JP3479224B2 - Preventive or therapeutic agent for postmenopausal osteoporosis containing herbal plant as active ingredient - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a prophylactic or therapeutic agent for osteoporosis, and more particularly to a prophylactic or therapeutic agent for postmenopausal osteoporosis containing an herbal plant component and optionally calcium.

[0002]

2. Description of the Related Art Osteoporosis is a systemic bone disease that causes a decrease in bone mass due to a decrease in calcium and collagen that form bone (mainly cancellous bone) and a regression of the fine structure of bone tissue. It is characterized by the risk of fracture. With the increase in the elderly population in recent years, this disease has been increasing year by year, and it has become an important disease in modern society like lifestyle-related diseases represented by diabetes. Also, due to osteoporosis, fractures of the lumbar spine and femur,
When the bedridden state is prolonged, there is a risk of causing dementia, so prevention and early treatment are important issues.

Postmenopausal osteoporosis, which is classified as primary osteoporosis, frequently occurs in postmenopausal women over the age of 50 and accounts for 90% or more of the number of patients with osteoporosis. It has become one of the intractable diseases for which the development of is desired. Postmenopausal osteoporosis is caused by a lack of endogenous estrogen secretion from the ovaries due to the abolition of the birth function, but some women do not develop osteoporosis despite the onset of menopause in all women. There are many unclear points, and the whole picture of the onset mechanism has not been clarified.

[0004] For the treatment of this disease, diet therapy, exercise therapy and supplementation of calcium deficiency with calcium preparations are carried out, and hormone replacement therapy with oral or transdermal estrogen preparations is adopted to achieve therapeutic effects. I am raising. In addition, calcitonin (CT) and parasolmon (PTH), which are bone metabolism regulating hormone preparations, vitamin K ₂ and active vitamin D ₃ , which are vitamin preparations, and more recently, bisphospho, which has a strong bone resorption inhibitory effect. Nate compounds and the like are also used depending on the progress of symptoms.

However, the higher the efficacy of these bone metabolism improving agents used clinically, the narrower the difference between the safe range and the toxic range, and the more difficult it is to control the dose. In particular, it is said that bisphosphonate preparations are difficult to set a protocol for drug administration / discontinuation, and at times, they may cause exacerbation of symptoms by suppressing calcification of bone constituents important for bone formation.

[0006] Estrogen preparations that are commonly used for the treatment of postmenopausal osteoporosis are no exception in terms of the possibility of causing side effects. During long-term administration for 6 months or more, flushing of the face, breast pain, uterus and vagina Frequent side effects such as irregular genital bleeding. Furthermore, if a luteinizing hormone preparation (progesterone) is not used in combination, there is a risk of developing uterine body cancer and breast cancer, and it has been pointed out that there is a problem in terms of safety.

On the other hand, among the components contained in soybean in recent years, isoflavones are basic skeletons such as isoflavone glycosides daidzin and genistin, and their aglycones daidzein and genistein. The estrogen activity was found in the above ingredients, and the usefulness as a food-derived safe estrogen against postmenopausal osteoporosis has been attracting public attention.

[0008]

SUMMARY OF THE INVENTION The present invention is intended to solve the above-mentioned conventional problems, and its object is to:
Unlike synthetic hormone preparations, there are few side effects, the estrogen action is alleviated, and by simply ingesting this component together with calcium in the form of drinks and foods on a daily basis, it is effective for the prevention and treatment of postmenopausal osteoporosis. It is intended to provide a preventive or therapeutic agent for post-osteoporosis.

[0009]

[Means for Solving the Problems] Today, for the evaluation of estrogen activity, a test substance is administered to female immature rats to measure an increase in uterine weight, and an assay method using the proliferation of estrogen-sensitive cells as an index. Is used. Cell lines MCF-7, T-47D and ZR established from human breast cancer tissue
-75-1 showed proliferative properties depending on estrogen concentration in in vitro culture system. Among them, MCF-7 [Soule
HD, Vazquez J., Long A., Albert S., Brennan M.,
J. Natl. Cancer. Inst., 51, 1409 (1973)] is highly sensitive to 17β-estradiol, which has high bioactivity among estrogen synthesized in vivo,
Estrogen agonists and anti-cancer drugs for the treatment of breast cancer, as well as endocrine disruptors that are being recognized as a social problem as artificially produced environmental pollutants that cause abnormalities in the ecosystem and human reproductive function It is also widely used for evaluation of (environmental estrogen).

The present inventors searched for a material (phytoestrogen) exhibiting estrogenic activity from safe and natural products and herbs that are normally used as foods, using the proliferation activity of MCF-7 as an index. As a result, the genus Petroselinum, the genus Carthamus, the genus Rubus, the genus Agrimonia, the genus Verbena, and the melissa family Melissa We found estrogenic activity in extracts obtained from herb plants of the genus.

That is, the present invention relates to the genus Petroselinum of the family Apiaceae, Carthamu of the family Asteraceae.
s) genus, Verbena genus Verbena genus, and Melissa genus Melissa genus of herbs selected from at least one raw or dried plant, and an extract obtained from these as an active ingredient. The present invention achieves the above object by providing a preparation effective for the prevention or treatment of postmenopausal osteoporosis in which calcium is added as necessary.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The plant which is a raw material of the present invention will be described.
In the genus m), native to Central and Northern Europe, so-called leaf-edible parsley (Petroselinum crispum MILL. NYMA)
N, Petroselinum Krispum Mill Niemann)
And its modified varieties of turnip root parsley (Petroselinum cri
spum MILL. NYMAN var. tuberosum CROVETTO, Petroselinum, Crispum, Mill, Niemann, Bar, Tuberosum, Clovette). These plants are edible as vegetables and tea, as well as raw or dried leaves, dried roots, seeds, and sometimes oil for diuresis, menstruation,
Used as a lactating, stomach and carminative.

Among plants belonging to the genus Carthamus of the Asteraceae family, safflower (Carthamus tinctorius) native to Egypt and cultivated in Akita Prefecture in China, India and Japan.
L., Curthamus Cinquetorius El) and the like. Pigments obtained from safflower flowers are used as colorants and dyes, and oils are used for cooking. It is also listed in the 13th revised Japanese Pharmacopoeia and is used as a gynecological drug to promote menstruation.

Among the plants belonging to the genus Rubus of the family Rosaceae, the European raspberry (Rubus idaeus L.), which is native to the Eurasian continent and is generically called Raspberry, and its subspecies, Rubus ulmi. Further improved varieties such as Rubus ulmifolius SCHOTT and Rubus ursinus CHAM & SCHLECHTEND are also available. While raspberries are widely used as a raw material for wine and liqueurs, confectionery, cooking, and cosmetics, for medicinal purposes, raw or dried leaves and fruits are used as astringent, labor-promoting and laxatives. There is.

Also, there are many Rubus genus in China, which are used as Chinese medicine. For example, Fuyu strawberry (Rubus buergeri MIQ)
The leaves and whole plants are called "whole-haired hanging hooks" and have a postpartum cold,
It is used for irregular menstruation, soreness in limbs, and paralysis.

In the genus Agrimonia of the family Rosaceae, Agrimony, which grows naturally in Asia, Europe, and North America, is also known as Agrimonia.
moniaeupatoria L., Agrimonia pilosa LEDEB, which is known as the Chinese medicine “Sentsurugusa” in China. Agrimony uses dried plants with flowers as astringents for hemostasis and menstrual suppression. In addition, Agrimony chinensis is used as an astringent drug as well as an anti-inflammatory tonic.

Among the plants of the genus Verbena of the genus Verbena, Verbena officinalis L., which is native to the Mediterranean region and widely distributed in Japan, China, Europe and North Africa, is mentioned. To be This plant is
Use the dried whole plant with flowers as a tonic, astringent, diuretic, sweating, milking, menstruation, wound and antispasmodic. Also, in China, it is called "Hymenochusococcus" and is used as a water reliever and a menstrual drug to promote menstruation and placenta detachment after delivery.

A plant belonging to the genus Melissa of the Labiatae family, which is native to Southern Europe and is widely distributed in temperate regions of the northern hemisphere, is called lemon balm, also known as yamaha mint.
Melissa officinalis L. (Melissa officinalis
Elle).

The leaves of this plant are used not only for flavoring foods and drinks by taking advantage of their unique lemon scent, but also for medicinal purposes such as carminative, sweating, antispasmodic and sedative. Furthermore, in China, Melissa axillaris BE
All plants of NETH BAKH.

As described above, the raw material plants of the present invention are already known as foods and folk medicines, but they have estrogen activity and are effective for the prevention and treatment of postmenopausal osteoporosis. There was no knowledge.

The herb plant of the present invention is a raw or dried product of the above-mentioned raw plant as it is or after crushed,
These are used alone or in combination of two or more. Also,
From plants of these raw material plants, hot water extraction, or lower alcohols such as ethanol or hydrous lower alcohols,
It also includes an extract obtained by organic solvent extraction represented by a lower ester such as ethyl acetate.

In addition to the calcium materials derived from animal bones, egg shells, seafood, whey and plants, calcium gluconate and carbonic acid, which are generally used as food additives, are used in the present invention. Calcium, calcium citrate, etc. can be used.

The herb plant of the present invention may be obtained by crushing raw or dried raw material plants as they are, but is prepared by using a solvent capable of effectively extracting the active ingredient from the above-mentioned plants. The extracted extract may be used.

The solvent used in this extraction is not particularly limited as long as it can effectively extract the active ingredient, and examples thereof include water, acetone, or a lower solvent such as methanol, ethanol or propanol. It is preferable to use an organic solvent miscible with water such as alcohol, a mixed solution thereof, a lower ester such as ethyl acetate, or the like.

In extracting the active ingredient, the raw materials can be directly extracted with these solvents, but in order to enhance the extraction efficiency, they are subjected to processing such as roasting, kneading, chopping, etc., and then with the solvent. It is desirable to extract. Further, the extraction efficiency can be increased by repeating the extraction 2 to 4 times.

A preferable extraction method is a method using 5 to 100 ml, particularly 10 to 20 ml of solvent per 1 g of shredded raw material. When an organic solvent is used as the extraction solvent, it is carried out at room temperature for 1 day to 1 month, preferably 2 to 5 days, or when water, methanol, ethanol or a mixed solvent thereof is used as the extraction solvent, Heating is desirable in order to improve extraction efficiency.

The extract contained in the present invention may be obtained from a single plant, but it is also possible to apply the above extraction method after mixing two or more plants. is there.

Raw or dried raw materials selected from raw material plants, or crushed raw materials, alone or in combination of two or more kinds, and an extract obtained from them as an active ingredient, calcium is added to this. In order to produce a preventive or therapeutic agent for postmenopausal osteoporosis, a pulverized product,
Alternatively, the extract obtained as described above may be used as an active ingredient and combined with a known food or pharmaceutical carrier according to a conventional method to prepare a formulation.

The prophylactic or therapeutic agent for postmenopausal osteoporosis of the present invention can be administered in various dosage forms. For example, as an oral administration agent, tea agents, capsules, tablets, granules, fine granules, Examples include syrups and dry syrups.

The amount of herb used as an active ingredient of the prophylactic or therapeutic agent for postmenopausal osteoporosis of the present invention is usually 1 to 100 g, preferably 3 to 30 g per adult per day as raw material dry matter.
May be a dosage unit that can be orally administered in 1 to 3 divided doses. In addition, the amount of the calcium material to be mixed is 600 to 1200 mg per day for an adult, and in the case of food additives, it may be used within the usage permission range. These doses can be appropriately increased or decreased depending on age, symptoms and the like.

It is also possible to add the plant extract obtained as described above to ordinary foods and drinks. The food and drink thus obtained can be ingested on a daily basis, so that it can be expected to have a preventive effect on postmenopausal osteoporosis and is useful as a health food.

The amount of the plant extract to be added to such food or drink may be added in a range that does not impair the original taste of the food, depending on the type of the food.
It may be added in the range of 0.01 to 10% by weight.

The herb plant used in the preventive or therapeutic agent for postmenopausal osteoporosis and the food and drink of the present invention has already been used as a food or a folk medicine as described above, and is a particular problem in safety. There is no.

Next, the method for evaluating the estrogen activity performed on the herb plant of the present invention will be described.

The estrogen activity test using MCF-7 cells is a method generally used for evaluation of pharmaceuticals and environmental pollutants as described above, and the outline thereof is as follows.

2 × 10 ⁴ was added to Dulbecco's modified Eagle medium (D-MEM) containing 5% of fetal calf serum (DCC-FCS) from which endogenous estrogen was removed with dextran / activated carbon.
100 μl of MCF-7 cells prepared so that the number of cells / ml will be seeded on a 96-well plastic culture plate. After culturing for 24 hours, the whole amount of the medium is exchanged, and after dissolving in dimethylsulfoxide (DMSO), 10 μl each of the test substance diluted with the medium is added, and the culturing is continued for another 4 days. After culturing, MC
Degree of proliferation of F-7 cells by MTT assay [Carmichael J., DeGraff
Wg, Gazdar AF, Minna JD, Mitchell JB,
Cancer Res., 47, 936 (1987).] Was used to determine the presence or absence of estrogenic activity by comparing the change in absorbance at wavelength 562 nm with cell proliferation with the group to which no test substance was added. is there.

In this test, the extract obtained from the herb plant of the present invention significantly promotes the growth of MCF-7. From this, it is clarified that the herb plant of the present invention exhibits estrogen activity, and is proved to be useful as a preventive or therapeutic agent for postmenopausal osteoporosis.

[0038]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples.

Example 1 Preparation of Parsley Extract 100 g of dried above-ground parsley from the United States was cut into small pieces, 2 liters of methanol was added, and extraction was carried out for 2 hours while heating to 70 ° C. After filtration, 2 liters of methanol was added to the residue again, the mixture was further extracted for 2 hours, and the filtrates obtained by the extraction twice were collected, concentrated under reduced pressure and freeze-dried to obtain an extract.
The yield of the obtained methanol extract was 32.4%.

Example 2 Preparation of safflower extract To 100 g of dried Chinese safflower flowers, 2 liters of methanol was added, and extraction was carried out for 2 hours while heating at 70 ° C. After filtration, add 2 liters of methanol again to the residue,
After a further 2 hours of extraction, the filtrates obtained from the two extractions are collected,
After concentration under reduced pressure and freeze-drying, an extract was obtained. The yield of the obtained methanol extract was 23.6%.

Example 3 Preparation of raspberry leaf extract 100 g of dried leaves of Hungarian raspberry were shredded, 2 liters of methanol was added, and extraction was carried out for 2 hours while heating at 70 ° C. After filtration, 2 liters of methanol was added to the residue again, the mixture was further extracted for 2 hours, and the filtrates obtained by the extraction twice were collected, concentrated under reduced pressure and freeze-dried to obtain an extract. The yield of the obtained methanol extract was 17.6%.

Example 4 Preparation of agrimony extract 100 g of dried agrimonies from the USA were finely chopped,
While adding 2 liters of methanol and heating to 70 ℃,
Extraction was carried out for 2 hours. After filtration, the residue is again methanol 2
After adding liter and further extracting for 2 hours, the filtrates obtained by the extraction twice were collected, concentrated under reduced pressure and freeze-dried to obtain an extract. The yield of the obtained methanol extract was 17.4%.

Example 5 Preparation of Verbain Extract 100 g of the above-ground part of Bulgarian verbain was sliced into 2 pieces.
Add 2 liters of methanol and heat to 70 ℃.
Time extraction was performed. After filtration, 2 liters of methanol was added to the residue again, the mixture was further extracted for 2 hours, and the filtrates obtained by the extraction twice were collected, concentrated under reduced pressure and freeze-dried to obtain an extract. The yield of the obtained methanol extract was 12.8%.

Example 6 Preparation of Lemon Balm Extract 100 g of dried leaves of Bulgarian lemon balm were finely chopped into 2 pieces.
Add 2 liters of methanol and heat to 70 ℃.
Time extraction was performed. After filtration, 2 liters of methanol was added to the residue again, the mixture was further extracted for 2 hours, and the filtrates obtained by the extraction twice were collected, concentrated under reduced pressure and freeze-dried to obtain an extract. The yield of the obtained methanol extract was 17.6%.

Example 7 Evaluation of Estrogenic Activity of Herbal Plant Extract To evaluate estrogenic activity, inactivated fetal bovine serum (10
%, Bio Whittaker, insulin (10 μg / m
l, Sigma) and non-essential amino acids (0.1 mM, Lifetech) in D-MEM (containing phenol red, Lifetech) MCF-7 (Tohoku University aging) subcultured 10 to 20 times Used from a medical research institute).

The cells 3 to 4 days after the passage were recovered using a phosphate buffered saline (PBS) treatment containing 0.02% trypsin (Difco) and 0.05% EDTA, and 5% DCC-FCS.
Containing D-MEM (Phenol Red-free, Life Tech Co., Ltd.)
And wash 3 times by centrifugation (× 80g), then 2 × 1
The concentration was adjusted to 0 ⁴ cells / ml.

100 μl of this was inoculated on a 96-well plastic culture plate (Corning Co.) and cultured at 37 ° C. in the presence of 5% CO ₂ for 24 hours. . To this, 10 μl of a test substance dissolved in DMSO and diluted with medium (DMSO diluted with medium for the control group) was added by 10 μl and stirred, and then the culture was continued for another 4 days. The final concentration of DMSO was unified to 0.1%.

After completion of the culture, 10 μl of MTT dissolved in PBS at 5 mg / ml was added to each well and incubated at 37 ° C. for 4 hours. After removing the medium by suction, 200 μl of mineral oil (Nacalai Tesque, Inc.) was added to each well, and the generated formazan was sonicated (Microson ^™ ,
Crush using "Hart Systems" and centrifuge (x15)
00g), 100 μl of the supernatant was transferred to another assay plate, and the absorbance at a wavelength of 562 nm was 660 nm using a microplate reader (model EL-340, Biotech).
Was measured as a reference wavelength.

The strength of the estrogen activity of the test substance was indicated by the absorbance in the control group, that is, the relative value when the proliferation of MCF-7 was taken as 100. The Dunnett's multiple comparison test was used for the significance test, and the risk rate p <0.05 or less was considered significant.

The results are shown in Table 1. As the extract, a methanol extract was used except for the soybean isoflavone glycoside fraction. The results (proliferation of MCF-7) were expressed as a relative value (%) with respect to the control group, with the cell growth rate of the control group being 100. An asterisk (*) represents a significant difference from the control group, * for p <0.05 and ** for p <0.01.

[0051]

[Table 1]

The herbal plant extract of the present invention significantly enhanced the growth of MCF-7. This result suggests that the herb plant of the present invention has estrogen activity and is effective in preventing or treating postmenopausal osteoporosis.

Example 8 Preparation of tea preparation

[0054]

[Table 2]

Dried products of each herb plant shown in Table 2 were
After shredding into 10 mm squares, add calcium lactate and mix well,
Filled the teabag.

Example 9 Preparation of drink

[0057]

[Table 3]

According to the formulation shown in Table 3, the components were distilled water 800 m.
Dissolve in 1 and add distilled water to make 1000ml, then 0.22m
Sterilized with a m. sterilization filter and aseptically filled in 100 ml amber bottles, and 200 mg of the extract of Example 1 and 6 per agent
A drink containing 00 mg of calcium was obtained.

[0059]

EFFECTS OF THE INVENTION It was confirmed that the herbal plants and their extracts, which are the active ingredients of the present invention, contain ingredients having estrogenic activity. Since these herbal plants have been traditionally used as foods and folk medicines for the treatment of gynecological diseases since ancient times, unlike estrogen preparations used clinically, they have few side effects and are extremely safe. Furthermore, by adding calcium to this, it is effective for the prevention and treatment of postmenopausal osteoporosis.

Therefore, the therapeutic agent for postmenopausal osteoporosis of the present invention is
It is useful for preventing or treating postmenopausal osteoporosis.
In addition, by adding the extract of the present invention to food and drink and ingesting it daily, it is also useful as a food for health and the like for preventing postmenopausal osteoporosis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI A61K 35/78 A61K 35/78 W 33/06 33/06 A61P 19/10 A61P 19/10 43/00 43/00 // A23L 1/30 A23L 1/30 B 2/38 2/38 C 2/52 2/00 F (58) Fields investigated (Int.Cl. ⁷ , DB name) A61K 35/78 A61K 33/06

Claims (2)

(57) [Claims] 1. Petroselinum (Petroselinum)
Sp., Asteraceae Kasamusu (Carthamus) genus, click Matsudzura Department of Verbena (Verbena) genus, and Labiatae Melissa (Mel
A prophylactic or therapeutic agent for postmenopausal osteoporosis, which comprises a raw or dried product of at least one plant selected from the herbs of the genus issa) or an extract obtained therefrom as an active ingredient. 2. The preventive or therapeutic agent for postmenopausal osteoporosis according to claim 1, further comprising calcium.