JP7630794B2 - Tumor promotion inhibitors - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Announced in the online abstracts of the 140th Annual Meeting of the Pharmaceutical Society of Japan on March 5, 2020

The present invention relates to a tumor promotion inhibitor.

The number of cancer patients is increasing year by year, and cancer is now the leading cause of death among Japanese people. In 2017, approximately 860,000 new cases of cancer were diagnosed, and approximately 370,000 cases of cancer death occurred. Cancer is treated by chemotherapy, surgical procedures, radiation therapy, etc., but the therapeutic effects are not always satisfactory, and this can lead to a decrease in the quality of life of patients. In addition, it is said that recurrence of cancer after treatment cannot be completely prevented. Therefore, as with other diseases, a preventive medical perspective is required, and methods of preventing carcinogenesis, including cancer recurrence, are attracting attention.

Generally, cancer prevention methods include lifestyle improvements such as quitting smoking and reviewing diet, as well as environmentally friendly measures such as dealing with stress, but as a more proactive measure, the development of substances that are effective in cancer prevention is desired. Cancer is believed to begin when the genes of normal cells are damaged (initiation), and then the characteristics of the cells change and unregulated proliferation is added (promotion). It is believed that inhibiting these processes will lead to cancer prevention.

Therefore, various preventive agents that inhibit or suppress the initiation and/or promotion activity of carcinogens have been proposed (for example, Patent Documents 1 to 3). Patent Documents 1 to 3 contain, as active ingredients, Acanthus arguta fruit juice or a highly polar organic solvent extract of Acanthus arguta fruit juice, a polymer composition derived from tea leaves, or a compound extracted from red yeast rice.

Recently, the Bhas 42 cell transformation test has been published as a test method capable of detecting substances that affect carcinogenesis promotion in vitro with high accuracy (e.g., Non-Patent Document 1). The Bhas 42 cells used in this test are a cell line established by introducing the v-Ha-ras oncogene into BALB/c 3T3 cells (whole mouse fetuses).

JP 2013-136556 A JP 2013-95694 A JP 2008-56618 A

GUIDANCE DOCUMENT ON THE IN VITRO BHAS 42 CELL TRANSFORMATION ASSAY, Series on Testing & Assessment No. 231 (2016)

The objective of the present invention is to provide a novel tumor promotion inhibitor.

In order to achieve the above object, the present inventors conducted extensive research and discovered that Enzigzag has the effect of suppressing carcinogenic promotion. In other words, the present invention is a carcinogenic promotion inhibitor that contains Enzigzag as an active ingredient.

The present invention provides a novel tumor promotion inhibitor.

FIG. 13 shows the effect of a herbal medicine preparation (Noukatsusei) on the increase in focus number caused by the addition of a tumor promoter (TPA). FIG. 1 shows the effect of Enshi on the increase in focus number caused by the addition of a tumor promoter (TPA).

The cancer promotion inhibitor of the present invention contains Polygalae Radix as an active ingredient. Polygalae Radix is the root or root bark of Polygala tenuifolia Willdenow (Polygalaceae) (17th Revised Japanese Pharmacopoeia). Representative pharmacological effects of Polygala have been known to be expectorant, antitussive, sedative, and memory and learning improvement. The present inventors have found that Polygalae Radix has an effect of suppressing cancer promotion, specifically, an effect of suppressing the change in the characteristics of tumor cells caused by tumor promoters and the unregulated proliferation of tumor cells with the changed characteristics.

As the polysaccharide, for example, a commercially available product, Onji dry extract (Alps Yakuhin Co., Ltd.), can be used. Alternatively, a dry extract prepared by a conventional method from the roots of Polygala tenuifolia may be used. The dry extract can be prepared by obtaining an extract from the roots of Polygala tenuifolia using a known method, concentrating, sterilizing, and drying the extract. Examples of the dry extract include a water-based dry extract and an ethanol-based extract.

The carcinogenesis promotion inhibitor of the present invention may contain saffron or red ginseng as an active ingredient in addition to Crocus sativus Linne. Saffron is the stigma and upper part of the style of Crocus sativus Linne, and its representative pharmacological actions have been known to be sedative, analgesic, and menstrual. Red ginseng is the root of Panax ginseng C.A. Meyer, which is steamed and then dried, and its representative pharmacological actions have been known to be nourishing and strengthening, improving blood flow, lowering blood sugar, and anti-stress. When such ingredients are contained, the carcinogenesis promotion inhibitory action is further enhanced.

Alternatively, the carcinogenesis promotion inhibitor of the present invention may contain both saffron and red ginseng in addition to Radix Candida. Radix Candida, saffron and red ginseng are contained in a herbal medicine preparation (Noukatsusei) together with Antelope horn and Agarwood, etc. This herbal medicine preparation is commercially available as an OTC drug, and has the efficacy of nutritional tonic (weak constitution, physical fatigue, during and after illness, gastrointestinal weakness, loss of appetite, poor complexion, poor circulation, and growth period).
The present inventors have discovered that a herbal medicine preparation (Noukatsusei) containing Solanum Root, Saffron and Red Ginseng has the effect of suppressing carcinogenesis promotion.

In the carcinogenesis promotion inhibitor of the present invention, Cornus officinalis, Saffron, and Red Ginseng can be used as raw herbal medicines, but it is preferable to use them as extracts extracted from the raw herbal medicines or as pulverized materials (powder) obtained by pulverizing the raw herbal medicines. For example, a dried water extract of Red Ginseng can be used. The methods of extraction and pulverization are not particularly limited as long as they are methods used in the manufacture of pharmaceuticals and foods, and any method can be used.

For example, each pulverized raw material can be extracted with hot water in an amount 5 to 30 times, preferably 10 to 20 times, its mass, and the resulting extract can be concentrated and dried for use. The raw materials can be pulverized using known means such as an impact mill or jet mill. Also, known means such as reduced pressure evaporation, spray drying, and freeze drying can be used for concentration and drying.

The carcinogenesis promotion inhibitor of the present invention can be prepared as a preparation containing, for example, 0.03 to 30 mg of dried onji extract, 0.1 to 100 mg of saffron powder, and 0.3 to 300 mg of dried red ginseng extract as active ingredients per kg of human body weight per day. In this case, ingredients other than the active ingredients can include 0.1 to 100 mg of antelope horn powder and 0.01 to 10 mg of agarwood. Furthermore, additives such as pearl, corn starch, and fragrance (ryunou) can be included. These ingredients can also be encapsulated in gelatin.

The carcinogenesis promotion inhibitor of the present invention can be prepared by a method known to those skilled in the art together with suitable excipients, etc., depending on the administration method. Examples of administration methods include oral administration, topical administration, rectal administration (suppository), and parenteral administration such as eye drops. Among these, oral administration and topical administration are preferred. When administered orally, the above-mentioned daily amount can be administered all at once or in several divided doses. Administration may be performed before, after, or between meals, and the administration period is not particularly limited.

The carcinogenesis promotion inhibitor of the present invention can be in any dosage form for oral or parenteral administration. Examples include granules, fine granules, tablets, powders, capsules, chewable agents, liquids, and suspensions. These can be formulated by conventional methods. Examples of forms for injection include direct intravenous injection and drip administration. If necessary, carriers such as lactose, glucose, D-mannitol, starch, crystalline cellulose, calcium carbonate, kaolin, and gelatin, and conventional additives such as solvents, solubilizers, and isotonicity agents may be appropriately blended.

The topical preparation containing the carcinogenesis promotion inhibitor of the present invention is of a transdermal absorption type. If necessary, known additives and the like can be mixed to prepare a topical preparation by a conventional method. Specific examples of topical preparations include creams, liquids, lotions, emulsions, tinctures, ointments, aqueous gels, oily gels, aerosols, powders, shampoos, and soaps. The daily dosage of such topical preparations can be appropriately set depending on the symptoms, purpose, age, and method of administration of the subject to be administered.

The cancer promotion inhibitor of the present invention can be contained in food. Food containing the cancer promotion inhibitor of the present invention can take the form of a general food or drink. For example, it can be made into a drinkable agent, such as a soft drink or powdered drink, either by itself or by adding an appropriate flavor to it. Specifically, it can be added in an appropriate amount to juice, milk, confectionery, jelly, etc. and consumed.

Foods containing the carcinogenic promotion inhibitor of the present invention can be provided as health functional foods, such as foods for specified health uses or foods with nutrient functions, for the prevention of cancer.

Furthermore, the food containing the carcinogenesis promotion inhibitor of the present invention can be used as a concentrated liquid diet or a food supplement. When used as a food supplement, it can be prepared in the form of, for example, tablets, capsules, powders, granules, suspensions, chewable agents, syrups, etc. Food supplements refer to those that are taken for the purpose of supplementing nutrition other than those taken as food, and include nutritional supplements and dietary supplements.
Foods can be processed and produced by mixing the tumor promotion inhibitor of the present invention with general food ingredients in a conventional manner.

When the cancer promotion inhibitor of the present invention is contained in food, an appropriate amount should be ingested so that a predetermined amount of the cancer promotion inhibitor is ingested. The amount of food to be ingested can be appropriately selected depending on the content of the cancer promotion inhibitor in the food.

Examples of nutritional supplements or functional foods include processed forms such as liquid foods, semi-digested nutritional foods, elemental nutritional foods, drinks, capsules, and enteral nutrients that contain sugars, fats, trace elements, vitamins, emulsifiers, and flavors. The above-mentioned various foods, such as sports drinks and nutritional drinks, can also contain nutritional additives such as amino acids, vitamins, and minerals, as well as sweeteners, spices, flavors, and colorings, in order to improve nutritional balance and flavor. Furthermore, tocopherol, L-ascorbic acid, BHA, rosemary extract, and the like can be used in the usual manner as antioxidants that are usually added to foods.

By adding the carcinogenesis promotion inhibitor of the present invention to the feed of livestock, poultry, and pets, it is possible to prevent cancer in these animals. In terms of form, it can be added to dry dog food, dry cat food, wet dog food, wet cat food, semi-moist dog food, chicken feed, and feed for livestock such as cows and pigs.

The carcinogenesis promotion inhibitor of the present invention can also be used appropriately in animals other than humans, for example, livestock mammals such as cows, horses, pigs, and sheep, poultry such as chickens, quails, and ostriches, and pets such as reptiles, birds, and small mammals.

The inhibitory effect of the tumor promotion inhibitor of the present invention on tumor promotion is an inhibitory effect on transformation, and can be confirmed with reference to the Bhas 42 cell transformation test method.

The carcinogenesis promotion inhibitors according to the present invention are specifically described below, but are not intended to limit the present invention.

(Test Drug 1)
As test drug 1, a herbal preparation (Noukatsusei) (0.313 mg/mL, 1.25 mg/mL, 5 mg/mL) was prepared. The herbal preparation (Noukatsusei) was subjected to ultrasonic extraction with 10% dimethyl sulfoxide for 30 minutes, and the supernatant obtained after centrifugation (1000 rpm for 3 minutes) was sterilized by filtration. The final solvent concentration was 1%.

The herbal medicine preparation (Nong Kao Ji) contains 30 mg of dried extract of Enzhi (a kind of herb) (four capsules) per day. 90 mg of powdered saffron, 330 mg of dried extract of red ginseng, 100 mg of powdered antelope horn, and 10 mg of agarwood. Additionally, it contains pearl, corn starch, and a fragrance (Ryuno), and the mixture containing these is encapsulated in gelatin.

(Test Drug 2)
As test drug 2, Enji (0.00390 mg/mL, 0.0130 mg/mL, 0.0390 mg/mL, 0.130 mg/mL) was prepared. Enji was prepared by redissolving dried extract of Enji (manufactured by Alps Pharmaceutical Co., Ltd.) in 10% dimethyl sulfoxide and sterilizing by filtration. The final solvent concentration was 1%.

[Cancer promotion inhibition test]
The tumor promotion inhibitory effect of the test drug was evaluated using a tumor promoter, with reference to the Bhas 42 cell transformation test method (Non-Patent Document 1). First, Bhas 42 cells (1.4 x ¹⁰⁴ cells) were seeded on a 35 mm dish and culture was started (day 0). After the start of culture, on days 4, 7, and 11, the medium was replaced with 10% FBS-containing DMEM:F12 medium containing the test drug at a specified concentration.

In addition to the prescribed concentrations of the test drugs, 50 ng/mL of 12-O-Tetradecanoylphorbol 13-acetate (TPA) was added to the 10% FBS-containing DMEM:F12 medium, which is well known to be a potent tumor promoter and to activate signaling pathways downstream of protein kinase C (PKC).
After changing the medium on day 14, the cells were cultured until day 21 and then fixed with methanol. The cells were stained with 5% Giemsa stain and observed under a microscope to count foci (transformation foci) to evaluate the inhibitory effect of the test drug on the transformation promotion by TPA.

(Example 1: Carcinogenesis promotion inhibitor containing herbal medicine preparation (Noukatsusei))
Figure 1 shows the effect of herbal medicines on tumor promotion in Bhas 42 cells. In Figure 1, data are shown as mean ± standard error (mean ± SE). A multiple comparison test (Dunnett's method) was used to analyze significant differences from the control group without the addition of herbal medicines. ** indicates p<0.01.
From the results in Figure 1, it was confirmed that the herbal preparation (Nokatsusei) at 0.313 mg/mL or more suppressed the increase in the number of foci caused by the addition of TPA, and in particular, the herbal preparation (Nokatsusei) at 1.25 mg/mL or more significantly suppressed the increase in the number of foci. In addition, when the herbal preparation (Nokatsusei) was contained at 5 mg/mL, the number of foci was reduced compared to the normal group. This suggests that the effect of the herbal preparation (Nokatsusei) on transformation in Bhas 42 cells is not a specific inhibition of the promoting effect of TPA, and furthermore, that this herbal preparation has the effect of widely suppressing the expression of transformation.

(Example 2: Tumor promotion inhibitor containing Enshi)
Figure 2 shows the effect of E. coli on tumor promotion in Bhas 42 cells. In Figure 2, data are shown as mean ± standard error (mean ± SE). A multiple comparison test (Dunnett's method) was used to analyze significant differences from the control group without E. coli. ** indicates p<0.01.
From the results in Figure 2, it was confirmed that 0.00390 mg/mL or more of schizophyllum suppressed the increase in the number of foci caused by the addition of TPA, and in particular, 0.0130 mg/mL or more of schizophyllum significantly suppressed the increase in the number of foci caused by the addition of TPA. Furthermore, when 0.0390 mg/mL or more of schizophyllum was contained, the number of foci was reduced compared to the normal group. This suggests that the effect of schizophyllum on the transformation of Bhas 42 cells is not a specific inhibition of the promoting effect of TPA, and furthermore, that schizophyllum has a broad effect of suppressing the expression of transformation.

Claims (1)

A carcinogenic promotion inhibitor containing saffron , saffron, and red ginseng as active ingredients.

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