KR101753560B1

KR101753560B1 - Composition containing naringenin and hesperetin for preventing, treating or improving pancreatic cancer

Info

Publication number: KR101753560B1
Application number: KR1020160032025A
Authority: KR
Inventors: 김재훈; 이중회; 윤정훈
Original assignee: 제주대학교 산학협력단
Priority date: 2016-03-17
Filing date: 2016-03-17
Publication date: 2017-07-05

Abstract

The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating pancreatic cancer containing naringenin and hesperetin as an active ingredient. It has been confirmed that the anticancer effect against pancreatic cancer is remarkably exhibited when the combination of naringenin and hesperetin in the absence of growth inhibition, metastasis inhibition and killing effect of pancreatic cancer cells, A pharmaceutical composition containing naringenin and hesperetin as an active ingredient can be usefully used for the treatment of pancreatic cancer. In addition, since naringenin and hesperetin are compounds derived from natural materials that can be separated from citrus peels, they are safe for use as anticancer drugs because they are unlikely to cause side effects when applied to human body.

Description

[0001] The present invention relates to a composition for preventing, treating or improving pancreatic cancer containing naringenin and hesperetin as an active ingredient,

The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating pancreatic cancer containing naringenin and hesperetin as an active ingredient.

Pancreatic cancer is a common primary malignancy, with less than 5% of all patients presenting with a 5-year survival rate. Compared to other carcinomas, it is known as the only cancer species that has not improved its average survival rate for decades. Currently, the most effective treatment for pancreatic cancer is surgical removal. Since the cancerous mass of pancreatic cancer is mainly composed of fibrous tissue and only a few cancer cells are present, it is difficult to evaluate the therapeutic response to cancer after chemotherapy. Since pancreatic cancer is known to be relatively uncomplicated cancer, Surgical resection is recommended rather than chemotherapy. Gemcitabine (Eli Lilly and Company) is a clinically used anticancer drug, but there is a constant question about its therapeutic effect. However, there is no substitute material or treatment method.

Thus, if a natural substance with fewer side effects is developed for the treatment of pancreatic cancer without effective treatment other than surgical removal, the possibility of side effects such as various symptoms, anemia, weight loss, hair loss, It is expected.

On the other hand, citrus peels such as citrus peel, orange, and citron include various physiologically active substances such as flavonoids, carninoids, coumarins, phenylpropanoides, and limonoids. Flavonoids are pale yellow or yellow pigmented compounds, most of which are present in the form of glycosides attached to sugars, and are known to have no side effects when ingested. The major flavonoid compounds derived from citrus fruits are naringin and hesperidin, and their aglycone forms, naringenin and hesperetin, as well as rutin, Deosmine, nobiletin, tangeretin, and the like. As a result, many compounds have been found in citrus fruits, and their evaluation of their function has been reported as antioxidant activity, prevention of circulatory diseases, anti-inflammation, antiallergic, antibacterial, antiviral, lipid lowering, immune enhancement, (Non-Patent Document 1).

Studies have been continuing to use citrus-derived components, naringenin and hesperetin, or derivatives thereof, for medicinal uses. For example, a pharmaceutical composition for the prevention and treatment of alcoholic liver disease comprising hesperetin, 7- O -glucoside, naringenin-7- O -glucoside or a combination thereof, obtained by hydrolyzing nesserine, (Patent Document 1). In addition, prior art patents relating to a health functional food composition for prevention and improvement of Helicobacter pylori containing hesperetin have been disclosed (Patent Document 2). However, there are no previous studies using naringenin and hesperetin to elicit anticancer effects.

Accordingly, the present inventors have studied to develop a therapeutic agent for pancreatic cancer derived from a natural substance having no side effects, and found that the combination of naringenin and hesperetin, a component derived from citrus peel, exhibits a synergistic effect on treatment of pancreatic cancer.

Korean Patent Registration No. 1268325. Korean Registered Patent No. 1133748.

Jae-Young Cha, "Physiological Functional Activity of Citrus Flavonoids", Proceedings of the Korean Society of Life Science Conference, Vol. 31, 2001.1, pp.19-24.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating pancreatic cancer, which comprises naringinin and hesperetin, or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or ameliorating pancreatic cancer, which comprises naringinin and hesperetin, or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a method of preventing or treating pancreatic cancer comprising administering to a subject in need thereof a composition containing naringinin and hesperetin, or a pharmaceutically acceptable salt thereof as an active ingredient .

One embodiment of the present invention provides a pharmaceutical composition for preventing or treating pancreatic cancer, comprising naringinin and hesperetin, or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "Naringenin" is a flavonoid flavanone nonpolar form which may be, but is not limited to, a compound of formula 1 or a derivative thereof:

[Chemical Formula 1]

As used herein, the term " Hesperetin "is a flavonoid, which may be, but is not limited to, a compound of formula (2)

(2)

As used herein, the term "pharmaceutically acceptable salt" may be prepared by any method conventional in the art, and specifically includes base addition salts formed by addition of bases. Wherein the base is selected from the group consisting of alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal bicarbonates such as sodium bicarbonate and potassium bicarbonate, alkali metal carbonates such as sodium carbonate, potassium carbonate and calcium carbonate, Such as an inorganic base, or an organic base such as a primary or secondary tertiary amine amino acid. The pharmaceutically acceptable salts should be low in toxicity to humans and should not adversely affect the biological activity and physico-chemical properties of the parent compound.

The naringenin and hesperetin may be isolated from the citrus peel.

The citrus fruits are plant leaves belonging to the dicotyledonous plant axilla and the citrus tree subspecies. Examples of the citrus fruits include lemon, kumquat, lime, orange, mandarin, bergamot, grapefruit, It may be, but is not limited to, citrus unshiu .

The naringenin and hesperretin are prepared by 1) dissolving citrus rind in the buffer solution; 2) treating the solution with cellulase, pectinase and beta-glucosidase enzyme; And 3) removing narirutin and hesperidin from the enzyme-treated citrus peel solution to obtain a solution containing naringenin and hesperetin. In addition, the method may further comprise: 4) removing the solvent from the solution comprising naringenin and hesperetin.

The buffer solution of step 1) may be a known buffer solution, but not necessarily limited thereto, for example, sodium acetate may be used.

The enzyme of step 2) may be used in an amount of 40 to 70 U / g of cellulase, 100 to 140 U / g of pectinase and 70 to 110 U / g of betaglucosidase, more specifically 50 to 60 U / for example, 54 U / g of cellulase, 120 U / g of pectinase and 90 U / g of pectinase, and 100 U / g of pectinase and 80 to 110 U / g may be used, but not always limited thereto, and a known enzyme treatment method for obtaining naringenin and hesperetin may be used.

In step 2), the enzyme may be reacted at a temperature of 30 to 60 ° C at 100 to 500 rpm for 10 to 50 hours after the enzyme treatment. For example, the reaction may be performed at 45 ° C at 150 rpm for 24 hours, , And known enzyme treatment reaction conditions for obtaining naringenin and hesperetin can be used.

In the step 3), the removal of naryutin and hesperidin can be carried out by using a prep-HPLC (Preparative-High Performance Liquid Chromatography) method, but the present invention is not limited thereto, and known tablets for removing naryutin and hesperidin Method can be used.

The above step 3) can be performed to obtain a solution containing high purity naringenin and hesperetin.

Removal of the solvent in step 4) may be carried out using methods known to those skilled in the art, including, but not limited to, drying, specifically lyophilization to remove the solvent have.

Therefore, in the present invention, naringenin and hesperetin can be obtained by removing the naringutin and hesperidin from the enzyme-treated citrus peel solution or removing the naringutin and hesperidin from the enzyme-treated citrus peel solution And the remaining sample may be used, or a commercially available purified sample may be used.

It is preferred that the pharmaceutical composition simultaneously release or treat naringenin and hesperetin, and naringenin or hesperetin may be released or treated earlier than hesperetin or naringenin.

The naringenin and hesperetin may be treated at a weight ratio of 1 to 100: 100 to 1, more specifically 1: 50: 50 to 1, more specifically 1 to 10:10 to 1 , More specifically from 1: 5: 5 to 1, more specifically from 2: 4: 1, and can be processed in a weight ratio of, for example, from about 3: 1, but the present invention is not limited thereto.

The naringenin can be treated at a concentration of 0.1 to 500 μM, specifically at a concentration of 0.1 to 100 μM, more specifically at a concentration of 1 to 100 μM, more specifically at a concentration of 0.1 To 50 [mu] M, more specifically at a concentration of 1 to 50 [mu] M, and may be treated at a concentration of, for example, 20 [mu] M, but is not limited thereto.

The hesperetin may be treated at a concentration of 0.1 to 500 μM, specifically at a concentration of 0.1 to 100 μM, more specifically at a concentration of 1 to 100 μM, and more specifically, Can be treated at a concentration of 0.1 to 50 μM, more specifically at a concentration of 1 to 50 μM, and can be treated at a concentration of, for example, 20 μM, but are not limited thereto.

The naringenin and hesperetin are used in a weight ratio of 1 to 100: 100 to 1, specifically 1 to 50:50 to 1, more specifically 1 to 10:10 to 1, more specifically 1 to 5 Kg of a daily dose of 0.1 to 1000 mg / kg, more specifically 1 to 1000 mg / kg, in a weight ratio of 5: 1 to 5: 1, more specifically 2: 4: More specifically from 1 to 500 mg / kg, more particularly from 1 to 100 mg / kg, even more specifically from 10 to 100 mg / kg, for example 50 mg / kg.

As used herein, the term "active ingredient" means a component that exerts the effects of a drug or quasi-drug. The term " active ingredient "

The pancreatic cancer is a tumor mass composed of cancer cells formed in the pancreas. The pancreatic cancer includes pancreatic adenocarcinoma, cystic adenocarcinoma (endocarcinoma) and endocrine tumor. However, pancreatic adenocarcinoma arising in the pancreatic duct is about 90% It is known. Pancreatic cancer is a very poor prognosis with a 5-year survival rate of less than 3%, which is less than 20% of patients who can be surgically resected at the time of diagnosis, and the survival rate is less improved by micro-metastasis after surgery. This is because the reaction is low. The primary chemotherapeutic agents for pancreatic cancer include gemcitabine, erlotinib, capecitabine, platinum, tegafur, gemeracil, Oteracil, etc. are used as a combination therapy or a secondary treatment. However, the use of such an anticancer agent has a problem in that the therapeutic effect remains questionable, and the possibility of side effects is very high. On the other hand, the pharmaceutical composition of the present invention is an anticancer agent derived from a natural substance and has excellent anticancer effect against pancreatic cancer as well as low possibility of side effects.

As used herein, the term "prevention" means any action that inhibits or delays pancreatic cancer by administration of a composition containing the naringenin and hesperetin.

As used herein, the term "treatment" refers to any action that improves or cures the symptoms of pancreatic cancer by administration of a composition containing the naringenin and hesperetin.

As used herein, the term "pharmaceutical composition" is intended to encompass a single dosage form, such as an oral or injectable, to be taken or administered at once, as well as a plurality of unit dosage forms Are also encompassed. For example, "pharmaceutical composition containing naringenin and hesperetin" is intended to encompass both single unit dosage forms containing these two active ingredients, as well as two unit dosage forms, each containing one active ingredient Are interpreted to include both. In addition, even a single unit dosage form containing two active ingredients may be manufactured and simultaneously administered to be released at a time, or two unit dosage forms may be administered simultaneously or at intervals of a predetermined time or less to be contained in each of these unit dosage forms When two active ingredients coexist in the body to cause a synergistic action, the pharmaceutical composition for preventing or treating pancreatic cancer containing "naringenin and hesperetin as an active ingredient" together with single or two unit dosage forms May be referred to as belonging to the category. The two active ingredients can be released or processed (or administered) simultaneously or in time. Accordingly, the pharmaceutical composition for preventing or treating pancreatic cancer, which comprises the naringinin and hesperetin, or a pharmaceutically acceptable salt thereof, as an active ingredient, can be used not only in a simple combination pharmaceutical composition capable of simultaneously treating (or releasing) Time release (or release) of the pharmaceutical composition.

As used herein, the term "time lag or time lag" means that a drug is released or administered so that it is not simultaneously absorbed and sequentially absorbed when each active ingredient is administered or administered. Such "time release or time delay administration" is intended to encompass not only the combination preparation in which the two components are contained in one formulation, but also the preparation of the formulation in such a manner that at least one component is released in a time- Thereby allowing the preparation or the administration unit to operate at the same time even if administered or administered at the same time. The above-mentioned "time delay administration" may also include a method of administering the two active ingredients at regular time intervals.

In the above pharmaceutical composition, naringenin and hesperetin are preferably used as such, but derivatives thereof may be prepared and used in consideration of solubility and stability characteristics.

Meanwhile, the pharmaceutical composition of the present invention may contain one or more pharmaceutically acceptable carriers in addition to the above-mentioned active ingredients. The term "pharmaceutically acceptable carrier " as used herein means a pharmaceutical additive that is useful when formulating a pharmaceutical composition for administration and is non-toxic and non-sensitive under the conditions of use. The specific content of such excipient The ratio can be determined by standard pharmaceutical practice, as well as the solubility and chemical properties of the active ingredient, the route of administration chosen. In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, phosphoric acid Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, stearic acid, stearic acid, sodium carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydrogenated calcium, lactose, mannitol, sugar, arabic gum, pregelatinized starch, cornstarch, powdered cellulose, Aluminum, calcium stearate, white sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

The pharmaceutical composition may be administered orally or parenterally in various clinical formulations. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or the like may be used &Lt; / RTI > 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 Calcium carbonate, Sucrose, Lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition may be administered orally or parenterally in accordance with the desired method. In the case of parenteral administration, the composition may be administered by external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, . The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

Another embodiment of the present invention is a health functional food composition for preventing or ameliorating pancreatic cancer, comprising naringinin and hesperetin, or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "improvement" means any action that improves or alters the symptoms of pancreatic cancer by administration of a composition containing the naringenin and hesperetin.

As used herein, the term "health functional food" refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, or mixing a specific ingredient contained in a food raw material , The health functional food composition can perform functions related to prevention of pancreatic cancer and recovery of pancreatic cancer.

The health functional food may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It can also contain natural fruit juices and pulp for the production of fruit juices and vegetable drinks. These components may be used independently or in combination. The health functional food may be any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin .

In addition, the health functional food may further include food additives, and the suitability of the food functional food as a "food additive" is not limited to the corresponding items in general rules and general test methods approved by the Food and Drug Administration Shall be determined according to the relevant standards and standards.

Examples of the products that have been used in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, sensory coloring matter, licorice extract, crystalline cellulose, high- - Mixed preparations such as a sodium glutamate preparation, a noodle-added alkaline agent, a preservative preparation, a tar coloring agent and the like.

At this time, naringenin and hesperetin, which are added to foods in the process of manufacturing a health functional food, can be added or decreased as needed, and may be added in an amount of 0.1 wt% to 50 wt% to 100 wt% of the food have.

Since the naringenin and hesperetin of the present invention are derived from natural materials, they can be used safely and usefully when they are used as a food composition or a pharmaceutical composition because they have fewer side effects than general synthetic compounds.

Another embodiment of the present invention is a method for preventing or treating pancreatic cancer comprising administering to a subject in need thereof a composition comprising naringinin and hesperetin, or a pharmaceutically acceptable salt thereof, as an active ingredient to provide.

As used herein, the term "individual" means all animals, including humans, who have already developed or can develop pancreatic cancer, and the composition of the present invention is effective for preventing and treating the disease by administration to a subject .

The composition is preferably a pharmaceutical composition and can be administered in a therapeutically effective amount.

As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dosage level will vary depending on the species and severity, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by a person skilled in the art.

It has been confirmed that the anticancer effect against pancreatic cancer is remarkably exhibited when the combination of naringenin and hesperetin in the absence of growth inhibition, metastasis inhibition and killing effect of pancreatic cancer cells, A pharmaceutical composition containing naringenin and hesperetin as an active ingredient can be usefully used for the treatment of pancreatic cancer. In addition, since naringenin and hesperetin are compounds derived from natural materials that can be separated from citrus peels, they are safe for use as anticancer drugs because they are unlikely to cause side effects when applied to human body.

Fig. 1 shows the results of four pancreatic cancer cell lines (AsPC-1, Miapac-1, and Asp-1) treated with enzyme-treated citrus pectin (EHCUP) at a concentration of 5 mg / Growth (A) and anti-metastasis (B) activity in the Panc-2, Panc-1 and SNU-213 cells.
FIG. 2 shows the results of the enzyme-treated citrus papillary component (Panc-1 and SNU-213 cells), except for AsPC-1 and Miapaca-2, which are the weakest anti-growth and anti- (A) and anti-metastasis (B) by treating the cells with various concentrations (0, 1, 2.5, and 5 mg / mL)
FIG. 3 is a graph showing the effect of the enzyme-treated citrus papule component (EHCUP) on the growth and metastasis of pancreatic cancer cell line by using human normal umbilical vein endothelial cells (HUVEC) The results of the comparative experiment are as follows:
A, HUVEC cells were treated with various concentrations (0, 2.5, 5 mg / mL) of citrus peel (EHCUP) and treated with 100 ng / mL of VEGF-A as a positive control. A graph showing a result;
B, simultaneous-culture system of Panc-1 cells and HUVECs; And
(EHCUP) treatment with various concentrations (0, 2.5, 5 mg / mL) after co-culturing C, Panc-1 or HUVEC cells.
Figure 4 shows the effect of varying concentrations (0, 1, 5 mg / mL) of gemcitabine (Gemzar) (A, C) or doxorubicin (Doxo.) (B) on Panc-1, SNU- (Viability), indicating that gemcitabine and doxorubicin have the effect of inhibiting the growth of HUVEC, unlike the enzyme-treated citrus peel component (EHCUP).
Figure 5 shows that the enzyme-treated citrus ploidy component (EHCUP) affects the signal transduction system in the pancreatic cancer cell line (SNU-213, HUVECs), and the major intracellular signal involved in the growth, penetration and survival of cancer cells Inhibits the phosphorylation of transporter proteins (FAK, Src, AKT, MEK1 / 2, ERK1 / 2, JNK. P38).
FIG. 6 is a result of identification of major constituents constituting the enzyme-treated citrus peel component, wherein Narirutin and Hesperidin are detected in the water-soluble citrus peel ingredient (A), and treatment with three enzymes (B) The results show that Naringenin and Hesperetin are detected additionally.
FIG. 7 is a chart showing the viability of a HUVECs cell after treatment with various samples; FIG.
Control, PBS treatment group;
EHCUP, enzyme treated citrus pectin component 5 mg / mL treated group;
-Hes., 5 mg / mL of hESPERIN alone group;
-Hes / Nar., Treated group of naringenin and hesperetin 5 mg / mL; And
# 3, treated with 5 mg / mL of naringenin and hesperetin mixed samples (-Hes / Nar.).
Figure 8 shows the effect of intracellular signaling proteins (FAK, Src, ERK, p38) on HUVECs cells after 0, 10, 30 and 60 minutes after concurrent treatment (-Hes./Nar.) Of naringenin and hesperetin And the results of Western blotting confirmed the degree of phosphorylation.
9 is a graph showing the viability of cells treated with Panc-1 cells at various concentrations (0, 1, 2.5 mg / mL) for 72 hours or 96 hours:
EHCUP, enzyme treated citrus pectin component 5 mg / mL treated group;
-Hes., 5 mg / mL of hESPERIN alone group;
-Hes / Nar., Treated group of naringenin and hesperetin 5 mg / mL; And
# 3, treated with 5 mg / mL of naringenin and hesperetin mixed samples (-Hes / Nar.).
10 shows the results of treatment of a mixture of naringenin and hesperetin at various concentrations (0, 1, 2.5, 5 mg / mL) in Panc-1 or SNU-213 cells, (Migrate cells) and SNU-213 cells were mixed with 5 mg / mL of naringin and hesperetin, respectively. FIG.
FIG. 11 shows the results of a single treatment with Hessperine (H), a treatment with Naringinin alone (N), and a treatment with Asp-1, Miapaca-2, SNU-213 or Panc-1 cells at various concentrations (0, 1, 5, 10, 20 μM) ) Or a combination of naringenin and hesperetin, followed by measurement of cell viability.
12 shows the results of confirming that the naringenin and hesperetin mixed sample (-Hes./Nar.) Shows the effect of inhibiting the growth of cancer even in vivo without special change in the weight of experimental nude mice:
(31, 34, 38, 41, 45) in the nude mice injected subcutaneously with SNU-213 cells in the PBS administration group (control) and the naringenin and hesperetin mixed sample administration groups (-Hes / (Tumor Volume, mm < ³ >);
(35, 39, 42, 46, 49) in PBS-administered group (control) and naringenin and hesperetin mixed group (-Hes / Nar.) In nude mice subcutaneously injected with Pan- (Tumor Volume, mm < ³ >); And
(SNU-213 / PBS or Pan-1 / PBS) or naringenin and hesperetin mixed samples (SNU-213 / PBS) in nude mice subcutaneously injected with SNU- Days after administration of samples of Pan-1 / -Hes./Nar., Pan-1 / -Hes. / Nar. (Body weight (g)) according to the number of days (35, 39, 42, 46, 49 days).

Hereinafter, the present invention will be described in more detail in the following Examples. It should be noted, however, that the following examples are illustrative only and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

< Example 1> Cell culture

All of the pancreatic cancer cell lines (AsPC-1, SNU-213, Miapaca-2, Panc-1) were incubated in the following medium containing 10% fetal bovine serum (Gibco) at 37 ° C in a 5% Lt; / RTI > AsPC-1 and SNU-213 cells were cultured in RPMI-1640 medium (Gibco), and Miapaca-2 and Panc-1 cells were cultured in DMEM (Dulbecco's modified Eagle's medium; Gibco). The pancreatic cancer cell lines used in the experiments were purchased from Korean Cell Line Bank (KCLB). Human Umbilical Vein Endothelial Cell (HUVEC), a normal cell, is purchased from ATCC and maintained at 5% carbon dioxide using EGM-2 (Hyclone) medium containing 10% fetal bovine serum (Gibco) And incubated at 37 ° C in a constant temperature incubator.

< Example 2> Enzyme-treated Citrus p Preparation of ingredients

Citrus bloom was obtained from the processing company (Samdasoo) as a byproduct remaining after citrus beverage processing. The citrus fruits were lyophilized and dissolved in 50 mM sodium acetate buffer solution. The cells were treated with cellulase (54 U / g; Sigma), pectinase (120 U / g; Sigma) and beta glucosides (90 U / g; Sigma) Lt; / RTI > Enzymatic Hydrolyzed Citrus Unshiu Peel (EHCUP) was used in the following experiments.

< Example 3> Enzyme-treated Citrus p Of Pancreatic Cancer Cell Growth

In order to investigate the effect of the enzyme-treated citrus pellet treatment on the growth of pancreatic cancer cells, cultured pancreatic cancer cells were treated with various concentrations of enzyme-treated citrus pectin for 72 hours, and WST-1 (manufactured by Nalgene Co., ) Technique to detect viability.

Specifically, the enzyme-treated citrus peel component (EHCUP) prepared in Example 2 was treated with AsPC-1, Miapaca-2, Panc-1 and SNU-213 cells at a concentration of 5 mg / Treated with 5 mg of enzyme treated citrus paw (EHCUP) per 1 mL of culture medium). In addition, Panc-1, SNU-213 cells were treated with the enzyme-treated citrus extract (EHCUP) prepared in Example 2 at concentrations of 0, 1, 2.5, and 5 mg / mL, respectively. Next, the survival rate of each cell treated with citrus peel components was measured by the WST-1 technique. The WST-1 method was performed by measuring the formation of a formazan coloring material in Tetrazolium Salts (WST-1) by mitochondrial dehydrogenase in cells by ELISA. Namely, the cells treated with citrus peel were treated with 5% (v / v) of Premix WST-1 solution and incubated for 5 to 10 minutes in an incubator, and absorbance was measured at 450 nm.

As a result, it was confirmed that the enzyme-treated citrus peel component (EHCUP) treated at various concentrations inhibited the growth of the pancreatic cancer cell line (FIGS. 1A and 2A).

< Example 4> Enzyme-treated Citrus p Of Pancreatic Cancer Cells

In order to examine the effect of the enzyme-treated citrus peel components on the migration of pancreatic cancer cells, cultured pancreatic cancer cells were treated with various concentrations of enzyme-treated citrus peel components for 6 hours, and then trans-well ) Migration technique.

Specifically, the enzyme-treated citrus peel component (EHCUP) prepared in Example 2 was treated with AsPC-1, Miapaca-2, Panc-1 and SNU-213 cells at a concentration of 5 mg / mL. In addition, Panc-1, SNU-213 cells were treated with the enzyme-treated citrus peel component (EHCUP) prepared in Example 2 at concentrations of 0, 2.5 and 5 mg / mL, respectively. Next, the migration ability of each cell treated with citrus peel component was measured by a trans-well migration technique. Trans-well migration was performed using 24-Transwell (Corning, MA, USA) according to the manufacturer's protocol. The cells were applied to the upper chamber containing serum-free RPMI for 6 hours and stained cells moved to the back of the filter for 24 hours. Eluted stained cells were measured at 560 nm with an ELISA reader (Bio-Rad, Richmond, Calif.).

As a result, it was confirmed that the enzyme-treated citrus peel component treated at various concentrations inhibited the migration of the pancreatic cancer cell line (FIG. 1B and FIG. 2B).

< Example 5> Enzyme-treated Citrus p Prove the non-toxic efficacy of the ingredient

In order to verify that the anticancer effect of the enzyme-treated citrus pomace is not due to the toxicity of the sample itself, the human umbilical vein endothelial cell (HUVEC), which is a normal cell line, is treated with the same concentration of enzyme- Metastasis was observed.

Specifically, the enzyme-treated citrus peel component (EHCUP) prepared in Example 2 was treated with Panc-1 and HUVECs cells at concentrations of 0, 2.5 and 5 mg / mL, respectively. Vascular endothelial growth factor-A (VEGF-A), a protein that stimulates angiogenesis and angiogenesis, was used as a positive control. Next, the survival rate of each cell treated with citrus peel was measured in the same manner as in the WST-1 method of Example 3, and the migration ability was measured in the same manner as the transwell migration method of Example 4.

As a result, unlike pancreatic cancer cells, human umbilical vein endothelial cells were not observed to have the effect of inhibiting growth and migration by enzyme-treated citrus pectin (Fig. 3). Therefore, it was confirmed that the effect of the enzyme-treated citrus peel component on growth and metastasis of pancreatic cancer cells was not due to the toxicity of the sample itself.

< Example 6> cancer cells and Human umbilical vein endothelial cells The control samples for growth effect

Gemcitabin (Sigma), which is currently being used in pancreatic cancer patients, and doxorubicin (Sigma), which is known to have anticancer activity against pancreatic cancer, are used to treat pancreatic cancer cells and human umbilical vein endothelial cell growth To investigate the effects of different concentrations of control samples on the Panc-1, SNU-213, and HUVEC cells cultured, various survival rates were determined by WST-1 (Nalgene) method after 72 hours of control.

Specifically, gemcitabine (Gemzar), doxorubicin (Doxo.) 0, 1, and 5 uM were treated with Panc-1, SNU-213 and HUVECs cells, respectively. The WST-1 method was carried out in the same manner as in Example 3.

As a result, the control samples gemcitabine and doxorubicin were simultaneously observed to inhibit growth of cancer cells and human umbilical vein endothelial cells (Figs. 4A to 4C). Therefore, it was confirmed that gemcitabine and doxorubicin inhibit growth not only in pancreatic cancer cells but also normal cells, but that the enzyme-treated citrus papillary component (EHCUP) specifically inhibits growth of pancreatic cancer cells.

< Example 7> Enzyme treated Citrus p Effect of Component Treatment on Intracellular Signal Transduction in Pancreatic Cancer Cells

Treatment of phosphorylated phosphatidylcholine components of intracellular signaling proteins (FAK, Src, AKT, MEK1 / 2, ERK1 / 2, JNK, p38; Cell Signaling) involved in the growth, penetration and survival of cancer cells The experiment was performed to verify whether it was decreased by the western blotting.

Specifically, the enzyme-treated citrus peel component (EHCUP) prepared in Example 2 was treated at a concentration of 5 mg / ml in SNU-213, which is a pancreatic cancer cell line, and HUVEC, which is a human umbilical vein endothelium, Were collected and dissolved in the cell lysis solution. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce). The same amount of each protein was developed on 10% SDS-PAGE and Western blotted with nitrocellulose membrane at 35 V for 12 hours . The membranes were blocked with 5% skim milk overnight at 4 ° C and reacted with 1% skimmed milk for 1 hour with secondary antibodies appropriate for the primary antibodies, respectively. In order to investigate the expression of non-phosphorylated proteins as comparative groups, FAK, Src, AKT, MEK1 / 2, ERK and JNK were added to the nitrocellulose membrane which was treated by the same treatment as above. p38 antibody for 1 hour. After incubation for 1 hour with a secondary antibody for each primary antibody, the cells were washed with TBST and the degree of phosphorylation of each protein was detected with ECL (Amersham Pharmacia Biotech).

As a result, phosphorylation of major proteins was reduced only in SNU-213 cells treated with enzyme-treated citrus pectin (EHCUP). On the other hand, it was observed that treatment of the enzyme-treated citrus pod components with the same conditions did not involve reduction of intracellular signal transduction of the human umbilical vein endothelial cell, HUVEC (FIGS. 5A and 5B).

Therefore, it was confirmed that the enzyme-treated citrus peel component (EHCUP) reduced the signal transduction protein specifically to pancreatic cancer cells.

< Example 8> HPLC Used Citrus p Identify the major components of the ingredient

Identify the major components of the citrus pox that are changed by the treatment of cellulase (54 U / g; Sigma), pectinase (120 U / g; Sigma) and beta glucosides (90 U / g; Sigma) The water - soluble citrus peel component and the enzyme treated citrus peel component (EHCUP) were identified by HPLC.

Specifically, HPLC (High Performance Liquid Chromatography) was performed using a Shimadzu HPLC system (manufactured by Shimadzu) equipped with a PDA (photo diode array) detector and a Luna C18 (2) column (5 μm particle size, 4.6 mm × 250 nm, Phenomenex, Torrance, Shimadzu Scientific Instruments, Columbia, MD). HPLC analysis was performed as follows: 20% acetonitrile at 0 min, 40% acetonitrile at 10 min, 70% acetonitrile at 20 min, 70% acetonitrile at 25 min, 20% acetone at 27 min Nitrile, 20% acetonitrile in 30 minutes at a column flow rate of 1 mL / min. The peak was detected at 270 nm. LC-MS / MS analysis was performed using an Accela HPlC system coupled with a Thermo LXQ ion trap mass spectrometer with an ESI negative interface (Thermo Scientific Waltham, MA, USA).

As a result, it was confirmed that narirutin and hesperidin were detected as water-soluble citrus peel components, but the components of Naringenin and Hesperetin were further detected by enzyme treatment ( 6A and 6B).

Therefore, it was confirmed that naringenin and hesperetin can be obtained by enzyme treatment of citrus peel.

< Example 9> Enzyme-treated with Prep-HPLC Citrus p Purification of ingredients

The results showed that HUVEC, a human umbilical vein endothelial cell, was slightly increased in growth and migration by treatment with enzyme - treated citrus pectin, suggesting that it may be accompanied by anticancer effect and neovascularization. Therefore, only Naringenin and Hesperetin components were purified using Prep-HPLC to remove them.

Specifically, hesperidin and narirutin were removed from the enzyme-treated citrus pectin component (EHCUP) using Prep-HPLC (Preparative-HPLC), so that only naringenin and hesperetin components were highly pure . A sample from which the hesperidin and nariroutin were removed in EHCUP was used as a mixed sample of naringenin and hesperetin (-Hes./Nar.). Prep-HPLC system (Sigma-Aldrich, St. Louis, MO) consists of pump (LC-20AP), photodiode array detector (SPD-M20A), autosampler (SIL-10AP) and system controller (CBM-20A) . Qualitative analysis was performed at a flow rate of 1 mL / min in a step gradient mode using a mixture of acetonitrile and water (2: 8, 4: 6, 7: 3 and 2: 8). The enzymatically treated citrus pectin component (50 mg) was dissolved in 1 mL of eluent and 250 μL of this solution was injected into the column. The total running time was 30 minutes. Detection was performed by monitoring the absorbance at 270 nm. Chromatographic analysis was performed by comparing the retention time of each peak with reference HPLC data. Also, it was confirmed that the weight ratio of naringenin and hesperetin in the mixed sample (-Hes./Nar.) Of naringenin and hesperetin was 74.57%: 25.42%.

The effects of Naringenin and Hesperetin (-Hes./Nar.) Obtained by Prep-HPLC on the growth and migration of cancer cells and HUVEC were observed. The viability of the cells was measured by the same method as the WST-1 method of Example 3, and the migration ability was measured by the same method as the transwell migration method of Example 4.

7 shows the result of measuring the cell survival rate in the same manner as in the WST-1 method of Example 3 after 72 hours from the treatment of each sample with HUVEC. As a control, PBS (used in the same volume as the other experimental groups) was used. EHCUP used the enzyme-treated citrus peel component prepared in Example 2 at a concentration of 5 mg / mL, and the sample treated with hESPERIN alone was purchased from Sigma A purified sample to be sold was purchased and used at a concentration of 5 mg / mL, and a mixture sample of naringenin and hesperetin was obtained from the above-mentioned Heis./Nar. The sample was used at a concentration of 5 mg / mL, and # 3 was -Hes./Nar. The same sample as the sample was used at a concentration of 5 mg / mL.

The experiment of FIG. 8 was performed by treating a mixture of Naringenin and Hesperretin (-Hes./Nar.) In a culture HUVEC at a concentration of 5 mg / mL with time (0, 10, 30 or 60 minutes) and Western blotting (FAK, Src, ERK, p38) involved in the growth, penetration and survival of cancer cells.

In the experiment of FIG. 9, each sample was cultured in the cultured Panc-1 cells at a concentration of 0, 1 or 2.5 mg / mL for 72 hours or 96 hours, and the cell viability was measured in the same manner as in the WST- . The EHCUP used the enzyme-treated citrus pectin component prepared in Example 2 at a concentration of 5 mg / mL, the purified sample sold by Sigma was used at a concentration of 5 mg / mL, and the naringenin And a hesperetin mixed sample were prepared by the method described in -Hes. The sample was used at a concentration of 5 mg / mL, and # 3 was -Hes./Nar. The same sample as the sample was used at a concentration of 5 mg / mL.

In the experiment of FIG. 10, the Nareningin and Hessperin content (-Hes./Nar.) Was treated at a concentration of 0, 1, 2.5 or 5 mg / mL in the cultured Panc-1 or SNU-213 cells, 4 shows the result of measuring the moving ability in the same manner as the trans-well migration method.

As a result, a mixture of Naringenin and Hesperetin (-Hes./Nar.) Differentially inhibited the growth and metastasis of pancreatic cancer cells, unlike the enzyme-treated citrus pectin (EHCUP) (Fig. 7 to Fig. 10).

< Example 10> With Narinenin Hesperetin Synergy effect by simultaneous processing

To investigate the effect of simultaneous treatment of Naringenin and Hesperetin, pancreatic cancer cell lines AsPC-1, Miapaca-2, SNU-213 and Panc-1 cell lines were treated with different concentrations (0, 1, 5, 10, and 20 μM), respectively, and the growth inhibitory activity was observed using WST-1 (Nalgene). Naringenin and hesperetin were purchased from Sigma and used as purified samples, respectively.

As a result, it was observed that treatment with naringenin and hesperetin alone did not inhibit specific cancer cell growth inhibition, but it was observed that co-treated cells excellently suppressed growth (Fig. 11). In particular, the simultaneous treatment of naringenin and hesperetin at a concentration of 20 [mu] M rapidly inhibited the growth of pancreatic cancer cells.

Thus, when combined with naringenin and hesperetin, significant synergistic effects were observed in the treatment of pancreatic cancer.

< Example 11> In using nude mice vivo Anti-cancer effect verification

In order to examine the anticancer effect of naringenin and hesperetin in vivo , a nude mouse was used for the anticancer test.

Specifically, ¹ x 10 ⁷ SNU-213 and Panc-1 cells were subcutaneously injected into 6-week-old female nude mice and administered for about 30 days. A mixture sample of naringenin and hesperetin was prepared as described in Hes./Nar. The sample was used by removing the solvent through lyophilization. When the volume of the initial cancer was about 400 mm ³ , a mixed sample of the naringenin and hesperetin was subcutaneously injected at a daily dose of 50 mg / kg. At this time, a mixed sample of naringenin and hesperetin was injected by dissolving in PBS (buffer solution). An experimental group injected with only PBS (buffer solution) was used as a control group.

Control comparison showed that the volume of the cancer was reduced without specific weight changes in rats injected with a mixture of naringenin and hesperetin (Figs. 12A-12C).

Claims

A pharmaceutical composition for preventing or treating pancreatic cancer, comprising naringenin and hesperetin, or a pharmaceutically acceptable salt thereof, as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the naringenin and the hesperetin are separated from the citrus peel.

The pharmaceutical composition according to claim 1, wherein the naringenin and the hesperetin are each contained at a concentration of 1 to 100 μM.

[Claim 2] The pharmaceutical composition according to claim 1, wherein the naringenin and the hesperetin are administered at a daily dose of 10 to 100 mg / kg in a weight ratio of 1: 10: 10-1.

Or a pharmaceutically acceptable salt thereof, as an active ingredient, and a composition for preventing or improving pancreatic cancer.