KR101477966B1 - 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 containing pine needle extract as an active ingredient. Specifically, the pine needle extract of the present invention inhibits human papillomavirus (HPV) and is useful as a medicine for prevention and treatment of cervical cancer caused by the above- Compositions and health functional foods.

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) , pp 458-462

[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 , pp 547-549, 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 , pp 584, 1995

[16] Yoo JS, Jang JS et al., KOR . J. Pharmacogn , 26 , pp 355, 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 cancer. It has the highest incidence and mortality rate in Korean women. In recent years, infection of human papillomavirus (HPV) Which is known to be a causative agent.

Cervical cancer is currently the most common cancer incidence in developing countries (Vanchieri, C., IARC Publishes Data on Worldwide Cancer Cases, Journal of the National Cancer Institute, 85 (13), 1028-1029 The number of patients has been reported to reach about 500,000 (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 the results of a 2-year cancer incidence study conducted between July 1991 and June 1993 in the Seoul area of Seoul National University Medical Research Team (Kim, Jin-bok, and An Yeon-oh, 1993), 123 women per 100,000 women per year had cancer, 22%, gastric cancer 18%, breast cancer 12.9%, the highest incidence of cervical cancer in Korea.

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 papilloma viruses are now identified in about 70 genotypes in humans, and several genotypes have been reported to be specific for the infected tissue and cause a variety of diseases (Broker et al., Papillomaviruses: Retrospectives and Prospectives, Cancer cells 4 / DNA tumor viruses, Cold Spraying Harbor Laboratory, USA, 17-36 (1989)).

Many of these genotypic papillomaviruses were not treated because they were not well treated and did not cause a fatal disease, although they inflicted great distress on infected patients. However, in recent years, certain types of the virus, especially human papillomavirus type 16 and 18, have been associated with malignant tumors in the genitalia, oral cavity, skin, etc., and are a major factor in causing cervical cancer, which accounts for more than 90% In addition, type 6b and type 11 were found to induce benign tumors of the male and female genital tracts, which are called condyloma acuminata. In addition, epidemiological studies have suggested that uterine cancer is caused mainly by factors 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 (85% to 100% of all cervical intraepithelial neoplasms (CINs)) were found in papilloma Viruses in human cancers, Science, 254, 1173-1187 (1991)), papilloma viruses (papillomaviruses), papilloma viruses (Galloway, DA et al., Human papillomaviruses and carcinomas, Adv. Virus Res., 37, 125-171 (1990)).

On the other hand, infection rates of human Papillomaviruses 16 and 18 in cervical cancer patients are generally reported to be about 50 to 70% in 16 type and 15 to 25% in 18 type. However, in the case of metastatic cancer, 25% of patients with type 16 and 50% of patients with type 18 infection were found to be metastatic (Lorincz, AT et al., Infection of the Cervix: Relative Risk Associations of 15 Common Anogenital Types, Obsterics and Gynecology, 79, 328-337 (1992)).

Among the plants that are native to Korea, pine leaves are widely distributed in Korea and have been used as a medicinal agent for insomnia, tension, excitement, diuretic, analgesic and insect repellent (Kim, TJ, Korean Resources Plants . II , pp194-195, 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, studies on pine needle components have shown that the flavonoids quercetin 3-O-galactoside, quercetin 3-O-galactoside, rhamnoside, 3,4,7-trihydroxyflavone and a-spinastery glucoside, several kinds of triterpenoid, saponin and lignan components (Kaneta M., Hikichi H. et al., &Quot; Agricultural and Biological Chemistry , 44 , pp1407, 1980; Woo WS, Kang SS et al., Journal of Natural Products , 49 , pp 547-549, 1984). In addition, fragonol glycosides quercitrin and isoquercitrin were 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 pines in the world, among them Pinus. Among them, P. palustris Miller (North America), P. pinaster Aiton (France), Pinus P. longifolia Rocvurgh (India), Pinus densiflora cepab (P. sylvestris L.), P. laricid poiret (Austria), P. longifolia Rocvurgh densiflora Sieb. et Zucc.: Korea, Japan), and P. thunberii Palatore (Haesong, Japan).

The main components of pine needles are terpentine oil, Cineole, Salinigrin, Coniferin, P-Cymen, Densipimaric acid, Protein, fat, phosphorus, iron, enzymes, minerals, fat-soluble vitamin A and vitamin C. The major components are somewhat different depending on species and season.

Essential oils obtained from the needles of Pinus densiflora Sieb. Et Zucc. Include alpha-pinene, beta-pinene, camphene, pellandrene, borneol, bornylacetate, caryophyllene, cadinene diterpene, sesquiterpen, sesquiterpenalcohol, cerylalcohol, ), Wax contains juniperic acid, sabinic acid, hexadecane-diol, triacontan-1-ol, etc., and matsusterin ), Phytosterin, chinic acid, shikimic acid, quercetine, kaempferol, etc. (Akamatsu Kimyoji, Shinjeonghwa Chinese medicine, pp664- 665, 1980).

Among the pine trees native to Korea, the pine tree leaves are used as a remedy for gonorrhea and syphilis in folk remedies (御 賀 雅 幸 じ ゃ, Nihon Scientific Journal, p 336, 1991), but the various pharmacological actions of pine leaves are not known, It is known that it has the effect of quickly recovering the blood clotted skin against the bruising area of the skin, and has an effect of healing the eczema, the omphalus and the heat rash (Park Jong-Kap, Broadcasting, Korean Folk Therapy, p21, 1988). In addition, it is said that there is an effect to prevent the hair from whitening, and it is mentioned that the grass of the grass is strengthening the swelling, the hair improvement, the interior decoration and lengthening the life span. 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, fractionated extracts or tablets.

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

The fraction extracts 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 a fraction extract obtained by removing the nonpolar solvent-soluble tablet and allowing the remaining crude extract to be fractionated with butanol or water.

(W / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times (w / w), to the crude extract, Of SP207, HP20SS, Diaion HP 20, and SP-850 resin, which are used for separation 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).

The extracts defined herein include crude extracts or non-polar solvent soluble extracts and can 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 a polar solvent-soluble extracted and purified product soluble in polar solvents such as butanol and water.

For example, the purified product of the present invention may be added to the crude extract in an amount of about 1 to 30 times (w / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times / w), and SP207, HP20SS, Diaion HP 20, and SP-850 resin, which are used for separation 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 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 relates to water, alcohol, lower alcohol having 1 to 4 carbon atoms containing purified water of about 1 to 200 times (w / w), preferably 10 to 100 times (w / w) Or a mixed solvent thereof, preferably water or a water and ethanol mixed solvent, more preferably water or a mixed solvent of 50-99% water and ethanol, for 12 hours to 1 week, preferably 48 hours to 72 hours, The extraction method such as cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction, preferably the cold extraction extraction method or the hot water extraction method, is performed at 10 to 150 ° C, preferably 20 to 100 ° C, more preferably at room temperature A first step of obtaining an extract; (W / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times (w / w) of purified water is added to the crude extract and the purified water SP207, HP20SS, Diaion HP 20, and SP-850 resin, which are used for the separation and purification of water- 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 may be performed (Harborne JB, Phytochemical methods: A guide to modern techniques of plant analysis , 3 rd Ed . , pp6-7, 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, Weight percent.

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 formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, 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 extract and the purified product of the present invention are preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg per day. 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 may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine 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 of the tablet form may be prepared as it is, or it may be prepared by putting an excipient, a binder, a disintegrant, or other additives into the granules by an appropriate method, Functional food may be prepared by directly compressing the mixture of the food or by mixing directly with an excipient, binder, disintegrant or other suitable additive, or by mixing the preformed granules with the proper food additives or the appropriate additives, Add an excipient, binder or other suitable additive to the functional food, wet the powder evenly with a solvent, mold the wet powder into a low-pressure mold, and dry it 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.

The hard capsule of the capsule type health functional food is usually prepared by mixing the capsule with an appropriate excipient such as a health functional food or a health functional food or by granulating it in a suitable manner or by granulating it into a granular form suitable for granulation, The soft capsules are usually capsules containing an excipient suitable for a health functional food or a health functional food. The capsules are coated with an appropriate capsule base such as gelatin or the like with glycerin or sorbitol, And if necessary, a coloring agent preservative or the like may be added to the capsule base.

The ring type health functional food is usually prepared by mixing an excipient, a binder and a disintegrant in an ordinary healthy functional food, shaping it into a spherical form by an appropriate method, and then adding a gelatin to starch or other suitable skin care agent, It may also be an objectionable substance.

The granular health functional foods are usually prepared by mixing the healthy functional food as it is or by adding the excipient, binder, disintegrant, etc. to the health functional food and then making it into granular form by proper method and evenly grinding the granular material as needed. , A mating agent, and the like. In the next granularity test using granules No. 12 (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm), all of the 12th tongue passed through and 14th And not more than 5.0% of the total amount and not more 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 Of 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 Fractional  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 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. Transfection

: 293TT cells were seeded in 75T flasks at 5 × 10 ⁶ cells and incubated at 37 ° C and 5% CO ₂ for 16 hours. 19 μg of p16L1 / L2 and 19 μg of pSEAP or pc-Luc plasmids were incubated with Lipofectin Reagent (18292-011, Invitrogen, CA, USA). After 6 hours of transfection, the cells were replaced with complete media and incubated at 37 ° C for 48 hours. Cells were harvested by trypsinization. The 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 5 × 10 ³ 96-well plates and incubated at 37 ° C and 5% CO ₂ for 16 hours. After 5-fold serial dilution of HPV PVs, cells were inoculated into each well and incubated for 72 hours. Titer measurements of HPV-SEAP PV were performed by ascertaining the activity of secreted alkaline phosphatase (SEAP) using the Great EscapE ™ SEAP Chemiluminescence Kit (631738, Clontech, CA, 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 performed using the Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA) and the relative light units (RLU) were calculated and the HPV PV titer was calculated.

1-3. In vitro antiviral assay

: Screening assays and HPV inhibition assays for natural substances were performed using Shaneyfelt's method (Shaneyfelt et al., 2006) and Schiller's method (Roden et al., 1996, Unckell et al., 1997, Touze et al., 1998, Selinka et al., 2003, and Klasse et al., 2002). Before the initiation of the in vitro antiviral assay, 293TT cells were seeded on a 96-well plate at 5 × 10 ³ and incubated at 37 ° C and 5% CO ₂ lt; / RTI > 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 the extract was treated for 16 hours, the culture 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. To determine antiviral activity, the cell culture supernatant was assayed for secreted alkaline phosphatase (SEAP) activity using the Great EscapE ™ SEAP Chemiluminescence Kit (Clontech, CA, USA). Relative light units (RLU) values were obtained by using a luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA) for SEAP activity, and the RLU values of extract- , And the decrease in SEAP activity was regarded as an inhibition 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. Viruses 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 the cell in a 96-well plate inoculated (seeding) with 5 x 10 ^3, and incubated for 16 hours. The next day, the sample is mixed with the medium at a concentration of 50 ug, 100 ug / ml and treated for 16 hours. After washing twice with PBS 100 μl, 100 μl of 10 ⁶ RLU / ml HPV pseudovirion was infected into the cells and incubated for 48 hours at 37 ° C, 5% CO ₂ Lt; / RTI >

Well plate (3912, Costar, NY, USA) with 45 μl of 1X lysis buffer and 15 μl of the cell culture medium, and incubated for 1 hour in a 5X lysis buffer of Great EscapE ™ SEAP Chemiluminescence Kit (631738, Clontech, CA, USA) . Add 60 μl of substrate in the Great Escape ™ SEAP chemiluminescence kit and obtain relative light units (RLU) using a Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA). We compared the RLU values in extract-treated cells versus extract-treated cells and considered the reduction in SEAP activity as an inhibition effect on HPV PVs.

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

Crystalline cellulose 3 mg

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 12} H 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 quantity

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 of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above ingredients were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the resulting solution was filtered and obtained in a sterilized 2 liter container, sealed, sterilized and refrigerated It is used in the production of the health 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)

(1) adding 1 to 200 times (w / w) of water or 50-99% water and ethanol mixed solvent to the pine needle sample of Pinus densiflora Sieb. Et Zucc. For 48 hours to 72 hours, A first step of obtaining a crude extract by performing a cold extraction method or a hot water extraction method at 20 ° C to 100 ° C; The methylene chloride fraction (S11-MC) soluble in methylene chloride obtained by adding water at a concentration of 0.05 to 0.5 times the volume of the crude extract obtained above (v / w%) and performing a conventional fractionation process using methylene chloride, ; HP20SS, Diaion HP 20, and SP-850 resin, which are used for the separation and purification of a water-soluble substance having the same weight as the weight of the purified water, is added to the crude extract 5 to 15 times (w / w) Further purification is carried out by adsorption chromatography using activated carbon or an adsorbent resin selected from Amberlite XAD-2,4, and the mobile phase is sequentially eluted with low polarity and eluted with ethanol to obtain a second purification step A pharmaceutical composition for the prevention and treatment of cervical cancer caused by infection with human papillomavirus (HPV) comprising 70% ethanol-eluted purified product (S11-HP70) as an active ingredient. delete delete delete delete delete delete delete delete (1) adding 1 to 200 times (w / w) of water or 50-99% water and ethanol mixed solvent to the pine needle sample of Pinus densiflora Sieb. Et Zucc. For 48 hours to 72 hours, A first step of obtaining a crude extract by performing a cold extraction method or a hot water extraction method at 20 ° C to 100 ° C; The methylene chloride fraction (S11-MC) soluble in methylene chloride obtained by adding water at a concentration of 0.05 to 0.5 times the volume of the crude extract obtained above (v / w%) and performing a conventional fractionation process using methylene chloride, ; HP20SS, Diaion HP 20, and SP-850 resin, which are used for the separation and purification of a water-soluble substance having the same weight as the weight of the purified water, is added to the crude extract 5 to 15 times (w / w) Further purification is carried out by adsorption chromatography using activated carbon or an adsorbent resin selected from Amberlite XAD-2,4, and the mobile phase is sequentially eluted with low polarity and eluted with ethanol to obtain a second purification step A health functional food for prevention and improvement of cervical cancer caused by infection with human papillomavirus (HPV) comprising 70% ethanol eluted purified product (S11-HP70) as an active ingredient. delete delete delete delete delete delete delete 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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