homogenate at 9,000^χg for 10 minutes. The obtained supernatant was S-9 fraction, which was distributed in microcentrifuge tubes by 1 mC/tube and then frozen rapidly in dry ice. The tubes were stored at -80 ^°C. S-9 mixture was prepared by mixing 1 m? of S-9 fraction, 0.2 ill? of MgCl₂-KCl salt (1.65 M KCl + 0.4 M MgCl₂) , 5 ill? of 0.2 M PBS (pH 7.4, 0.2 M NaH₂P0₄ + 0.2 M NaH₂P0₄- H₂0) , 0.04 ill? of 0.1 M NADP, 0.05 ill? of 1 M glucose-6-phosphate and 3.35 ill? of distilled water.

<3-l-5> Preparation of minimal glucose agar plate, top agar and histidine-biotin solution In order to prepare minimal glucose agar plate, 15 g/f of agar, 850 ml/ 1 of distilled water, 100 ml/ l of 10 X VB salt and 50 ml/ 1 of 40% glucose solution were mixed together and the mixture was autoclaved (121^°C, 1 a. p., 20 minutes), followed by cooling to 70^°C. Then, the mixture was distributed to petridishes by 20 in?, leading to the preparation of solid plates. Top agar was prepared by sterilizing and mixing Nacl (5 g/ l ) and agar (6 g/ t ) . Vogel-Bonner medium-E (10X) necessary for the composition of minimal glucose agar plate was prepared by mixing 2 g of MgS0₄, 20 g of citric acid monohydrate, 100 g of KH₂P0₄ (potassium phosphate) dibasic and 35 g of sodium ammonium phosphate in 1 < of warm distilled water, which was then stored at a refrigerator. Histidine/biotin solution, required to confirm histidine auxotroph, was prepared with D-biotin (122.15 g/ t ) and L-histidine-HCl (77.58 g/l), and the solution was stored at a 4 ^°C refrigerator. Whenever necessary, histidine/biotin solution was mixed with top agar at the ratio of 10 ill? : 100 111?.

<3-l-6> Mutagenicity test Mutagenicity test was performed by using TA98 and TA100, mutants of Salmonella typhimuri um, with preincubation method modified from Ames test, for which S-9 mixture was added as a metabolism activator. Particularly, samples were distributed in dry heat- sterilized glass tubes at different concentrations of 0, 50, 100, 150 and 200 μl. /plate , to which preincubated Salmonella typhimurium culture solution was added by 100 /? per each plate. 0.2 M PBS (pH 7.4) was added so as to make the total volume 700 μl.. The reaction solution was shaking-cultured at 37 ^°C for 20 minutes, then top agar (45^°C) supplemented with histidine/biotin was added by 2 in?. Upon being completely mixed, the solution was distributed onto minimal glucose agar plate, followed by further culture at 37 ^°C for 48 hours. The resultant his⁺ revertant colony was counted to determine mutagenicity of the extract. As a result, the number of his⁺ revertant colony of a negative control was 18+3 in TA98 and 176+7 in TA100, indicating that the sample itself has no mutagenicity.

<3-l-7> Anti-mutagenicity test The sample solution was distributed to each dry heat-sterilized glass tubes at different concentrations of 0, 50, 100, 150 and 200 f /plate, to which MNNG and 4NQ0, which have been known as direct mutagens, and B(α)P and Trp-P-1, which are indirect mutagens, were added by 50 μl respectively. In case a metabolism activator was needed, 250 μl. of S-9 mixture was added. 100 μl of preincubated Salmonella typhimurium culture solution was added thereto, and then 0.2 M sodium phosphate buffer was added to make the final volume 700

/(' . The solution was shaking-cultured at 37 ^°C for 20 minutes and revertant colony formation was induced in analogy to the procedure as described in example <3-l-6>.

The number of generated revertant colony was counted to investigate anti-mutagenecity . The contents of each sample and mutagens were determined by the pre-tests and anti-mutagenicity activity was calculated by inhibition rate (%) of the sample to the activity of a mutagen (Mathematical Formula 3) .

[Mathematical Formula 3]

Inhibition Rate (%) = -S_x / M-S₀ 100 M; Number of revertant colony in the presence of mutagen only, So; Number of revertant colony generated spontaneously, Si; Number of revertant colony in the presence of sample

As a result, sample of the present invention showed 45.9% and 85.5% inhibition rate to MNNG (0.4 /g/plate) and 4NQO (0.15 /zg/plate) , direct mutagens, respectively with the concentration of 200 C/plate in TA100 strain. The sample showed 88% inhibition rate to B( )P (10 /tg/plate ) , indirect mutagen, with the equal concentration to the above in the same strain. In the meantime, the sample of the present invention showed 85% inhibition rate to Trp-P-1 (0.15 g/plate) , indirect mutagen, even with the low concentration of 50 /R/plate and 91% inhibition rate with the concentration of 200 ?/plate, indicating that the sample has very strong dose-dependent anti-mutagenicity activity. Therefore, it was confirmed that inhibiting activity of the sample was much more active to indirect mutagens than to direct mutagens and such activities were dose-dependent (Fig. 3) - In the case of TA98 strain, the sample solution showed 62% inhibition rate to Trp-P-1 (0.15 /zg/plate) with the concentration of 200 ? /plate, which was much lower than that showed in TA100 strain although it was dose-dependent (Fig. 4).

<3-2> Cancer cell growth inhibiting effect All the cell lines used in this invention were purchased from Korea Cell Line Bank, (KCLB) . A549 and AGS cell lines were maintained in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS (fetal bovine serum, Gibco, USA) and 100 μl /mt of penicillin (Sigma, USA) . MCF7, HeLa and HepG2 cell lines were cultured in DMEM medium (Gibco, USA) supplemented with 10% FBS and 100 μt /ml of penicillin. 293 cell line was cultured in MEM (Minimum Essential Medium) supplemented with 10% FBS and 100 μl / l of penicillin, at 37 ^°C in a 5% CO incubator. In order to investigate the effects of the sample on the growth of various human cancer cell lines, cytotoxicity was measured by SRB (sulforhodamine B) test. Particularly, both cancer cells and normal cells (A549, MCF-7, AGS, HeLa, HepG2 , 293) were inoculated to RPMI 1640, DMEM and MEM, which were supplemented with 10% FBS, with the inoculum size of 5^χl0⁴ cells/ml', followed by distribution to each well of a 96-well plate by 100 //('/well. The plate was cultured in a 37 ^°C , 5% C0₂ incubator for 24 hours. 100 μl of sample was added to each well by the different concentrations of 0, 0.25, 0.5, 0.75 and 1.0 mg/ml, followed by further culture for 48 hours. The supernatant was removed carefully with an aspirator. 10% (w/v) TCA, which was being stored in a refrigerator, was added by 100 μl per each well, which was just left at 4^°C for one hour. The plate was washed with distilled water five times. After drying at room temperature, 100 μl. of 0.4% (w/v) SRB solution in 1% (v/v) acetic acid was added, leading to staining for 30 minutes. Unreacted SRB solution was removed by washing four times with 1% (v/v) acetic acid. After drying, 100 μl of 10 mM Tris buffer was added to dissolve the sample completely, then OD₅₄o was measured. As a result, when the extract was added to A549 cells by 50 μ /well , 78.7% inhibiting effect was observed. Likewise, when the extract was added by 50 /?/well to each AGS cells, MCF-7 cells, HeLa cells and HepG2 cells, 90.8%, 62.3%, 93.7% and 90% inhibiting effects were observed, respectively. In conclusion, the extract of the present invention inhibited cancer cell growth dose-dependently (Fig. 5). Cytotoxicity to human normal 293 cells by the extract was investigated at different concentrations. While the extract, treated to cancer cells, showed about 90% inhibiting rate, it showed just under 34% growth inhibiting rate to normal 293 cells (Fig. 6) . The results indicate that the extract has strong cytotoxicity to cancer cells but not to normal cells.

<Exampie 4> In vivo anticancer activity <4-l> Weight gaining in small animals Male SD (Sprague-Dawley) rats (Biogenomics, Korea) weighing 270-300 g were adapted to animal facility for one week. Each experimental group is composed of 6 rats in similar weights. The temperature was maintained as 21-26^°C and 45-55% humidity was kept in the animal facility. Lights turned on at 9:00 am and turned off at 9:00 pm, which was automatically regulated. Commercial formula feed (crude protein 22.1%, crude fat 3.5%, crude fiber 5.0%, ash 8.0%, calcium 0.6%. phosphorous 0.4%) for mouse, purchased from Samyang Oil & Feed Corporation, was given to the rats. As drinking water, distilled water was given. Feed and water were freely given to rats. The weights of rats were measured twice a week at fixed time. Statistic significance of the result was represented as mean + SEM by SAS (statistical analysis system) program, and statistic significance of difference in mean values among experimental groups was examined by Duncan's multiple range test (p < 0.05). As a result, it was confirmed that the administration of the extract of the present invention resulted in significant weight gaining in rats (Table 6 and Fig. 7) .

[Table 6]

Effect on weight gaining in rats Initial Final Weight

Group Sample weight ( g)_ _weight (g) gaining (g) Clear extract of 357.912.0 451.4+33.0 93.4^*±23.6 Rat pine needles Distilled water 341.715. 415.7+15.7 74.0+12.7 p < 0.05

<4-2> Organ weight gaining in small animals Rats were raised under the same conditions as provided in example <4-l> for 4 weeks, then fasted for 12 hours. They were anesthetized with ether to open the abdomen. Liver, kidney, spleen, and heart were taken out and washed with saline. Moisture on them was eliminated with a filtering paper. The organs weighed, which were converted into the weight of organ per 100 g of body weight. As a result, significant weight gainings in 4 different organs, comparing to those of a control treated with distilled water, were not observed after the administration of the extract of the present invention (Table 7). [Table 7]

Effect on organ weight gaining in rats Clear extract of Distilled Organ _ pine ieedles _water Total weight (g) 14.693+3.62 13.294+1.68 Liver g/100 g b.w. 3.620+0.18 3.198+0.49 Total weight (g) 3.243+0.28 3.380+0.28 Kidney g/100 g b.w. 0.602+0.12 0.813+0.08 Total weight (g) 0.843+0.09 0.708+0.09 Spleen g/100 g b.w. 0.187+0.06 0.170+0.07 Total weight (g) 1.340+0.22 1.354+0.18 Heart g/100 g b, w, 0.297+0.04 0.326+0.02 b.w Body weight

<4-3> Genotoxicity inhibition test <4-3-l> MNNG induced genotoxicity MNNG was used as an inducer of micronucleus formation. MNNG was dissolved in distilled water at different concentrations of 50, 100, 150 and 200 mg/kg. The MNNG solution was injected in abdominal cavity of a mouse by 0.2 ill-?, per mouse (25 g) with 1 ill?, disposable syringe. After 36 hours from the injection, animals were sacrificed and dose response of MNNG was investigated. As a result, frequency of micronucleus formation (MNNG/1,000 cells) was each 1.5+0.4, 3.9+0.5, 4.6+0.45, 12.710.6, and 15.8+0.29. However, animals treated with the dose of 200 mg/kg were not healthy in general and weight changes were not significant, either, comparing to a control (Fig. 8) . Thus, the dose of MNNG to confirm genotoxicity inhibiting effect was determined to be 150 mg/kg.

<4-3-2> Inhibiting effect on MNNG-induced micronucleus formation In order to investigate genotoxicity inhibiting effect of clear extract of pine needles of the present invention to MNNG, clear extract of pine needles prepared in the above preparative example 1 was orally administered at different concentrations of 0, 25, 50, 100 and 200 illCJkg simultaneously with MNNG (150 mg/kg, intraperitoneal injection). 36 hours later, animals were sacrificed, and the dose showing the highest genotoxicity inhibiting effect was investigated in each administration groups. As a result, while the frequency of micronucleus formation in a positive control was 12.7+0.6, the frequencies in experimental groups each treated with the dose of 25, 50, 100 and 200 niC/kg were 9.7+0.5, 8.3+0.8, 6.8+0.6 and 3.5+0.5, and inhibition rates of them were 23.7, 34.2, 46.3 and 72.4%, respectively (Table 8 and Fig. 9) .

[Table 8] Inhibiting effect of clear extract of pine needles on MNNG-induced micronucleus formation

^υ Mean+SD in 6 mice (p<0.05)

^2> 100-(MNPCE of Sample/MNPCE of Positive Control) ^χl00 ³⁾ Distilled DMSO (0.2 Hl-C/25 g) ⁾ A7-methyl -i\J ' -nitro-7J-nitros

<4-3-3> Collection of bone marrow and preparation of specimen Mice treated with MNNG and the extract of the invention were sacrificed by cervical dislocation according to Schmid method, then their femurs were taken out. Muscles were completely removed by using gauze from the femurs, which were then cut by a scissors. 0.2 in? of FBS in a syringe was injected in marrow canal repeatedly. The washed out bone marrow cell suspension was centrifuged at 1,000 rpm for 5 minutes to separate bone marrow cells. Small amount of serum was added to the separated bone marrow cells, followed by suspension by smooth shaking. Small amount of bone marrow cell suspension was taken by a Pasteur pipette, and then smeared on slide glass, which was washed and dried earlier, followed by air-drying. The dried slide was fixed with 100% methanol for 10 minutes, which was then stained with 5% Giemsa solution (Gurr 66) diluted in Sorensen buffer (1/15 M KH₂P0₄ + 1/15 M Na₂HP0₄, pH 6.8) for 30 minutes. After washing with distilled water several times, it was dried. Observation under microscope (^χl,000) was carried out to investigate formation frequency (%) of micronucleated polychromatic erythrocyte (MNPCE) among 3,000 polychromatic erythrocyte (PCE) in a mouse. As a result, contrary to a positive control treated with only mutagen (MNNG: 150 mg/kg, intraperitoneal injection), experimental groups treated with different concentrations of the extract (25, 50, 100, 200 ιn£/k ) along with the mutagen showed dose- dependent genotoxicity inhibiting effect. And, in particular, the group treated with the dose of 200 C/kg showed 72.4% inhibiting effect. <4-4> Solid tumor growth inhibiting effect in sarcoma- 180 cell line Male Balb/c mice weighing about 25 g were used as test animals in the present invention. The temperature was maintained as 21~26^°C and 45-55% humidity was kept in the animal facility. Lights turned on at 9:00 am and turned off at 9:00 pm, which was automatically regulated. Commercial formula feed (crude protein 22.1%, crude fat 3.5%, crude fiber 5.0%, ash 8.0%, calcium 0.6%. phosphorous 0.4%) for mouse, purchased from Samyang Oil & Feed Corporation, was given to the mice. As drinking water, distilled water was given. Feed and water were freely given to the mice. Sarcoma-180 cancer cell line was sub-cultured in the abdominal cavity of Balb/c mouse at 7-10 days interval. Particularly, sarcoma-180 cell line, which was sub-cultured in abdominal cavity of the test animal at 7-10 days interval, were collected by using a syringe, and then centrifuged (1,200 rpm, 10 minutes) together with PBS to separate cancer cells. The cells were centrifuged again with PBS to remove supernatant. The concentration of the cancer cell suspension was adjusted to l.OxlO⁶ cells/inC, which would be injected in abdominal cavity by 1 in?,. 5-6 mice were grouped for the experiments investigating tumor growth inhibiting effect of the extract. 0.2 in?, (6^χl0⁶ cells/mouse) of sarcoma-180 suspension was hypodermically injected in left groin of each mouse. From 24 hours after the injection, 200 μl of the extract was administered into abdominal cavity once a day for 20 days. The test animals were sacrificed on the 26^th - 30^th day from the cancer cell injection. The developed solid tumor was taken out and weighed. Tumor growth inhibition ratio (I.R:%) was calculated according to the below mathematical formula 4 (LEE YOUNG-SUK etc, anti-cancer and immunity-activity for sarcoma-180 cell of sugar-protein extracted from sea lettuce and brown seaweed, 21, 544-550, 1992) .

[Mathematical Formula 4]

I.R. (%) = C_w - T_w / Cw X 100

C_w ; Average tumor weight of control group mice, T_w ; Average tumor weight of experimental group mice

As a result, while the average tumor weight in control group mice transplanted with tumor cells was

3.53+0.84 (g) , the average tumor weight of experimental group mice treated with 100 [ /day of clear extract of pine needles was 2.83+0.47 (g) and the tumor weight of experimental group mice treated with 200 μl/day of clear extract of pine needles was 2.58+0.72 (g) . Although there was no significant difference in weights between the groups, solid tumor growth inhibiting effect was the highest (26.9%) when undiluted clear extract of pine needles was treated (Table 9) .

[Table 9]

Anticancer effect of clear extract of pine needles

¹¹ Mean+SD of 6 mice (p<0.05)

^Ξ) Sample 1 ; Clear extract of pine needles (100 ('./day, intraperitoneal injection)

³⁾ Sample 2 ; Clear extract of pine needles (200 //('/day, intraperitoneal injection)

<Preparative Example> Preparation of health food containing clear extract of pine needles as an effective ingredient <!-!> Preparation of tea containing clear extract of pine needles The present inventors prepared health promoting tea containing clear extract of pine needles produced in the above manufacturing example 1 by the conventional tea producing method. Tea was prepared in two different forms of liquid and granules containing clear extract of pine needles. First, tea in the form of liquid containing clear extract of pine needles was prepared as canned beverage, to which food additives were added in order to improve taste and flavor. As additives, vitamin C (0.02 - 0.04%, w/v%) and liquid flavor (0.02 - 0.06%, v/v%) were added, which was then heated to 90+5^°C in a mixed plate type heat exchanger. Can was filled with tea as being hot. Nitrogen gas was injected through headspace of the can to reduce dissolved oxygen therein. After being tightly sealed up, the can was sterilized and cooled down, resulting in the preparation of liquid type tea containing clear extract of pine needles. Second, tea in the form of granules containing clear extract of pine needles was prepared. Particularly, as additives, lactose, corn starch, glucose and vitamin C were quantified by 60.0%, 20.0%, 1.5% and 1.0%, respectively (making 82.5% of total content of granule tea) . Mixing, pulverizing, and gauging with 80-mesh gauge followed. The pulverized additive mixture and clear extract of pine needles of the present invention were mixed at the ratio of 82.5% : 17.5%, which was molded into granules by using a backspin granulator. The resultant granules were reformed by an extrusion granulator. Granules were put on a tray 2 cm thick, which was put in a drying oven, followed by drying at 60~80^°C for 24 hours to reduce moisture under 5%. The dried granules were put in a size-sorter in which 250 mesh size granules were prepared by using #35 and #80 mesh. The granule tea containing clear extract of pine needles was packed in Al foil tea bags by 3.0 g/bag.

<l-2> Preparation of beverage containing clear extract of pine needles The present inventors prepared health promoting beverage containing clear extract of pine needles produced in the above manufacturing example 1 by the conventional beverage producing method. Honey 522 nig Thioctic acid amide 5 g Nicotinic acid amide 10 mg Sodium riboflavin hydrochloric acid 3 mg Pyridoxine hydrochloric acid 2 mg Inositol 30 g Ortho acid 50 mg Clear extract of pine needles 0.5 - 1.5 g Water 200 ml

<l-3> Preparation of Saeng-sik containing clear extract of pine needles The present inventors prepared health promoting Saeng-sik containing clear extract of pine needles produced in the above manufacturing example 1 by the conventional Saeng-sik producing method. Clear extract of pine needles 10% Brown rice 40% Job' s tears 12% Sorghum 15% Black soybean 8% Kale 5% Perilla 10%

<l-4> Preparation of candy containing clear extract of pine needles The present inventors prepared health promoting candy containing clear extract of pine needles produced in the above manufacturing example 1 by the conventional candy producing method. Sugar 50 ~ 60 % Millet jelly 39.26 ~ 49.66 % Clear extract of pine needles 0.24 ~ 0.64 % Orange flavor 0.5 %

<l-5> Preparation of chewing gum containing clear extract of pine needles The present inventors prepared health promoting chewing gum containing clear extract of pine needles produced in the above manufacturing example 1 by the conventional chewing gum producing method. Gum base 20 % Sugar 76.36 ~ 76.76 % Clear extract of pine needles 0.24 ~ 0.64 % Fruit flavor 1 % Water 2 %

[industrial Applicability] The present invention relates to health food containing clear extract of pine needles as an effective ingredient, more precisely, health food for anticancer and/or anti-aging containing clear extract of pine needles having excellent anti-oxidative and anticancer activities as an effective ingredient.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

[CLAIMS] [Claim 1] Health food for anti-oxidation containing clear extract of pine needles as an effective ingredient.

[Claim 2] Health food for anticancer containing clear extract of pine needles as an effective ingredient.

[Claim 3] Health food as set forth in claim 1 or claim 2, wherein the clear extract of pine needles is prepared by the following steps: 1) Water and pine needles cut by a cutter are put in a steamer at the ratio of 1:1, and the steamer is heated by a boiler until the temperature of the steamer is raised to 90^°C. Then, the boiler is turned off, followed by cooling down for 10 hours, which is the primary heating stage; 2) The steamer is heated for 1 hour and 20 minutes at 100 ^°C under the pressure of 2 kg/cilf, which is the secondary heating stage; 3) The temperature is raised to 110 ^°C and the pressure is kept at 2.7 kg/cilf during heating for another one hour, which is the third heating stage; 4) Heating is performed for 40 minutes at 120 ^°C under the pressure of 3.4 kg/cilf, which is the forth heating stage; 5) The first obtained vapor is passed through distilled water concentration line for 30 minutes, and then the vapor is drained out; 6) After being passing through distilled water concentration line, the vapor is cooled down in a cooler; and 7) The extract cooled down above is filtered by a filter.

[Claim 4] Health food as set forth in claim 1 or claim 2, wherein the health food is selected from a group consisting of tea, Saeng-sik, meat, sausages, bread, chocolate, candies, snacks, cookies, pizza, ramyun, noodles, gums, dairy products, soups, beverages, or drinks.