KR20130134663A - A composition comprising the extract of pine tree leaf for the prevention and treatment of cervical cancer - Google Patents

Abstract

The present invention relates to a composition which contains a pine needle extract as an active ingredient and, more specifically, a composition which is useful for a pharmaceutical composition and health function foods for preventing and treating cervical cancer caused by the viral infection by controlling human papillomavirus (HPV) using the pine needle extract. [Reference numerals] (AA) Hydratase

Description

TECHNICAL FIELD The present invention relates to a composition for preventing and treating cervical cancer containing pine needle extract,

It is intended to provide a composition for the prevention and treatment of cervical cancer comprising a pine needle extract having a cervical cancer inhibitory activity by inhibiting human papillomavirus (HPV).

[0005] Vanchieri, C., IARC Publishes Data on Worldwide Cancer Cases, Journal of the National Cancer Institute, 85 (13), 1028-1029 (1993)

[Document 2] Munoz, N. and Bosch, F. X., Epidemiology of cervical cancer. In: Human Papillomaviruses and Cervical Cancer, eds. Munoz, N., Bosch, F. X. and Jensen, O. M. IARC Scientific Publications, Lyon, 9-40 (1989)

[Patent Document 3] Broker et al., Papillomaviruses: Retrospectives and Prospectives, Cancer cells 4 / DNA tumor viruses, Cold Spraing Harbor Laboratory, USA, 17-36 (1989)

[4] Durst, M. et al., Papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographical regions, Proc. Natl. Acad. Sci., USA, 80. 3812-9-2 1019950024886, 3815 (1983)

[Literature 5] Hausen, H., Viruses in human cancers, Science, 254, 1173-1187 (1991)

[Literature 6] Galloway, D. A. et al., Human papillomaviruses and carcinomas, Adv. Virus Res., 37, 125-171 (1990)

[Literature 7] Lorincz, A. T. et al., Human Papillomavirus Infection of the Cervix: Relative Risk Associations of 15 Common Anogenital Types, Obsterics and Gynecology, 79, 328-337 (1992)

[Document 8] Kim, TJ, Korean Resources Plants . II , pp194-195, 1996

[Literature 9] Kang, TH, Jeong, ST, et al., Journal of Ethnopharmacology , 71 , pp 321-323, 2000

[Literature 10] Jung, MJ, Chung, HY et al., Archives of pharmacal research , 26 (6) , pp458-462

[Document 11] Kaneta M., Hikichi H. et al. Agricultural and Biological Chemistry , 44 , pp1407, 1980

[Document 12] Woo WS, Kang SS et al., Journal of Natural Products , 49 , pp547-549, 1984

[Literature 13] Woo WS, Lee EB et al., Kor . J. Pharmacogn , 12, pp 153, 1981

[14] Moon CK, Lee BG et al., Archives of Pharmacal Research , 8 , pp 277, 1985

[Literature 15] Yang MS, Ha YL et al., Agricultural Chemistry and Biotechnology, 38 , pp584, 1995

[16] Yoo JS, Jang JS et al., KOR . J. Pharmacogn , 26 , pp355, 1995

[Literature 17] Zou K., Zhao Y. et al., Carbohydrate Research , 324 , pp 182-188, 2000

[Literature 18] Kim, Akamatsu, Shin Jeonghwa Chinese medicine, pp664-665, 1980

[Literature 19] Masayoshi Ogaga, Journal of the Japanese Institute of Pharmaceutical Sciences, p 336, 1991

[Literature 20] Park, Jong-gap, I, pp. 134, 1984; Cultural Broadcasters, Korean Folk Therapy, p21, 1988

[Document 21] Harborne JB, Phytochemical methods: A guide to modern techniques of plant analysis , 3 rd Ed . , pp6-7, 1998

[Literature 22] Korean Pharmacopoeia Commentary, Mun Sung-sa, Korea Pharmacy College Council, 5th edition, p33-48, 1989

[23] Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nat Med 13; 857-861.

Cervical cancer is a malignant tumor-causing virus that accounts for more than 90% of uterine cancers, and it is the most common cancer disease among Korean women. In recent years, infection with human papillomavirus (HPV) is the most important in carcinogenesis. It turns out to be a factor that acts.

Uterine cancer is currently the highest incidence among women in developing countries (Vanchieri, C., IARC Publishes Data on Worldwide Cancer Cases, Journal of the National Cancer Institute, 85 (13), 1028-1029 (1993)). It has been reported that there are about 500,000 patients (Munoz, N. and Bosch, FX, Epidemiology of cervical cancer.In: Human Papillomaviruses and Cervical Cancer, eds. Munoz, N., Bosch, FX and Jensen) , OM IARC Scientific Publications, Lyon, 9-40 (1989). According to a two-year study of cancer incidence from Seoul National University Medical School (Kim Jin-bok and Ahn Yun-ok, 1993) from July 1991 to June 1993 in Korea, 123 women per 100,000 people get cancer annually. The highest incidence of uterine cancer in Korea was 22%, stomach cancer 18%, and breast cancer 12.9%.

Papilloma viruses have been known to infect epithelial cells of various tissues of animals and cause benign tumors, often called warts, on the hands, feet, skin, and larynx. These papillomaviruses are currently known to have about 70 genotypes in humans, and several genotypes have been reported to be specific for infectious tissues and cause various diseases (Broker et al., Papillomaviruses: Retrospectives and Prospectives, Cancer cells 4 / DNA tumor viruses, Cold Spraing Harbor Laboratory, USA, 17-36 (1989).

Most of these different genotype papillomaviruses were not noted because they were poorly treated and caused great pain in infected patients but did not cause fatal diseases. Recently, however, certain types of the virus, particularly human papillomavirus types 16 and 18, are associated with malignant tumors in the genitals, mouth, and skin of men and women and may act as a major factor in cervical cancer, which accounts for more than 90% of female uterine cancers. In addition, 6b and 11 have been shown to cause benign tumors of the male and female genitalia, called condyloma acuminata. Epidemiological studies have also suggested that uterine cancer is caused primarily by factors that are transmitted by sexual contact (Durst, M. et al., Papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographical regions, Proc). Natl.Acad.Sci., USA, 80. 3812-9-2 1019950024886, 3815 (1983)), papilloma in 85-100% of the prostate cancer lesions named cervical intraepithelial neoplasm (CIN). Because of the large body of evidence that papillomaviruses are involved in the development of uterine cancers, including the presence of viruses (Hausen, H., Viruses in human cancers, Science, 254, 1173-1187 (1991)), papillomaviruses In addition, studies on the carcinogenic mechanisms of cancer cells have been required as well as the development of diagnostic and therapeutic agents (Galloway, DA et al., Human papillomaviruses and carcinomas, Adv. Virus Res., 37, 125-171 (1990)).

On the other hand, the infection rate of human papillomavirus type 16 and 18 in uterine cancer patients is generally reported that type 16 to 50 to 70% and type 18 to 15 to 25%. However, in metastatic cancer, 25% of cancer patients infected with type 16 and more than 50% of cancer patients infected with type 18 were metastatic cancers (Lorincz, AT et al., Human Papillomavirus Infection of the Cervix: Relative Risk Associations of 15 Common Anogenital Types, Obsterics and Gynecology, 79, 328-337 (1992).

Among the plants native to Korea, pine needles are widely distributed in Korea, and have been used as folk medicines for insomnia, tonic, excitement, diuresis, pain relief, and insect repellent since ancient times (Kim, TJ, Korean). Resources Plants . II , pp 194-195, 1996).

Recently, many chemical components have been isolated from pine needles and their structure has been revealed (Kang, TH, Jeong, ST, et al., Journal of Ethnopharmacology , 71 , pp 321-323, 2000; Jung, MJ, Chung, HY et al., Archives of pharmacal research , 26 (6) , pp 458-462). For example, the study of pine needle components showed that quercetin 3-O-galactoside, a flavonoid, in the skin, quercetin-3-O-lamnoside, rhamnoside, 3,4,7-trihydroxyflavone and alpha-spinastery glucoside, several triterpenoids, saponin and lignan components And the like (Kaneta M., Hikichi H. et al., Agricultural and Biological Chemistry , 44 , pp1407, 1980; Woo WS, Kang SS et al., Journal of Natural Products , 49 , pp547-549, 1984), and flavonol glycosides, quercitrin and isoquercitrin, were also isolated from flowers. In addition, studies on the pharmacological action of pine needle components have shown that the saponin fraction increases the uterine contraction (Woo WS, Lee EB et al., Kor . J. Pharmacogn , 12, pp 153, 1981), polysaccharide fraction (Moon CK, Lee BG et al., Archives of Sarcoma 180 solid form) of Pharmacal Research , 8 , pp 277, 1985), antimicrobial activity (Yang MS, Ha YL et al., Agricultural Chemistry This and Biotechnology, 38, pp584, 1995 ), such as diarrhea inhibitory effect (Yoo JS, JS Jang et al., KOR. J. surface effect by Pharmacogn, 26, pp355, 1995) , and flavonol glycosides (flavonol glycoside) have. Among these compounds, saponin is known to exhibit strong lethal activity in cancer cell lines (Zou K., Zhao Y. et al., Carbohydrate Research , 324 , pp182-188, 2000), but the cytotoxic mechanism at the molecular level has not yet been elucidated.

There are about 80-90 species of pine in the world of Pinus, but among them, P. palustris Miller (North America), P. pinaster Aiton (France), Pinus Sylvestris (P. sylvestris L. (European)), Pinus laricid Poiret (Austria), Pinus longifolia Rocvurgh (India), Pinus densiflora sieb (P) densiflora Sieb. et Zucc .: Korea, Japan) and terpenes from P. thunberii Palatore (Haesong, Japan) are mainly used in industry.

The main components of pine needles are Terpentine oil, Cineole, Salinigrin, Coniferin, P-Cymen, Densipimaric acid, Retten (Retene) and chlorophyll, protein, no fat, phosphorus, iron, enzymes, minerals, fat-soluble vitamin A and vitamin C, etc. The main ingredients are somewhat different depending on the species and the season harvested.

Essential oils obtained from the leaves of Pinus densiflora Sieb. Et Zucc. Include alpha-Pinene, beta-Pinene, camphene and pelanden ( phellandrene, borneol, bornyllacetate, caryophyllene, cadinene diterpene, sesquiterpene, sesquiterpenalcohol, cerylalcohol ), Beeswax contains juniperic acid, sabinic acid, hexadecane-diol, triacontan-1-ol, etc., and other machusterin ), Phytosterin, chinic acid, shikimic acid, quercetine, and kaempferol. (赤松 金 芳 ers, 신 정화 한 제, pp664- 665, 1980).

Among pines growing in Korea, pine needles are used as a medicine for gonorrhea and syphilis in folk medicine (御 影 雅 幸 雅, 日本 生 藥學 雜誌, p 336, 1991), but the pharmacological effects of pine needles are not known. And it is known to have the effect of restoring the coagulated skin to the original to the bruises of the skin quickly, and to heal eczema, scabies and sweat bands (Park Jong-gap, Korean Medicine, p134, 1984; Culture Broadcasting, Korean Civil Therapy Exhibition, p21, 1988). It is also mentioned in the appendix that it has the effect of preventing the whitening of hair. The herb wood is said to strengthen swelling, hair improvement, intestines and prolong life. Deodorant effect, insomnia healing effect, skin cosmetic effect, etc. However, none of the above documents have taught or disclosed that pine needle has therapeutic effect on uterine cancer.

Accordingly, the present inventors completed the present invention by confirming the inhibitory effect of human papillomavirus (HPV) on pine needle extract or purified water.

According to the above object, the present invention provides a pharmaceutical composition for preventing and treating cervical cancer comprising pine needle leaf extract as an active ingredient.

Extracts as defined herein include crude extracts, fraction extracts or purified products.

The crude extract as defined herein is soluble in lower alcohols such as methanol, ethanol and butanol or a mixed solvent thereof, preferably a mixed solvent of 50-70% water and C1-C4 lower alcohol, more preferably 60% ethanol. One extract.

Fractional extracts as defined herein include nonpolar solvent soluble fraction extracts and polar solvent soluble fraction extracts.

The nonpolar solvent soluble fraction extract as defined herein is prepared by suspending the crude extract in water followed by treatment with a nonpolar solvent selected from hexane, methylene chloride, dichloromethane, chloroform or ethyl acetate, preferably hexane, methylene chloride fraction, or ethyl acetate , More preferably a fraction extract which is soluble in a non-polar solvent obtained by fractionation with a methylene chloride nonpolar organic solvent; The polar solvent soluble fraction extract includes the fraction extract soluble in the polar solvent obtained by removing the non-polar solvent soluble tablet and fractionating the remaining crude extract with butanol or water.

The purified product as defined herein is about 1 to 30 times (w / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times (w / w) to the crude extract. SP207, HP20SS, Diaion HP 20, SP-850 resin, used for separating and purification of a water-soluble substance having the same weight as the weight of the purified water Activated carbon, Amberlite XAD-2,4, preferably Diaion HP 20, SP-850 resin, Amberlite XAD-2,4, and the mobile phase was eluted sequentially with ethanol, acetone, or methylene chloride solvent to lower the polarity, followed by elution (Hereinafter referred to as S11-HPO), 30% ethanol eluted tablets (hereinafter referred to as S11-HP30), 50% ethanol eluted tablets (hereinafter referred to as S11-HP50), 70% ethanol elution Purified water (hereinafter referred to as S11-HP70), 95% ethanol-eluted purified product (hereinafter referred to as S11-HP95), acetone and methylene chloride eluting purified product (hereinafter referred to as S11-HPAM).

   Pine needles, as defined herein, are selected from the group consisting of Pinus densiflora Sieb. Et Zucc., Pinus rigida, Pinus taeda, Pinus thunbergii parlatore or Pinus koraiensis Sieb. Et Zucc The leaves of Pinus, preferably Pinus densiflora Sieb. Et Zucc., Pinus rigida, or Pinus taeda.

The cervical cancer includes cervical cancer, preferably cervical cancer caused by infection with human papillomavirus (HPV).

Extracts as defined herein include crude extracts or nonpolar solvent soluble extracts and may be prepared by the following process.

For example, the crude extract of the present invention may contain water, alcohol, and water containing 1 to 200 times (w / w), preferably 10 to 100 times (w / w) To 4% by weight of a lower alcohol or a mixed solvent thereof, preferably water or a mixed solvent of water and ethanol, more preferably water or a mixed solvent of 50% -99% water and ethanol, for 12 hours to 1 week, preferably 48 hours The extraction method such as cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction at a temperature of from 10 to 150 ° C, preferably from 20 to 100 ° C, more preferably at room temperature, preferably, And then conducting a hydrothermal extraction method to obtain an extract.

For example, the non-polar solvent and polar solvent-soluble extract of the present invention may be used in an amount of about 0.0005 to 0.005 times, preferably 0.05 to 0.5 times (v / v) of the crude extract, preferably 60 to 90% w%) of water, followed by a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate and butanol to obtain nonpolar solvent-soluble extraction tablets (n-hexane, water; And polar solvent soluble extract purified products which are soluble in polar solvents such as butanol and water, respectively.

For example, the purified product of the present invention is about 1 to 30 times (w / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times (w) to the crude extract. / w) purified water and SP207, HP20SS, Diaion HP 20, SP-850 resin, which is used for separation and purification of water-soluble substances having the same weight as the purified water. Activated carbon, Amberlite XAD-2,4, preferably Diaion HP 20, SP-850 resin, Amberlite XAD-2,4, and the mobile phase is sequentially eluted with a polarity lowering solvent such as ethanol, acetone or methylene chloride, followed by eluting with a water eluant (Hereinafter referred to as S11-HPO), a 30% ethanol eluate (hereinafter referred to as S11-HP30), a 50% ethanol elution solution (hereinafter referred to as S11- HP50), a 70% ethanol elution solution 95% ethanol eluate (hereinafter referred to as S11-HP95), acetone and methylene chloride elution solution (hereinafter referred to as S11-HPAM).

Accordingly, the present invention provides water, spirits, lower alcohols having 1 to 4 carbon atoms, including purified water of about 1 to 200 times (w / w), preferably 10 to 100 times (w / w) of the total weight of pine needle samples. Or a mixed solvent thereof, preferably water or water and ethanol mixed solvent, more preferably water or 50-99% water and ethanol mixed solvent, for 12 hours to 1 week, preferably 48 hours to 72 hours, 10 ℃ to 150 ℃, preferably 20 ℃ to 100 ℃, more preferably at room temperature extraction methods such as cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, preferably cold extraction or hot water extraction A first step of obtaining an extract; About 1 to 30 times (w / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times (w / w) of purified water are added to the crude extract. SP207, HP20SS, Diaion HP 20, SP-850 resin, used for the separation and purification of water-soluble substances of the same weight Activated carbon, Amberlite XAD-2,4, preferably Diaion HP 20, SP-850 resin, Amberlite XAD-2,4, and the second step of eluting the mobile phase with a polarity lowering solvent such as ethanol, acetone or methylene chloride solvent by sequentially performing an additional purification process using an adsorption chromatograph method using an adsorption resin such as Amberlite XAD- (Hereinafter referred to as S11-HPO), 30% ethanol eluted tablets (hereinafter referred to as S11-HP30), 50% ethanol eluted tablets (hereinafter referred to as S11-HP50) (Hereinafter referred to as S11-HP70), 95% ethanol-eluted tablets (hereinafter referred to as S11-HP95), acetone and methylene chloride-eluted tablets (hereinafter referred to as S11-HPAM) And a manufacturing method thereof.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of cervical cancer containing the pine needle extract or the purified product obtained by the above-described manufacturing process and the above-described manufacturing process as an effective ingredient.

In addition, conventional fractionation processes can also be carried out (Harborne JB, Phytochemical methods: A guide). to modern techniques of plant analysis , 3 rd Ed . , pp 6-7, 1998).

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of cervical cancer, which comprises the pine needle extract obtained from the above-mentioned production method as an active ingredient.

The human papillomavirus inhibitory activity of the pine needle extract and the purified product of the present invention obtained by the above method was tested by a bioluminescence (SEAP) detection method in 293TT cells infected with HPV16 pseudovirion, It was found that the inhibition of contact with viruses was increased and it was confirmed that the inhibition of C57BL / 6 mouse cervical cancer induced by pseudovirion in vivo is useful as a composition for prevention and treatment of cervical cancer It can be used.

In addition, the pine needle has been used as a herbal medicine for a long time, and the extract of the present invention extracted therefrom has no problems such as toxicity and side effects.

The pharmaceutical composition for the treatment and prevention of cervical cancer according to the present invention comprises 0.02 to 50% of the above extract and purified product, It is included by weight percentage.

Pharmaceutical compositions comprising the extracts and tablets of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extracts and tablets of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition containing the extract and the purified product according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories, And the like. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, water, and the like in the extracts and tablets. Prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The preferred dosage of the extract and the purified product of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extracts and tablets of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract and the purified product of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or Intracerebroventricular injection.

The present invention also provides a health functional food for prevention and improvement of cervical cancer which contains pine needle leaf extract as an active ingredient.

&Quot; Health functional food "as defined herein means food prepared and processed using raw materials or ingredients having functionality useful to the human body in accordance with Law No. 6727 on Health Functional Foods." Functional " Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.

The health functional food for prevention and improvement of cervical cancer according to the present invention contains 0.01 to 95% by weight, preferably 1 to 80% by weight, of the extract and the purified product, based on the total weight of the composition.

For the purpose of improving and preventing cervical cancer, it can be manufactured and processed into a health functional food in the form of tablet, capsule, powder, granule, liquid, ring and the like.

For example, the health functional food in the form of tablets may be prepared as it is or granularly mixed with an excipient, a binder, a disintegrating agent or other additives in a suitable manner, and then compressed into a glidant, etc. It is made by directly compressing the functional food as it is or by mixing it evenly with an excipient, binder, disintegrant or other suitable additives, or mixing the health functional food as it is or evenly adding the appropriate additive to the prepared granules, The excipient, binder or other suitable additives are added to the functional food, and the powder mixed evenly is wetted with a solvent, the wet powder is molded into a mold at low pressure, and then dried and prepared by a suitable method. In addition, a mating agent or the like may be added to the health functional food in the form of tablets, if necessary.

Among the health functional foods in the form of capsules, the hard capsules are usually prepared by mixing the capsules evenly with the health functional foods or excipients suitable for the health functional foods, granulated by a suitable method, or granulated with a suitable epidermal agent as it is or Soft capsules are prepared by filling them, and soft capsules are usually filled with capsules containing glycerin or sorbitol in a capsule form containing gelatin or appropriate excipients suitable for health functional foods or health functional foods. It is molded and prepared, and a coloring agent preservative etc. can be added to the said capsule base as needed.

Circular functional foods are usually mixed with excipients, binders, disintegrants, etc., and then molded into a spherical form in a suitable manner, and the coating is carried out with white sugar or other suitable coating agent, or starch, talc or You can also be greeted with a suitable substance.

The health functional food in the form of granules is usually made by mixing the health functional food as it is or by adding excipients, binders, disintegrating agents, etc., evenly and then granulating it in a proper way to make the particles as even as possible. , Mating agent, etc. can be added. For the health functional food in the form of granules, No. 12 (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm) are used for the next particle size test. It should be less than 5.0% and pass through No.45 to less than 15.0% of the total amount.

The definitions of the excipients, binders, disintegrants, lubricants, mating agents, flavoring agents, etc. of the present invention are described in documents known in the art and include the same functions or the like (see, for example, , Council of Korean Pharmacy College, 5th ed., P. 33-48, 1989).

The pine needle extract and purified product of the present invention exhibit inhibitory activity against human papillomavirus (HPV), and thus can be effectively used in pharmaceutical compositions and health functional foods for the prevention and treatment of cervical cancer caused by the virus infection have.

Fig. 1 is a graph showing the virus-inhibiting ability when the pine needle leaf extract, the fraction and the purified water sample were pretreated.
FIG. 2 is a graph comparing the protective ability against an attack inoculation using HPV16 pseudovirion of pine needle leaf fraction and purified water sample,
Figure 3 compares the protective ability against attacking inoculation using HPV16 pseudovirion according to the administration method of pine needle leaf fraction and purified water samples.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1 Preparation of Ethanol Extract of Pine Needle

2 kg of pine needle purchased from Kyungdong market was repeatedly extracted with 20 L of 95% ethanol (alcohol) at 50 캜 by warming and concentrated under reduced pressure to obtain 338.85 g (hereinafter referred to as S11-T) of an ethanol extract.

Example  2. Pine needles Fractions  Produce

The ethanol extract of Example 1 was suspended in 1.8 L of distilled water, and the fraction was sequentially fractionated three times with the same amount of hexane, methylene chloride, ethyl acetate and n-butanol according to the conventional method to obtain a hexane fraction (95.18 g, hereinafter referred to as S11-H) , A methylene chloride fraction (46.11 g, hereinafter referred to as S11-MC), an ethyl acetate fraction (19.42 g, hereinafter referred to as S11-E), an n-butanol fraction (57.94 g, hereinafter referred to as S11- 90.2 g, hereinafter referred to as S11-W).

Example  3. Ethanol extract of pine needles HP -20 column Small fraction  Produce

30 g of the pine needle ethanol extract obtained as in Example 1 was suspended in 30 ml of water and subjected to DIAION HP-20 column chromatography. First, 2 L of water was added to the mobile phase to obtain an eluate, and 2 L of 30% ethanol, 2 L of 50% ethanol, 2 L of 70% ethanol, 2 L of 95% ethanol, 2 L of acetone and 1.5 L of methylene chloride were added respectively. S11-HP0 (water, 7.95 g), S11-HP30 (30% ethanol, 3.44 g) and S11-HP50 (50% ethanol, 2.97 g) were obtained by mixing acetone eluent and methylene chloride eluent from 7 eluents ) S11-HP70 (70% ethanol, 1.75 g), S11-HP95 (95% ethanol, 6.35 g) and S11-HPAM (acetone and methylene chloride, 6 g) respectively.

References 1. Experimental preparation

1-1. Cell preparation

: 293TT (human embryonic kidney cell used for HPV pseudovirus production and in vitro assay) was supplied from Schiller Lab, a cell line expressing SV40 large T antigen on 293T cells transformed with adenovirus E1a) Dulbecco's modified Eagles medium (DMEM ( SH30243, Hyclone, UT, USA)) in 10% inactivated FBS was incubated with heat the medium supplemented with (26140079, Hyclone, UT, USA ) in 5% CO ₂ Lt; RTI ID = 0.0 > 37 C. < / RTI >

1-2. HPV -16 Of pseudovirus  production

1-2-1. Plasmid

: HPV-SEAP pseudovirus was produced for in vitro antiviral assay and HPV-Luc PV was produced for in vivo challenge test. PC-Luc and p16L1L2 plasmids were used to produce p-SEAP and p16L1L2 plasmids and HPV-Luc PV for HPV-SEAP PV production. Each plasmid was provided from Schiller Lab (Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, USA).

1-2-2. Infection

: Seed 293TT cells in a 75T flask with 5 X 10 ⁶ incubation at 37 ° C, 5% CO ₂ for 16 hours, and then use 19 μg of p16L1 / L2 and 19 μg of pSEAP or pc-Luc plasmid for Lipofectin Reagent. (18292-011, Invitrogen, CA, USA) was used for co-transfection. After 6 hours of transfection, the cells were exchanged for complete media and cultured at 37 ° C. for 48 hours. The cells were harvested by trypsinization. Harvested cells were washed with Dulbecco's Phosphate-Buffered Saline (DPBS, 14190-250, Invitrogen, CA, USA).

1-2-3. Cell harvesting and virion maturation

: 0.2 ml of a 5% Triton X-100 (9002-93-1, Sigma, MO, USA), 25 mM ammonium sulphate (pH 9) (A4418, Sigma-Aldrich, MO, USA) was resuspended in 1 ml of DPBS Benzonase (9025-65-4, Benzonas, Sigma, UK) was added and incubated at 37 ° C for 24 hours to maturation of the virus.

1-2-4. Salt extraction

: The maturated virion is cooled on ice for 5 minutes, then added with 0.17 volume of 5N NaCl and incubated on ice for 20 minutes. Then collect all virion solutions and transfer them to an e-tube. Centrifuge at 4 ° C and 12,000 rpm for 10 min. Collect the supernatant, and store at -80 ° C by opti-prep ultracentrifugation.

1-2-5. refine

: Prepare a 46% optiprep gradient solution by mixing 23 ml of DPBS / 0.8M NaCl in 77 ml of SIGMA density gradient medium and make 37%, 33%, and 39% gradient solution with DPBS / 0.8M NaCl solution (27 %: 9.3 ml DPBS / 0.8 M NaCl + 13.2 ml 46% Optiprep, 33%: 6.4 ml DPBS / 0.8 M NaCl + 16.1 ml 46%, 39%: 3.4 ml DPBS / 0.8 M NaCl + 19 ml 46%). Carefully insert 39 ml, 33%, and 27% gradient solutions of 1 ml each into a 5 ml Beckman ultracentrifuge tube (361625, Beckman Coulter, USA) to prevent the layer from breaking and load 1 ml of virion supernatant onto it. do. And centrifuged at 47,800 rpm, 16 ° C for 4 hours using an Ultracentrifuge (Optima L 90K, Beckman Coulter Ultracentrifuge, USA). The virion fractions were collected and stored at -80 ° C.

1-2-6. Titration of HPV PVs

: 293TT cells were seeded in 96 × well plates at 5 × 10 ³ each and incubated at 37 ° C and 5% CO ₂ for 16 hours. After 5-fold serial dilution of HPV PVs, the cells of each well were infected and incubated for 72 hours. The titer measurement of HPV-SEAP PV was made by taking cell culture supernatants and confirming the activity of secreted alkaline phosphatase (SEAP) using the Great EscAPE ™ SEAP Chemiluminescence Kit (631738, Clontech, Calif., USA). In addition, titer measurement of HPV-Luc PV was performed by using BioLuxGaussia Luciferase Assay Kit (0301008, New England biolabs, MA, USA). Chemiluminescent detection was carried out using the Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA) to obtain the relative light units (RLU) value, each calculated HPV PVs titer.

1-3. In vitro antiviral assay

: Screening assays and HPV inhibition assays for natural substances were performed by Shaneyfelt (Shaneyfelt et al., 2006) and Schiller Lab (Roden et al., 1996, Unckell et al., 1997, Touze et al., 1998, Selinka et al., 2003, and Klasse et al., 2002). Before starting the in vitro antiviral assay, seed 293TT cells in a 96-well plate by 5 × 10 ³ at 37 ° C, 5% CO ₂ incubation. After 16 hours, the culture medium was removed and each extract was diluted to a concentration of 100 μg / ml or 50 μg / ml and dispensed into cells in a volume of 100 μl. After treating the extract for 16 hours, the culture medium was removed and the cells were washed twice with PBS. Cells were infected with 100 μl of HPV PVs at 106 RLU / ml and incubated at 37 ° C and 5% CO ₂ for 48 hours. Antiviral activity was measured using cell culture supernatants to determine the activity of secreted alkaline phosphatase (SEAP) using the Great EscAPE ™ SEAP Chemiluminescence Kit (Clontech, CA, USA). SEAP activity was obtained by using relative light units (RLU) using Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA) and comparing RLU values in cells not treated with extracts. The reduction of SEAP activity was considered to be an inhibitory effect on HPV PVs.

1-4. administration( Administration )

: Male Balb / C mice (Orientbio Co., Korea) were used at 6 weeks of age and S11-MC and S11-HP (70-95) were orally or externally administered as follows. For oral administration, extract was mixed with 0.5% methyl cellulose (Sigma, MO, USA) and fed at 300 mg / kg once a day for 5 days. Four hours after the last oral dose, HPV16 PVs were injected into the abdomen subcutaneous area. For external use, extract was used as a topical agent in the genital tract, mixed with 0.5% methyl cellulose and administered at a dose of 150 mg / kg once a day for 3 days. Four hours after the last oral administration, HPV16 PVs were injected into the genital tract. A mouse without extract administration as a negative control was used.

1-5. Pseudovirus  Attack inoculation ( challenge )

: HPV PVs were used for the inoculation test, and HPV16-Luc PVs expressing the luciferase gene were produced, maturation, extraction, purification and titration (http://home.ccr.cancer.gov/lco/). Four weeks before challenge, mice were injected with 3 mg depot medroxyprogesterone acetate (Depo-Provera ™) (Pharmacia, Belgium) and 3% carboxymethylcellulose (Sigma Aldrich, MO, USA) 4% nonoxinol-9 (Sigma Aldrich, MO, USA) was used (Roberts et al., 2007). The mice were orally injected with S11-MC and S11-HP (70-95) at 5 × 10 ⁶ RLU of HPV 16-Luc PVs by subcutaneous injection. Mice were treated intravaginally with 20 μl of 4% nonoxynol-9 6 hours before challenge with HPV16-Luc PVs, and then HPV16-Luc PVs mixed with 20 μl of 3% carboxymethylcellulose was injected into 5 × 10 ⁶ RLU were injected into the vaginal tract. Three days after challenge, all mice were anesthetized and injected intraperitoneally with 30 μl of luciferin (caliper, MA, USA) at a concentration of 7 mg / ml. HPV16-Luc PVs can be pseudoinfection by encapsidating the plasmid, pLucf, which carries the luciferase gene (Jeff Roberts, 2007). After 10 minutes of incubation, luciferase expression was detected in each mouse using IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA), and images were compared and analyzed. In addition, the expression of luciferase was quantified using Living Image 2.20 software (Xenogen, NJ, USA) and the HPV defense efficacy of extract was compared and analyzed.

Experimental Example  1. Virus Low performance  Experiment

In order to confirm the ability of the samples obtained in the above Examples to inhibit the contact with luciferase-containing HPV virus, the method described in the literature was applied as follows (Shaneyfelt et al., 2006, Roden et al., 1996, Unckell et al , 1997, Touze et al., 1998, Selinka et al., 2003, and Klasse et al., 2002).

293TT cells are seeded at 5 × 10 ³ in 96-well plates and incubated for 16 hours. The next day, the sample is mixed in the medium at a concentration of 50ug, 100ug / ml and treated for 16 hours. After washing twice with 100ul of PBS, 10 ⁶ RLU / ml of HPV pseudovirion was infected with 100 μl of cells, and 37 ° C, 5% CO ₂ for 48 hours. Incubated.

Prepare 1X 5X lysis buffer of Great EscAPE ™ SEAP Chemiluminescence Kit (631738, Clontech, CA, USA), and then take 45 ul of 1X lysis buffer and 15 ul of cell culture medium (3912, Costar, NY, USA) Put it in. Add 60 ul of substrate in the Great EscAPE ™ SEAP Chemiluminescence kit and use a Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA) to obtain relative light units (RLU) values. The reduction of SEAP activity was considered as inhibition effect on HPV PVs by comparing the RLU values in the extract-treated cells and the extract-treated cells.

As shown in FIG. 1, 13 samples were able to confirm the virus-inhibiting ability. S11-B, S11-E, S11-M, S11-HP70, and S11-HP95 showed more than 50% virus inhibitory effect at a sample concentration of 100 μg / Showed 50% inhibition of HPV16 PVs even at a sample concentration of 50 μg / ml.

Experimental Example  2. Virus according to preprocessing time Low performance  Comparative experiment

Among the extracts of Examples, S11-MC and S11-HP (70-95), which were highly effective in inhibiting viruses, were selected as representative materials, and In In vitro antiviral assay was used to measure the virus inhibition by varying the treatment time of two samples.

Each sample was diluted to a concentration of 100 μg / ml or 50 μg / ml, and treated for 29, 4, 8, 12, and 16 hours with 293TT cells. The culture medium was removed from each well, washed twice with PBS, and infected with 10 ⁶ RLU / ml of HPV16 PVs. After 48 hours, the inhibition of HPV16 PVs was assessed by measuring SEAP activity from each cell culture.

Figure 2 shows the percent inhibition of S11-MC and S11-HP (70-95) against HPV16 PVs. S11-MC showed a decrease in HPV16 PVs at 8%, 12% at 12 hours, and 80% at 16 hours treatment at a concentration of 50 ug / ml for 4 hours to 8 hours, and at a concentration of 100 μg / ml 4 hours, 34% at 8 hours, 68% at 12 hours, and 94% at 16 hours (FIG. 2 (A)). S11-HP (70-95) showed almost no virus reduction effect (0 - 2%) at the concentration of 50 μg / ml for 4 hours to 12 hours, 42% for 8 hours treatment at 100 μg / 47% in 12 hours treatment, and 88% in HPV16 PVs treatment in 16 hours treatment (Fig. 2 (B)).

Therefore, HPV16 PVs could be effectively inhibited by treatment with S11-MC or S11-HP (70-95) for 16 hours or more before infection with HPV16 PVs.

Experimental Example  4. Mouse in vivo HPV16 Pseudovirus  Inhibitory efficacy test

In order to confirm the inhibitory activity against cervical cancer in mice treated with the luciferase-containing pseudovirion of the sample obtained in the above example, the method described in the literature was applied as follows (Roberts, JN, et al (2007). Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan, Nat Med 13: 857-861.).

4-1. Sample treatment

Six-week old female Balb / C mice (Orientbio Co., Korea) were used and S11-MC or S11-HP (70-95) were administered orally or externally as described below.

For oral administration, extract was mixed with 0.5% methyl cellulose (Sigma, MO, USA) and fed at 300 mg / kg once a day for 5 days. Four hours after the last oral dose, HPV16 PVs were injected into the abdomen subcutaneous area.

For external use, extract was used as a topical agent in the genital tract, mixed with 0.5% methyl cellulose and administered at a dose of 150 mg / kg once a day for 3 days. Four hours after the last oral administration, HPV16 PVs were injected into the genital tract. A mouse without extract administration as a negative control was used.

Luciferase-containing pseudovirion Four days prior to challenge, Depot medroxyprogesterone acetate (Depo-Provera ™, Pharmacia, Belgium) was injected subcutaneously into the back of a 6-week-old female Balb / C mouse (Orientbio Co., Korea).

4-2. stomach challenge  exam

For the external mouse, the sample was mixed with 0.5% methyl cellulose and administered once a day at 150 mg / kg for 3 days to the vaginal tract. Each mouse was injected with HPV16-Luc PVs in a 1: 1 ratio of 1% carboxymethylcellulose (Sigma Aldrich, MO, USA) for 4 hours after external administration on the third day. After 3 days, mice were anesthetized and luciferin (caliper, MA, USA) was injected into the peritoneal cavity at a concentration of 7 mg / ml at 30 μl and 10 minutes later, IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA) The images were taken.

In the case of oral administration mice, the samples were mixed with 0.5% methyl cellulose and orally administered at 300 mg / kg once a day for 5 days. On the last day 5, 4 hours after oral administration, HPV16-Luc PVs were mixed with 1% carboxymethylcellulose (Sigma Aldrich, MO, USA) at a ratio of 1: 1 and injected into the abdomen subcutaneous area. The subsequent imaging procedure was performed in the same manner as the external mouse.

4-3. In vagina challenge  exam

The mice were treated with 20 μl of 4% nonoxynol-9 (Sigma Aldrich, MO, USA) vaginally 6 hours before challenge with HPV 16-Luc PVs and then with 20 μl of 3% carboxymethylcellulose (Sigma Aldrich, MO, USA) were injected into the vagina at a dose of 5 × 10 ⁶ RLU.

Three days after challenge, all mice were anesthetized and injected intraperitoneally with 30 μl of luciferin (caliper, MA, USA) at a concentration of 7 mg / ml. HPV16-Luc PVs can be pseudoinfection by encapsidating the plasmid, pLucf, which carries the luciferase gene (Jeff Roberts, 2007). After 10 minutes of incubation, luciferase expression was detected in each mouse using IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA), and images were compared and analyzed. In addition, the expression of luciferase was quantified using Living Image 2.20 software (Xenogen, NJ, USA) and the HPV defense efficacy of extract was compared and analyzed.

4-4. Challenge  Test result

As a negative control, 6-week-old Female Balb / C mice without extract were used, and the method of attack inoculation was the same as described above.

In the mice challenged through the vaginal tract, the expression of luciferase was localized to the genital part. In the subcutaneous adventitious mice, luciferase expression was observed in the injection site and whole body of the mouse. Therefore, luciferase activity was detected by infection with HPV16 PVs. In addition, since luminescence in the negative control group was confirmed, it was possible to compare and confirm the protective effect in the experimental group.

The mice treated with S11-MC externally showed no luminescence and showed complete protection against HPV16 PVs. In addition, S11-MC was mixed with 0.5% methyl cellulose and orally administered at 300 mg / kg once a day for 5 days. On the last day of the 5th day, 4 hours after oral administration, HPV16-Luc PVs were injected into the abdomen subcutaneous area. The subsequent imaging method was performed as described above. S11-MC orally administered mice showed that luminescence was observed in the abdominal area, which did not protect HPV PVs.

S11-HP (70-95) was mixed with 0.5% methyl cellulose and challenged with HPV by external application to 150 mg / kg once a day for 3 days in the vaginal tract. It was also confirmed that luminescence was not observed even in mice administered S11-HP (70-95) externally, thus completely defending HPV16 PVs. In addition, S11-HP (70-95) was mixed with 0.5% methyl cellulose and administered orally at a dose of 300 mg / kg once a day for 5 days, and HPV16-Luc PVs were injected into the abdomen subcutaneously to image. S11-HP (70-95) mice were orally administered with luminescence in the abdomen and the whole body, and it was confirmed that HPV16 PVs were not defended at all.

As a result of quantifying luciferase-induced luminescence, mice treated with S11-MC and S11-HP (70-95) externally showed less than 2% of luminescence compared to mice without extract (Fig. . In the case of oral administration, the amount of luminescence to HPV 16 PVs attacked vaccine was similar to that of negative control mice or rather high. Therefore, S11-MC and S11-HP (70-95) were able to kill or infect HPV virus when administered externally, thus confirming complete protection against attack. In other words, external administration of S11-MC and S11-HP (70-95) seems to be the most effective way to inhibit HPV.

Hereinafter, formulation examples of the pharmaceutical composition containing the extract and purified product of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

S11-B 300 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

S11-HP70 300 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

S11-MC 300 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

S11-H 300 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

S11-T 300 mg

10 g per isomer

5 g mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

S11-HP70 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin B ₁

0.15 mg of vitamin B ₂

0.5 mg of vitamin B ₆

Vitamin B ₁₂ 0.2 g

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

S11-MC 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

After mixing the above components in accordance with the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed, sterilized and refrigerated It is used to prepare a healthy beverage composition of the present invention. Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (18)

Cervical cancer prevention and treatment pharmaceutical composition comprising pine needle extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the pine needle extract is a crude extract, a fraction extract or a purified product. The pharmaceutical composition according to claim 2, wherein the pine needle crude extract is an extract soluble in lower alcohols such as methanol, ethanol and butanol, or a mixed solvent thereof. The pharmaceutical composition according to claim 2, wherein the pine needle fraction extract is a nonpolar solvent soluble fraction extract or a polar solvent soluble fraction extract. The method of claim 4, wherein the pine needle non-solvent soluble fraction extract is a fraction soluble in a non-polar solvent obtained by suspending the crude extract with water and then fractionating it with a non-polar solvent selected from hexane, methylene toroid, dichloromethane, chloroform or ethyl acetate. Pharmaceutical composition, characterized in that the extract. 5. The pharmaceutical composition according to claim 4, wherein the pine needle soluble fraction extract of pine needles is a fraction extract soluble in a polar solvent obtained by removing the non-polar solvent soluble tablet and fractionating the remaining crude extract with butanol or water. The method according to claim 2, wherein the pine needle purified product SP207, HP20SS, which is used for the separation and purification of water-soluble substances having the same weight as the weight of the purified water in 1 to 30 times (w / w) of purified water of claim 2 Diaion HP 20, SP-850 resin, Further purification is carried out using the adsorption chromatograph method using activated carbon, adsorbent resin such as Amberlite XAD-2,4, and the mobile phase is eluted with ethanol, acetone or methylene chloride solvent which sequentially lowers the polarity. Water eluted purified (S11-HPO), 30% ethanol eluted (S11-HP30), 50% ethanol eluted (S11-HP50), 70% ethanol eluted (S11-HP70), 95 A pharmaceutical composition comprising% ethanol eluting purification (S11-HP95), acetone and methylene chloride eluting purification (S11-HPAM). The method of claim 1,
The pine leaves include leaves of Pinus densiflora Sieb. Et Zucc., Pinus rigida, Pinus taeda, Pinus thunbergii parlatore or Pinus koraiensis Sieb. Et Zucc. ≪ / RTI > The pharmaceutical composition according to claim 1, wherein the cervical cancer is cervical cancer due to human papillomavirus (HPV) infection. Health functional food for cervical cancer prevention and improvement containing pine needle extract as an active ingredient. The health functional food according to claim 10, wherein the pine needle extract is a crude extract, a fraction extract or a purified product. 12. The dietary supplement of claim 11, wherein the crude extract of pine needles is an extract available in a lower alcohol such as methanol, ethanol and butanol or a mixed solvent thereof. 12. The health functional food according to claim 11, wherein the pine needle fraction extract is a nonpolar solvent soluble fraction extract or a polar solvent soluble fraction extract. The non-solvent soluble fraction extract of pine needles is a fraction extract soluble in a non-polar solvent obtained by distilling the crude extract with water and then fractionating it with a non-polar solvent selected from hexane, dichloromethane, chloroform or ethyl acetate. Health functional food. 14. The health functional food according to claim 13, wherein the pine needle soluble fraction extract of pine needles is a fraction extract soluble in a polar solvent obtained by removing the non-polar solvent soluble tablet and fractionating the remaining crude extract with butanol or water. 15. The method according to claim 13, wherein the pine needle purified product SP207, HP20SS, which is used to separate and purify 1-30 times (w / w) of purified water in the crude extract of claim 2 and the water-soluble substance of the same weight as the weight of the purified water; Diaion HP 20, SP-850 resin, Further purification is carried out using the adsorption chromatograph method using activated carbon, adsorbent resin such as Amberlite XAD-2,4, and the mobile phase is eluted with ethanol, acetone or methylene chloride solvent which sequentially lowers the polarity. Water eluted purified (S11-HPO), 30% ethanol eluted (S11-HP30), 50% ethanol eluted (S11-HP50), 70% ethanol eluted (S11-HP70), 95 A dietary supplement comprising% ethanol eluting tablets (S11-HP95), acetone and methylene chloride eluting tablets (S11-HPAM). The health functional food according to claim 10, wherein the cervical cancer is cervical cancer caused by human papillomavirus (HPV) infection. 11. The health functional food according to claim 10, wherein the health functional food form is powder, granule, tablet, capsule or beverage.

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