JPH07188038A - Therapeutic agent for hypercholesteremia - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for hypercholesteremia which contains, as an active ingredient, an acidic heteropoly-saccharide which is extracellularly secreted by the callus induced from a plant belonging to the genus Polianthes and has hypocholesteremic action with high safety and manifests the effect with less dosage than that in natural polysaccharides. CONSTITUTION:This therapeutic agent contains an acidic heteropoly-saccharide which is extracellularly secreted by the callus induced from a plant belonging to the genus Polianthes such as tuberose, preferably composed of arabinose, galactose, mannose, glucuronic acid and xylose as constituents (the formula gives the linking pattern and the constitution proportion, while the molecular weight is 1X10<4> to 2X10<7>), as an active ingredient.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly safe therapeutic agent for hyperlipidemia having a blood cholesterol lowering effect.

[0002]

2. Description of the Related Art In recent years, by ingesting indigestible polysaccharides, epidemiological studies have revealed prophylactic and therapeutic effects on myocardial infarction, hyperlipidemia, arteriosclerosis, diabetes and the like. From nutritional surveys, the effect of suppressing elevation of blood cholesterol level has been revealed. Based on such investigations, polysaccharides derived from plants or microorganisms and crustaceans are currently used for the prevention or treatment of diseases such as hyperlipidemia. Known polysaccharides include plant-derived guar gum, konjac mannan, pectin, lignin, hemicellulose, cellulose, chitin derived from crustaceans and chitosan which is a derivative thereof.

However, all of these polysaccharides are derived from natural products and it is difficult to supply them in stable quality and quantity. Further, in order to reduce blood cholesterol level by these polysaccharides, it is necessary to administer a large amount of several g to several tens of g per day in the case of an adult male,
Under the present circumstances, the physical burden on the patient is increased and the sufficient effect is not obtained.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems, exhibits a low blood cholesterol lowering effect even in a small amount, is highly safe, and is capable of stably supplying a homogeneous product for hyperlipidemia. The purpose is to provide an agent.

[0005]

The inventors of the present invention have investigated the blood cholesterol-lowering effects of many polysaccharides. As a result, a small amount of heteropolysaccharides obtained from callus of a plant belonging to the genus Polyantes They found that they have a cholesterol lowering action and high safety, and completed the present invention.

The present invention is directed to the genus Polianthes.
The present invention relates to a therapeutic agent for hyperlipidemia, which comprises an acidic heteropolysaccharide secreted extracellularly by callus derived from a plant belonging to L. ) as an active ingredient.

The acidic heteropolysaccharide, which is the active ingredient of the therapeutic agent for hyperlipidemia of the present invention, is disclosed in, for example, JP-A 64-10997.
According to the method described in No. 3, page 3, upper left column, line 4 to page 4, lower right column, line 4, callus derived from explants of plants belonging to the genus Polyanthes is cultured in a plant hormone-containing medium. It can be obtained by collecting from the culture.

Examples of the plant belonging to the genus Polyanthes include Tuberose ( Polianthes tuverosa L. ). Explants include flowers, stems, leaves, bulbs,
Examples include organs such as roots or a part of their tissues, and among these, flowers or a part of their tissues are preferable.

Callus can be induced from a callus induction medium by a conventional method. As this callus induction medium, a basal medium supplemented with plant hormones and sugar (carbon source) can be used. The callus induction medium may be either solid or liquid, but solid medium is usually preferred.

Examples of the basal medium include Murasige-Skoog medium, Linsmaier-Skoog medium, Gamborg medium, White medium, Tuleeke medium, Nitsch & Nitsch medium, etc. which are usually used for plant tissue culture. it can.

2,4-dichloro is a plant hormone.
Phenoxyacetic acid (2,4-D), α-naphthalene acetic acid
(NAA), indole acetic acid (IAA), indole dairy
Auxins such as acid (IBA); furfurylaminop
Phosphorus (Kinetin), Benzyl Adenine (BA), Dime
Cytokinins such as tilaminopurine (2iP)
Can you name some? Among these, 2,4-D
Alone, a combination of NAA and BA or kinetin is preferred
Good The concentration of phytohormones required for callus induction depends on the species.
However, in the case of 2,4-D alone, 5 ×
10^-Four~ 1 x 10 ^-7M, NAA and BA or kine
In case of combination of Chin, NAA concentration is 5 × 10 -4
~ 1 x 10-7M, BA or kinetin concentration is 1 x 10^-7
It is M.

As the sugar, glucose, fructose,
Mannose, xylose, sucrose, rhamnose,
Although fucose, starch, etc. can be mentioned, sucrose is usually preferable among these.

The induced callus can be subcultured for 10 or more generations while maintaining the same morphology in the callus induction medium. The subculture medium is usually Linsmaier-Skoog medium, Murasige-Skoog medium, as the basic medium.
The media, as a plant hormone, 1 × 10 ^-4 ~1 × 10
^-7 M 2,4-D or 1 × 10 ^-4 to 1 × 10 ^-7 M NA
It is possible to use a combination of A and 1 × 10 ⁻⁴ to 1 × 10 ⁻⁷ M BA, and further added the above-mentioned sugar as a carbon source, preferably sucrose (1 to 6% by weight).

As a plant hormone-containing medium used for culturing a polysaccharide from callus, a plant hormone-containing medium in which the types and concentrations of the following plant hormones and sugars are appropriately set can be used according to a callus induction medium. The medium may be either solid or liquid, but is preferably a liquid medium.

As plant hormones, 2,4-D and NA
Auxins such as A, IAA, IBA; kinetin,
BA, 2iP and other cytokinins; gibberellin A3
Examples thereof include gibberellins such as (GA3).
Of these, 2,4-D or NAA alone, or a combination of NAA and BA or kinetin is preferable. Although the concentration of plant hormones varies depending on the type, for example, 2,4-
In the case of D or NAA alone, 5 × 10 ⁻⁴ to 1 × 10
^-7 M, preferably 5 × 10 ^{-5 to} 5 × 10 ^-6 M, N
In the case of the combination of AA and BA or kinetin, N
AA concentration is 1 × 10 -4 to 1 × 10 ^-7 M, preferably 1 ×
It is 10 ^{−4 to} 5 × 10 ⁻⁶ M, and the BA or kinetin concentration is 5 × 10 ⁻⁵ to 1 × 10 ⁻⁹ M, preferably 1 × 10 ⁻⁵ .
It is 1 × 10 ⁻⁷ M.

As the sugar as a carbon source, the same ones as mentioned above can be mentioned, and among them, sucrose is preferable. The amount of sugar added is generally 1 to 6% by weight.

The culturing method is not particularly limited, but may be 20 to 3
A method of shaking culture at 0 ° C. for 15 to 30 days can be applied.

As a method for collecting the polysaccharide from the culture thus cultivated, for example, the following method can be applied. First, cells are removed from the culture by centrifugation or filtration, and then the culture solution is concentrated by a rotary evaporator or the like. Next, ethanol is added to the concentrated liquid to cause precipitation, and the precipitate is collected and freeze-dried. Further, this freeze-dried product (crude refined product) can be further purified by a usual means. For example, by dissolving the crude purified product in water and removing the insoluble matter by centrifugation, by subjecting it to dialysis or ion exchange chromatography, gel filtration chromatography, etc.,
A highly purified product can be obtained.

The acidic heteropolysaccharide, which is the active ingredient of the therapeutic agent for hyperlipidemia of the present invention, is hydrolyzed with 2N sulfuric acid at 10 ° C. for 8 hours, and then ethyl acetate: pyrimidine: acetic acid: water = 5: Thin layer chromatography was performed using a developing solvent having a mixing ratio of 5: 1: 3, and aniline: diphenylamine:
Acetone: Arabinose when colored with formic acid reagent,
Galactose, mannose, glucuronic acid and xylose are detected. Further, the analysis results by gas chromatography also confirm that these sugars are contained as constituent sugars. That is, the binding mode and the composition ratio according to the result of the analysis by the GC-MS method after methylation by the Hakomori method are:

[0020]

[Chemical 2]

[0021] Further, 0 to 50% of the carboxyl group of glucuronic acid exists as a methyl ester, and this polysaccharide is acidic because it is adsorbed on an anion exchange resin or the like. Further, high performance liquid chromatography (column: TSK Gel 4000PW, 5 manufactured by Toso Co., Ltd.
000 PW and 6000 PW), and the molecular weight is 1 × 10 ⁴ to 2 × 10 ⁷ .

Further, the acidic heteropolysaccharide has the following physical properties. Soluble in solvent Soluble in water but insoluble in ethanol, ether, and acetone. Coloring reaction Anthrone reaction: Positive Carbazole reaction: Positive Elson-Morgan reaction: Negative Color and shape Those that have undergone ethanol precipitation are white to off-white powders. The product purified by ion exchange through dialysis and freeze-dried is white cotton-like or fibrous. Specific rotation [α] _D ²⁵ : 0 to + 20 ° (c = 1.0, aqueous solution)

The therapeutic agent for hyperlipidemia of the present invention can be constituted by an acidic heteropolysaccharide alone, but if necessary, a known formulation carrier can be used to obtain a desired dosage form. Known formulation carriers include diluents or excipients such as fillers, fillers, binders, disintegrants, surfactants, lubricants, etc., which are appropriately selected according to the dosage form. Can be used. Further, the therapeutic agent for hyperlipidemia of the present invention can further contain a colorant, a preservative, a flavoring agent, a flavoring agent, a sweetening agent, other pharmaceuticals and the like together with the above-mentioned preparation carrier regardless of its dosage form. . The dosage form is not particularly limited, tablets,
It can be made into a desired dosage form such as pills, powders, solutions, suspensions, emulsions, granules, capsules and injections. The therapeutic agent for hyperlipidemia of the present invention can be orally or parenterally administered to mammals including humans, depending on its dosage form.

The content of the acidic heteropolysaccharide in the therapeutic agent for hyperlipidemia of the present invention varies depending on the dosage form. For example, in the case of a liquid preparation, it is preferably contained in an amount of 0.5 to 1% by weight.

The dosage of the therapeutic agent for hyperlipidemia of the present invention is appropriately selected according to the usage, the age and sex of the patient, etc., but it is usually 0 as an acidic heteropolysaccharide for adult males. 6 to 6 g / day.

[0026]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 (Production of Acidic Heteropolysaccharide) From buds of sterilized tuberose 2 to 7 days before flowering, 1 × 10 ^-5 M NAA and 1 × 10 ^-6 M as plant hormones were used.
Callus was induced using Linsmaier-Skoog's medium (agar medium) containing 3% by weight of BA and 3% by weight of sucrose as a carbon source. The induced callus was subcultured in a medium having the same composition. A stable callus that has been passaged for several generations or more contains 1 × 10 ⁻⁵ M 2,4-D as a plant hormone and 5% by weight of sucrose as a carbon source Linsmaier-Skoo
g medium (liquid medium) was inoculated to a concentration of 5% by weight. Then, this medium was cultivated for 30 days under the conditions of a shaking frequency of 120 rpm and a temperature of 27 ± 1 ° C. using a rotary shaker in the dark. After completion of the culture, cells were removed from the culture solution by filtration and centrifugation, and the residual solution was concentrated by a rotary evaporator. To this concentrated solution was added 3-fold amount of ethanol and the mixture was allowed to stand at 5 ° C. for 24 hours to obtain a precipitate. The precipitate was collected by centrifugation, washed with 70% ethanol three times, and then freeze-dried to remove water, thereby obtaining a target polysaccharide.

This operation was repeated 5 times in total, and changes in polysaccharide yield, total sugar content, uronic acid content, protein content, water content, and neutral sugar composition ratio were measured and compared for each lot. As a result, all the values had little fluctuation and the quality and quantity were stable.

As a result of thin layer chromatography of the thus obtained polysaccharide by the above-mentioned method, arabinose, galactose, mannose, glucuronic acid and xylose were detected. Moreover, it was confirmed from the analysis results by gas chromatography that these sugars were contained as constituent sugars. The binding mode and constituent ratio based on the result of analysis by the GC-MS method after methylation by the Hakomori method were those shown by the above formula.
As a result of measurement by high performance liquid chromatography, the molecular weight was 1 × 10 ⁴ to 2 × 10 ⁷ .

Example 2 (tablet) 70 mg of the acidic heteropolysaccharide of Example 1 and 100 of starch
mg, magnesium stearate 15 mg and lactose 45
Using mg, tablets were produced by a conventional method.

Example 3 (solution) 1000 mg of the acidic heteropolysaccharide of Example 1 and 1 of distilled water
A liquid preparation was produced by a conventional method using 00 ml.

Example 4 (Blood Cholesterol Reduction Test) Eight 5-week-old male SD rats (manufactured by CLEA Japan, Inc.) were used as one group, and a standard diet group, a high cholesterol diet group, and a high cholesterol diet 10 mg / kg were used. Four groups were prepared: an acidic heteropolysaccharide administration group and a high cholesterol diet 100 mg / kg acidic heteropolysaccharide administration group. That is, high cholesterol diet 1
For the 0 mg / kg acidic heteropolysaccharide administration group, 2 ml of a 0.1% aqueous solution of acidic heteropolysaccharide (= 2 mg acidic heteropolysaccharide) was added to the high cholesterol diet 100 mg / kg acidic heteropolysaccharide administration group. For each, 2 ml of a 1% aqueous solution of acidic heteropolysaccharide (= 20 mg of acidic heteropolysaccharide) was orally administered by force using an ordinal probe. 2 ml of distilled water was similarly administered to the standard diet group and the high cholesterol diet group. The sample compositions of the standard diet and the high-cholesterol diet are shown in Table 1 below.

[0033]

[Table 1] Feed ingredients Standard diet (wt%) High cholesterol diet (wt%) Casein 20 20 Mineral 3.5 3.5 3.5 Vitamin 11 Fat 10 10 Cholesterol-1 Deoxycholic acid-0.3 Potato starch 65.5 64.2

The test period was 10 days, during which the feed and water were freely ingested, and the feed intake and body weight change were measured every other day. The test was started under such conditions, and in the non-fasted state on the 7th day, a part of blood was collected from each individual by blood sampling under ether anesthesia, and the serum lipid amount was measured. The measurement items are total cholesterol (TCHO),
The five items are free cholesterol (FCHO), high density lipoprotein cholesterol (HDLC), phospholipid (PL) and triglyceride (TG). From these serum lipid amounts, the cholesterol ester (CE) index and the atherogenic index are calculated by the following formulas;

[0035]

[Equation 1]

It was calculated based on These results are shown in Table 2.
Shown in. After that, the test is continued and 10
After a lapse of days, the rats were opened under ether anesthesia,
Whole blood was collected from the abdominal aorta. In addition, the liver, kidney and spleen were removed and the wet weight was measured. The results are shown in Table 3.

[0037]

[Table 2]

The total cholesterol amount was significantly increased in the high-cholesterol diet group as compared with the standard diet group, and the suppressive effect by the administration of the acidic heteropolysaccharide was recognized. The amount of free cholesterol showed an increase tendency similar to the amount of total cholesterol, but it was significantly suppressed by the administration of 10 mg / kg of acidic heteropolysaccharide (p <0.05). High-density lipoprotein cholesterol is also called good cholesterol and has an action of removing cholesterol accumulated in organs. Compared to the standard diet group, the serum level was decreased in the high cholesterol diet group, but acidic heteropolysaccharide 100 mg / kg
Was significantly increased (p <0.05). A decrease in the cholesterol ester index is an index of severe liver parenchymal disorder. The index value of the high-cholesterol diet group was lower than that of the standard diet group, but the index value clearly showed an increasing tendency by the administration of the acidic heteropolysaccharide. The atherogenic index, which is also called the arteriosclerosis index, indicates the ratio of cholesterol in the lipoprotein fraction and is used for the risk factor evaluation of hyperlipidemia treatment for the prevention of arteriosclerosis. Compared with the standard diet group, the high cholesterol diet group had an increased atherogenic index, which clearly showed a tendency to decrease due to the administration of the acidic heteropolysaccharide. A dose of 5 g / kg is required for guar gum, which has a cholesterol-lowering effect, to exhibit the same effect as that of acidic heteropolysaccharide on cholesterol-loaded rats (Shiro Takeno et al., Japan Society of Nutrition and Food Science). See Vol.43, No.6, 421-425, 1990). On the contrary,
Acidic heteropolysaccharides showed comparable cholesterol lowering effects at doses of about 1/50 of guar gum. The clinical dose of guar gum is 12 g / day for adult males (Noboru Irie et al., Effect of dietary fiber guar gum on serum lipoprotein and fat absorption, “Dietary Fiber-Food Science and Nutritional Approach- , Shinohara Publishing, 157-169, 1983),
In comparison, a small amount of acidic heteropolysaccharide may be used as described above (0.6 to 6 g / day).

[0039]

[Table 3]

The kidney and spleen showed no difference in weight between the groups, but the liver weight was significantly increased in the high cholesterol group as compared with the standard diet group (p <0.01). The liver of the standard diet group had a light brown color, while the high-cholesterol group had a discoloration of ocher, clearly showing signs of fatty liver. On the other hand, the administration of acidic heteropolysaccharides obviously suppressed the increase in liver weight due to the high cholesterol diet.

Example 5 (Acute toxicity test for oral administration) 500 mg / kg of the aqueous solution of the acidic heteropolysaccharide of Example 1 was applied to one group of male and female mice (one group consisting of 5 rats).
, A single oral dose. As a result of observation for 14 days after administration, all individuals survived and no abnormal symptoms were observed. Therefore, LD ₅₀ by oral administration is 5000 m
It is estimated to be g / kg or more.

Example 6 (Intraperitoneal Acute Toxicity Test) To 7 week-old ICR male mice, 19.7 mg / ml aqueous solution of the acidic heteropolysaccharide of Example 1 was administered 4 times at 2 hour intervals. It was administered intraperitoneally. As a result, a total of 1000m
Although the dose was g / kg, all the individuals survived and no abnormal symptoms were observed even 7 days after the administration.

[0043]

INDUSTRIAL APPLICABILITY The therapeutic agent for hyperlipidemia of the present invention comprises an acidic heteropolysaccharide secreted extracellularly by callus derived from a plant belonging to the genus Polyanthes ( Polianthes L. ) as an active ingredient, It has an excellent cholesterol-lowering effect and high safety, and is useful for treating hyperlipidemia. Further, compared with known natural polysaccharides known to exhibit a cholesterol-lowering effect, the same effect is exhibited at a far lower effective dose. Furthermore, since the acidic heteropolysaccharide, which is the active ingredient of the therapeutic agent for hyperlipidemia, is produced by the application of plant cell culture technology, a stable and homogeneous product can be supplied.

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Claims (2)

[Claims] 1. A therapeutic agent for hyperlipidemia, which comprises as an active ingredient an acidic heteropolysaccharide secreted extracellularly by callus derived from a plant belonging to the genus Polianthes L. 2. It contains arabinose, galactose, mannose, glucuronic acid and xylose as constituent sugars, and their binding modes and constituent ratios are as follows: And a therapeutic agent for hyperlipidemia, which comprises an acidic heteropolysaccharide having a molecular weight of 1 × 10 ⁴ to 2 × 10 ⁷ as an active ingredient.