KR102558650B1 - 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 mixed extract of hippocampus and herbal medicine as an active ingredient, and more particularly, since the mixed extract of the present invention has an excellent effect of inhibiting the growth, migration, and invasion of prostate cancer cells and inducing apoptosis, it can be usefully used as a composition for preventing, improving, or treating prostate cancer containing the same as an active ingredient.

Description

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 containing a mixed extract of hippocampus and herbal medicine as an active ingredient.

Prostate cancer is a malignant tumor that occurs in the male prostate. Most of the gland cells in the prostate become cancerous and metastasize to lymph nodes and bones. About 90% of prostate cancer is proliferated by male hormones produced in the body. In the West, prostate cancer is the most common cancer among men and shows a high incidence, and in Korea, the frequency of prostate cancer is rapidly increasing due to the influence of Western eating habits and an increase in life expectancy. In the case of prostate cancer, unlike the past when patients in their 70s were the majority, these days, the number of patients undergoing prostate cancer surgery in their 40s is rapidly increasing. 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 inhibits testosterone secretion or blocks hormone production through surgery because a significant portion of prostate cancer cells proliferate in an androgen-dependent manner. In more than 80% of patients, this method shows temporary therapeutic effects such as suppression of cancer growth or shrinking of lesions. However, even patients who respond well to temporary hormone therapy can progress to hormone-refractory prostate cancer (HRPC) after a certain period of time, and such 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.

Seahorse is a protected fish whose international trade is restricted, but consumption is steady in Southeast Asia, mainly in China, and seahorses are cultivated artificially in Australia, neighboring countries, and China. Seahorses produced by aquaculture are helping to restore the biological resources of seahorses that are gradually becoming extinct. In Korea, the Big Valley seahorse ( Hippocampus abdominalis ) is mainly farmed. According to China's representative pharmacological book, Herbal Medicine, it is described that when seahorse is used as medicine, it boosts yang and treats diseases. Thus, dried seahorses have been used medicinally in traditional Chinese medicine and are still used to treat a wide range of diseases, including asthma, heart disease and bone fractures.

On the other hand, Korea Patent Patent No. 2012-0020640 discloses 'Prostate Cancer Treatment Composition' including Mok-hyang, and Korean Patental Patent No. 2019-0065647 discloses 'prevention and treatment of kidney diseases containing the maritime extract as an active ingredient' and a health functional food composition. The composition for preventing, improvement or treatment of prostate cancer contained as an active ingredient.

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

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 hippocampus, rhubarb, cow, dermis, angelica, chamomile and soybean as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating prostate cancer containing a mixed extract of hippocampus, rhubarb, cow, dermis, angelica, chamomile and soybean as an active ingredient.

The present invention relates to a composition for preventing, improving, or treating prostate cancer containing a mixed extract of hippocampus and herbal medicine as an active ingredient, and since the active ingredient has excellent effects of inhibiting growth, migration, and invasion of prostate cancer cells and inducing cell death, it can be usefully used as a health functional food for preventing or improving prostate cancer.

1 is a result of confirming the cell growth inhibitory ability of 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 mixed extract (HMT) and culturing for 24 hours, and B is a result showing cell growth rate through absorbance measurement. *** indicates a statistically significant decrease in the cell growth rate of the hippocampus and herbal medicine mixed extract (HMT)-treated group compared to the control group that was not treated with anything, p<0.001.
Figure 2 is a result of confirming the cell migration (A) and invasion (B) inhibition ability of the hippocampus and herbal medicinal mixture extract (HMT) in prostate cancer cells (PC-3). ### indicates a statistically significant increase in cell migration and invasion in the serum-containing medium-treated group compared to the serum-free medium-treated group (FreeM), p<0.001, * indicates a statistically significant decrease in cell migration in the hippocampus and herbal medicine mixed extract (HMT) treated group at 50 μg/ml compared to the FBS (fetal bovine serum) alone treated group, p<0.05, ** is FBS (fetal bovine serum) alone Compared to the treatment group, the cell invasion of the hippocampus and herbal medicine mixed extract (HMT) treatment group at 50 μg/ml was statistically significantly reduced, p<0.01.
Figure 3 is a result of 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 protein expression level in the case of no treatment was set as 1, and the expression level of each protein was shown.
Figure 4 is a result of measuring the death of three-dimensional spheroid prostate cancer cells (PC-3) by hippocampus and herbal medicine mixture extract (HMT). A was observed under an optical microscope and a fluorescence microscope, and green fluorescence represents apoptotic cells. B represents the fluorescence intensity of apoptotic cells, * indicates a statistically significant increase in apoptotic cells in the hippocampus and herbal medicine mixed extract (HMT)-treated group compared to the untreated control group, 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 containing a mixed extract of hippocampus, rhubarb, cowhide, dermis, angelica, chamomile and soybean as an active ingredient.

The breed of the hippocampus is preferably the 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 may preferably be a mixture of 80 to 120 parts by weight of rhubarb, 80 to 120 parts by weight of cow, 80 to 120 parts by weight of dermis, 80 to 120 parts by weight of Angelica gigas, 40 to 60 parts by weight of saffron, and 40 to 60 parts by weight of leek, more preferably 100 parts by weight of hippocampus, based on 100 parts by weight of hippocampus. It may be a mixture of 90 to 110 parts by weight of rhubarb, 90 to 110 parts by weight of cowhide, 90 to 110 parts by weight of dermis, 90 to 110 parts by weight of Angelica quail, 45 to 55 parts by weight of chamomile, and 45 to 55 parts by weight of green onion, It may be a mixture of 100 parts by weight of Angelica quail, 50 parts by weight of Mokhyang, and 50 parts by weight of green beans, but is not limited thereto.

The mixed extract of the present invention is preferably extracted using C ₁ ~ 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 the effect of inducing proliferation, migration, invasion inhibitory effect and cell death of prostate cancer cells.

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

When using the health functional food composition of the present invention 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 conventional methods. Active ingredients can be appropriately used depending on their purpose of use (prevention or improvement). In general, when preparing 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 health functional food. Examples of foods to which the functional food composition may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages and vitamin complexes, and the like, including all health foods in the conventional sense.

In addition, the health functional food composition of the present invention can be made into food, particularly functional food. The functional food of the present invention may contain ingredients that are commonly added. Examples include proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared as a drink, natural carbohydrates or flavors may be included as additional ingredients in addition to active ingredients. The natural carbohydrate is preferably a monosaccharide (eg, glucose, fructose, etc.), a disaccharide (eg, maltose, sucrose, etc.), an oligosaccharide, a polysaccharide (eg, dextrin, cyclodextrin, etc.) or a sugar alcohol (eg, xylitol, sorbitol, erythritol, etc.). As the flavoring agent, natural flavoring agents (eg, thaumatin, 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, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. may be further contained. The ratio of the components to be added 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 hippocampus, rhubarb, cow, dermis, angelica, chamomile and soybean 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 and used in the form of oral formulations such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods.

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, methylhydroxybenzoate, propylhydroxybenzoate, various compounds or mixtures including talc, magnesium stearate and mineral oil, and the like.

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

A suitable dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and response sensitivity.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can 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 Preparation Examples and Examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

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

With respect to 100 parts by weight of seahorse, 100 parts by weight of rhubarb, 100 parts by weight of cow, 100 parts by weight of dermis, 100 parts by weight of Angelica gigas, 50 parts by weight of Mokhyang, and 50 parts by weight of soybean were mixed and pulverized with a mixer. Then, 10 times 50% (v / v) ethanol was added and extracted at room temperature for 72 hours. Thereafter, the mixture was filtered using a filter paper, concentrated in a vacuum evaporator, and lyophilized to prepare a mixed extract (HMT) of hippocampus and herbal medicine.

Example 1. Measurement of cell growth inhibition ability

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

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. Measurement of cell migration and invasion inhibition ability

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

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

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 of hippocampus and herbal extracts (HMT) diluted in RPMI-1640 medium without FBS were seeded into the upper chamber containing a Matrigel-coated filter, and RPMI-1640 medium without FBS was added to the lower chamber. After culturing for 24 hours, the cells were stained using Diff-Quick (Sysmax), and then the number of cells infiltrating into the lower chamber was measured through photographs taken under 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

The PAK1 gene is known to be overexpressed in cancer cells, and in particular, when the PAK1 gene is knocked down in prostate cancer cells, the expression of MMP9 is reduced, the expression of TGF-β is increased, and cell motility and growth rate are known to decrease. Therefore, the anticancer effect can be evaluated through analysis of PAK1 expression inhibitory ability. It is known that the caspase-3 gene induces apoptosis by being converted into 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 the hippocampus and herbal medicine mixed extract (HMT) inhibited the expression of PAK1 protein, thereby having an anticancer effect against prostate cancer. In addition, it was found that 100 and 200 μg/ml of hippocampus and herbal medicine mixture extract (HMT) could induce apoptosis of prostate cancer cells (PC-3) by increasing c-cas3 protein expression (FIG. 3).

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

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

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

Claims (4)

delete delete delete A pharmaceutical composition for preventing or treating prostate cancer, which contains, as an active ingredient, a mixed ethanol extract prepared by mixing 80 to 120 parts by weight of rhubarb, 80 to 120 parts by weight of cowpea, 80 to 120 parts by weight of dermis, 80 to 120 parts by weight of Angelica gigas, 40 to 60 parts by weight of chamomile, and 40 to 60 parts by weight of green soybean, with respect to 100 parts by weight of Big Valley seahorse, and inhibits proliferation and metastasis of prostate cancer. .