JPH06340542A - Bone disease-preventing and treating agent originated from medicinal plant - Google Patents

Abstract

PURPOSE:To obtain an absorptive bone disease-preventing and treating agent containing the extract of at least one plant selected from Berberidaceae Podophyllum plants, Umbelliferae Notopterygium plants and Thujopsis dolabrata as an active ingredient. CONSTITUTION:This bone disease-preventing and treating agent contains as an active ingredient an extract obtained by extracting at least one plant selected from Berberidaceae Podophyllum plants, Umbelliferae Notopterygium plants, and Thujopsis dolabrata with an organic solvent. The plant has widely been used as a galenical drug, is excellent in safety, prevents hypercalcemia, and inhibits a bone absorption action. The agent is useful as a preventing and treating agent for malignant hypercalcemia, bone Paget's disease, osteoporosis, etc., and administered in a dose of 1-100g/day for an adult, the dose being calculated in terms of the dry raw material. The agent is also useful as healthful food for preventing absorptive bone diseases, etc., when the agent is added to a vegetable or food and subsequently ingested daily.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a prophylactic / therapeutic agent for bone diseases, and more specifically, a plant of the genus Podfilm of the family Barberry,
Resorbable bone diseases such as malignant hypercalcemia, Paget's disease of bone, or osteoporosis containing as an active ingredient an extract obtained from at least one plant selected from plants of the genus Notopteridium or Asunaro of the cypress family Related to preventive and therapeutic agents for

[0002]

2. Description of the Related Art In recent years, so-called geriatric diseases have increased along with a rapid increase in the elderly population. Among them, bone diseases such as osteoporosis frequently cause bone fractures, and as diseases that lead to bedridden elderly people, development of preventive and therapeutic methods thereof is desired.

Once bone is formed, it is not a construct that does not change at all, and its structure and quantity are maintained on the balance between bone formation and bone resorption. Therefore, when the balance is lost due to aging or other causes, various bone diseases develop.

Among the bone diseases, malignant hypercalcemia caused by myeloma and lymphoma, Paget's disease of bone caused by local bone resorption, and the cause are unknown as diseases caused by abnormally increased bone resorption. However, osteoporosis and the like, in which bone mass decreases with age, can be mentioned.

Bone is composed mainly of collagen fibers which are organic and calcium salts which are inorganic, and these two are combined to form a strong structure which is strong against both tension and pressure. In particular, calcium salts account for 70% of total bone weight, but in bone-resorbing bone diseases such as osteoporosis,
With its progress, calcium salt is eluted from the bone into the blood with its progress, and as a result, calcium salt is gradually lost from the bone.

Up to now, a method for maintaining calcium has been adopted for the prevention of such diseases, and a method for supplementing calcium has been adopted for the therapy, and active vitamin D ₃ preparations and calcium preparations have been used. In addition, hormone agents such as estrogen preparations and calcitonin preparations have been used for the purpose of suppressing demineralization from bone.

[0007]

However, although the effects of these drugs are found to have some effects in reducing pain, preventing the progress of medical conditions, etc., the effects are not satisfactory, and are more reliable. There is a need for a medicinal component that exhibits various effects. Furthermore, when the component is routinely ingested for the purpose of preventing absorbable bone disease, oral intake is preferable, and it is particularly desirable that the component be in a form that can be taken together with food and drink as a blended component of food and drink. There is.

[0008]

Means for Solving the Problems The present inventors have previously described PT
We know that calcium and inorganic phosphate release from cultured neonatal mouse calvaria induced by Hrp (parathyroid hormone-related protein) or its active fragments have a high correlation with the mechanism of absorbable bone diseases such as osteoporosis. It was And using this technique, when searching for extractives from various medicinal plants for preventive / therapeutic effects on resorbable bone diseases, plants of the genus Podfilm of the barberry family,
The inventors have found that an extract obtained from a plant of the genus Notopteridium or the asunaro of the Cypress family exhibits a calcium and inorganic phosphate release inhibiting action in this evaluation system, and completed the present invention.

That is, an object of the present invention is to provide a plant of the genus Podfilm of the barberry family, a plant of the genus Notopteridium of the family Seriaceae, or the asunaro of the cypress family.
The present invention provides a prophylactic / therapeutic agent for absorbable bone diseases, which comprises an extract obtained from at least one plant selected from ata) as an active ingredient.

The plant extract of the present invention means an extract obtained from these plants by hot water extraction or organic solvent extraction represented by lower alcohol such as ethanol or hydrous lower alcohol, lower ester such as ethyl acetate. Means

[0011] As described above, the raw material plants used to obtain the plant extract of the present invention are plants of the genus Podfilm of the barberry family, plants of the genus Notopteridium of the family Aceraceae or asunaro of the cypress family, which have been used since ancient times. It is widely used as a folk medicine.

[0012] For example, for plants belonging to the genus Podfilm of the barberry family, Podofilm
um Peltatum) is used as a laxative in North America. In addition, the rhizome of Podophyllumemodi of the same genus is also used in the United Kingdom. Furthermore, Miyaso (Podo)
Phyllum pleianthum rhizome (Taiwan name: Octagonal) and Podfilm Versipyre (Pod)
The rhizome (Chinese name: Onitsu) of Ophyllum versipelle) is also used in the same manner.

On the other hand, regarding plants of the genus Notopteridium of the family Apiaceae, the name of the Chinese medicine, Notopterygium
insium)
ygium forbesii), Kawakatsu (Not)
rhizomes such as Opterygium franchetii) are used as a Chinese medicine for colds and the like.

Further, with respect to Asunaro of the cypress family, the hot water extract of its leaves is used as a folk medicine for strong liver.

As described above, the raw material plant suitably used for obtaining the plant extract of the present invention is already known as a crude drug, but its component has hitherto been the preventive or absorbable bone disease. There was no finding that it was effective for treatment.

The plant extract which is the active ingredient of the present invention can be obtained by extracting from the raw materials of the above-mentioned plants with a solvent capable of effectively extracting the active ingredient, or by partially purifying these.

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 may be directly extracted with these solvents, but it is preferable to cut these raw materials finely and then extract with the solvent in order to enhance the extraction efficiency. . Further, the extraction efficiency can be increased by repeating the extraction 2 to 4 times.

The preferred extraction method is 5 to 100 ml, especially 10 to 20 m, per 1 g of finely cut raw material.
A method using about 1 solvent can be mentioned. When an organic solvent is used as the extraction solvent, it is desirable to carry out the extraction at room temperature for 1 day to 1 month, preferably 2 to 5 days. Further, when water is used as the extraction solvent, it is desirable to heat it in order to enhance the extraction efficiency.

Furthermore, in order to increase the concentration of the active ingredient in the extract, the obtained extract is further subjected to means such as concentration, liquid-liquid partitioning, adsorption chromatography, normal phase or reverse phase chromatography, if desired. It is also possible to attach.

The active ingredients used in the present invention may be those obtained from a single plant, but it is also possible to mix and use the active ingredients obtained from various plants.

In order to produce the preventive / therapeutic agent for absorbable bone diseases of the present invention, the plant extract obtained as described above is used as an active ingredient, and it is formulated according to a conventional method in combination with a known pharmaceutical carrier. Just do it.

The preventive / therapeutic agent for absorbable bone diseases of the present invention is
Administration in various dosage forms is possible, and examples of oral administration agents include capsules, tablets, granules, fine granules, syrups, and dry syrups. Parenteral administration agents include injections. Other examples include suppositories such as suppositories and vaginal suppositories, nasal administration agents such as sprays, ointments, and percutaneous absorbents such as transdermal tapes.

The preventive / therapeutic agent for absorbable bone diseases of the present invention is
As a dry matter equivalent to raw materials, usually 1 to 100 per adult per day
g, preferably 3 to 30 g, may be divided into 1 to 3 times to give a dosage unit that can be administered orally or parenterally. 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 and take it on a daily basis. The type of food that can be added is not particularly limited,
Since the plant extract has a unique flavor, it is preferably added to beverages such as drinks and foods with strong sweetness such as candy.

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 absorbable bone diseases and is useful as a food for health.

The amount of the plant extract to be added to such foods and drinks may be such that it is added within 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 1% by weight.

Since the plant extract used in the preventive / therapeutic agent for absorbable bone diseases and the food and drink of the present invention is obtained from a plant already used as a herbal medicine or the like as described above, it is safe. There is no particular problem with sex.

Next, with respect to the plant extract which is the active ingredient of the present invention, a method for evaluating the calcium and inorganic phosphate release inhibiting action will be described.

PTHrp (parathyroid hormone-related protein) is a protein identified as a causative agent of human hypercalcemia, and PTHrp (1-34), which is a fragment consisting of amino acid residues 1-34. Is the active form. And this PTHrp (1-34) is
Since it has been shown to promote bone resorption in vitro (Journal of Clinical Investigation, 81, No. 2, 596-600, 1988; End Clinology, 123, 2841-2848). ，
1988), and can be used as an evaluation system for osteoporosis.

Therefore, the in vitro bone resorption inhibitory effect of the active ingredient of the present invention was evaluated using cultured newborn mouse calvaria and PTHrp (1-34). This evaluation system is based on the method of Royce using forearm bones of fetal rats and ⁴⁵ Ca (Journal of Clinical Investigation, 44, No. 1, 103-116, 1).
965), and has the advantage that many specimens can be safely evaluated without using radioisotopes.

This improved method has already been widely used as a screening system for therapeutic agents for bone diseases (Journal of the Japanese Society for Bone Metabolism, Vol. 8, No. 3, page 221, 1990; 9;
Volume 3, Issue 239, 1991; Japanese Journal of Pharmacology, Volume 55, Addendum 1,
120 pages, 1991), but the outline is as follows.

That is, calvaria of 4 to 6-day-old ICR mice were collected, pre-cultured for 1 day, and then replaced with a medium to be assayed and a medium containing 10 ⁻⁸ M PTHrp (1-34). After further culturing for 2 days, the calcium and inorganic phosphorus concentrations in the culture supernatant are measured after completion of the culture, and PTHr
This is a method for comparison with the addition group containing only p (1-34).

In this in vitro model of calcium and inorganic phosphorus release from mouse calvaria induced by PTHrp (1-34), the active ingredient of the present invention was used to determine the concentration of calcium and inorganic phosphorus in the culture supernatant. Lowered. Further, the active ingredient of the present invention is PTHrp (1-3
In an in vivo model of rat hypercalcemia caused by 4), the serum calcium concentration was significantly reduced. From this, the extract of the present invention is
Since it suppresses the bone resorption promoting action caused by p (1-34), it was found to be useful as a preventive or therapeutic agent for bone diseases.

[0035]

The plant extract and its purified product, which are the active ingredients of the present invention, are cultured neonatal mice caused by PTHrp (1-34), which is an active fragment of the accessory thyroid line-related protein, as shown in the Examples below. It has the effect of suppressing the release of calcium and inorganic phosphorus from the calvaria, and PTHr
In a hypercalcemia model induced by P (1-34), the serum calcium concentration is reduced. This is
It is speculated that the plant extract and the purified product thereof, which are the active ingredients of the present invention, effectively prevent and treat resorbable bone diseases such as malignant hypercalcemia, Paget's disease of bone and osteoporosis.

[0036]

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

Example 1. Extraction of Active Ingredients from Kawara-Katsu 100 g of bamboo-bamboo paste (dried rhizome of Kawa-Koi-Kai, obtained from Kampo medicine dealer) was finely cut, immersed in 200 ml of 50% methanol at room temperature for 2 days, and filtered. 5 to the residue again
200 ml of 0% methanol was added, and the mixture was further immersed at room temperature for 2 days and then filtered. 200 ml of 50% methanol in the residue again
Was added, and the mixture was similarly immersed and then filtered. The filtrates obtained by the three extractions were collected, concentrated under reduced pressure and then freeze-dried to obtain 1.2 g of the extract.
Got

Example 2. Extraction of active ingredient from asunaro 1 kg of asunaro leaves (raw leaves) is finely cut to 2000 m
3 times as in Example 1 with 1 of 75% methanol,
Then, the same treatment was carried out to obtain 10 g of the extract.

Example 3. Effective formation from pod film resin
100 mg of partially purified Podfilm resin (Podfilm, alcohol extract of Pertatum rhizome, manufactured by Merck) was dissolved in 5 ml of chloroform, and the filtrate was separated with SEPPAKSiO _2.
Adsorbed on a cartridge (made by Millipore Limited), 10 ml each of (1) chloroform, (2) chloroform / methanol 9: 1 mixture, (3) chloroform / methanol 8: 2 mixture, (4) ethyl acetate,
(5) Chloroform / methanol 1: 1 mixture and (6) methanol were eluted in this order. Bioassay revealed activity in the chloroform elution section of (1), so the solvent was removed from this section under reduced pressure to obtain 75.5 m.
g of partially purified product was obtained.

Example 4. Of the active ingredients from Kawakatsu extract
100 mg of the extract obtained in the partially purified Example 1 was treated in the same manner as in Example 3 to obtain 65.9 mg of a chloroform-eluted fraction (fraction 1). This was dissolved in 5 ml of methanol, and the filtrate was adsorbed on a SEPPAK C ₁₈ cartridge (manufactured by Millipore Limited), and (1) methanol 10 was added.
Elution was performed with 10 ml of (2) chloroform, followed by 10 ml of (2) chloroform.
By the bioassay, activity was observed in the methanol elution section of (1), so the solvent was removed from this section under reduced pressure to obtain 55.9 mg of a partially purified product (fraction 2).

The obtained fraction 2 was dissolved in 10 ml of methanol, and Develosil ODS-10 was dissolved.
Preparative high performance liquid chromatography using a / 20 (φ50mn × 400mn) column (manufactured by Nomura Chemical Co., Ltd.) and developed with a linear concentration gradient of 20-80% acetonitrile in methanol for 120 minutes at a flow rate of 32 ml / min. did. When monitored by UV absorption at 210 nm, 7 peaks were obtained. By bioassay, the 4th peak was collected and the solvent was removed under reduced pressure to obtain 2.5 mg of partially purified product (fraction 3). It was

Evaluation Example 1. By PTHrp (1-34)
Calcium and calcium from the neonatal calvaria induced in cultured mice
Measurement of Inhibitory Effect on Inorganic Phosphorus Release ICR mice (4 to 6 days old) are cut out from the calvaria to remove soft tissue, and then punched out to a diameter of 4 mm. Fitton containing 5% fetal bovine serum (FBS)
-Jackson modification-BGJ
b medium (manufactured by Sigma, catalog number: B664
4. In the following, each piece is placed in a 48-well plate containing BGJb medium) and precultured for 24 hours under the conditions of 37 ° C., 5% carbon dioxide / air. After completion of the pre-culture, the medium is replaced with a 5% FBS-BGJb medium containing 10 ₋₈ M PTHrp (1-34) and 100 μg / ml of the sample obtained in Examples 1 to 4, and further cultured for 48 hours.

After completion of the culture, VX10 manufactured by JEOL Ltd.
Using an 00 type biochemical automatic analyzer, the calcium concentration of the culture supernatant was measured by the OCPC method, and the inorganic phosphorus concentration was measured by the molybdic acid direct method, and compared with the value of the control group to which only PTHrp (1-34) was added. Then, the bone resorption inhibitory effect was examined.

The results are shown in [Table 1], and the plant extract of the present invention and its partially purified product suppressed the release of calcium and inorganic phosphorus. This result strongly suggests that the plant extract of the present invention and its partially purified product can prevent or suppress malignant hypercalcemia. It is shown to be useful as a preventive or therapeutic agent for diseases.

[0045]

[Table 1]

Evaluation Example 2. PTHrp (1-34) -induced LA
Blood Calcium Concentration in a Hypercalcemia Model
Suppression effect Wistar system (5 weeks old, male, 70-90g, 5 animals per group)
Was intraperitoneally administered 10 mg / kg of the fraction 3 of Example 4 and 2 hours later, 5 nmol of PTHrp (1-34)
e / kg intravenous injection induces hypercalcemia, part 1
Blood is collected after a lapse of time and blood calcium concentration is measured by JEOL Ltd.
OC using a VX1000 type biochemical automatic analyzer manufactured by
The hypercalcemia inhibitory effect was measured by the PC method and compared with the value of the control group to which only PTHrp (1-34) was added.

The results are shown in [Table 2]. Fraction 3 of Example 4 significantly suppressed hypercalcemia.

[0048]

[Table 2]

From these experimental facts, it is clear that the active ingredient of the present invention has the effects of preventing the release of calcium from bone and lowering the blood calcium concentration. Then
Formulations were prepared for various dosage forms.

Formulation Example 1. Production of Capsule [Formulation] Fraction 2 of Example 4 100 parts (parts by weight) Potato starch 148 parts 〃 Magnesium stearate 2 parts 〃 [Manufacturing method] After thoroughly mixing the above ingredients in accordance with the prescription, No. 1 Each 250 mg of hard gelatin capsules was filled to obtain a capsule containing 100 mg of the fraction 2 of Example 4 in one capsule.

Formulation Example 2. Production of Rectal Suppository [Production Method] Witepsol H-15 was heated and melted, and the partially purified product of Example 3 was added thereto to a concentration of 12.5 mg / ml, and the mixture was uniformly mixed. 2 ml each was poured into a suppository mold, cooled, and 25 mg of one formulation in Example 3
A rectal suppository containing the partially purified product was obtained.

Formulation Example 3. Preparation of Drink [Formulation] Extract of Example 2 2 g DL-sodium tartrate 10 mg Succinic acid 1 mg Liquid sugar 80 g Citric acid 1.2 g Vitamin C 1 g Flavor 1 ml Potassium chloride 0.1 g Magnesium sulfate 50 mg [Production method] Dissolve the components in 800 ml of distilled water, add distilled water to make the total volume 1000 ml,
Sterilization was performed using a 0.22 μm sterilization filter, and 100 ml of each was aseptically filled in a brown bottle to obtain a drink preparation containing 200 mg of the extract of Example 2 per preparation.

Formulation Example 4. Preparation of candy [Formulation] Partially purified product of Example 3 1 g Purified water 1 g Granulated sugar 49 g Syrup 48 g Citric acid 0.5 g Lemon flavor 0.5 g [Production method] Granulated sugar and starch syrup are heated and melted according to a conventional method, and other components Was suspended in purified water and added, and then uniformly mixed to produce 2 g of a candy. One particle contains 20 mg of the partially purified product of Example 3.

[0054]

Industrial Applicability The plant extract and its partially purified product, which are the active ingredients of the present invention, suppress the release of calcium and inorganic phosphorus, suppress hypercalcemia, and suppress the bone resorption effect. confirmed. The plants that are the raw materials for these plant extracts have been widely used as crude drugs since ancient times, and there is no particular problem in safety.

Therefore, the preventive / therapeutic agent for absorbable bone diseases of the present invention is useful for preventing or treating absorbable bone diseases such as malignant hypercalcemia, Paget's disease of bone, osteoporosis and the like. Further, by adding the active ingredient of the present invention to food and drink and ingesting it daily, it is also useful as a health food or the like for the prevention of absorbable bone diseases. (that's all)

Continued Front Page (72) Koichi Nakahara 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Prefecture Suntory Biomedical Research Institute

Claims (2)

[Claims] 1. A plant of the genus Podfilm of the barberry family, a plant of the genus Notopteridium genus of the Aceraceae or an asunaro of the Cypress (Thuj).
A preventive / therapeutic agent for resorbable bone disease, which comprises an extract obtained from at least one plant selected from Opsis dorabrata) as an active ingredient. 2. The absorbable bone disease is malignant hypercalcemia,
The preventive / therapeutic agent according to claim 1, which is Paget's disease of bone or osteoporosis.