JP4976031B2 - Cartilage formation promoter containing galacturonic acid as an active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To discover a cartilage formation promoter useful as a preventive or therapeutic agent or the like of a disease originated from cartilage damage, for example, arthritis and rheumatism, and having little toxicity, side effect or the like. <P>SOLUTION: The cartilage formation promoter contains galacturonic acid or a salt thereof as an active ingredient. The preventive or therapeutic agent of the disease originated from the cartilage damage, and the formation promoter of glycosaminoglycan and/or proteoglycan are also provided. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

The present invention relates to a cartilage production promoter containing galacturonic acid as an active ingredient, a prophylactic or therapeutic agent for diseases caused by cartilage damage, and a glycosaminoglycan and / or proteoglycan production promoter.

Galacturonic acid is a component such as pectin, various plant mucilages, and bacterial polysaccharides, and is known to have antibacterial action. It is clear that pectin degradation products based on galacturonic acid are used as preservatives for foods and beverages, and that plant extracts based on galacturonic acid have anti-inflammatory and free radical scavenger effects (Non-patent Document 1).
Pectin, which is composed of a chain structure of 1,4-glycosidic bonds of galacturonic acid, constitutes the cell wall of plants and has effects such as lowering serum total cholesterol concentration, reducing active oxygen, and inhibiting colon cancer. Used and consumed as a dietary fiber and food additive in daily life. Pectin has also been confirmed to have beneficial effects on the body, such as activating phagocytic cells, anti-inflammatory effects and effects on immune responses.
However, there are few reports on the effects on the living body of administration of galacturonic acid, which is a component of pectin.
Nergard et al., J. Ethnopharmacol. 96; 255-269, 2005

  Many diseases such as arthritis and rheumatism are known as diseases caused by cartilage damage, and many drugs have been developed and marketed. However, since there are individual differences in effects and side effects, it is not fully satisfactory. Moreover, it is more preferable to have a composition such as food or drink that can be taken preventively. The inventors of the present invention have completed the present invention in consideration of development of various uses of galacturonic acid, paying attention to the safety of galacturonic acid.

Galacturonic acid, in particular α-D-galacturonic acid, is known as a component of pectin present in many plants such as fruits or vegetables. Pectin has various physiological activities as plant fiber, is often used as a food additive, is a highly safe substance, and has antibacterial activity for galacturonic acid. Therefore, it is a highly safe compound.
Galacturonic acid is a saccharide classified into the same uronic acid as glucuronic acid and iduronic acid. Glucuronic acid and iduronic acid are known as main substances constituting glycosaminoglycan, which is a constituent of cartilage. For example, glucuronic acid constitutes hyaluronic acid and chondroitin sulfate, and iduronic acid constitutes dermatan sulfate and the like, which are indispensable for synthesizing a cartilage matrix. However, although classified into the same uronic acid, galacturonic acid is a component of pectin present in plants, and its relevance to bone in animals is not known at all. As a result of investigating the effects of galacturonic acid on oral cartilage when orally administered, the inventors of the present invention studied the development of various applications of galacturonic acid, focusing on the safety of galacturonic acid. In addition, the inventors have found that galacturonic acid has an action of promoting the production of glycosaminoglycan and proteoglycan and an action of promoting the production of cartilage, thereby completing the present invention.

That is, the present invention is (1) a cartilage production promoter containing galacturonic acid or a salt thereof as an active ingredient, except food and drink,
(2) A preventive or therapeutic agent for diseases caused by cartilage damage containing galacturonic acid or a salt thereof as an active ingredient, excluding food and drink,
(3) Glycosaminoglycan and / or proteoglycan production promoter containing galacturonic acid or a salt thereof as an active ingredient, except food and drink,
(4) a prophylactic or therapeutic agent for diseases derived from cartilage damage according to chondrogenesis promoter or (2) according to galacturonic acid or the containing enzymatic degradation product of pectin containing the salt (1), where Excluding food and drinks,
It is about.
And (5) a food or drink for promoting cartilage production or treating or preventing cartilage damage containing galacturonic acid or a salt thereof as an active ingredient, and (6) a glycosaminoglycan for promoting cartilage production of galacturonic acid or a salt thereof. And / or use for promoting production of proteoglycan or preventing or treating wounds caused by cartilage damage,
Is also disclosed for reference.

  The galacturonic acid used in the present invention has no toxicity, side effects, etc., and has an excellent action for promoting the production of glycosaminoglycan and proteoglycan and an excellent action for promoting the production of cartilage despite being a substance that is not originally present in the animal body. is doing. Therefore, galacturonic acid is promoted as a prophylactic or therapeutic agent for diseases caused by cartilage damage, such as arthritis and rheumatism, as a cartilage formation promoter, and further, the production of glycosaminoglycan and proteoglycan in cartilage is promoted. Therefore, it is useful as a production accelerator for glycosaminoglycan and / or proteoglycan. Furthermore, since the production of proteoglycan, which is a water retention component contained in the skin tissue, is promoted, skin beautifying action and the like can be expected.

The invention is described in more detail below.
The galacturonic acid used in the present invention may be a free acid or a salt, particularly a pharmacologically acceptable salt, or a hydrate thereof. In addition, oligogalacturonic acid that is absorbed into the blood as galacturonic acid by oral administration (where galacturonic acid is bound by about 2 to 6) is also included in galacturonic acid in the present invention. However, in the examples, galacturonic acid means a monosaccharide galacturonic acid.
Usually, a free acid or a hydrate thereof is used. The salt of galacturonic acid is not particularly limited as long as it is a pharmacologically acceptable salt, and any salt with an inorganic base or an organic base can be used. The salt generally includes an alkali metal salt such as a sodium salt.

In the present invention, galacturonic acid or a salt thereof (hereinafter simply referred to as galacturonic acid) may be used alone as the cartilage production promoter, the prophylactic or therapeutic agent for diseases caused by cartilage damage, and the glycosaminoglycan and / or proteoglycan production promoter. However, usually, galacturonic acid is mixed according to a conventional method together with additives used for pharmaceuticals or foods such as carriers, excipients, auxiliary agents (flavoring agents, fragrances, sweeteners, binders), It may be a formulated composition for promoting cartilage production, a composition for preventing or treating diseases caused by cartilage damage, and a composition for promoting the production of glycosaminoglycan and / or proteoglycan. The preparation may be either a liquid agent or a solid agent, and examples thereof include tablets, granules, powders, capsules, and jelly agents. Examples of the carrier or excipient include water and saccharides. The content of galacturonic acid in these is not particularly limited, and is usually 0.2% (mass%: the same hereinafter) or more, preferably 1% or more, and may be up to 100%.
These formulated compositions or galacturonic acid can be used as it is or taken as a health food or the like for the purpose of promoting cartilage formation, preventing or treating diseases resulting from cartilage damage, and promoting the production of glycosaminoglycans and / or proteoglycans. Can be ingested. Therefore, in the present invention, as long as it is used for these purposes, galacturonic acid alone, a mixture prepared by mixing excipients, etc., or a galacturonic acid-containing food or drink, etc. by adding galacturonic acid to a food or drink These are all included. Moreover, it may be used in combination with other drugs. When used in combination, galacturonic acid and other pharmacologically active substances may be mixed and formulated with excipients as necessary, or each may be formulated separately. You may use things together.
As one of the preferable pharmacologically active substances to be used in combination, there can be mentioned other pharmacologically active substances having the same action as galacturonic acid of the present invention, such as glucosamine. By using both in combination, an additive or synergistic effect of both can be expected.
The administration method is not particularly limited, and any method such as oral, injection, enteral, and other transdermal methods can be used, but oral administration is preferable.

When the cartilage production promoter, the preventive or therapeutic agent for diseases caused by cartilage damage, and the glycosaminoglycan and / or proteoglycan production promoter in the present invention are foods and drinks, galacturonic acid is appropriately mixed with food and drink. What is necessary is just to make food and drink containing galacturonic acid. Foods and drinks that can be mixed with galacturonic acid are not particularly limited, and may include milk drinks such as milk and lactic acid drinks, drinks such as other drinks, and foods such as ham, sausage, confectionery, and other processed foods. it can.
The content of galacturonic acid in these foods and drinks is not particularly limited, but is usually 0.1% (mass: the same unless otherwise specified) or more, preferably 0.3% or more, more preferably 0, based on the whole food. The upper limit is not particularly limited, but is usually 10% or less, preferably 5% or less, more preferably 4% or less from the viewpoint of taste and the like.
The dose of galacturonic acid for use in the present invention is usually 0.3 g or more per adult per day, more preferably 0.5 g or more, more preferably 1 g or more, and the upper limit is not particularly limited because there is almost no toxicity. It is 20 g or less, preferably 10 g or less, more preferably about 5 g or less.

The galacturonic acid used in the present invention may be crude or purified to a purity of 95% or more. For example, a crude or partially purified pectin degradation product containing galacturonic acid obtained by enzymatic degradation of pectin with an enzyme such as pectinase is a safety and production cost for health foods used in the application of the present invention. It is one of the preferable also from such points. At this time, the content of galacturonic acid contained in the pectin degradation product is not particularly limited as long as the effect of the present invention can be achieved, and is 1% or more, preferably 10% or more, more preferably 25% in terms of dry matter. % Or more is sufficient. As a raw material used for a preparation or the like, the content is 30% or more, more preferably 40% or more, and most practically about 50 to 90%.
In the present invention, when galacturonic acid is used as a cartilage formation promoter, a prophylactic or therapeutic agent for diseases caused by cartilage damage, it is preferably used in combination with glucosamine (salt) known as an arthritis therapeutic agent. The amount and dosage of glucosamine (salt) in the preparation when used in combination are substantially the same as in the case of galacturonic acid. The glucosamine salt used in combination is not particularly limited as long as it is pharmacologically acceptable, and both inorganic acid salts and organic acid salts can be used. In general, inorganic salts such as sulfates or hydrochlorides, and organic acid salts such as acetates, citrates and malates are used. In the present invention, hydrochlorides and citrates are preferred.
In addition, the glycosaminoglycan and / or proteoglycan production promoter of the present invention can be used in various applications that can be expected to have a favorable effect on the living body due to their production. For example, it is known that proteoglycan has a preferable action such as beautiful skin on the skin tissue due to the effect of water retention, etc., and the production promoter of the present invention is administered orally or transdermally to give beautiful skin. It can also be used for applications such as medical drugs, cosmetics or foods and drinks. The dosage and the like are the same as described above.

Next, the present invention will be specifically described by way of examples.
In the examples, galacturonic acid and pectin degradation products were used as test substances.
As galacturonic acid, D (+)-galacturonic acid monohydrate (Wako Pure Chemical Industries, Ltd.) was used. A pectin degradation product having the following composition ratio provided by Koyo Chemical Co., Ltd. was used.
Composition ratio (mass) of pectin degradation products: galacturonic acid 65.0%, glucose 14.3%, galactose 5.7%, arabinose 5.0%, Na 3.2%, moisture 5.0%, other 1.8%.

Example 1
Absorption confirmation test from the intestinal tract of galacturonic acid Three white females (weight approximately 2.0-3.0 kg) were used as test rabbits, and after acclimatization to the environment for 1 week after acquisition, galacturonic acid 3 g / The head was orally administered and blood was collected over time. Blood samples were collected before administration (pre), 10 minutes, 30 minutes, 60 minutes, 180 minutes, 360 minutes, 720 minutes, and 1440 minutes after administration.
The concentrations of galacturonic acid, glucuronic acid and acetic acid contained in plasma were measured by high performance liquid chromatography using a column for carboxylic acid analysis (Shim-pack SCR-102 X 3 linked). The results are shown in FIGS.

  From FIG. 1, the galacturonic acid concentration increased immediately after oral administration, and reached a maximum at 60 minutes. After that, it rapidly decreased and returned to the value before administration in 720 minutes. On the other hand, no glucuronic acid was detected. In addition, after the administration of galacturonic acid, the plasma acetic acid concentration tended to increase gradually. That is, from the absorption experiment of galacturonic acid from the intestinal tract, it is considered that this substance was rapidly absorbed in the body, some metabolism was performed on the carboxyl group of galacturonic acid, and acetic acid was synthesized.

Example 2
Healing test of damaged cartilage (1) After using 9 female white rabbits (body weight approximately 2.0-3.0 kg) (3 birds in each group) and acclimatizing the environment for 1 week after acquisition, control group , C group), galacturonic acid group (hereinafter referred to as GalU group), and pectin degradation product group (hereinafter referred to as P group). General anesthesia was performed by intramuscular administration of ketamine hydrochloride 25 mg / kg (Sankyo Yale Co., Ltd.) after intramuscular administration of xylazine 10 mg / kg (Bayer Co., Ltd.).
The left knee joint of the test rabbit under such anesthesia was shaved, disinfected with chlorhexidine gluconate (Dainippon Sumitomo Pharma Co., Ltd.) and 50% isopropyl alcohol (Nihon Shinyaku Co., Ltd.), then approached from outside the knee joint The skin was then longitudinally incised from the center of the femur toward the rough tibial surface. Further, the inside of the joint capsule was incised, and the patella and femoral joint were completely exposed by dislocation of the patella outward. And with reference to the experiment of Tamai et al. (Carbohydro-Polym. 48, 369-378, 2002), the diameter of 2 mm in the inner pulley ridge at the distal part of the femur and the upper and lower parts of the thigh pulley groove, A hole having a depth of 4 mm was produced by a drill using a micro engine. Thereafter, the surgical wound was thoroughly washed with physiological saline, and the joint capsule, subcutaneous tissue, and skin were sutured and closed using a synthetic absorbent thread 3-0PDS (polydioxanone, Johnson & Johnson Co., Ltd.). For 3 days after surgery, 10 mg / kg of oxytetracycline hydrochloride (Pfizer Inc.) was administered subcutaneously twice a day for the purpose of preventing infection.

All test substances and water were administered using rabbit water bottles. The rabbits in the GalU group were given 3 g / head / day of galacturonic acid, and the rabbits in the P group were given 3 g / head / day of the pectin degradation product. For the GalU group and P group, prepare an aqueous solution in which the daily amount of each test substance is dissolved and give it to the rabbits in each group. After confirming that the daily amount of the day has been completely drunk, tap water. Water was allowed to drink freely. Group C was allowed to drink tap water freely.
The administration period was 2 weeks each, and appetite, skin condition, body weight, presence of diarrhea, etc. were also observed during the administration period.

After the end of the administration period, euthanasia was performed by overdose (80 mg / kg iv) of pentobarbital Na (Dainippon Sumitomo Pharma Co., Ltd.), and the change in body weight at the time of surgery and autopsy was measured. In addition, after collecting the left and right lateral vastus muscles and biceps femoris, weigh the wet weight, determine the weight ratio (%) of the surgical muscle weight to the non-surgical muscle weight, An indirect evaluation was made of the degree of atrophy.
Regarding the degree of repair of the damaged hole created in the distal part of the femur, the surgical site was exposed, the degree of healing with the naked eye was observed, and the repair rate for the operation was measured by measuring the depth of the damaged hole.
In addition, after decalcifying the left femur, a tissue sample of the repair site, normal articular cartilage and growth zone was taken, and hematoxylin and eosin heavy staining (HE staining), safranin O staining and Alcian blue staining were performed. Microscopic observation and image analysis were performed.
The numerical values obtained above were subjected to statistical processing (Student t test), and if it was 5% or less, it was determined that a significant change had occurred.

The measurement and analysis methods and results are described below.
(1) Body weight measurement results Table 1 shows the body weight measurement results of each group. After surgery, weight gain was seen in group C. In the sample administration group, there was an increase in body weight and a slight decrease, but no significant difference was observed. In addition, no abnormal changes were observed in the general state in all groups, and pectin containing galacturonic acid as a constituent was used as a food additive, confirming that this dose is safe. It was.

(2) Weight comparison of hind limb muscles The muscle weight of each of the outer vastus and biceps femoris of the limb on which the operation was performed was measured, and the weight ratio to the muscle weight on the non-operative side was determined. The results are shown in Table 2. As a result of the significant difference test, a significant increase in the lateral vastus muscle was observed in all sample groups with respect to the C group. There was no significant change in the biceps femoris in all sample groups.
In group C, post-surgical waste atrophy was prominent, suggesting that pain persisted. In rabbits, the hind limbs are usually bent, and when the amount of exercise decreases due to pain, the hind limbs do not extend, so it is thought that atrophy occurred mainly in the outer vastus muscles, which are extensors. In addition, since the flexor and extensor muscles function in concert, atrophy was also observed in the biceps femoris, which is a flexor, accompanying the absence of the outer vastus. In the GalU group and the P group, since pain disappeared or was reduced at an early stage, it was considered that there was less pain, the amount of exercise increased, and atrophy of the outer vastus muscle, which is the extensor muscle, decreased.

(3) Observation of degree of healing with the naked eye The surgical site of the collected left femur was well observed with the naked eye. Looking at the degree of healing after 2 weeks, the defect hole of group C had the same appearance as that at the time of preparation, and inflammation was still continuing in the surrounding tissues. In the other two groups, inflammation in the surrounding tissue was observed as in Group C, but the defect holes were repaired to some extent. However, all the traces were clear.
The criteria for determining the degree of defect hole repair were classified as follows, and each was scored for statistical processing.
Less than 50% healing rate 0 point healing rate 50 to less than 60% 1 point healing rate 60 to less than 80% 2 point healing rate 80 to 100% 3 points Table 3 shows the degree of macroscopic repair. GalU group scored significantly higher than C group. In group P, the scores were almost unchanged compared to group C, and no significant difference was observed.

(4) Histological observation The left femur was sampled and fixed with a 10% neutral buffered formalin aqueous solution (Wako Pure Chemical Industries, Ltd.). After fixation, it was decalcified with 5% formic acid (Wako Pure Chemical Industries, Ltd.). The tissue piece that had been decalcified was cut out so that the repaired site was in the longitudinal section. After that, paraffin embedding was performed as usual, and sliced to a thickness of 5 μm with a microtome.
For staining, hematoxylin and eosin double staining (HE staining) was performed. Also, Safranin O staining, which stains PG red for the purpose of staining proteoglycan (PG), is ion-bonded to carboxyl and sulfate groups for the purpose of staining glycosaminoglycan (GAG), and acidic GAG is stained blue. Alcian blue staining (pH 2.5) was performed.

H. As a result of histological observation by E staining, safranin O staining and Alcian blue staining, infiltration of neutrophils, lymphocytes and macrophages was observed in the injured part, and an inflammation image was observed. Also, the defect pores were filled with fibrous connective tissue, with increased capillary growth and fibroblasts. In Safranin O and Alcian Blue staining, the darkly stained part in the damaged part could not be confirmed.
In the GalU group, it was confirmed that there was an increase in capillaries and inflammatory cells in the damaged part as in the C group. However, although the central part of the defect hole was filled with fibrous connective tissue, the growth of chondrocytes was confirmed in the deep part and the margin of the defect hole. As in the GalU group, chondrocyte proliferation was also observed in the P group. In both the GalU group and the P group, in the safranin O and Alcian blue staining, the deep part and the peripheral part of the damaged part were observed, indicating that the synthesis of PG and GAG was promoted.

(5) Repair rate measurement Under a microscope, the depth of the damaged hole was measured using a micrometer, and the degree of repair after 2 weeks with respect to the initial depth of 4 mm was determined as a percentage (%). The results are shown in Table 4. Both the GalU group and the P group showed a significant increase in the degree of repair compared to the C group. There was no significant difference between the GalU group and the P group.

(6) Image analysis using specially stained specimens At the repaired site where safranin O staining and Alcian blue staining (pH 2.5) were performed, 200 times the image was used with Photograb ab-300 version 1.0 (Adobe Systems Inc.) The image is captured and digitized using Adobe Photoshop 5.0 (Adobe Systems Inc.), and the number of pixels occupied by the target color tone is processed for 120,000 pixels (20,000 pixels at random). It was measured by.
Table 5 shows the image analysis results at the cartilage damage site. From the results of the image analysis, it was confirmed that the amount of PG was significantly increased in the GalU group and the P group with respect to the C group in the Alcian blue staining. In Safranin O staining, it was confirmed that the GalU group showed a significant increase in GAG relative to the C group, and the P group showed a tendency to increase the GAG relative to the C group.
That is, this shows the same result as when glucuronic acid or glucosamine necessary for GAG synthesis is administered, and it is considered that galacturonic acid acts in some way on GAG synthesis. It is done.

(1) Weight measurement result, (2) Weight comparison result of hind limb muscles, (3) Healing degree observation result with the naked eye, (4) Histological observation, (5) Repair rate measurement, (6) Special stained specimen Image analysis using galacturonic acid and pectin degradation products revealed that a good cartilage repair effect can be obtained by oral administration of galacturonic acid and pectin degradation products even though galacturonic acid is a substance that does not originally exist in the animal body.
In addition, although the increase in blood concentration of glucuronic acid due to the administration of galacturonic acid was not confirmed, the increase in acetic acid was confirmed after the administration of galacturonic acid, which contributed to the formation of chondrocytes and cartilage matrix through some metabolism It is thought to produce some kind of substance.

  The repair promotion effect was recognized in both GalU group and P group, but the repair rate was slightly inferior in P group, although there was no significant difference. The pectin degradation product used this time has a galacturonic acid content of 65%, and it is considered that the P group consumed less galacturonic acid than the GalU group.

  As is apparent from the above, galacturonic acid has no toxicity and side effects, has the effect of promoting the production of glycosaminoglycan and / or proteoglycan, which is a component of cartilage, and promotes the production of damaged cartilage. It is useful as a prophylactic / therapeutic agent for diseases caused by injury.

The time-dependent change of plasma galacturonic acid concentration and the time-dependent change of glucuronic acid concentration by oral administration of galacturonic acid are shown. The time-dependent change of the acetic acid concentration in plasma by oral administration of galacturonic acid is shown.

Explanation of symbols

In FIG. 1, ♦ indicates the galacturonic acid concentration, and ■ indicates the glucuronic acid concentration. Further, the vertical axis indicates the respective concentrations, and the horizontal axis indicates the elapsed time (units). Also, the vertical axis shows the respective concentrations, and the horizontal axis shows the elapsed time (units).

Claims (4)

Cartilage formation promoter containing galacturonic acid or a salt thereof as an active ingredient , except food and drink . A preventive or therapeutic agent for diseases caused by cartilage damage containing galacturonic acid or a salt thereof as an active ingredient , except food and drink . Glycosaminoglycan and / or proteoglycan production promoter in cartilage containing galacturonic acid or a salt thereof as an active ingredient , except food and drink . The agent for promoting cartilage formation according to claim 1 or the agent for preventing or treating diseases caused by cartilage damage according to claim 2, which contains an enzymatic degradation product of pectin containing galacturonic acid or a salt thereof , but food and drink Goods are excluded .

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