KR20220078740A - Composition for preventing, ameliorating or treating prostate cancer comprising Hippocampus abdominalis and herbal medicine mixed extract as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, improving, or treating prostate cancer containing a seahorse and herbal medicine mixed extract as an active ingredient, and more particularly, the mixed extract of the present invention has the effect of inhibiting growth, migration, invasion and cell growth of prostate cancer cells Since the death-inducing effect is excellent, it can be usefully used as a composition for preventing, improving or treating prostate cancer containing it as an active ingredient.

Description

The composition for preventing, ameliorating or treating prostate cancer comprising Hippocampus abdominalis and herbal medicine mixed extract as effective component

The present invention relates to a composition for preventing, improving or treating prostate cancer, which contains a seahorse and herbal medicine mixed extract as an active ingredient.

Prostate cancer is a malignant tumor that occurs in the prostate gland in men. Most of the gland cells in the prostate become cancerous and metastasize to lymph nodes and bones. . In the West, prostate cancer is the most common cancer among male cancers and shows a high incidence. Unlike in the past, when patients in their 70s were the mainstay of prostate cancer, the number of patients undergoing prostate cancer surgery in their 40s is increasing rapidly these days. Prostate cancer treatment methods include radical prostatectomy, radiation, chemotherapy, and hormone therapy. The most widely used treatment method is hormone therapy, which is a so-called androgen removal method that suppresses male hormone secretion or blocks hormone production through surgery, since a significant portion of prostate cancer cells proliferate in a male hormone-dependent manner. This method shows temporary therapeutic effects such as suppression of cancer growth or reduction of lesions in more than 80% of patients. However, even in patients who have responded well to temporary hormone therapy, most of them can progress to hormone-refractory prostate cancer (HRPC) after a certain period of time, and these HRPC patients die within one year of diagnosis on average. In the case of HRPC, existing anticancer drugs, chemotherapy, or radiation therapy do not show a great effect, so a new treatment method is required.

Although seahorse is a protected fish with limited international trade, it is consumed steadily in Southeast Asia, mainly China, and artificially cultivated seahorse in Australia and neighboring countries and China. Seahorses produced by aquaculture are helping to restore the biological resources of seahorses, which are gradually becoming extinct. In Korea, Big Valley hippocampus ( Hippocampus abdominalis ) is mainly cultured. According to the main Chinese pharmacopoeical book, Bonchogangmok, when seahorse is used as a medicine, it is said that it boosts yang and cures diseases. Therefore, in traditional Chinese medicine, the dried hippocampus has been used as a medicine, and the hippocampus is still used to treat asthma, heart disease, fractures, as well as a wide range of diseases.

On the other hand, Korea Patent Application Publication No. 2012-0020640 discloses 'a composition for treating prostate cancer containing mulberry', and Korean Patent Publication No. 2019-0065647 discloses 'Prevention and treatment of kidney disease containing seahorse extract as an active ingredient'. Although a pharmaceutical composition and a health functional food composition for use have been disclosed, there is no description of the 'composition for preventing, improving or treating prostate cancer containing a seahorse and herbal medicine mixed extract as an active ingredient' of the present invention.

The present invention has been derived from the above needs, and the present inventors have completed the present invention by confirming that the hippocampus and herbal medicine mixed extract is excellent in the effect of inhibiting growth, migration, invasion and apoptosis of prostate cancer cells.

In order to solve the above problems, the present invention provides a health functional food composition for preventing or improving prostate cancer containing a mixed extract of seahorse, rhubarb, silkworm, dermis, angelica, mokhyang and green onion as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating prostate cancer containing a mixed extract of seahorse, rhubarb, silkworm, dermis, angelica, mokhyang and green onion as an active ingredient.

The present invention relates to a composition for preventing, improving, or treating prostate cancer containing a seahorse and herbal medicine mixed extract as an active ingredient, wherein the active ingredient is excellent in the effect of inhibiting growth, migration, invasion and apoptosis inducing effect of prostate cancer cells Therefore, it can be usefully used as a health functional food for preventing or improving prostate cancer.

1 is a result confirming the cell growth inhibitory ability of the hippocampus and herbal medicine mixed extract (HMT) in prostate cancer cells (PC-3). A is a photograph after treating prostate cancer cells (PC-3) with 50, 100 and 200 μg/ml of hippocampus and herbal medicine mixture extract (HMT) and culturing for 24 hours, B is the cell growth rate through absorbance measurement. is the result *** indicates that the cell growth rate of the hippocampus and herbal medicine mixed extract (HMT) treated group was statistically significantly decreased compared to the control group that was not treated with anything, p<0.001.
2 is a result confirming the cell migration (A) and invasion (B) inhibitory ability of the hippocampus and herbal medicine mixed extract (HMT) in prostate cancer cells (PC-3). ### is a statistically significant increase in cell migration and invasion of the medium treated group containing serum (serum) compared to the medium treatment group containing no serum (FreeM), p <0.001, * is FBS (fetal Compared to the bovine serum alone group, the cell migration of the hippocampus and herbal medicine mixed extract (HMT) treated group at 50 μg/ml was statistically significantly reduced, p<0.05, and ** denotes FBS (fetal bovine serum) alone treatment. Compared to the group, the cell infiltration of the hippocampus and herbal medicine mixed extract (HMT) treated group at 50 μg/ml was statistically significantly reduced, p<0.01.
3 is a result confirming the expression levels of PAK1 and c-cas3 (cleaved caspase-3) in prostate cancer cells (PC-3) by hippocampus and herbal medicine mixed extract (HMT) treatment. The expression level of each protein was indicated by setting the protein expression level to 1 when nothing was treated.
4 is a result of measuring apoptosis of three-dimensional spheroid prostate cancer cells (PC-3) by hippocampus and herbal medicine mixed extract (HMT). A is observed with an optical microscope and a fluorescence microscope, and green fluorescence indicates apoptotic cells. B indicates the fluorescence intensity of the killed cells, * indicates that the number of dead cells in the hippocampus and herbal medicine mixed extract (HMT) treated group increased statistically significantly compared to the control group not treated with anything, p<0.05.

In order to achieve the object of the present invention, the present invention provides a health functional food composition for preventing or improving prostate cancer, which contains a mixed extract of sea horse, rhubarb, silkworm, dermis, angelica, mokhyang and green onion as an active ingredient.

The breed of the hippocampus is preferably Big Valley hippocampus ( Hippocampus abdominalis ), but is not limited thereto.

In the health functional food composition for preventing or improving prostate cancer of the present invention, the mixed extract preferably contains 80 to 120 parts by weight of rhubarb, 80 to 120 parts by weight of rhubarb, 80 to 120 parts by weight of dermis with respect to 100 parts by weight of hippocampus. , 80 to 120 parts by weight of angelica, 40 to 60 parts by weight of mulberry, and 40 to 60 parts by weight of green onions by weight, more preferably, with respect to 100 parts by weight of seahorse, 90 to 110 parts by weight of rhubarb, 90 to 110 weight It may be a mixture of cow silkworm, 90 to 110 parts by weight of dermis, 90 to 110 parts by weight of angelicae, 45 to 55 parts by weight of mulberry, and 45 to 55 parts by weight of green beans, and most preferably 100 parts by weight based on 100 parts by weight of seahorse. It may be a mixture of rhubarb, 100 parts by weight silkworm, 100 parts by weight of dermis, 100 parts by weight of angelicae, 50 parts by weight of mulberry, and 50 parts by weight of green onions, but is not limited thereto.

The mixed extract of the present invention is preferably extracted using a C ₁ to C ₄ lower alcohol, water, or a mixture thereof as a solvent, and more preferably extracted using ethanol as a solvent, but is not limited thereto.

In one embodiment of the present invention, the mixed extract has an effect of inhibiting proliferation, migration, invasion and inducing apoptosis of prostate cancer cells.

The health functional food composition of the present invention may be in one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The active ingredient may be used appropriately depending on the purpose of its use (prevention or improvement). In general, in the production of food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of the health functional food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the ordinary sense.

In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention may include ingredients that are usually added. Examples include proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols (eg, , xylitol, sorbitol, erythritol, etc.) is preferable. As the flavoring agent, natural flavoring agents (eg, taumartin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent and the like used in beverages. The ratio of these added ingredients is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention provides a pharmaceutical composition for preventing or treating prostate cancer containing a mixed extract of seahorse, rhubarb, silkworm, dermis, angelica, mokhyang and green onion as an active ingredient.

In the pharmaceutical composition for preventing or treating prostate cancer of the present invention, the method for preparing the mixed extract is the same as described above.

The pharmaceutical composition according to the present invention may be formulated in the form of oral dosage forms such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. can

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. prepared by mixing In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used.

A suitable dosage of the pharmaceutical composition of the present invention may be prescribed variously depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient. can

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

Hereinafter, the present invention will be described in more detail using Preparation Examples and Examples. These preparations and examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Preparation Example 1. Preparation of seahorse and herbal medicine mixture extract

With respect to 100 parts by weight of the seahorse, 100 parts by weight of rhubarb, 100 parts by weight of silkworm, 100 parts by weight of dermis, 100 parts by weight of angelicae, 50 parts by weight of mulberry and 50 parts by weight of green onions by weight were mixed and pulverized with a mixer, 10 times 50% (v/v) ethanol was added and extraction was performed at room temperature for 72 hours. Then, it was filtered using filter paper, concentrated with a vacuum evaporator, and freeze-dried to prepare a seahorse and herbal medicine mixed extract (HMT).

Example 1. Measurement of cell growth inhibitory ability

PC-3 cells, a human prostate cancer cell line, were purchased from the American Type Culture Collection (ATCC) and used in 10% fetal bovine serum (FBS), 2 μM L-glutamine and penicillin in RPMI (Roswell Park Memorial Institute)-1640 medium. / After the addition of streptomycin, 37 ℃, 5% CO ₂ Incubated in conditions. After dispensing in a 96-well plate at 1×10 ⁴ cells/well, 50, 100 and 200 μg/ml of hippocampus and herbal medicine mixed extract (HMT) were added and cultured for 24 hours. Then, 10 μl of CELLOMAX ^TM reagent (Precaregene, Korea) was added, reacted for 2 hours at 37° C. under dark conditions, and absorbance was measured at 450 nm to calculate the cell growth rate according to the following formula.

Cell growth rate (%) = {(S-B)/(C-B)}×100

(S: absorbance of sample, B: absorbance of blank, C: absorbance of control)

As a result, it was confirmed that the growth of prostate cancer cells (PC-3) was inhibited in a concentration-dependent manner by the hippocampus and herbal medicine mixed extract (HMT). In particular, it was confirmed that the growth rate of prostate cancer cells was inhibited by about 50% or more by 100 μg/ml of hippocampus and herbal medicine mixed extract (HMT) (FIG. 1).

Example 2. Cell migration (migration) and invasion (invasion) inhibitory ability measurement

In order to measure the ability of the hippocampus and herbal medicine mixture extract (HMT) to inhibit the migration and invasion of prostate cancer cells, a wound healing assay and an analysis using a matrigel coating chamber were performed.

Dispense PC-3 cells in a 6-well plate at 1×10 ⁶ cells/ml and incubate at 37° C. and 5% CO ₂ condition to reach 50% confluence. Scratch with a 200 μl pipette tip. (scratched). Thereafter, 50 μg/ml of hippocampus and herbal medicine mixed extract (HMT) was treated for 24 hours and stained using Diff-Quick (Sysmax), and the number of cells that migrated to the scratched area through a photo taken under a microscope. was measured.

As a result, it was confirmed that the migration of cancer cells induced by FBS (fetal bovine serum) was reduced by about 50% by the hippocampus and herbal medicine mixed extract (HMT) (FIG. 2A).

PC-3 cells treated with 50 μg/ml hippocampus and herbal medicine mixed extract (HMT) diluted with RPMI-1640 medium without FBS were dispensed into the upper chamber containing the Matrigel-coated filter, and FBS was included RPMI-1640 medium was added to the lower chamber. After culturing for 24 hours, the cells were stained using Diff-Quick (Sysmax), and the number of cells infiltrating into the lower chamber was measured through photographs taken with a microscope.

As a result, it was confirmed that the infiltration of cancer cells induced by FBS (fetal bovine serum) was reduced by about 57% by the hippocampus and herbal medicine mixed extract (HMT) (FIG. 2B).

Example 3. PAK1 and c-cas3 (cleaved caspase-3) Expression regulation analysis

It is known that the PAK1 gene is overexpressed in cancer cells. In particular, when the PAK1 gene is knocked down in prostate cancer cells, the expression of MMP9 is decreased, the expression of TGF-β is increased, and the motility and growth rate of cells are reduced. Therefore, it is possible to evaluate the anticancer effect through the PAK1 expression inhibitory ability analysis. The caspase-3 gene is known to induce apoptosis while being converted to a cleaved form. Therefore, in order to confirm the expression of PAK1 and c-cas3 (cleaved caspase-3) in PC-3 cells by the hippocampus and herbal medicine mixed extract (HMT) of the present invention, Western blotting was performed.

As a result, it was found that 100 and 200 μg/ml of seahorse and herbal medicine mixed extract (HMT) could have an anticancer effect on prostate cancer by inhibiting the expression of PAK1 protein. In addition, it was found that 100 and 200㎍ / ㎖ of hippocampus and herbal medicine mixed extract (HMT) can induce apoptosis of prostate cancer cells (PC-3) by increasing the expression of c-cas3 protein (Fig. 3).

Example 4. Measurement of apoptosis of three-dimensional (3D) spheroid cells

The three-dimensional spheroid cancer cell model is used for effective drug screening in an environment similar to in vivo conditions. PC-3 cells were seeded at 2,000 cells per well in 96-well round bottom ultra-low attachment plates (Corning, USA), and 37 in RPMI medium containing 10% fetal bovine serum (FBS). Incubated at °C for 5 days to induce three-dimensional spheroid formation. Thereafter, 200 μg/ml of hippocampus and herbal medicine mixed extract (HMT) was treated for 48 hours, and 2 μM of CellEvent (Invitrogen) was put into each well for apoptosis analysis and reacted for 1 hour, then using a fluorescence microscope. Images were acquired and the fluorescence intensity was calculated using the ImageJ program.

As a result, it was confirmed that the hippocampus and herbal medicine mixed extract (HMT) induces apoptosis of three-dimensional spheroid prostate cancer cells (PC-3) (FIG. 4).

Claims (4)

A health functional food composition for preventing or improving prostate cancer, which contains a mixed extract of seahorse, rhubarb, silkworm, dermis, angelica, mulberry and green onion as an active ingredient. According to claim 1, wherein the mixed extract is based on 100 parts by weight of the seahorse, 80 to 120 parts by weight of rhubarb, 80 to 120 parts by weight of silkworm, 80 to 120 parts by weight of dermis, 80 to 120 parts by weight of Angelica, 40 to 60 parts by weight A health functional food composition for preventing or improving prostate cancer, characterized in that it contains a mixture of Mokhyang and 40 to 60 parts by weight of onion. The health functional food composition for preventing or improving prostate cancer according to claim 1, wherein the solvent of the extract is C ₁ to C ₄ lower alcohol, water, or a mixture thereof. A pharmaceutical composition for preventing or treating prostate cancer, comprising a mixed extract of seahorse, rhubarb, silkworm, dermis, angelica, mokhyang and green onion as an active ingredient.

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